Shared and unique mechanisms of macrophage-like ... · students, Rajiv and Yodit. I thank all of them for their experimental contributions to the work. I thank as well Drs. Poubelle,

© Ryder Whittaker Hawkins, 2019

Shared and unique mechanisms of macrophage-like neutrophils in EAE

Mémoire

Ryder Whittaker Hawkins

Maîtrise en microbiologie-immunologie - avec mémoire

Maître ès sciences (M. Sc.)

Québec, Canada

ii

Shared and Unique Mechanisms of Macrophage-Like Neutrophils in EAE

Mémoire

Ryder Whittaker Hawkins

Sous la direction de :

Luc Vallières, directeur de recherche

iii

Résumé

L’ensemble des maladies démyélinisantes (e.g. la sclérose en plaques et la neuromyélite optique)

représente un fardeau majeur sur la société et sur le bien-être des citoyens affectés. Quoique des progrès aient

été fait dans la compréhension des mécanismes biologiques qui y sont sous-jacents, les causes ultimes ne sont

pas connues et il y a un besoin de développer des traitements plus pointus. Le symptôme caractéristique de

toute maladie démyélinisante est la perte de la myéline qui isole les fibres nerveuses du système nerveux central

(SNC). Cette perte est effectuée par l’action néfaste et non-contrôlée de cellules du système immunitaire : les

lymphocytes T qui réagissent contre les protéines de la myéline, les cellules B qui sécrètent des autoanticorps,

les macrophages qui phagocytent des débris de myéline, et les cellules dendritiques qui orchestrent tout.

Cependant, il est clair aujourd’hui d’après le modèle animal de la sclérose en plaques, l’encéphalomyélite

autoimmune expérimentale (EAE), que les neutrophiles sont indispensables au développement complet de la

maladie. Ainsi, la déplétion des neutrophiles prévient l’apparition des symptômes. Néanmoins, le mécanisme

d’action des neutrophiles reste à être élucidée. Le présent mémoire résume d’abord les connaissances actuelles

sur les neutrophiles dans l’EAE et la sclérose en plaques et présente ensuite des données originaux permettant

de mieux comprendre les fonctions des neutrophiles dans l’EAE. Nous démontrons que les neutrophiles infiltrant

le SNC subissent des changements moléculaires qui les activent; que leur transcriptome devient plus similaire

à ceux des macrophages et des cellules dendritiques; que les neutrophiles interagissent physiquement avec les

lymphocytes T et B in situ dans la moelle épinière enflammée; et que, dans un nouveau modèle de la sclérose

en plaques dépendant de cellules B, les neutrophiles utilisent la protéase ASPRV1 pour prolonger l’inflammation

à long terme. Ces observations améliorent notre compréhension des maladies démyélinisantes et servent de

base pour de prochaines expériences sur le rôle des neutrophiles en général.

iv

Abstract

Autoimmune demyelinating diseases (ADDs) are a leading cause of neurological disability in youth and

adults, especially in Canada; the best-known of which is multiple sclerosis (MS); others include neuromyelitis

optica spectrum disorder. These diseases are characterized by destruction of myelin and loss of nerve

conductivity leading to motor deficits and deteriorating quality of life. ADDs have been studied for nearly two

centuries and disease-mitigating therapies are now available for patients; however, a cure has not yet been

found. Demyelination proceeds largely via the reaction of autospecific T cells with endogenous myelin proteins,

in cooperation with B cells, macrophages and dendritic cells; the root cause of this autoreactivity is still unknown.

Yet it is becoming increasingly clear from the study of animal models of MS that depletion of neutrophils, an

abundant innate leukocyte population, has the potential to block the development of disease symptoms. We

therefore aim to comprehend the molecular reasons behind this phenomenon in mice and translate our findings

to the human case. This work aims, firstly, to summarize the facts known about neutrophils in MS, neuromyelitis

optica, and other ADDs, as well as in mouse models of demyelination; secondly, to present the results of

experiments on neutrophils with the model system experimental autoimmune encephalomyelitis (EAE). We have

found that neutrophils in EAE that migrate to the central nervous system undergo transcriptional and proteomic

changes that leave them in a putatively activated state. These activated neutrophils physically interact with T

and B lymphocytes in the inflamed spinal cord. Furthermore, we use an improved model of EAE, that better

describes MS, to show that neutrophils act through the novel gene Asprv1 to prolong and worsen inflammation.

This study sheds light on the subtleties of neutrophils in a societally relevant context and provides data for the

continued investigation into neutrophil biochemistry and systems biology.

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Table of Contents

Résumé ................................................................................................................................................................ iii

Abstract ................................................................................................................................................................iv

Table of Contents ................................................................................................................................................. v

List of Figures ...................................................................................................................................................... vii

List of Tables ...................................................................................................................................................... viii

Acknowledgments ................................................................................................................................................xi

Foreword ............................................................................................................................................................. xii

Introduction......................................................................................................................................................... 1

The Development of Autoimmunity ............................................................................................................. 2

Current State of Treatment .......................................................................................................................... 3

Studying Demyelination in the Laboratory ................................................................................................... 5

Neutrophils are Essential for Demyelination................................................................................................ 7

Chapter 1: Neutrophils in ADDs and EAE: Current State of Knowledge ..................................................... 9

The mechanism of neutrophil recruitment in EAE ..................................................................................... 10

Expansion and mobilization ....................................................................................................................... 13

Rolling, adhesion and crawling .................................................................................................................. 14

Extravasation ............................................................................................................................................. 15

Are Neutrophils Essential for EAE? ........................................................................................................... 16

BBB disruption ........................................................................................................................................... 16

Immunomodulation .................................................................................................................................... 18

Antigen presentation .................................................................................................................................. 18

Myelin degradation and phagocytosis ....................................................................................................... 19

How do neutrophils contribute to human ADDs?....................................................................................... 19

Clinical Potential for Neutrophil-Related Biomarkers................................................................................. 21

Next Steps ................................................................................................................................................. 22

Chapter 2: ICAM1 Identifies Transcriptionally Unique Macrophage-Like Neutrophils in EAE ................. 25

ICAM1 distinguishes extra- from intravascular neutrophils in the CNS of EAE mice ................................ 26

ICAM1+ neutrophils have a distinctive transcriptional profile revealing a potential for antigen presentation

and immunostimulation .............................................................................................................................. 31

ICAM1+ neutrophils form immunological synapses with T and B cells ..................................................... 37

Methods ..................................................................................................................................................... 40

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Chapter 3: The Protease ASPRV1 is a Neutrophil Gene and Worsens EAE via B Cells .......................... 43

ASPRV1 is specific to neutrophils and increases in the CNS during EAE and severe forms of MS ......... 46

ASPRV1 is required for the chronic phase of a B cell-dependent EAE model .......................................... 47

Methods ..................................................................................................................................................... 50

Chapter 4: Discussion and General Conclusions ......................................................................................... 53

Catchup ..................................................................................................................................................... 54

ICAM1 ........................................................................................................................................................ 54

Transcriptomes .......................................................................................................................................... 56

MHCII......................................................................................................................................................... 57

bMOG ........................................................................................................................................................ 59

ASPRV1..................................................................................................................................................... 59

Future Directions ....................................................................................................................................... 60

References ......................................................................................................................................................... 62

vii

List of Figures

Introduction

Fig. 0.1: Appearance of multiple sclerosis lesion ................................................................. 1

Fig. 0.2: Transfer of neuromyelitis optica ............................................................................. 2

Fig. 0.3: Macrophage APCs infiltrate the spinal cord in EAE............................................... 6

Fig. 0.4: Neutrophils infiltrate the spinal cord in EAE ........................................................... 7

Fig. 0.5: Depletion of neutrophils reduces EAE severity...................................................... 8

Chapter 1: Neutrophils in ADDs and EAE: Current State of Knowledge

Fig. 1.1: Neutrophils expand during EAE and depend on Th17 cells and Cxcr2 ............... 11

Fig. 1.2: Recruitment and functions of neutrophils in EAE: a working model .................... 12

Fig. 1.3: The effects of G-CSF in EAE ............................................................................... 13

Fig. 1.4: BBB destruction in EAE and its rescue by depleting neutrophils......................... 17

Chapter 2: ICAM1 Identifies Transcriptionally Unique Macrophage-Like Neutrophils

Fig. 2.1: The spinal cord of EAE mice contains two subsets of neutrophils ...................... 27

Fig. 2.S1: Generation of Catchup × Ai6 mice .................................................................... 27

Fig. 2.2: ICAM1+ and ICAM1− neutrophils are differently distributed................................ 28

Fig. 2.S2: Validation of the Catchup × Ai6 system............................................................. 29

Fig. 2.S3: Sort scheme ...................................................................................................... 32

Fig. 2.3: ICAM1+ neutrophils have a distinct transcriptional profile ................................... 33

Fig. 2.4: Protein-protein interaction networks .................................................................... 34

Fig. 2.5: Co-stimulatory molecule expression .................................................................... 35

Fig. 2.6: Neutrophils form immune synapses with lymphocytes ........................................ 37

Fig. 2.S4: Excision of H2-Ab1 ............................................................................................ 39

Chapter 3: The Protease ASPRV1 is a Neutrophil Gene and Worsens EAE via B Cells

Fig. 3.1: ASPRV1 is a neutrophil-specific marker in mouse and human ........................... 44

Fig. 3.2: Molecular dynamics simulation of murine ASPRV1............................................. 46

Fig. 3.S1: B cells are required for bMOG EAE .................................................................. 47

Fig. 3.3: ASPRV1 is required for the chronic phase of EAE .............................................. 48

Fig. 3.4: Stereological investigation of macrophages in Asprv1 mice................................ 49

Chapter 4: Discusssion & General Conclusions

Fig. 4.1: Neutrophil transcriptomes in other diseases ........................................................ 57

viii

List of Tables

Introduction

Table 0.1: Comparison of demyelinating autoimmune diseases ......................................... 4

Chapter 1: Neutrophils in ADDs and EAE: Current State of Knowledge

Box 1.1: Key questions to answer to understand and exploit neutrophils ......................... 22

ix

Dedicated to my sweetheart

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Epigraph : Nullius in verba

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Acknowledgments

First of all, I thank my supervisor, Dr. Luc Vallières, for his supervision and direction. Dr. Vallières

conceived of the project idea and supported me all the way, from applying for the scholarship that helped fund

the project, to the interpretation and presentation of results. My thanks go equally to Drs. Alexandre Patenaude

and Aline Dumas, my coauthors on this work. Drs. Dumas and Patenaude’s expertise not only in technical

matters, but in laboratory best practices, data analysis, planning of experiments, and long hours in the laboratory

were invaluable to the success of this project. I really couldn’t have done it without them. As well, this work was

the fruit of international collaboration. Thanks to Dr. Matthias Gunzer for consultation on the manuscript, on our

healthy collaboration and on providing the mice that drove this paper forward. Although we never met, thanks to

Dr. Takeshi Matsui for his insights into the ASPRV1 side of the work. Our work was also helped by a collaboration

with some fellow MS researchers at Western University, the team of Dr. Steven Kerfoot and his two wonderful

students, Rajiv and Yodit. I thank all of them for their experimental contributions to the work. I thank as well Drs.

Poubelle, Larochelle and Pelletier, for their local collaboration. For the review chapter, thanks to my coauthors

Drs. Courtney Casserly and Julia Nantes, who wrote the review with myself and Dr. Vallières. This thesis was

supported by an endMS studentship from the Multiple Sclerosis Society of Canada, who also sponsored the

review paper as part of their SPRINT program. Finally, I thank the Université Laval for the supportive, well-

connected, and encouraging environment, which supported my scientific career through conferences, seminars,

courses and close networking between labs.

xii

Foreword

The Introduction is an original work by the student. Chapter 1 is taken from the published review

article in Autoimmunity Reviews, on which the student was third author, entitled “Neutrophil perversion in

autoimmune demyelinating diseases: mechanisms to medicine” (1). The student worked in close collaboration

in the researching and writing of this review paper on neutrophils. Only the passages and figures that were

directly relevant to the content of the thesis were included. As well, figures were added by the student during the

writing of the thesis in order to improve legibility and to better summarize the progress in the field.

Chapters 2 and 3 are taken from the student’s first-author published article in JCI Insight (2). The

student contributed both to the experimental and writing aspects of this paper. The introductions and conclusions

of the article were shortened for continuity. To better introduce each subject of the article, the paper was divided

into two logically coherent sections. As well, new original figures have been added to give a better idea of the

effort that went into the project, even those parts that were considered not pertinent for formal publication. All

supplementary information to the published article can be found on the journal website.

Chapter 4 is original and situates the conclusions and results of the two included articles in the greater

scientific context. This chapter extends and replaces much of the discussion sections of the two published

articles.

A full list of references is included at the end of the thesis.

For any specific details about publication dates, coauthors, etc., please see the first two references.

1

Introduction

Multiple sclerosis (MS) is a leading cause of neurological disability that strikes spontaneously in early

age and persists throughout life. The disease is common, with a Canadian incidence of around 200 per hundred

thousand for a total of around 100,000 in the nation with a population near 35 million. Patients can expect a

chronic worsening of symptoms, being dependent on a cane or wheelchair after 15 years, and a reduced life

expectancy (3). The malady is defined by cm-sized lesions (Fig. 0.1) on the nerve fibres of the central nervous

system (CNS), which must be disseminated across anatomical space and evolve throughout time (4). The

lesions lack myelin* as visualized by a Luxol fast blue stain (5). Often the brainstem and spinal cord are affected,

and the location of lesions will dictate the symptoms experienced: ocular disorders, unsteadiness or ataxia, or

paresthesia or loss of sensation (4).

Fig. 0.1 Appearance of multiple sclerosis lesion in human white matter. a, Perivascular infiltration of immune cells; b, Loss of myelin stained dark by Luxol Fast Blue. The plaque appears as a white area surrounding the blood vessel. From the author’s own unpublished work.

Lymphocytes infiltrate in perivascular cuffs in man, where a majority of said cells are CD8+ and a

variable number are CD4+. MHC class II-positive macrophages and some few B cells are present in lesions

themselves (6). Within these lesions, the myelin sheath surrounding axons is extensively eroded, which results

in the observed neurological symptoms (3,4). In chronic cases, plaques become “inactive”, with most of the

cellular infiltrate dispersing and leaving behind a totally demyelinated area; the cycle is then repeated at other

sites in the CNS. In addition, some re-myelination can occur, and has the appearance of light myelin staining in

an obviously damaged area (3). Within lesions, concomitant with demyelination, the oligodendrocytes are

destroyed and microglia and astrocytes proliferate and adopt ‘reactive’ character (7-11).

* The cellular organelle that encircles and insulates nerve fibers.

2

MS belongs to the broader family of autoimmune demyelinating diseases (ADDs), which differ in their

specific histological details, but which all share the characteristic of loss of myelin and an autoimmune

component. Notable other ADDs are NMOSD (neuromyelitis optica spectrum disorder), a relapsing disease with

prominent inflammation of the optic nerve and large lesions in the spinal cord (3), and ADEM (acute disseminated

encephalomyelitis), a serious acute, monophasic disorder that often complicates childhood measles, chickenpox,

or smallpox or vaccinations (Table 0.1)(3,4). The NMOSD autoantigen is known: the aquaporin (AQP)-4 water

channel protein, a part of the dystroglycan complex (12,3). Autoantibodies targeting AQP-4, which is expressed

on astrocytic end-foot processes, localize to the cerebral capillary endothelium and cause the degradation of the

blood-brain barrier (BBB) (3). Importantly, antibodies collected from NMOSD-patient cerebrospinal fluid are

capable of transmitting demyelination to rats (13,14), a functional proof reminiscent of Koch’s postulates (15),

which furthermore seems to depend on neutrophils (16)(Fig. 0.2). Cellular infiltrates in NMOSD comprise

macrophages, neutrophils and eosinophils (17). On the contrary, ADEM plaques are mainly perivascular and

contain macrophages, T and B cells, and occasional plasma cells and granulocytes (3). Additionally, ADEM

patients are sometimes seropositive for IgG autoantibodies towards myelin basic protein (MBP), proteolipid

protein (PLP), MOG and crystallin epitopes (18). It should be noted that oligoclonal IgG in the CSF is a hallmark

of MS, but the targeted antigen is not known. These two examples, along with the case of MS above, serve

to highlight the diversity of clinical pictures associated with a similar demyelinating process.

Fig. 0.2 Neuromyelitis optica can be transferred to animals by injection of NMO-patient serum. a-e, disease is worsened by the injection of anti-AQP4 NMO patient serum, but not by serum from NMO patients without anti-AQP4. Statistics tabulated at right. From (13).

The Development of Autoimmunity

For autoimmunity to develop, several conditions must be met. First, mutations in susceptibility genes

are often noted in patients with familial autoimmune diseases, including MS and arthritis, and the most salient

of these such mutations lie in the major histocompatibility complex* (or HLA genes) (19,20). These mutations

can lead to the loss of self-tolerance, whether central (by the elimination of autoreactive T cells) or peripheral

(by the action of the regulatory cells—Tregs and Bregs). Once tolerance fails, autospecific T cells can persist in

the body. Upon triggering by an infection, environmental shift, injury or other immunologically pertinent change,

* class of antigen-presenting cell-surface glycoproteins (encoded by the HLA genes in human and the H-2 genes in mice).

3

the autoantigen can be brought into contact with the self-reactive T cells. Due to non-functional mechanisms of

tolerance, the self-antigen activates the autospecific cell. One theory, the shared epitope hypothesis, holds that

the nature of the mutation is to produce an MHC that inappropriately binds self-antigen. The structure of the

major MS-predisposing MHCII allele, HLA-DRB1*1501, with its antigen was solved by Wucherpfennig and

colleagues in 1998 (21); yet, we are no closer to an understanding of the precise effects of genetics on MS.

Mutations in virtually any step of the tolerance machinery, such as a reduction in the T-cell activation threshold

or a failure in apoptosis of autoreactive cells, as well as molecular mimicry by a pathogen, could lead to

autoimmunity. The final result is an immune response directed against self-proteins (22). A provocative

hypothesis is that MS is caused by a lack of neonatal exposure to Epstein-Barr virus (EbV) (which would be

asymptomatic and protective), but instead an infection by EbV in adolescence, leading to mononucleosis and,

later on, MS; this is supported only on the basis of statistics (23). This would explain partly why the incidence of

MS is inversely correlated with prevalence of infectious disease and with GDP (24,25). Bearing the above points

in mind, it is important to further investigate any and all genetic effects on ADDs, using animal models if

necessary, in order to chart the molecular steps that lead to disease.

Current State of Treatment

While disease-mitigating therapies are available for patients with MS and other ADDs (26-37)(Table

0.1), there is currently no cure. Desensitization with myelin basic protein was tried and yielded poor results

(38,39). Some landmark therapies were the development of glatiramer (copolymer-1) in the 1990s, and more

recently, the application of immunomodulatory biologics such as rituximab and ocrelizumab. In Chapter 1 we will

discuss possible neutrophil-focused therapies in more detail.

4

Table 0.1. Comparison of demyelinating autoimmune diseases (adapted from (1).)

MS NMOSD* ADEM

Epidemiology†

Mean age at onset (years)

25-46 (can occur at any age) 30-46 (can occur at any age) 5-9 in children (rare in adults)

Sex ratio (♀:♂) 2-3 2-10 ~1

Incidence (per 100,000/year)

4-9 (CAN, EUR, USA) Unknown in CAN and USA 0.2-0.4 in children (CAN, USA)

0.1-2.6 (Asia, tropical countries) 0.05-0.4 (EUR, JPN, tropical countries) 0.1-0.6 in children (EU, JPN)

Prevalence (per 100,000)

43-350 (CAN, EUR, USA) Unknown in CAN and USA Unknown

1-27 (Asia, tropical countries) 0.5-5 (EUR, JPN, tropical countries)

Risk factors Genetics (e.g. HLA-DRB1 and NR1H3 alleles), vitamin D deficiency (low sunlight exposure), Epstein-Barr virus infection, smoking, obesity in early life

Largely unknown (probably genetic and environmental)

Recent infection or vaccination

Clinical manifestation

Common symptoms

Fatigue, weakness, numbness, balance problems, spasticity, tremor, vision impairment, pain, cognitive problems, depression, bladder/bowel/sexual dysfunction

Fatigue, weakness, numbness, balance problems, spasticity, tremor, vision impairment, pain, cognitive problems, depression, bladder/bowel/sexual dysfunction, paralysis, nausea/vomiting, hiccups, respiratory failure, narcolepsy, coma

Fatigue, weakness, numbness, balance problems, vision impairment, paralysis, headache, nausea/vomiting, fever, confusion, coma, seizures

Main forms Relapsing-remitting (85%), primary progressive (15%), secondary progressive (66% of RRMS patients convert to SPMS within 30 years)

Relapsing (90-95%), monophasic (5-10%) Monophasic

Variants Marburg’s disease, Balo’s concentric sclerosis, tumefactive MS

None (historic variants of NMO are now classified under the umbrella of NMOSD)

Recurrent or multiphasic disseminated encephalomyelitis, acute hemorrhagic leukoencephalitis, ADEM with PNS involvement

Diagnosis†

Lesion distribution

Usually ≥ 2 lesions disseminated in time and across the brain, spinal cord and/or optic nerves; are predominantly in white matter, often asymmetrical.

Lesions in the optic nerves and/or spinal cord (extending continuously over ≥ 3 vertebral segments); brain often has white matter lesions, but usually of different appearance than MS − common locations include area postrema, brainstem, diencephalon and cerebrum; affect the white and grey matter and can be bilateral

Lesions disseminated across the brain, spinal cord and optic nerves; typically large with poorly defined margins; affect the white and grey matter; less frequent in the periventricular area and corpus callosum as compared to MS

Serum aquaporin-4 IgG (% of + patients)

0 (false positive < 5%) 70-90 (using cell-based assays) 0

CSF oligoclonal IgG bands (% of + patients)

82-89 (by isoelectric focusing on agarose gel and immunodetection)

14-35 0-37 (higher in adults)

Pathology

Proposed mechanism

Autoreactive T cell-mediated cytotoxicity toward oligodendrocytes

Antibody-dependent myeloid cell-mediated cytotoxicity toward astrocytes, leading to secondary oligodendrocyte damage

Immune reaction triggered by infection or vaccination that cross-reacts with myelin or that causes secondary demyelination

Infiltrating leukocytes

CD8+ T cells, CD4+ T cells, B cells, macrophages

CD4+ T cells, B cells, macrophages, neutrophils, eosinophils

T cells, B cells, macrophages, neutrophils

Contribution of neutrophils

Perhaps in certain subtypes or phases Yes Probably yes, at least in the hemorrhagic form

Therapy

Acute (managing relapses)

Glucocorticoids (e.g. prednisone, methylprednisolone), plasmapheresis

Glucocorticoids, plasmapheresis Glucocorticoids, plasmapheresis, intravenous immunoglobulin

Chronic (modifying the disease course)

Interferon β, glatiramer acetate, dimethyl fumarate, teriflunomide, fingolimod, natalizumab (anti-ITGα4), alemtuzumab (anti-CD52), daclizumab (anti-CD25), ocrelizumab (anti-CD20), mitoxantrone, cyclophosphamide

Rituximab (anti-CD20), azathioprine, mycophenolate mofetil, methotrexate, mitoxantrone, cyclophosphamide

None

*Statistics for NMOSD are generally based on NMO and are therefore underestimates. †Numerical data are ranges of means reported in different studies from around the

world. Abbreviations: CAN, Canada; EUR, Europe; IgG, immunoglobulin G; JPN, Japan; PNS, peripheral nervous system; USA, United States of America.

5

Studying Demyelination in the Laboratory

Much of our knowledge about ADDs comes from the animal model experimental autoimmune

encephalomyelitis (EAE), in which animals (chiefly rodents) are immunized with a myelin antigen or brain extract

in order to reproduce the aforementioned symptoms of immune-driven demyelination and neurological deficits

(40,41). This paradigm can be manipulated in three distinct strategies, depending on the mechanism step

studied:

1) active EAE, induced by direct injection of myelin peptide or protein (such as MOG35-55*) with

an immunogenic adjuvant – to study antigen-mediated effects (42,43);

2) passive EAE, caused by the transfer of autospecific T cells from an active-EAE animal to an

unimmunized animal – to study the encephalitogenic potential of T-cell subsets (44,42); and

3) transgenic EAE models, in which EAE is induced spontaneously by virtue of a hard-coded

myelin-specific receptor – to study T-cell specificities, single-epitope immune responses, or

epitope spreading, or for combining EAE to other mouse models. For example, in the 2D2

mouse, the TCR † from a MOG35-55-reactive T-cell clone was cloned and knocked in to

generate a strain with monospecific autoreactive T-cells (45); in another case, an

immunoglobulin heavy chain knock-in mouse, IgHMOG, produces a B-cell repertoire with a high-

frequency of anti-MOG antibody-secreting cells and is hypersensitive to injection of MOG (46).

The pathological profile of EAE mirrors that of human ADDs, particularly ADEM (47): perivascular

infiltration of immune cells including neutrophils and lymphocytes; the symptoms comprise ascending flaccid

paralysis of the limbs and occasional motor incoordination (48). Since the causative agent in EAE is defined,

while that of ADDs is unknown, EAE does not perfectly mimic human disease. Rather, by attempting to recreate

the symptoms instead of the cause, EAE provides a reproducible test-bed in which to pick apart the immune

cascades responsible for demyelination.

* Myelin oligodendrocyte glycoprotein peptide. † T-cell antigen receptor, encoded in two chains by the Tcra and Tcrb genes.

6

Fig. 0.3 Macrophages, which can serve as APCs, infiltrate the ventral median fissure of the spinal cord in EAE. Immunohistochemistry for F4/80 antigen.

EAE in the C57BL/6 mouse is induced by injection of MOG35-55 using pertussis toxin as an adjuvant,

and since genetic manipulation in C57BL/6 mice is commonplace, this model can be used to understand the

effects of gene knockouts on EAE and hence autoimmunity. As outlined above, autoimmunity is the

(mis)direction of normally homeostatic immune processes towards destroying the structures of the self. As such,

the processes of immunity and autoimmunity are largely conserved. Self-peptide presentation to naïve CD4+ T

cells bearing cognate TCRs stimulates T-cell proliferation and, depending on the local cytokine environment, the

commitment to one of two lineages, Th1 or Th17 (1). The MOG35-55 peptide is presented by MHC class II*, and

so the resulting T-cell response is (at least initially) CD4+ and class II-restricted. Th1 and Th17 cells accumulate

in the meninges and perivascular spaces of the CNS, where they re-encounter APCs† presenting the myelin

antigen for which they are specific. Upon restimulating the T cell, the APC releases the pro-inflammatory factors

interleukin (IL)-12 and IL-23 that engender the secretion of growth factors and chemokines and further

proliferation of the autospecific T cell (49). These secreted factors mobilize classical, Ly-6Chi, monocytes from

the bone marrow to the CNS and give rise to macrophages (Fig. 0.3)(shown originally by the fact that bone-

marrow chimeras bear MHC complexes of the haplotype of the donor on meningeal monocytes and

macrophages in (50)).

These macrophages can initiate demyelination through phagocytosis (51) and seem to be required for

EAE, since disease is blocked in Ccr2 mice that are unable to recruit macrophages to the spinal cord (52).

Monocytes also give rise to CD11c+ DCs that amplify and perpetuate the immune response. Microglia, in

contrast, are not replaced by hematopoietic cells, but also contribute in their own way in EAE (50). B cells aid in

* class of MHC proteins responsible for presentation of infectious and autoimmune epitopes, borne only on immune cells. † antigen-presenting cells: canonically, dendritic cells (DCs), B lymphocytes or macrophages, bearing MHC class II complexes.

7

the generation of autospecific Th cells and promote their migration into the CNS; however, B cells also suppress

the early stages of EAE by secreting anti-inflammatory interleukin-10 (53).

Fig. 0.4 Neutrophils infiltrate the spinal cord and brain in EAE. a, b, neutrophils stained for Ly6G or marked with fluorescent reporter infiltrate the median fissure of the spinal cord (a) and brain ventricles (b) during EAE. c, neutrophils marked for Ly6G infiltrate the spinal cord beginning at day 7 post-immunization (d.p.i.) and are abundant by day 14. d, e, early observations of neutrophils in EAE; anti-Gr-1. f, g, neutrophils are abundant in the onset and peak phases of EAE and decline afterwards. From (54), (55), (56), and author’s own observations.

Neutrophils are Essential for Demyelination

Notably, neutrophils are abundantly present in CNS lesions in EAE (Fig. 0.4). These short-lived innate

immune effector cells originate from the myeloid lineage and are stockpiled in great numbers in the bone marrow

in preparation for infection or injury (57,58). Neutrophils are the first cellular line of defense towards damage to

the body, and are mobilized both to destroy pathogens and to repair organs after sterile insult (59). Neutrophils’

presence in EAE has been known from the investigations of Hoenig (60) with dyestuffs, of Määttä (55) with the

anti-Gr-1 antibody RB8-6A5, and of Segal (61) using the more specific anti-Ly6G 1A8 antibody. More details on

what is known of neutrophils in EAE are given in Chapter 1.

An important study showed that anticipatory depletion of neutrophils using anti-Ly6G could suppress

the symptoms of EAE modestly to almost totally, depending on the dose (62) (Fig. 0.5). They found that neither

myeloperoxidase nor elastase were responsible for the observed function, but hypothesized neutrophils

influenced the local environment to stimulate the expression of MHCII and co-stimulatory molecules on

macrophages and microglia. This finding has been reproduced independently (63,64,54). Work from Segal and

colleagues has shown correlative evidence for neutrophil-derived and -activating factors in the blood and

cerebrospinal fluid (CSF) of MS patients (65); yet the relevance of these observations on human disease is not

yet functionally clear.

8

Fig. 0.5 Depletion of neutrophils reduces EAE severity. a, depletion with anti-Gr-1; bcdef, depletion with anti-Ly6G; g, h, inhibition of CXCR2 has an effect reminiscent of neutrophil depletion. From (63), (62), (54), (64), and (61).

To address this lack of knowledge into neutrophils’ exact functions, we performed a study on the cell-

surface characteristics, transcriptional landscape, anatomical distribution, and cellular interactions of neutrophils

in EAE (Chapter 2). We found that neutrophils that enter the CNS parenchyma during active MOG35-55-induced

EAE are distinguished by their high surface expression of ICAM1*. Transcriptomic analysis suggested that

neutrophils expressing ICAM1 are substantially different from those that do not, with ICAM1+ neutrophils more

resembling macrophages and DCs. Various aspects of the transcriptomic data were tested in vitro and in vivo

using knockout mice. Finally, a novel model for EAE that more accurately mimics MS was developed: the bMOG

model, a collaboration with Dr. Steven Kerfoot and colleagues at the University of Western Ontario (Chapter 3).

Study of this model revealed the protease ASPRV1 as a neutrophil effector molecule, previously uncharacterized

in the immune system, that contributes to the development of chronic inflammation in EAE, and so possibly in

human ADDs.

* Intercellular adhesion molecule 1, encoded by the Icam1 gene in mouse and human; a member of the immunoglobulin superfamily.

9

Chapter 1:

Neutrophils in ADDs and EAE:

Current State of Knowledge

Adapted in part from ‘Neutrophil perversion in demyelinating autoimmune diseases: mechanisms to medicine’ (1)

Résumé : Les neutrophiles sont essentiels pour la santé et l’unité du corps, mais constituent un danger si mal contrôlé. La perversion de neutrophiles à une fonction autre que celle de l’homéostasie est bien documenté dans plusieurs maladies autoimmunes, telles le lupus, le psoriasis et l’arthrite, mais peu connu dans le cadre de la démyélinisation. Grâce au modèle animal encéphalomyélite autoimmune expérimentale (EAE), certaines molécules qui déterminent l’invasion de neutrophiles dans le système nerveux central ont été identifiées. Des mécanismes par lesquels les neutrophiles pourraient empirer la démyélinisation ont également été proposés. Chez l’humain, les neutrophiles sont abondantes dans le système nerveux dans des cas de neuromyélite optique et d’autres maladies démyélinisantes sévères; cependant, l’évidence concrète pour la présence de neutrophiles dans la sclérose en plaques manque. En dépit de ce fait, des molécules de recrutement et d’activation de neutrophiles (e.g. CXCL1, CXCL5, élastase) restent des indicateurs de sévérité pour la sclérose en plaques. L’effet des traitements courants sur les neutrophiles est discuté.

Neutrophils are essential to a healthy life, yet pose a threat if improperly controlled. Neutrophil perversion is well documented in a variety of inflammatory disorders (e.g. arthritis, lupus, psoriasis), but is only beginning to be demystified in autoimmune demyelination, the most common cause of neurological disability in young adults. Using the animal model experimental autoimmune encephalomyelitis (EAE), several molecules that help neutrophils invade the central nervous system (CNS) have been identified. Mechanisms by which neutrophils may contribute to demyelination have also been proposed (e.g. secretion of endothelial/leukocytic modulators, antigen presentation to T cells, myelin degradation and phagocytosis). In human, neutrophils are seen in the CNS of people with neuromyelitis optica spectrum disorder and other severe variants of autoimmune demyelinating diseases. At the time of autopsy for multiple sclerosis (MS) — often many years after its onset — neutrophils appear to have escaped the scene of the crime. However, new clues implicate neutrophils in MS relapses and progression. This warrants further investigating 1) the differential importance of neutrophils among demyelinating diseases, 2) the largely unknown effects of current MS therapies on neutrophils, and 3) the potential of neutrophil proteins as clinical biomarkers or therapeutic targets.

Studies on injury and infection tell us that neutrophils quickly invade the affected tissues to execute

different functions (e.g. phagocytosis, degranulation, production of reactive oxygen species, extracellular trap

formation, antigen presentation) (58). Is the same true in the context of ADDs? In this chapter, we take a

comprehensive look at how neutrophils gain access to the CNS and contribute to demyelination in EAE. We

highlight the importance of neutrophils in human diseases, such as MS and NMOSD, and propose clinical

directions. Finally, we outline future challenges in this emerging area of research.

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The mechanism of neutrophil recruitment in EAE

Under normal physiological conditions, neutrophils, like most other leukocytes, are excluded from the

CNS parenchyma* by the blood-brain barrier (BBB) (reviewed in (66)), a functional obstacle of specialized

endothelial cells interconnected by tight junctions (67). Despite, the CNS vasculature is constantly patrolled by

leukocytes that crawl on its luminal surface. These sentinel cells were discovered in 2003 (68) by transplantation

of GFP-tracked blood cells into lethally irradiated mice and were later found to develop into perivascular

macrophages under inflammatory stress (69). These cells comprise 25% neutrophils (CD11b+Ly6G+) and 75%

monocytes (CD11b+Ly6G−) (70), expand under the influence of TNF-α, interleukin-1β, and angiopoietin-2, and

are rod-shaped when patrolling through capillaries. Indeed, their movement on the endothelium is driven by a

leading edge (where actin polymerizes to push the cell front forward) and by a uropod (where microtubules

reorganize to allow retraction of the rear edge). While some rod-cells cross the BBB to replenish perivascular

macrophage pools, neutrophils do not reside in the healthy CNS.

In EAE mice (which, the reader will recall, are mice injected with myelin antigen and adjuvants), or in

mice treated with pertussis toxin (PTX), rod-shaped neutrophils expand at least four-fold in the brain circulation

(70). In contrast, the infiltration of neutrophils into the CNS parenchyma is a more specific event that occurs in

EAE, but not in response to toxin alone (as will be clear from the experiments in Chapter 2), nor to an irrelevant

antigen, nucleocapsid (61) (and see also (56,55)). These neutrophils appear in meningeal and perivascular

inflammatory foci shortly before the onset of clinical symptoms, and at the peak of disease are observed in the

CNS parenchyma within areas of demyelination and axonal damage (Fig. 0.4, Fig. 1.1a). CNS infiltration of

neutrophils (and expression of neutrophil chemokines) is also observed in passive EAE, but only when

transferring Th17 cells and not Th1 cells (61) (71)(Fig. 1.1bd). In other words, neutrophils can be engaged during

certain forms of EAE depending on the nature of the effector T cells (and thus the overall character of the

response).

Fig. 1.1 (next page) Neutrophils expand during EAE and their recruitment depends on Th17 cells and Cxcr2. a, Ly6G-expressing neutrophils expand during EAE; b, Passive transfer of Th17 cells induces a neutrophil-centric EAE; c, EAE is absent in Cxcr2 mice and is restored by transfer of Cxcr2+ neutrophils; d, Passive transfer of Th17 cells results in upregulation of the neutrophil chemokines CXCL1 and CXCL2; e, the blood-brain barrier is permeabilized in EAE, but not when neutrophils are pre-emptively depleted using anti-Ly6G. From (61) and (71).

* The grey and white matter substance of the CNS, as opposed to the connective-tissue meninges, which overlie it. The parenchyma is on the other side of the BBB.

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While the mechanism of neutrophil recruitment has been more extensively studied in peripheral tissues

and ex vivo models, it appears to be similar in the CNS. As illustrated in Figure 1.2 and reviewed elsewhere

(72), neutrophil recruitment includes the following general steps: 1) under the influence of pro-inflammatory

cytokines, neutrophils emigrate from the bone marrow, while endothelial cells up-regulate the expression of

chemokines and adhesion molecules; 2) these chemokines support the conversion of integrins to a high-affinity

state allowing the anchorage of neutrophils to the endothelium; 3) these adherent neutrophils adopt a polarized

shape and monitor the extracellular milieu as they crawl along the luminal wall; and 4) specific guiding cues

signal neutrophils to cross the endothelium towards the parenchyma. Molecules in each of these important steps

have been identified in the context of CNS inflammation, and are discussed below.

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Fig. 1.2 Recruitment and functions of neutrophils in EAE: a working model. a, Neutrophils sense PTX — and the other bacterial adjuvants used for immunization — through pattern-recognition receptors. This leads to the production of IL-1β, which triggers a systemic inflammatory cascade involving stromal cell-derived IL-6. At the same time, DCs take up myelin peptides and are activated by the adjuvants and neutrophil-derived IL-1β. b, Myelin-antigen-exposed DCs migrate to secondary lymphoid organs to activate T cells; the direct and indirect production of IL-1β and IL-6 by neutrophils favors the differentiation of Th0 cells into Th17 and Tfh cells. IL-6 also induces the differentiation of B cells. c, G-CSF and ELR+ chemokines (e.g. CXCL1 and CXCL2) — generated by the inflammatory cascade downstream of adjuvant-activated neutrophils and differentiated T cells — stimulate the expansion and mobilization of pools of neutrophils harbored in the bone marrow. d, Neutrophils roll on the cerebral vascular luminal wall by selectin-selectin receptor interaction. IL-6-activated endothelium allows neutrophil firm adhesion through ELR+ chemokines and adhesion molecules (e.g. ICAM1, Mac1). Thereafter, neutrophils crawl and can leave the blood through diapedesis in response to an unknown (Th17-derived) signal. e, Under the control of T cells and together with other myeloid cells (macrophages, DCs), infiltrating neutrophils participate in inflammation and demyelination by exerting immunoregulatory and effector functions (e.g. secretion of molecules disrupting the BBB and modulating other leukocytes, presentation of myelin antigens to T cells, and myelin degradation and phagocytosis via antibody-dependent and -independent mechanisms). Abbreviations: B, B cells; FcγR, Fc-gamma receptor; GAGs, glycosaminoglycans; Mac, macrophage; P, plasma cells; SELPLG, selectin P ligand. From (1).

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Expansion and mobilization

Born from stem cells in the red bone marrow, neutrophils are retained there through the

CXCL12−CXCR4 signaling pathway. They egress into the bloodstream when stimulated by CXCL1 and CXCL2,

the principal ELR motif* chemokines (73,74), acting through CXCR2. Upon inflammation, the production of

neutrophils must be scaled up to meet the higher demand for these short-lived cells, a response referred to as

emergency granulopoiesis (75). The granulocyte colony-stimulating factor (G-CSF or CSF3) plays a central role

in this process by downregulating CXCL12 and CXCR4 while upregulating CXCL1, CXCL2 and CXCR2 (76,77).

G-CSF also augments granulopoiesis by stimulating the proliferation and differentiation of neutrophil precursors

(75). G-CSF is produced by brain endothelium in response to IL-1β (78).

In agreement, neutrophils expand in the bone marrow and accumulate in peripheral hematopoietic

compartments (blood, spleen) during the presymptomatic phase of active EAE (65)(Figs. 1.1a, 1.3a). This

response is induced via the release of G-CSF and CXCL1 into the blood, since it is blocked in Csf3r mice (Fig.

1.3bc) and by anti-CXCR2 (Fig. 1.1ce). The related cytokine GM-CSF is not involved since even neutrophil-

specific Csf2rb knockout does not alter EAE (79)(Fig. 1.3d). Furthermore, adoptively transferred Th17 cells

increase the number of neutrophils in the blood (65)(Fig. 1.3e), providing convincing evidence that neutrophils

are mobilized not only in response to adjuvants, but secondary to the presence of autoreactive T cells.

Fig. 1.3 The effects of G-CSF in EAE—literature review. a, The bone marrow produces neutrophils in EAE; b, c, EAE is absent in Csf3r mice or Csf3r-to-wild-type bone marrow chimeras; d, EAE does not depend on the GM-CSF receptor, since EAE develops normally in Csf2rb mice; e, Neutrophils are increased in the blood when EAE is induced by passive transfer of Th17 cells (grey), but less so when induced by transfer of Th1 cells (black); compared with no injection (white). From (65) and (79).

* Glu-Leu-Arg, a tripeptide sequence found just before the characteristic CXC (Cys-X-Cys) chemokine motif in this subfamily. The presence of the ELR motif makes these chemokines chemotactic for neutrophils (57).

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As mentioned above, G-CSF is produced by brain endothelium upon exposure to IL-1β in culture; it is

also detected in the meninges ten days after MOG or adjuvant injection (80). Since G-CSF production is

downstream of T-cell activation, the T-cell factors that regulate G-CSF are also important to study. The Th17

cytokine IL-17 can mobilize neutrophils in EAE (71) and stimulates tissue cells to produce neutrophil-active

chemokines (57). It promotes neutrophil mobilization via G-CSF and CXCR2 ligands in other contexts. The G-

CSFR is expressed on myeloid progenitor cells, neutrophils, platelets, monocytes, and some nonhematopoietic

cell types, including endothelium (81). EAE depends on G-CSFR in hematopoietic cells, since WT mice with

Csf3r bone marrow do not develop EAE (65)(Fig. 1.3c). Since the opposite chimeras were not tested,

nonhematopoietic cells (e.g. microglia) may also respond to G-CSF. It remains to be seen whether neutrophil

mobilization in EAE is modulated by other cytokines such IL-6, which is expressed in EAE and can synergize

with G-CSF in other circumstances (69,70,82).

Rolling, adhesion and crawling

To access sites of inflammation, circulating neutrophils must first attach to the vasculature. This is

normally prevented by the glycocalyx, a layer of glycoproteins and polysaccharides that coats the luminal

endothelial surface and masks potential binding sites. Under the action of inflammatory mediators (e.g. IL-1β,

TNF, IL-6, histamine), this protective layer is partially shed by proteases (e.g. heparanase), while endothelial

cells produce transmembrane adhesion proteins (e.g. E-selectin, ICAM1, VCAM1) and chemoattractants (e.g.

CXCL1, CXCL2). The latter can be released into the circulation or retained on the thinned glycocalyx by binding

to heparan sulphate proteoglycans (72). Consequently, circulating neutrophils roll on the endothelium in a stop-

and-go manner through the glycoprotein selectin P ligand (SELPLG), which binds transiently and with low affinity

to endothelial selectins. This rolling motion slows down the flow of neutrophils, giving them more time to interact

with glycocalyx-bound chemokines via CXCR2 accumulated at the leading edge (83). The CXCR2 signaling

pathway, among other neutrophil-specific signals, then stimulates heterodimeric integrins (e.g. Mac-1, LFA-1) to

adopt an active conformational state that has high affinity for the corresponding ligands (e.g. ICAM1, VCAM1).

This allows neutrophils to firmly adhere to the vasculature, and subsequently spread and crawl along the

chemokine gradient (84). This process is promoted by collisions of platelets with the neutrophil’s uropod (83).

Evidence to date suggests that the recruitment of neutrophils follows the same general principles in the

CNS as in the periphery, although few studies have directly demonstrated the specific nature and dynamics of

the molecules involved in EAE. The increased number of crawling neutrophils in the CNS of EAE mice can be

attributed to two causes: the microbial adjuvants used for immunization (Freund’s adjuvant, PTX) and the

antigen-specific T cell response within the CNS (Fig. 1.2). In the periphery, the adjuvants are detected by

immune and non-immune cells through membrane-bound and cytoplasmic pathogen recognition molecules (e.g.

Toll-like and Nod-like receptors). The signaling pathways of these receptors converge to induce the transcription

15

factor NF-κB, which drives the expression of inflammatory mediators, notably IL-1β. The latter is a master

regulator that triggers a systemic molecular cascade, leading to increased neutrophil patrolling. This recruitment

can be further enhanced by IL-1β produced locally in the CNS by different cells after reactivation of myelin-

specific T cells (78).

The bacterial toxin PTX from B. pertussis is commonly used as an immunogenic adjuvant to potentiate

EAE and can be considered a simplified system for studying neutrophil activation and recruitment. PTX’s ADP-

ribosyltransferase activity modifies α subunits of G proteins, leading to recognition via TLR4. Myeloid-cell

activation of TLR4 occasions the synthesis of IL-1β under an inactive form (85). In parallel, PTX activates,

through GTPase modifications, the inflammasome sensor pyrin, which in turn induces the activation of caspase-

1 and the cleavage of pro-IL-1β (85,86). Now in its active form, IL-1β stimulates nearby stromal cells to release

IL-6, which circulates to induce expression of ICAM1 and CXCL1 on the cerebral endothelium (70,82).

Neutrophils can then roll on the endothelium via P-selectin and adhere via CXCR2 and Mac-1 (discussed above).

In sum, upon activation in peripheral tissues, the innate immune system can induce neutrophil crawling at distant

sites, including the CNS vasculature, via a pyrin−IL-1β−IL-6 cascade (Fig. 1.2).

Alternative mechanisms have also been found to induce the recruitment of crawling neutrophils in the

CNS vasculature. In the presence of bacterial lipopolysaccharide (LPS), neutrophil adhesion is not mediated by

IL-6, but rather involves a direct action of IL-1β and TNF on the endothelium (69). LPS also induces the synthesis

of angiopoietin-2 (69), which potentiates the action of IL-1β and TNF on the endothelium. Angiopoietin-2 acts by

antagonizing the inhibitory effect of its receptor, Tie2, on the NF-κB pathway.

Other alternative mechanisms of neutrophil recruitment include as a feature the engagement of diverse

pathways leading to IL-1β processing. Besides pyrin, four other sensors have been established so far to form

functional inflammasomes, each responding to a variety of stimuli (87) (88). The Freund’s adjuvant used to

induce EAE is a mixture of bacterial components comprising such stimuli. IL-1β can also be processed by

inflammasome-independent mechanisms; it is therefore not surprising that EAE is not attenuated (or only

partially (89)) in mice lacking inflammasome sensors. Further research is required to elucidate the complex

mechanism of neutrophil recruitment in EAE. Special emphasis should perhaps be placed on convergence

points among the various inflammatory pathways, which may represent more optimal therapeutic targets.

Extravasation

During intraluminal crawling, neutrophils screen the vascular surface for signs of inflammation. From

there, they can leave the circulation by a process termed extravasation. Two routes have been described:

paracellular (between endothelial cells) and transcellular (through an endothelial cell). Extravasation requires an

intricate combination of transmembrane (e.g. PECAM1, ICAM1, VCAM1, CD99, CD99L2, JAMs, ECAM, VE-

16

cadherin, Mac1, VLA4) and cytoplasmic proteins (e.g. LSP1, cortactin, GTPases, kinases) (90,91). Very little

research has investigated this process for neutrophil entrance into the CNS. Ultrastructural studies on cultured

cerebral endothelial cells support the notion that neutrophils primarily take a transcellular route to penetrate the

BBB (92,93). Future therapies should aim to address the following two questions:

1) does CNS extravasation involve BBB-specific molecules; and

2) what signals inform neutrophils where to extravasate during EAE?

One could expect that these signals include CXCR2 ligands; however, these ligands probably influence

extravasation indirectly by acting on earlier steps (mobilization, adhesion, crawling). This is supported by the

observation that CXCR2 ligands are abundantly produced by cerebral endothelial cells under other types of

inflammatory conditions (e.g. endotoxemia) in which neutrophils do not infiltrate the brain (82). Alternatively, it is

possible that neutrophils mainly extravasate in BBB-devoid regions (e.g. the meninges), from where they could

cross the parenchyma-limiting membrane (glia limitans). This would be consistent with the fact that neutrophils

are mainly found in submeningeal inflammatory foci at the peak of EAE (56,80).

Are Neutrophils Essential for EAE?

Are neutrophils a necessary condition for EAE? The literature so far indicates “yes”. Neutrophils have

long been overlooked in the field of autoimmunity, possibly since functional studies have been hampered by the

paucity of tools for neutrophil-specific identification and manipulation. However, recent observations

demonstrate the critical importance of neutrophils in active EAE: administration of anti-Ly6G or anti-Gr1 antibody

impedes EAE development (Fig. 0.5). This effect is due to the elimination of neutrophils by antibody-dependent

phagocytosis in macrophages (94,95) and not to a loss of function of Ly6G, since Ly6G-deficient mice normally

develop EAE (96). Interfering with the mechanism of neutrophil recruitment at any level blocks or attenuates

EAE. This is seen with inhibition of CXCR2 (61,65,64,97), CXCL1 (82,65), G-CSF and G-CSFR (65), IL-1R (98),

pyrin (85) and ceramide synthase 2 (99), and the resistance of Cxcr2 mice to EAE can be overcome by transfer

of Cxcr2+ neutrophils (Fig. 1.1c). Furthermore, EAE is exacerbated by factors that positively influence neutrophil

recruitment and action, such as a deficiency in ceramide synthase 6 (100) and administration of G-CSF between

immunization and disease onset (101). Overall, these studies demonstrate the harmful nature of neutrophils in

EAE. Although the underlying mechanism is still obscure, four non-mutually exclusive hypotheses are described

below.

BBB disruption

The BBB is temporarily altered in EAE, allowing the intrusion of leukocytes and blood-borne molecules

(e.g. immunoglobulin, fibrinogen, and test materials such as fluorescent tracers, Evans blue dye or gadolinium)

17

into the CNS (Fig. 1.4) (102). This likely results from the breakdown of endothelial tight junctions and substrate-

specific transporters as well as the degradation of the vascular wall matrix and basement membrane (103).

While several mechanistic explanations have been proposed, recent studies suggest that neutrophils are the

main culprit. As one of the first responders in EAE, neutrophils appear in the CNS shortly before the emergence

of clinical symptoms and concomitantly with BBB disruption (61,98) (Figs. 0.4, 1.1, 1.4). Further, depleting

neutrophils in circulation mitigates both BBB permeability and clinical severity of EAE (61,98) (Figs. 0.5 and

1.4). In theory, neutrophils could contribute to BBB disruption via contact-dependent mechanisms and by

releasing a variety of molecules, including cytokines (IL-1β, TNF, IL-6, IL-23), free radicals (nitric oxide, reactive

oxygen species) and proteases (e.g. MMP8). However, while neutrophils are known to express these molecules

in EAE, a functional experiment has yet not been performed. As BBB disruption can also occur in EAE models

without neutrophil infiltration, it seems that neutrophils are not the only ones with the key to unlock the BBB.

Fig. 1.4 BBB destruction in EAE and its rescue by depleting neutrophils. a, The BBB is permeabilized (as judged by release of blood Alexa-594 into parenchyma) starting ~1 day before onset (panel 2) and concomitantly with LysM+ neutrophil recruitment. Panels: 2 days before disease onset, 1 day before onset, day of onset, 2 days after onset and 4 days after onset. b-d, Quantification of leakiness of the BBB to Alexa-594 (b); to Alexa-594 and to cells (c); and its correlation with EAE score (d). e, BBB permeability is inhibited by depletion of neutrophils with anti-Ly6G (left) or with anti-Gr-1 (right). From (98) and (61).

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Immunomodulation

To mount coordinated attacks, immune cells must communicate with each other through contact-

dependent mechanisms and extracellular chemical messengers (e.g. interleukins, chemokines, growth factors,

lipid mediators, alarmins). Accordingly, neutrophils have been shown to interact with DCs, T cells and B cells in

different experimental settings (104-107). In EAE, a recent study presents neutrophils as stimulators of DCs (62),

which is in agreement with their overall deleterious effect. The authors found that neutrophil depletion does not

affect the peripheral priming and CNS infiltration of T cells, but reduces the number and activation of CD11c+

cells (62). Providing more precise support that neutrophils promote APC maturation, these authors found that

neutrophils isolated from the CNS of EAE mice could stimulate the expression of MHCII and co-stimulatory

molecules (CD80, CD86) on in vitro-differentiated bone marrow-derived DCs. Using a transwell assay, it was

demonstrated that this effect is mediated by a yet unknown soluble mediator that is not IL-6, IL-12, IFNγ or a

reactive oxygen species (ROS) (62).

The deleterious interaction of neutrophils with DCs does not exclude the possibility that neutrophils

interact with other immune cells, nor that they have immunosuppressive effects. For example, neutrophils, upon

stimulation with G-CSF, release TNF ligand superfamily member 13b (BAFF), a molecule implicated in B cell

proliferation and maturation (108,109). As B cells contribute to MS and NMOSD (110), it would be relevant to

investigate the possible interaction of neutrophils with B cells using appropriate EAE models, in which B cells

are pathogenic. Furthermore, neutrophils, under the influence of IFNγ, suppress myelin-reactive T cell

proliferation via the production of nitric oxide (NO) (111). Although this effect may seem contradictory to the

deleterious nature of neutrophils in EAE, it may be part of a negative feedback loop that might coordinate the

actions of T cells and neutrophils. This would explain why EAE is more severe in mice lacking IFNγ, IFNγR or

NO synthase (NOS2 or iNOS) (111). Another study has shown that adoptive transfer of spleen Ly6G+

neutrophils (referred to as granulocytic myeloid-derived suppressor cells) attenuates EAE by inhibiting T cell

priming in lymph nodes via PD-L1 (112). However, the question of why exogenous splenic neutrophils behave

differently than endogenous CNS-infiltrating neutrophils remains unanswered. It is possible that splenic

neutrophils have an immature phenotype or that the act of transferring them modifies their fate. In sum, there is

compelling evidence that neutrophils exert immunomodulatory functions in EAE. This provides hope that

manipulating these functions with specificity may influence the course of human demyelinating diseases.

Antigen presentation

Presentation of antigens to Th cells by APCs via MHCII molecules is a critical step in adaptive immunity.

It is therefore not surprising that MHCII alleles are the strongest genetic risk factors for MS (113). First, the

process involves the uptake of extracellular proteins by APCs and their processing into short peptides (14-20

amino acids), which are then loaded onto MHCII molecules. These steps are mediated by proteases (e.g.

19

cathepsins, legumain, IFI30) and chaperones (e.g. CD74 and H2-DM) in a series of endosomal compartments

(57). Second, the APCs must establish physical contacts with Th cells to form immunological synapses. At these

structures, cell-surface receptors and signaling components gather to generate intracellular signals that dictate

cytoskeletal and transcriptional changes, leading to T cell activation. Monocyte-derived CD11c+ DCs are

considered the main APCs in EAE (discussed in Introduction). However, a recent study challenged this view by

showing that depletion of DCs interferes with immune tolerance and aggravates EAE (114), and DCs are not

always required for the development of Th responses and can be replaced by other APCs.

An interesting possibility is that neutrophils serve as alternative APCs in EAE or contribute to epitope

spreading, whereby distinct epitopes become new targets in an ongoing immune response (115). Many studies

suggest that neutrophils can present antigens to T cells (116). While not equipped for this in steady state

conditions, neutrophils can express MHCII and co-stimulatory molecules during inflammation (62,117-120) or

when cultured with cytokines such as GM-CSF or IFNγ (117,118,120-129). Functional studies have shown that

neutrophils can process and present antigens to prime Th cells (119,127,130,131). Others have shown that

neutrophils can also cross-present exogenous antigens to CD8+ cytotoxic T cells (120,132,133). Chapter 2 will

deal more explicitly with a characterization, and an exciting finding, of MHC class II on neutrophils. The

acquisition of antigen-presenting capacity by neutrophils may well be a missing link between innate and adaptive

immune responses that causes or influences autoimmune disorders.

Myelin degradation and phagocytosis

A widely held concept is that demyelination in EAE results from the action of myeloid cells, such as

macrophages under the command of Th cells. The mechanistic details are unknown, but the final stage is

ostensibly phagocytosis of oligodendrocyte remains. A recent study has sparked renewed interest in this process

by reporting that monocyte-derived macrophages, in the CNS of EAE mice, exhibit an inflammatory phenotype

and initiate demyelination, often at nodes of Ranvier (51). This conclusion is based, in part, on 3D ultrastructural

images showing macrophages engulfing myelin debris. Interestingly, this study has also provided a few images

of neutrophils phagocytosing myelin. Another study has reported that EAE develops in Ccr2 mice, even though

CNS-infiltrating macrophages are largely replaced by neutrophils (134,135). It seems possible that neutrophils

and macrophages act similarly to degrade myelin in an antibody-dependent or independent manner.

Phagocytosis is an established function of neutrophils that deserves to be scrutinized in EAE in terms of

molecular mechanism and pathophysiological significance.

How do neutrophils contribute to human ADDs?

There is growing evidence that neutrophils are implicated in various human autoimmune demyelinating

diseases. The most compelling data comes from the study of NMOSD patients, in whom neutrophils have often

20

been directly observed in the CSF (136,137) and CNS lesions (136,17,138,16). Furthermore, injecting

autoantibodies extracted from NMOSD patients into the CNS of rodents causes neutrophil infiltration alongside

demyelination (Fig. 0.1). It has been proposed that neutrophils contribute to NMOSD through antibody-

dependent cell-mediated cytotoxicity (ADCC) (139), but this remains to be proven experimentally and does not

exclude other mechanisms. For MS and ADEM, direct evidence of CNS neutrophil infiltration has been limited

to rare variants of these illnesses that are severe and often fatal (i.e. Marburg's disease and acute hemorrhagic

leukoencephalitis (17)). However, recent studies suggest that neutrophils play a role even in the more prevalent

forms of these diseases.

As explained in the Introduction, MS is characterized by acute inflammatory brain lesions, and often

first presents with a relapsing-remitting course (RRMS), such that lesions coincide with focal neurological

symptoms that subsequently improve. There are also the primary and secondary progressive forms of MS

(PPMS and SPMS, respectively), in which irreversible neurodegeneration is thought to be the driving force

behind clinical disability. In these main forms, direct evidence for CNS neutrophil infiltration has generally not

been found (17), but this does not necessarily exclude a role for neutrophils in MS. It may be that available tissue

samples from autopsies of people with this chronic, multiphase disease do not capture the transient role

neutrophils could have had in the initial formation of CNS lesions. Another possibility is that neutrophils have

been missed due to their plastic nature, allowing them to adopt macrophage and DC characteristics under

inflammatory conditions (116). This may make neutrophils difficult to distinguish from other myeloid cells using

conventional histological methods, especially given that there is no known neutrophil-specific marker for humans.

A further possibility is that, in MS, neutrophils have their effect mainly in the periphery, rather than directly within

the CNS. For example, by producing inflammatory mediators in response to innate immune stimuli, they may

reduce the activation threshold for autoreactive T and B cells in lymphoid organs (140). All things considered, it

is important to look beyond histopathology when studying the possible contribution of neutrophils in MS.

Several lines of indirect evidence suggest that neutrophils play a role in human multiple sclerosis. First,

the levels of many neutrophil-related molecules (G-CSF, CXCL1, CXCL8, CXCL5, neutrophil elastase) increase

in the brain (141), CSF (142) and blood (65,143,144,145) of MS patients, although probably less than in NMOSD

(146,147) and ADEM patients (148). At least some of these markers (CXCL1, CXCL5, neutrophil elastase)

positively correlate with neurological disability and radiological measures of lesion burden and activity (65). One

study even reports higher blood levels of CXCL1 and neutrophil elastase over the course of SPMS compared to

that of RRMS (145). Second, neutrophils extracted from the blood of MS patients are more primed (i.e.

potentiated and ready to fully respond to further stimulation). This was evidenced by increases in their expression

of inflammatory markers, resistance to apoptosis, and propensity for degranulation, oxidative burst and

extracellular trap formation (149,150,151). Third, a recent study found that neutrophils are present in the CSF of

21

MS patients during relapse at an early stage of the disease, and correlate with the levels of IL-17A (152), a

cytokine that supports neutrophil activation and recruitment. This is in agreement with other studies that also

report evidence of increased neutrophil activity during relapse relative to the stable remission phases of MS.

Fourth, gain-of-function mutations in Mefv, the gene encoding pyrin, an inflammasome predominantly expressed

in neutrophils and mediating the effect of PTX on EAE (85), are associated with a higher susceptibility of

developing a more progressive or severe form of MS (153,154,155,156). Finally, and not least importantly,

administration of the neutrophil growth factor G-CSF exacerbates RRMS (157,158,159,160), as has also been

observed in NMOSD (159). Collectively, these observations could be unified by the hypothesis that neutrophil

activity is a factor that influences MS development and severity.

Clinical Potential for Neutrophil-Related Biomarkers

The human-based literature summarized above brings hope that neutrophils and associated proteins

may serve as clinically useful biomarkers. At present, some paraclinical tools are used to aid differential

diagnosis and management of demyelinating autoimmune diseases (Table 0.1). For example, magnetic

resonance imaging (MRI) is the gold standard to determine the spatiotemporal pattern of demyelination

(161,162), while serological testing for the anti-aquaporin-4 antibody is important to definitively distinguish

NMOSD from MS (162,163). These tools are nevertheless far from perfect. For example, the specificity and

sensitivity vary greatly according to the serological test used (163), which could lead to a delay in diagnosis or

misdiagnosis. Furthermore, there is no reliable biomarker to identify MS subtypes and predict clinical course

(e.g. relapses, conversion of clinically isolated syndrome to RRMS or of RRMS to SPMS). We have also limited

means to predict whether a patient will respond to a given pharmacological therapy or have an adverse reaction

to the drug.

While there is a need for additional biomarkers, we are currently several steps away from translating

emerging theory on neutrophils into clinically useful tools. To assess the potential of neutrophil-related markers,

larger-scale studies are required to confirm the previously observed differences among MS subtypes and the

other autoimmune demyelinating diseases. If the presence of neutrophils in the CNS is only confirmed in certain

subsets of MS patients, this may provide an opportunity to classify patients in ways that have important

implications for clinical care. Furthermore, it might be important to assess the impact of factors such as age, sex,

disease duration and medications on the prospective biomarkers, as these factors have not always been

accounted for in prior investigations. Controlled interventional studies are also needed to determine if neutrophil

markers could be useful to predict individual treatment response. We also must assess the added value of such

biomarkers compared to those currently available or in the development pipeline.

22

Next Steps

The sum of the research over the last decade demonstrates the importance of neutrophils not only in

the animal model EAE, but also in human ADDs. Direct evidence of neutrophil perversion has primarily been

found for severe forms of diseases, including NMOSD and rare variants of MS. Indirect evidence supports the

hypothesis that neutrophils participate, or at least exert an influence, in common forms of MS. The involvement

of neutrophils in ADEM also seems likely, but remains to be firmly established.

Neutrophil neutralization thus emerges as a logical and potentially feasible goal, at least for NMOSD

(Box 1.1), and should be achieved preferably using disease-modifying therapies (DMTs) that would modulate

neutrophil activity with specificity, since wholesale depletion of these key players of innate immunity is too

hazardous. The little we know of the currently available DMTs for MS and NMOSD suggests that they affect

neutrophils in a partial and/or non-specific manner. Neutrophils should be more closely examined in future

clinical trials to better understand their relevance to treatment effect. Moreover, neutrophil-specific drugs, like

sivelestat, should be developed and tested.

We anticipate that neutrophil-related molecules will not offer a significant advantage over the

established biomarker anti-aquaporin-4 antibody in distinguishing NMOSD from MS. Rather, neutrophils could

prove useful to identify variants or stages of MS that may require a different therapeutic approach. In such a

scenario, neutrophils could be exploited to guide the choice of treatment and predict the patient’s response to

therapy.

New neutrophil-based targets and biomarkers could flow from the study of EAE. This model has already

taught us much about the mechanisms of neutrophil recruitment and action in the CNS (Fig. 1.2). It appears so

far that these mechanisms are comparable to those observed in other tissues, although differences exist (e.g.

in EAE, neutrophils do not appear to form extracellular traps like those seen in other autoimmune diseases such

as lupus). Although EAE is an imperfect representation of its human counterparts, it remains a valuable tool to

Box 1.1. Key questions to answer to understand and exploit neutrophils in demyelinating autoimmune diseases.

• What molecule(s) indicate neutrophils when and where to infiltrate the CNS in EAE? While ELR+ chemokines are

involved in earlier steps of recruitment, the molecule(s) triggering extravasation remain unknown.

• Are BBB-specific molecules involved in neutrophil recruitment?

• By which mechanisms do neutrophils contribute to demyelination in EAE?

• Could new tools for neutrophils-specific gene deletion (e.g. Catchup mouse) be exploited to study the biology of

neutrophils in EAE?

• Do neutrophils influence MS and how? For example, considering that B cell activation and CSF oligoclonal bands

are hallmarks of MS, can neutrophils influence MS indirectly via B cells as they do in other contexts?

• Could neutrophils or related molecules be exploited as biomarkers to 1) identify variants or stages of MS, 2) guide

treatment, and 3) predict patient responses?

• Could new drugs be designed to specifically neutralize neutrophil functions and mitigate demyelinating diseases

without causing neutropenia?

• Do neutrophil-specific proteins exist in human? These could facilitate the identification of neutrophils from other

myeloid cells in histopathological samples.

23

gain mechanistic insights. Studying neutrophils in EAE is relevant, at least to understand how EAE differs from

MS, which is crucial to evaluate the translational potential of preclinical findings. Historically difficult, genetic

studies of neutrophils should be facilitated in the future by the Catchup mouse, a new model that allows for

neutrophil-specific gene deletion.

In Chapter 2, we answer some of the unknowns in Box 1.1 by identifying a novel marker for

encephalitogenic neutrophils in EAE, profiling the transcriptomes of said cells, and tracing their fate using a

Catchup fluorescent reporter line. In Chapter 3, we identify one conserved molecular mechanism by which

neutrophils exert this pathogenic effect—neutrophils use the effector ASPRV1 to promote chronic inflammation

in the spinal cord in EAE.

25

Chapter 2:

ICAM1 Identifies A Transcriptionally Unique

Macrophage-Like Neutrophil Population in EAE

Adapted in part from (2).

Résumé : Le neutrophile est un type cellulaire ubiquitaire qui est nécessaire pour la réponse aux pathogènes et aux blessures. Malgré le rôle démontré des neutrophiles dans plusieurs maladies démyélinisantes, leurs fonctions détaillées ont peu été étudiées et leur phénotype cellulaire est inconnu. Nous démontrons que les neutrophiles qui infiltrent le système nerveux central au cours de l’encéphalite autoimmune expérimentale (EAE) gagnent l’expression d’un marqueur de surface, ICAM1, qui les distingue de neutrophiles « normaux » intravasculaires. L’analyse transcriptomique de cette première population montre que les neutrophiles extravasées acquièrent l’expression de plusieurs gènes de macrophage, dont le CMH de classe II et de nombreux cytokines. Appuyant cette observation, les neutrophiles observés en microscopie de super-résolution forment des synapses serrées avec des cellules T et B dans le système nerveux central. On postule que les neutrophiles démontrent un phénotype unique semblable au macrophage qui leur est adaptatif dans le milieu du CNS enflammé. Il s’agit de la première étude poussée sur les phénotypes et génomes des neutrophiles dans un modèle clinique important.

The neutrophil is a ubiquitous innate immune cell that acts to perpetuate inflammation in infection, sterile injury, & other insults. As described in Chapter 1, they are also essential for the full symptoms of EAE. Yet, beyond these exploratory findings of a general place for neutrophils in EAE development, little has been established as to the mechanisms of pathogenesis or of the molecular characteristics of neutrophils in EAE. Here we identify ICAM1 expression as a diagnostic feature of CNS neutrophils in EAE, profile the transcriptomic differences between circulating and CNS-infiltrating neutrophils, identify differentially-expressed pathways active in CNS neutrophils, and document (using 3D STED super-resolution microscopy) physical interactions between neutrophils and lymphocytes in CNS tissue. This represents the first in-depth treatment of the features, genomes, and anatomy of two neutrophil species in a mouse model of MS.

EAE proceeds, generally, through five phases, namely, preclinical (where naïve T lymphocytes are

presented with MOG antigen, are thereby primed, and traffic to the CNS (164)), onset (licensing and reactivation

of T cells; initiation of inflammation), attack (where myeloid cells execute effector functions leading to

demyelination (165)), and chronic (perpetuation by corrupted immune-cells (166)). A fifth possible phase is the

resolution of inflammation when the scales are tipped in favor of the regulatory immune system. The mechanism

responsible for the progression of acute to chronic inflammation is unknown, yet critical for understanding and

treating autoimmune diseases.

The observation that neutrophils first appear in the CNS by day 7 (Fig. 0.3) post-immunization suggests

that neutrophils act at latest in the preclinical & onset phases. Indeed, BBB breakdown dependent on neutrophils

is obvious already by day 14 (61). As detailed in the preceding chapters, it is all but certain that neutrophils are

required for full EAE. In the following Chapter, we build on the work of previous authors in investigating the

precise genes through which neutrophils exert this effect in EAE.

26

ICAM1 distinguishes extra- from intravascular neutrophils in

the CNS of EAE mice

While other studies have used depletion as a means of establishing the functional contribution of

neutrophils to the development of EAE, little about the genetic mechanisms of these phenomena has been

studied. We undertook to find neutrophil-specific genes that explain their role in EAE. As a first step towards

addressing this problem, we induced EAE by immunication of C57BL/6 mice with MOG35-55 myelin peptide in

adjuvants, and recorded the numbers and cell-surface antigens of neutrophils by flow cytometry at day 15 (Fig.

2.1). This represents a time point at which all mice had developed signs of EAE (clinical scores ranging from 0.5

to 3; mean, 1.9 ± 0.2) and at which neutrophils were expected to be mobilized to the CNS.

27

Fig. 2.1 (previous page) The spinal cord of EAE mice contains two subsets of neutrophils distinguishable by ICAM1 surface expression. a, Flow cytometry gating strategy used to analyze neutrophils (CD45+CD11b+Ly6G+CD19−CD3ε−) from the spinal cord or blood of mice euthanized 15 days after immunization either with or without MOG35-55 (EAE or sham, respectively). At this time point, the EAE mice had clinical scores ranging from 0.5 to 3 (mean, 1.9 ± 0.2). Naïve mice were used as additional controls. Dead cells and doublets were excluded. b, Quantification of the data in a revealing an increase of neutrophils in the spinal cord and blood of EAE and sham-treated mice. For the spinal cords, counts were normalized to CD45− cells as an internal control. Stars indicate

significant increases from both the naïve and sham-treated mice (✦) or from the naïve mice only (✧),

as determined by ANOVA and post hoc Student’s t test (P ≤ 0.0089). Sample size for spinal cord: 21

(EAE), 10 (sham, naïve). Sample size for blood: 13 (EAE), 7 (sham), 6 (naïve). c, Flow cytometric analysis of ICAM1 on neutrophils from the spinal cord or blood of EAE and control mice. Data were gated as in a. d, Quantification of the data in c revealing a strong increase of ICAM1 expression on neutrophils isolated from the spinal cord of EAE mice. Left charts, counts of ICAM1+ and ICAM1− neutrophils in the spinal cord and blood. Right charts, median fluorescence intensity (MFI) obtained for ICAM1 when gated on the whole population of neutrophils or only on those positive for ICAM1. Stars indicate significant increases of ICAM1+ neutrophils from both the naïve and sham-treated mice

(✦) or from the naïve mice only (✧), as determined by ANOVA and post hoc Student’s t test

(P ≤ 0.0009). Sample size as in b. e, Flow cytometric quantification of CD11b and CD45 on ICAM1+

and ICAM1− neutrophils from the spinal cord of EAE mice. Stars indicate significant increases according to Student’s t test (P < 0.0001). The right chart shows a positive correlation between CD11b and CD45 expression, as calculated with the Pearson correlation test. Sample size: 15 per group. f, Confocal images showing different nuclear morphologies in spinal cord neutrophils isolated by FACS and stained with DAPI. Scale bar: 1 µm. g, Frequency of the different nuclear morphologies in ICAM1+ and ICAM1− neutrophils separately purified from the spinal cord of EAE mice by FACS.

No intergroup difference was observed (Student’s t test, P ≥ 0.1). Sample size: 50-160 nuclei were

counted per cell subset and per mouse (total of 5 mice). From (2).

We compared these samples to cells from mice unimmunized (naïve) or injected only with adjuvant

emulsion without myelin (sham immunization). In blood, total neutrophils (CD45+CD11b+Ly6G+) were

expanded after injections both in sham and EAE (Fig. 2.1a, b). In samples of total CNS cells, however, 4.3×

more neutrophils were measured in EAE than in sham-immunized mice. These results are consistent with the

prior observation that neutrophils crawl more frequently on the CNS endothelial surface upon exposure to

adjuvants, but infiltrate the parenchyma only during EAE (70).

Interestingly, ICAM1 (CD54) was highly expressed on neutrophils from the spinal cord in EAE but not

from the blood, nor the sham-immunized mice (Fig. 2.1c, d). In addition, ICAM1+ neutrophils expressed higher

levels of CD45 and CD11b, both markers of activation (Fig. 2.1e) (167,168,169). Yet, both populations were

identical in terms of forward and side-scatter properties and, when isolated and observed microscopically with

DAPI, nuclear morphology (Fig. 2.1f, g).

Fig. 2.S1 (next page) Generation of Catchup × Ai6 mice. Crossing the Ly6G-cre line Catchup to the potent reporter strain Ai6 produces mice with fluorescent green neutrophils amenable to fate tracing via a lox-STOP-lox system.

28

To facilitate the anatomical localization of ICAM1-positive and -negative neutrophils, we generated

reporter mice with green fluorescent neutrophils by crossing (Fig. 2.S1) heterozygous Catchup (Ly6G-cre) mice

(96) to the fluorescent lineage-tracing reporter line Ai6 (170). Catchup replaces the first exon of Ly6G with a

cassette containing cre recombinase and tdTomato. The Catchup strain is generally used in heterozygous to

preserve one copy of Ly6G for identification purposes. However, the loss of Ly6G was shown not to affect

neutrophil migration capacity or viability nor cause any developmental defects (96). The tdTomato reporter built

into Catchup is not sufficiently brilliant to use except in flow cytometry, hence the use of the brighter Ai6 reporter.

The Ai6 strain contains a cre-activated ZsGreen fluorescent protein* at the ROSA26 locus. A stop codon is

flanked by loxP recombination sites preceding the fluorescent protein gene, so upon cre expression, the reporter

is permanently expressed in that cell. In this way any cells expressing from the Ly6G promoter will be ZsGreen+,

even with only transient transcription from Ly6G.

Fig. 2.2 (next page) ICAM1+ and ICAM1− neutrophils are differently distributed in the spinal cord during EAE. a, Confocal images of a spinal cord section from a Catchup × Ai6 mouse with EAE (day 15) showing ZsGreen+ neutrophils (arrows) infiltrated in meningeal and submeningeal inflammatory foci. Note the multilobed morphology of their nucleus stained with DAPI. The right images are higher magnification views of the box in the left image. The dashed line delineates the leptomeninges. Scale bar: left, 100 µm; right, 10 µm. b, High magnification of a ZsGreen+ neutrophil immunostained for Ly6G. Scale bar: 5 µm. c, An ICAM1− neutrophil with a rod-shaped morphology crawling on the luminal surface of an ICAM1+ capillary (white, immunostaining for laminin revealing the endothelial basal membrane). Right images are y-z sections taken at the dashed line. Scale bar: 5 µm. d, Extravasated ICAM1+ neutrophils with an amoeboid morphology. Scale bar: 5 µm. From (2).

* Very similar to GFP.

Ai6

B6.Cg-Gt(ROSA)26Sortm6(CAG-ZsGreen1)Hze/J

STOP ZsGreenlox lox

ZsGreenlox

×Catchup

C57Bl/6-Ly6gtm2621(Cre-tdTomato)Arte

Ly6G-KO

cre+

Ly6G-KO

cre+

ZsGreen+

STOPlox lox

Ly6G+

Ly6g

Ly6g cre

Catchup × Ai6

cre

cre

Gt(ROSA)26Sor

Chr. 6 E3 [template strand]

wild-type

Ai6

(not to scale)

STOPlox lox ZsGreenCAG WPREFRT

E1 E2

E2

E3

E1 E2

E1

E1

E2

pA

Ly6g

Chr. 15 D3

wild-type

Catchup

(not to scale)

E3cre tdTomatoT2A pAFRT

E1 E2 E3 E4

E1 E3E2

E2

E1 E2

Ly6g

cre ZsGreen

29

We validated the model to be certain of its suitability. First, Catchup × Ai6 mice with EAE show abundant

ZsGreen-positive cells in the meninges and parenchyma (Fig. 2.2a, right). These cells have the cellular and

nuclear morphology characteristic of neutrophils and are Ly6G-positive in immunofluorescence (Fig. 2.2b).

Further, Ai6 (cre-negative) mice do not express any ZsGreen, so the model is not leaky, at least in the spinal

cord (Fig. 2.S2a). In most neutrophil cre systems, an efficiency (neutrophil expression of the reporter) of around

70% is obtained along with a selectivity (exclusivity to neutrophils) of around 60% (171). In the Catchup mouse,

the most advanced neutrophil cre system to date, Hasenberg et al. reported a 100% efficiency of expression of

the marker tdTomato in Gr1-bright, Ly6G-positive neutrophils and a 98% selectivity (Fig. 2.S2e-h). The 2% was

supposedly made up of eosinophils, as no expression was detected in other leukocytes. However, their analysis

was confined to the blood, spinal cord, spleen and liver. Further, when they measured the characteristics of their

IVM-red × Catchup line (a more powerful reporter for microscopy imaging), they obtained an efficiency of 93%

(blood), 89% (spleen), or 78% (spinal cord) at best, with a selectivity of 99.5% (96).

Fig. 2.S2 (next page) Validation of the system for fluorescent neutrophil tracking. a, No ZsGreen expression is seen in the parent Ai6 line. b, distribution of ZsGreen labeling in Ai6 x Catchup mice among various myeloid subsets. c, proportion of cells in each myeloid subset positive for ZsGreen. d, False positives could be due to phagocytosis of ZsGreen+ neutrophil debris by Iba1+ macrophages. e, Efficiency of IVM-red reporter expression in the Catchup model. f, Specificity of the Catchup model in spleen and minimal expression in liver. g, h, minimal stray expression in eosinophils or basophils. i, Almost no double-positive Iba1+ZsGreen+ cells were found from lineage-tracing experiments. j, the rare Iba1+ZsGreen+ cells are neutrophils and not transdifferentiated. From (2). Panels e-h from (96).

DAPILy6G

ZsGreenMerge

ZsGreen/DAPI ZsGreen/DAPI DAPI

ICAM1

DAPI

Merge

Laminin

ZsGreen

ICAM1 Laminin DAPIMerge ZsGreen

30

In our hands, we measured a 78% expression of ZsGreen on total spinal cord neutrophils, with minor

expression in DCs, macrophages, and microglia, but negligible expression in B and T lymphocytes (Fig. 2.S2b,

c). The selectivity was around 70%. Yet, upon confocal microscopical observation, many of the Iba1-positive

macrophages and microglia contained ZsGreen-positive granules within their cell bodies (Fig. 2.S2d). We take

this as evidence that the aberrant ZsGreen fluorescence was in part due to phagocytosis. Scarcely any double-

positive ZsGreen+Iba1+ cells were observed by stereology on spinal-cord sections from EAE mice (mean Iba1+,

4,380; mean ZsGreen+, 1,150; mean ZsGreen+Iba1+, 6 cells per section, n = 8 from 4 biological replicates)(Fig.

2.S2i). Plus, Iba1 is not precisely specific to macrophages – often, these double-positive cells more resembled

neutrophils that had picked up anti-Iba1 immunoglobulin than macrophages expressing ZsGreen (Fig. 2.S2j).

While Ly6G is known to be transiently expressed in monocyte progenitors during development (96), we did not

see this represented in our data. We conclude that transdifferentiation of mature neutrophils into monocytic cells

is not consistent with the present findings.

In the EAE spinal cord, ICAM1 was detected on capillaries, but not on intravascular neutrophils that

exhibited the rod-shaped morphology typical of crawling leukocytes (69) (Fig. 2.2c). In contrast, ICAM1 was

detected on the vast majority (> 90%) of extravascular neutrophils in the meninges and parenchyma (Fig. 2.2d).

Neutrophils were also observed in the vasculature of naïve and sham-treated mice (where they were negative

for ICAM1), but never in the parenchyma, as previously reported (70,85).

We conclude the existence of two populations of neutrophils in the CNS of EAE mice: one patrols the

CNS vasculature by crawling on its inner surface and is characterized by the absence or very low levels of

ICAM1; the other, more abundant, is recruited into the meninges and parenchyma by an antigen-driven

mechanism and is characterized by strong expression of ICAM1 and higher levels of CD11b and CD45. This

phenotype suggests a state of increased activation. We propose that circulating ICAM1− neutrophils (non-

31

activated) have the potential to transmigrate across the CNS vasculature and to mature into ICAM1+ neutrophils

(activated) that contribute to EAE development.

ICAM1+ neutrophils have a distinctive transcriptional profile

revealing a potential for antigen presentation and

immunostimulation

To measure the differences between ICAM1-positive and -negative neutrophils at a deeper level with

an eye to understanding their functions and the origins of their anatomic characteristics, we performed a

comparative transcriptomic analysis with Affymetrix Mouse Gene 2.0 ST arrays, interrogating 28,137 coding

transcripts. Cells from the spinal cord of EAE mice at day 15 post-immunization (at peak EAE) were collected

and purified by fluorescence-activated cell sorting in the following way (Fig. 2.S3): After selecting for live single

cells, CD45– non-immune cells were excluded. CD3+ T and CD19+ B lymphocytes were excluded. Neutrophils

were selected with Ly6G+. These were further divided into CD11bhi ICAM1hi and CD11blo ICAM1lo; macrophages

and dendritic cells were Ly6G–, CD11bhi and (in the case of DCs) CD11chi. Total RNA weighing 75 ng was

isolated from each cell type. The GeneChip measures transcript abundance by hybridation of cellular mRNA to

microprinted chips coated with wells of predefined primers towards common genes. An alternative method that

is now arising is RNA-seq, which has the advantage of being unrestricted to already known genes or

chromosomal regions; yet it brings with it its own particular issues, such as cost and underrepresentation of

certain transcripts. Thus, for our purposes, that of providing an overview of neutrophil transcriptional changes at

a global level, the microarray method was appropriate and powerful. Data were normalized and quantified using

the RMA algorithm. Raw and processed data have been deposited in the ArrayExpress database at EMBL-EBI

(www.ebi.ac.uk/arrayexpress) under accession number E-MTAB-6160.

32

Fig. 2.S3 Sort scheme for Figure 2.3. Four myeloid cell subsets were isolated from EAE spinal cord for microarray analysis: ICAM1+ neutrophils (N+), ICAM1– neutrophils (N-), macrophages (Mc) and DCs (DC).

Surprisingly, unsupervised hierarchical clustering revealed that ICAM1+ neutrophils shared more in

expression with macrophage and DC than with ICAM1– neutrophils (Fig. 2.3a, top). Expectedly, the biological

replicates clustered as most closely related. Genes were filtered according to the following criteria: fold change

≥ 3, mean hybridization signal ≥ 200 in at least one subset, and inter-group P-value ≤ 0.05 comparing

ICAM1+ and ICAM1– groups. These criteria are quite stringent and are stricter than what is generally found in

the literature (see e.g. (172)). The differentially expressed genes could be divided into three modules as follows:

479 genes in total modulated, of these 343 upregulated in ICAM1+ neutrophils and 136 downregulated (Fig.

2.3a). Hence, neutrophils are functionally plastic in EAE: after extravasation into the inflamed CNS, they acquire

distinct properties through a substantial transcriptional remodeling.

33

Fig. 2.3 ICAM1+ neutrophils have a distinct transcriptional profile suggestive of a capacity for immunomodulation and antigen presentation. a, Heat map of mRNAs differentially expressed in ICAM1+ and ICAM1− neutrophils (N+, N−) compared to each other or to CD11b+CD11c−Ly6G− macrophages (Mc) and CD11b+CD11c+Ly6G− dendritic cells (DC). These cells were simultaneously purified from the spinal cord of EAE mice by FACS and analyzed by DNA microarray. The hierarchical clustering dendrogram shows the degree of similarity among the samples (biological duplicates). The color scale indicates the hybridization signal intensity. The criteria used for comparison were as follows: fold

change ≥ 3; hybridization signal ≥ 200; Student’s t test, P ≤ 0.05. b, Frequency distribution, according

to biological function, of the 343 mRNAs identified as enriched in ICAM1+ neutrophils. c, Fold difference in the hybridization signals for mRNAs of three selected categories (blue text in b), as compared between ICAM1+ and ICAM1− neutrophils. d, Fold difference in the hybridization signals for neutrophil-specific mRNAs, as compared between neutrophils (ICAM1+ and ICAM1−) and macrophages. e, Signaling proteins expressed (light blue) or upregulated (dark blue) in ICAM1+ neutrophils, supporting the concept that these cells acquire immunostimulatory capacities. f, Schematic of the MHCII pathway showing that all of the

Ccl2

Ccl3

Ccl4

Ccl5

Ccl9

Ccr5

Ccrl2

Cmklr1

Csf1

Csf1r

Cxcl10

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Cxcl16

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Flt1

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Lifr

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Expressed but not upregulated in ICAM1+ neutrophils

MHCII pathway

H2-Aa

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34

proteins are upregulated (dark blue) in ICAM1+ neutrophils, suggesting the acquisition of antigen-presenting capacity. Color legend as in e. Abbreviations: ER, endoplasmic reticulum; MIIC, MHCII

compartment. From (2).

Fig. 2.4 Protein-protein interaction networks underlying ICAM1+ neutrophil differentiation. A map was generated using Cytoscape of the result of the computation using PPICompare. This map represents the protein-protein interactions gained (+, green) and lost (−, red) during the transition from ICAM1− to ICAM1+ neutrophils in EAE, based on the microarray analysis in Fig. 2.3.

To explain the initiating event responsible for these changes, I used the software package PPICompare

(173) to extract a minimal list of protein-protein interaction changes sufficient to explain the transcriptomic

differences (Fig. 2.4). PPICompare draws on data from the mentha and IntAct databases for networks of both

physical and genetic interactions. The minimal list of reasons predicted the gain of many interactions in ICAM1+

neutrophils for the proteins 14-3-3 epsilon, Polycomb protein EED, histone deacetylase Kat2a, cyclin-dependent

kinase CDK4, and kinase Fyn, as well as a loss of interactions with c-Kit, Lin28a, and MAP1LC3A. TLR4, MAPK8,

CD40, 4-1BBL and RANK all gained interactions. Ubiquitin C and the ubiquitin-like gene ISG15 gained

interactions, and this specific ubiquitin gene is known to be a stress-responsive form. These suggest a presence

35

for chromatin modifications (via Kat2a and EED) and for phosphoserine-mediated signaling (via 14-3-3 epsilon).

These results also suggest that more cytokine pathways than those in Fig. 2.3 may be affected, and in particular,

that Toll-like receptors may have a subtle role in guiding ICAM1+ neutrophils to the CNS during EAE. This

method is powerful in the sense that it can detect interactions that are not necessarily up- or downregulation of

transcript level, and sheds light on the bigger picture of neutrophil proteomes in disease. A more complete

analysis of this transcriptome is forthcoming.

Fig. 2.5 A fraction of ICAM1+ neutrophils exhibit surface proteins involved in antigen presentation. a, Representative cytometry plots showing the gates used to analyze MHCII and co-stimulatory molecules on neutrophils (CD11b+Ly6G+CD11c−CD19−CD3ε−) from the spinal cord of EAE mice. Dead cells, debris and doublets were excluded (see Materials and Methods). b, Quantification of the results in a revealing increases of MHCII and co-stimulatory molecules on subpopulations of ICAM1+ neutrophils. Stars indicate significant differences from the corresponding

36

ICAM1− population (✧) or from all the other populations (✦), as determined by the Kruskal-Wallis

test and post hoc Wilcoxon test (P ≤ 0.0051). Sample size: 6 mice. From (2).

Of the 343 genes that were upregulated in ICAM1+ neutrophils, 328 (96%) were also expressed by

macrophages and/or DCs at similar or higher levels. Further, 232 (68%) had a known function and could be

manually divided into 12 functional categories, including cell−cell or cell−matrix interaction, cytokine−cytokine

receptor interaction and antigen presentation (Fig. 2.3b). These three latter categories also stood out as over-

represented when the classification was done with software tools (KEGG and Ingenuity Pathway Analysis,

P < 0.0001). Notably, they included many genes coding for cytokines (e.g. IL-1α, CSF1), leukocyte

chemoattractants (e.g. CXCL9-11, CCL2-5), cell-surface receptors (e.g. CCR2, CCR5, HAVCR2, NIACR1) and

adhesion molecules (e.g. VCAN) (Fig. 2.3c). Remarkably, ICAM1+ neutrophils upregulated genes at every step

along the antigen-processing and -presentation pathway (Fig. 2.3c): proteases that intracellularly process

protein antigens (e.g. LGMN, IFI30, CTSB); subunits of the immunoproteasome (e.g. PSMB10); chaperones

necessary for MHCII complex formation (e.g. CD74/Ii, H2-DMa); MHCII subunits themselves (H2-Aa, H2-Ab1,

H2-Eb1); and co-stimulatory molecules (e.g. CD40, CD48, CD83, CD86).

We confirmed the presence of MHCII and co-stimulatory molecules by flow cytometry on a considerable

proportion of ICAM1+ neutrophils: 12.5% expressed high levels of MHCII with co-stimulatory molecules, 50.7%

expressed moderate levels of MHCII, and 36.8% were MHCII-negative (Fig. 2.5). These results indicate that

neutrophils, after infiltrating the CNS, acquire macrophage/DC properties, including the potential ability to

secrete immuno-stimulatory factors and present antigen to lymphocytes (Fig. 2.3e, f).

Another 104 genes were predominantly expressed in neutrophils and not or weakly in the other myeloid

cells (Fig. 2.3a). Among these neutrophil-unique genes were cell surface antigen Ly6G, chemokine receptor

Cxcr2, matrix metalloproteinase Mmp9, all known markers for neutrophils the recently discovered neutrophil

cytokine Il1f9 (174) (also called IL-36γ), the enzyme histidine decarboxylase (Hdc) that synthesizes histamine,

and new potential neutrophil markers with unclear functions (e.g. Asprv1, Chi3l1) (Fig. 2.3d). 13 of these 104

neutrophil-unique genes were enriched ≥3-fold in ICAM1+ neutrophils, including Mreg and Il23a.

To determine whether these ICAM1+-specific genes played a role in EAE pathology, we induced EAE

in the knockout models Mregdsu and Il23a. However, no substantial differences were seen in any of the disease

parameters. We conclude that while expressed by ICAM1+ neutrophils, melanoregulin and IL-23α are not

required for neutrophils’ action in the development of EAE. In summary, it appears that neutrophils in EAE are

equipped with a specific set of molecules allowing them to execute unique functions (e.g. via Asprv1), in addition

to roles redundant with those of macrophages and DCs (e.g. via MHCII and cytokine pathways).

37

ICAM1+ neutrophils form immunological synapses with T and B

cells

Our microarray results indicate that infiltrating neutrophils upregulate proteins known to be important

for physical and functional interactions with lymphocytes (e.g. ICAM1, MHCII and co-stimulatory molecules)

(Figs. 2.3, 2.5). To determine whether such interactions occur in our model, spinal cord sections from

Catchup × Ai6 mice with EAE were stained for the T-cell marker CD3ε and the B-cell marker B220, then the

number of neutrophils physically contacting lymphocytes was estimated. Figure 2.6a gives an overview of one

such section, where neutrophils and T cells were localized both in the parenchyma and surrounding meninges,

whereas most B cells were restricted to the meninges. At higher magnification, we observed, across the tissue,

many neutrophils that were juxtaposed to T and B cells (Fig. 2.6b). In all individuals studied (four), these contacts

were frequent: in the meninges, for example, ~36% of ZsGreen+ neutrophils contacted CD3ε+ T cells, while

another ~14% contacted B220+ B cells (Fig. 2.6c).

Fig. 2.6 (next page) Neutrophils form immune synapses with lymphocytes in the spinal cord of EAE mice. a, Low-magnification confocal image of the spinal cord and meninges in a EAE mouse at day 16 post-immunization. Neutrophils (green, ZsGreen) and T cells (red, CD3ε) infiltrated the subpial parenchyma, while B cells (blue, B220) remained in the meninges (under the dashed line). Scale bar: 10 µm. b, Close-up images of neutrophils making synapses (arrows) with T or B cells (top and bottom panels, respectively). Scale bar: 2 µm. c, Frequency of immune synapses in the spinal cord parenchyma and meninges. Data are expressed as the percentage of a given synapse (y axis) among a given leukocyte population (x axis). Sample size: 359-2167 cells were counted per region per animal (total of 4 EAE mice). d, Super-resolution micrographs of a neutrophil−T cell synapse (only 3 optical sections are shown). ICAM1 and CD3ε were acquired in STED mode, whereas ZsGreen and DAPI were acquired in confocal mode. Scale bar: 2 µm. e, Three-dimensional rendering of the cells in d (all optical sections are shown). Note the absence of ICAM1 at the point of synapse (dashed line). Scale bar: 2 µm. From (2).

38

T cells can contact APCs to form immunological synapses, which are characterized by a ring of ICAM1

surrounding a central zone devoid of ICAM1 (175). The occurrence of immunological synapses in CNS tissue

has only been thus far documented between cytotoxic T cells and astrocytes during infection (176). To

investigate the fine-scale structure of neutrophil−T cell doublets, we performed super-resolution microscopy by

ZsGreen/CD3 /B220

Merge DAPI

Merge DAPI

ZsGreen

ZsGreen B220

Merge ZsGreen DAPIICAM1

ZsGreen/ICAM1/CD3 /DAPI ZsGreen/ICAM1ZsGreen/ICAM1/

39

stimulated emission-depletion (STED) with a pulsed depletion beam and time-gated detection (Fig. 2.6d). 3D

reconstructions revealed the presence of a zone devoid of ICAM1 on neutrophils at the plane of contact with T

cells (Fig. 2.6e). To our knowledge, this represents the first in vivo evidence that neutrophils form immunological

synapses with T cells. Our observations suggest that neutrophils recruited to the mouse CNS during EAE can

physically engage with T and B cells and form immunological synapses with T cells, reminiscent of antigen

presentation.

To directly test the importance of the antigen presenting capability of neutrophils in EAE, we crossed

Catchup mice to a conditional null strain for H2-Ab1 (177), the only extant MHCII β-chain allele in C57BL/6 mice

and among those genes upregulated in ICAM1+ neutrophils, in order to abolish MHCII expression specifically in

neutrophils. Gene deletion (> 80%) was confirmed in genomic DNA obtained from spinal-cord neutrophils (Fig.

2.S4a), yet, perplexingly, H2-Ab1 mRNA and protein were unchanged in the same samples (Fig. 2.S4b-d). Thus,

H2-Ab1 adds to a list of genes that cannot be studied in the Catchup model, perhaps because the deletion is

partial, occurs after mRNA transcription or is bypassed by the acquisition of molecules from other cells (e.g. via

trogocytosis or vesicular transfer). This confounding result could be explained by longevity of mRNA transcripts

in neutrophils, which are known to be transcriptionally and translationally repressed (172).

Fig. 2.S4 Cre-mediated excision of the H2-Ab1 gene in neutrophils does not reduce H2-Ab1 mRNA and surface MHCII. a, Quantification of the H2-Ab1 gene exon 1 (floxed) by qPCR in neutrophil subsets purified by FACS from the spinal cord of Ly6gcre/cre (Catchup) and Ly6gcre/creH2-Ab1fl/fl mice with EAE. Data were normalized to exon 3 (not floxed). Stars indicate significant excision (> 80%), as determined by Wilcoxon test (P < 0.0001). Sample size: 4-6 mice per group. b, Quantification of H2-Ab1 mRNA by RT-qPCR in spinal cord neutrophils using primers directed at exon 1. Data were normalized to Hprt mRNA and revealed no intergenotype difference. Sample size: 5-6 mice per group. c, d, Flow cytometric analysis of MHCII showing no difference in the percentage of neutrophils that were positive for MHCII (c) nor in the amount of MHCII on the surface of these cells (d). MFI, median fluorescence intensity. Sample size: 5-6 mice per group. From (2).

In conclusion, this study has provided a tool with which to identify activated neutrophils in EAE (the

surface marker ICAM1), has measured their transcriptomes and found startling similarities to macrophages and

DCs as well as novel effector molecules; and has documented the formation of immune synapses in vivo in EAE.

These all point to an emerging picture of neutrophils as important effectors, communicators and initiators in EAE

and in general. A full discussion is reserved for Chapter 4. In the next Chapter we focus on one gene found to

have an important role in mediating the effect of these neutrophils in EAE.

Ly6gcre/creH2-Ab1

Ly6gcre/cre (control)

40

Methods

Animals: C57BL/6, Ai6 (170) and H2-Ab1tm1Koni (177) mice were obtained from Jackson Laboratory.

Il1f9−/− (174), Mregdsu (178) and Catchup (96) mice were derived from previously established colonies. Genotypes

were confirmed by PCR. Experiments were performed under specific pathogen-free conditions on mice aged 8-

10 weeks with the approval of our institutional animal protection committee.

EAE induction: Mice were subcutaneously injected into both flanks with a total of 200 μL of emulsion

containing either 300 μg of MOG35-55 (Synpeptide) or 500 μg of bMOG (see above) dissolved in saline and mixed

with an equal volume of complete Freund's adjuvant containing 500 μg of killed Mycobacterium tuberculosis H37

RA (Difco Laboratories). Mice were also intraperitoneally injected with 20 μg/kg of PTX (List Biological

Laboratories) immediately and 2 days after immunization.

EAE scoring: Mice were weighed and scored daily as follows: 0, no visual sign of disease; 0.5, partial

tail paralysis; 1, complete tail paralysis; 1.5, weakness in one hind limb; 2, weakness in both hind limbs; 2.5,

partial hind limb paralysis; 3, complete hind limb paralysis; 3.5, partial forelimb paralysis; 4, complete forelimb

paralysis; 5, dead or killed for humane reasons.

Flow cytometry: Blood samples were harvested by cardiac puncture in EDTA-treated cuvettes and

treated with ammonium chloride solution (Stemcell Technologies) to remove erythrocytes. Mice were then

anesthetized and exsanguinated by cardiac perfusion with saline. Spinal cords were harvested, minced with

razor blades in Dulbecco's PBS, digested for 45 min at 37°C with 0.13 U/ml Liberase TM (Roche Diagnostics)

and 50 U/ml DNase (Sigma-Aldrich) in Dulbecco's PBS, filtered through 40-μm cell strainers, then separated

from myelin debris by centrifugation at 1,000 ×g in 35% Percoll (GE Healthcare). For immunostaining, cells were

incubated on ice for 5 min with rat anti-CD16/CD32 antibody (BD Biosciences, clone 2.4G2, 5 μg/ml) and Fixable

Viability Dye eFluor 506 or 455UV (eBioscience, 1:1000), then for 30 min with combinations of primary antibodies.

Cells were washed and resuspended in PBS before being analyzed/sorted with a FACSAria II flow cytometer

(BD Biosciences). Before analysis, the following quality control checks were performed using FlowJo (Tree Star):

debris were removed using FSC-A and SSC-A, doublets were removed using FSC-A and FSC-H, and all dead

cells were removed that were positive for the fixable viability dye eFluor 455UV (eBioscience). Gates were based

on fluorescence-minus-one or unstained negative controls. Cell counts were normalized to CD45– cells, whose

numbers are proportional to the sample volume, in order to reduce error due to cell isolation and sample variation.

Histology: Mice were transcardially perfused with saline, followed by ice-cold 4% paraformaldehyde in

phosphate buffer, pH 7.4, over 10 min. Spinal cords were removed, postfixed for 4 h at 4°C, then cryoprotected

overnight in 50 mM potassium phosphate-buffered saline supplemented with 20% sucrose. Series of sections

were cut at 14 μm using a cryostat and stored at –20°C until analysis. Immunofluorescence was performed, as

41

described previously (68), using combinations of primary antibodies. Slides were counterstained for 1 min with

2 µg/ml DAPI, then mounted with coverslips no. 1.5H and ProLong Diamond medium (Molecular Probes;

refractive index, 1.47). Cell counts were estimated using a Stereo Investigator system (Microbrightfield) on a

Nikon E800 microscope by the optical fractionator method and Gundersen error coefficients were less than 0.07.

Microarrays: Total RNA was extracted from FACS-purified cells using the miRNeasy Micro Kit (Qiagen).

RNA (75 ng) was used to produce biotinylated single-strand cDNAs using the Affymetrix GeneChip WT Plus

Reagent Kit. The cDNAs were hybridized to Affymetrix Mouse Gene 2.0 ST arrays for 16 h at 45°C with constant

rotation at 60 rpm. The arrays were washed and stained using the Affymetrix GeneChip Fluidics Station 450,

then read using the Affymetrix GeneChip Scanner 3000 7G. Data were processed using the RMA algorithm in

Affymetrix Expression Console and filtered in Microsoft Excel. Using the MeV software (TIGR), filtered data

(intensity > 100 in at least one group; standard deviation > 100) were log2-transformed and analyzed by

hierarchical clustering using Spearman correlation. KEGG and Ingenuity Pathway Analysis were used to identify

enriched pathways.

RT-qPCR: Total RNA was extracted from cells, spinal cord and blood using EZ-10 Spin Column Animal

Total RNA Mini-preps Kit (Bio Basic), TRI-reagent (Sigma-Aldrich) and RNeasy Protect Animal Blood Kit

(Qiagen), respectively. First strand cDNA was generated from 1-5 μg of RNA using Superscript III (Invitrogen)

with random hexamers and 20-mer oligo-dT primers, then purified using the GenElute PCR Clean-Up Kit (Sigma-

Aldrich). The product (20 ng) was analyzed using the LightCycler 480 system with the SYBR Green I Master mix

and primers according to the manufacturer’s instructions (Applied Biosystems). The PCR conditions consisted

of 45 cycles of denaturation (10 s at 95°C), annealing (10 s at 60°C), elongation (14 s at 72°C) and reading (5

s at 74°C). The number of mRNA copies was determined using the second derivative method (179).

Confocal and STED microscopy: Confocal images were acquired with a Leica TCS SP8 STED 3X

microscope (equipped with white-light laser, 405-nm diode laser and HyD detectors) by sequential scanning

using the following settings: objective, HC/PL/APO 63×/1.40 oil or 10×/0.4 dry; immersion oil, Leica Type F

(refractive index, 1.518); scan speed, 600 Hz; line average, 2-4; time gate, 0.3-6.0 ns. Laser power and gain

were set to optimize signal-to-noise ratio and avoid saturation using the QLUT Glow mode. Sizes of pixel, pinhole

and z-step were set to optimize resolution or to oversample in the case of images to be deconvolved. STED was

performed with a 775-nm depletion laser and the following adapted settings: objective, PL/APO 100×/1.40 oil

STED White; line average, 6-8; time gate, 0.5-6.0 ns; STED 3D, ~50-75%; depletion laser intensity, 60% (Alexa

Fluor 594) or 30% (Atto 647N). Deconvolution was performed with Huygens Professional (Scientific Volume

Imaging) using a theoretical point spread function, manual settings for background intensity and default signal-

to-noise ratio. 3D reconstruction was performed with LAS X 3D Visualisation (Leica). Color balance, contrast

and brightness were adjusted with Photoshop (Adobe).

42

Statistics: Unless otherwise indicated, data are expressed as mean ± standard error. In general, means

were compared with non-parametric (Wilcoxon, Kruskal-Wallis) or parametric tests (Student’s t test, ANOVA)

when data were continuous, normally distributed (Shapiro-Wilk W test) and of equal variance (Levene’s test).

EAE incidence curves were constructed using the Kaplan-Meier method and compared by Wilcoxon test. EAE

severity curves were compared by two-way ANOVA with repeated measures using rank-transformed scores,

followed by Wilcoxon test for pairwise comparisons using untransformed scores. Correlation between variables

was determined using the Spearman’s or Pearson’s test. All these analyses were performed with JMP (SAS

Institute) using a significance level of 5%.

43

Chapter 3:

The Protease ASPRV1 is a Neutrophil Gene and

Worsens EAE via B Cells

Adapted in part from (2).

Résumé : À la suite de la caractérisation génomique des neutrophiles du Chapitre 2, nous avons identifié de nouveaux gènes spécifiques aux neutrophiles. Le premier d’entre eux était la protéase Asprv1. Nous démontrons que l’ASPRV1 est exprimée spécifiquement dans le neutrophile humain et murin dans le contexte du système immunitaire. Il corrèle également avec l’infiltration de neutrophiles dans le CNS. En utilisant une EAE dépendante des cellules B, nous démontrons que les souris déficientes en Asprv1 ne développent pas les symptômes d’envergure. La phase chronique de la maladie est freinée, et l’incidence de symptômes est également diminuée. Ceci représente peut-être une stratégie pour cibler la progression des maladies démyélinisantes dans leur enfance. En somme, les neutrophiles dans l’EAE exercent des fonctions aussi bien spécifiques que communes aux autres types cellulaires.

Our work in Chapter 2 showed that neutrophils are activated in EAE, infiltrate the central nervous system and assume macrophage-like characteristics. A closer analysis of microarray data revealed the presence of expressed genes that were neutrophil-specific but not known to date. The most salient, the aspartic retroviral-like protease ASPRV1, is specifically expressed by neutrophils in the mouse and human immune system, and correlates with CNS neutrophil infiltration in EAE. Mice lacking ASPRV1 are still susceptible to EAE induced with MOG35-55 peptide. However, the use of a new EAE model induced with the B cell-dependent myelin antigen bMOG revealed that Asprv1 mice are EAE-resistant, especially with regards to the chronic phase, and many recover completely. Thus, while neutrophils in EAE may fill roles redundant with macrophages in some aspects, they also fulfil unique functions through ASPRV1.

The results of Chapter 2 provided us with ample characterization of neutrophils in the context of CNS

autoinflammation, and also furnished some new neutrophil-specific molecules that could be used as markers.

Seeking to discover a unique function of neutrophils in EAE, we chose to follow up on ASPRV1, because it was

the most highly expressed neutrophil-specific gene with a human homolog that had not yet been studied in the

immune or nervous system, and because it encodes a little-known protease that could play a novel effector role

in inflammation. Here we demonstrate that ASPRV1 is:

1) only expressed by neutrophils in the immune and nervous systems both in mouse and human;

and

2) essential for the progression of acute to chronic inflammation, specifically when EAE is

induced with bMOG, a new MOG antigen that involves, like in MS (180) and contrary to the

traditional MOG35-55 peptide, a deleterious action of B cells.

ASPRV1 (aspartic peptidase, retroviral-like, 1) is an aspartate protease conserved in mammals down

to Rodentia. It is encoded by ASPRV1 on human chromosome 2 and by Asprv1 on mouse chromosome 6. It

was first isolated from skin epidermis electrophoreses and identified as a protease which processes the

44

extracellular matrix proteoglycan profilaggrin into filaggrin, thus maintaining hydration and barrier function

(181,182,183). ASPRV1 is believed to have been acquired from a retrovirus and incorporated into the host

genome (184). Indeed, ASPRV1 contains only a single exon and is not spliced; it also has homology to HIV-1

protease as well as to yeast Ddi1p (DNA-damage inducible 1). ASPRV1 has so far only been detected in

stratified epithelia where it cleaves its only known substrate, profilaggrin (183,182). ASPRV1’s catalytic activity

is dependent on a conserved protease domain with a catalytic aspartate residue (181). However, this form is not

always detectable by all antibodies and the relative abundance of the two cleavage variants is unknown. Its

knockdown or overexpression causes no major physiological defect (although the skin of adult ASPRV1-deficient

mice shows fine wrinkles and reduced hydration).

Fig. 3.1 ASPRV1 is a neutrophil-specific marker increased in the CNS during EAE and severe forms of MS. a, Quantification of Asprv1 mRNA by RT-qPCR in leukocytes isolated by FACS from the spinal cord of EAE mice at day 15 post-induction. CD3ε+ T cells (T) were from either mice immunized with MOG35-55 (active EAE), mice transplanted with encephalitogenic T cells (passive EAE), or 2D2 mice that developed EAE after PTX injection. CD19+ B cells (B), CD45loCD11b+ microglia (Mic), CD45hiCD11b+CD11c+ dendritic cells (DC), CD45hiCD11b+ macrophages (Mc) and Ly6G+ neutrophils expressing or not ICAM1 (N+ and N−, respectively) were from mice with active EAE. Sample size: 4 per group. b, ASPRV1 protein (~32 kDa) detected by Western blotting in Percoll-enriched neutrophils from the bone marrow of wild-type mice, but not of Asprv1–/– mice, nor in the mononuclear cell fraction from either genotype. Total loading per well: 20 µg protein. Actin (~42 kDa) was used as a control for protein loading. c, Quantification of Asprv1 mRNA by RT-qPCR in whole

45

spinal cord from mice with different forms of EAE or from controls, i.e. mice treated with PBS, PTX or CFA, and 2D2 mice that did not develop EAE after PTX injection (2D2 without [w/o] EAE). Stars indicate significant differences from PBS, PTX and CFA (for active and passive EAE) or from 2D2 without EAE (for 2D2 mice) at the same time point (Wilcoxon test, P < 0.018). Sample size: 6 (PBS), 7-13 (PTX), 3-8 (CFA), 8 (active EAE), 8 (passive EAE), 6 (2D2 with EAE), 5 (2D2 without EAE). d, Spearman analysis showing a strong positive correlation between Asprv1 and Ly6g mRNA

expression in the spinal cord during active EAE. Sample size: 32. e, RT-qPCR analysis of ASPRV1 mRNA in freshly isolated human blood cells. Star indicates a significant difference from the other groups (Wilcoxon test, P = 0.0005). Sample size: 2-10 per group. f, RT-qPCR analysis of ASPRV1 mRNA in post-mortem brain samples from control individuals (normal) or patients with MS of varying degrees of severity (low, moderate or severe). NAWM: normal-appearing white matter. Star indicates a significant difference from the other groups (Wilcoxon test, P = 0.006). Sample size: 13-16 per group. From (2).

No structure of ASPRV1 has been solved, although the protease’s primary structure contains a

canonical protease middle domain and, at its N-terminal, a predicted transmembrane segment. One homology

model was constructed by French researchers in 2005 (181). Despite this, N- and C-terminal protein sequencing

analysis had revealed that both termini are cleaved from the protein, leaving behind a soluble protease middle

domain; yet, this form is not detectable by all antibodies, and the relative abundance of the two cleavage variants

is unknown. In our hands, ASPRV1 is detected at a mass of around 31 kDa by reducing SDS-PAGE (Fig. 3.1b).

The antibody used, mG2-C (182), was raised against the C-terminal of ASPRV1, and therefore this band likely

represents the full- or nearly full-length protein. Since N- or C-terminal sequencing was not performed, we are

uncertain of the exact identity of ASPRV1 in neutrophils. ASPRV1 is also predicted to form dimers by analogy

with other viral aspartate proteases; however, these latter enzymes are constrained by the crowded environment

inside viral particles which favors dimerization. Matsui et al. also hypothesize that a similarly crowded

environment exists in the stratum corneum of the skin. Since we observed only a 31-kDa band and were unable

to detect a 15-kDa processed monomer, we cannot conclude whether ASPRV1 in neutrophils exists as a

monomer or dimer.

A previous study in 2005 constructed a homology model of ASPRV1 monomer using equine anemia

virus as a template (181). Currently, however, more sophisticated tools are available for homology modelling as

well as more abundant template structures. I constructed a model of ASPRV1 homodimer based on PDB model

4RGH (human DDI1) using SWISS-MODEL (185). The results are shown in Figure 3.2a. The mechanism of

reaction catalyzed by aspartate proteases is thought to proceed by general acid-base catalysis involving two

aspartic acid residues (186,187). A water molecule would be coordinated by the four oxygen atoms of the two

aspartate residues and deprotonated, forming hydroxide. The reactive hydroxide anion would then attack the

peptide bond to effect proteolysis. No enzyme-substrate covalent intermediate would be therefore formed. This

theory in itself lends credence to the idea that ASPRV1 is naturally dimeric, since no other suitable aspartate

Asp-Ser-Gly or Asp-Thr-Gly motif (188), common to aspartate proteases, is found in the protein. In the homology

model, the two aspartate residues formed hydrogen bonds with two bridging ligands at the dimer interface. In

46

addition, a 1-ns molecular dynamics simulation was performed using GROMACS (189) using the OPLS-AA all-

atom forcefield on the equilibrated and energy-minimized homology model structure. Throughout the simulation,

the dimers remained stably connected by hydrogen bonding. As well, the coordinated water molecules were

stable within a solvent network (contrary to the solvent molecules in other positions in the simulation) between

the two active-site aspartic acid residues (Figure 3.2b).

Fig. 3.2 Molecular dynamics simulation of murine ASPRV1 supports a dimeric structure. a, Homology modeling based on a larger PDB allowed the updating of the homology model from (181). In simulation, the protein’s middle protease region is stable as a dimer. b, The dynamics of the active site revealed two stably (> 1ns) bound water molecules, lending credence to a dimeric catalytic mechanism.

Interestingly, the molecular dynamics simulation did not reveal the presence of canonical “flap” domains.

In HIV-1 protease, and in several other aspartate proteases, these protrusions cover the active site, providing a

hydrophobic binding environment for the substrate. In contrast, as ASPRV1’s processed form is exceedingly

short, it may lack these domains and therefore prefer more hydrophilic substrates. Alternatively, as the homology

model only represents the protease domain with homology to other known structures, the unmapped regions of

ASPRV1’s structure may contain more extensive flaps as found in HIV-1 protease.

ASPRV1 is specific to neutrophils and increases in the CNS

during EAE and severe forms of MS

To study the functions of ASPRV1 in a disease-relevant context, we started by measuring its

expression levels in the mouse during EAE. By RT-qPCR, we found that Asprv1 mRNA was indeed expressed

in neutrophils (ICAM1+ and ICAM1−) extracted by FACS from EAE spinal cords, but not in any other immune

cell types tested (Fig. 3.2a). ASPRV1 protein was also expressed in neutrophils from mouse bone marrow, as

revealed by Western blotting with the same antibody used to first detect ASPRV1 in skin, mG2-C (182) (Fig.

3.2b). Specificity was confirmed by the absence of signal in mononuclear leukocytes and Asprv1−/− neutrophils

(Fig. 3.2b). In whole spinal cord extracts, Asprv1 mRNA was barely detectable in the absence of inflammation

and upon adjuvant injection (Fig. 3.2c); however, it was highly expressed during EAE and strongly correlated

47

with Ly6G mRNA (Fig. 3.2d). The abundance of Asprv1 mRNA was remarkable, i.e. higher than or equal to

Ly6G mRNA, both in spinal cord extracts and purified neutrophils (Fig. 3.2d).

To translate our findings to human, we analyzed, by RT-qPCR, blood fractions and post-mortem brain

samples from people with or without MS. ASPRV1 mRNA was abundant in blood neutrophils in the steady state,

but not in B cells, Th cells, monocytes or unfractionated mononuclear cells (Fig. 3.2e). Furthermore, ASPRV1

mRNA was detected in higher amounts in brain lesions from patients with severe MS compared to both normal-

appearing white matter and brain lesions from patients with low or moderate forms of MS (Fig. 3.2f). Altogether,

our results demonstrate that ASPRV1 is a neutrophil-specific protein in the immune system and can serve as a

neutrophil marker in the nervous system both in the mouse and human.

Fig. 3.S1 B cells are essential for EAE induction with bMOG. a, EAE score over time in B1-8+/+Jκ–/– mice and wild-type controls (WT) immunized with bMOG. Star indicates a significant intergenotype difference per time point identified by post hoc Wilcoxon test (P < 0.0001). Sample size: 6 (WT), 4 (B1-8+/+Jκ–/–). b, Detection of MOG1-125-specific plasma cells by ELISpot in bone marrow (BM) from WT and B1-8+/+Jκ–/– mice at day 29 post-immunization. Star indicates a significant difference, as determined by Student’s t test (P < 0.0001). Sample size: 6 (WT), 3 (B1-8+/+Jκ–/–). Each mouse was tested in triplicate. From (2).

ASPRV1 is required for the chronic phase of a B cell-dependent

EAE model

To determine whether ASPRV1 contributes to EAE, we immunized ASPRV1-deficient and wild-type

mice with two different antigens: the standard MOG35-55 peptide and bMOG. bMOG is a novel “humanized”

mouse MOG1-125 protein, developed by members of our group, that bears the S42P mutation abolishing the

immunodominant T-cell epitope (amino acids 35-55). bMOG can still induce EAE with prominent neutrophilic

infiltration, but contrary to MOG peptide (190), it acts through a B cell-dependent mechanism. This was

demonstrated in B1-8+/+Jκ–/– mice expressing a single B cell receptor to an irrelevant antigen, which mice did not

develop EAE in response to bMOG (Fig. 3.S1). Therefore, compared to MOG35-55, and similar to human MOG,

bMOG induces a form of EAE that is more similar to MS as it involves pathogenic B cells.

48

Fig. 3.3 ASPRV1 is required for the chronic phase of EAE induced with a B cell-dependent myelin antigen (bMOG), but not for initial neutrophil recruitment or T cell priming. a, Kaplan-Meier plot of EAE incidence in wild-type (Asprv1+/+) and ASPRV1-deficient mice (Asprv1−/−) after immunization with either MOG35-55 or bMOG. Shaded area represents the 95% pointwise confidence interval. For sample size and statistical testing, see incidence in c. b, Severity of EAE over time by group versus time from appearance of first symptoms. P-value shown on graph was calculated by two-way ANOVA with repeated measures using rank-transformed scores. Stars indicate differences in post-hoc testing by time point (Wilcoxon test, P < 0.0386). Only mice that had developed EAE are included (see incidence in c for sample size). c, Statistical data extracted from the clinical score of knockout and wild-type mice. Means were compared by Fisher’s exact test (for recovery), by the log-rank test (for incidence and mortality), or otherwise by Wilcoxon test. d, Flow cytometric counts of immune cells in the spinal cord of Asprv1−/− and wild-type mice before immunization with bMOG (naïve) or after at day 8 (EAE pre-onset), 13 (EAE peak) or 21 (EAE chronic phase). Counts were normalized to CD45− cells as an internal control. Stars indicate significant differences from Asprv1+/+ mice (Wilcoxon test, P < 0.0454). Abbreviations: Mic, CD45loCD11b+ microglia; N, Ly6G+ neutrophils; Mc, CD45hiCD11b+CD11c− macrophages; DC, CD45hiCD11b+CD11c+ dendritic cells; T, CD3ε+ T cells; B, CD19+ B cells. Sample size per group: 4-6 (naïve), 7 (pre-onset), 9-22 (peak), 7-10 (chronic). e, Counts of the ICAM1– (intravascular) and ICAM1+ (extravascular) subpopulations of macrophages and neutrophils showing no intergroup difference, except for ICAM1+ macrophages at the peak of bMOG EAE. Star indicates a significant difference from Asprv1+/+ mice (Wilcoxon test, P = 0.0374). Sample size as in d. f, Counts of blood neutrophils showing that bMOG immunization induces a similar mobilization of neutrophils between the genotypes. Sample size per group: 3-5 (naïve), 7 (pre-onset), 7-10 (peak). g, Counts of Th17 (IL-17+) and Th1 (IFNγ+) cells in inguinal lymph nodes (LN) at day 9 post-immunization with bMOG suggesting that there were no intergroup difference in T cell priming. Intracellular staining was performed on freshly collected LN cells after a 4-h stimulation with phorbol myristate acetate and ionomycin in the presence of brefeldin A. Sample size: 14-17 per group. From (2).

After immunization with MOG35-55, no difference was observed in EAE incidence and severity between

Asprv1–/– and wild-type mice (Fig. 3.3a-c). In contrast, with bMOG, EAE incidence and acute phase were slightly

reduced in the absence of Asprv1 (Fig. 3.3a-c). More importantly, the chronic phase was blunted in Asprv1–/–

MOG35-55

bMOG

Incidence

Day of onset

Day at peak score

Peak score

Median score

Cumulative score

Total dead/euthanized

Asprv1+/+

17/18 (94%)

13.7 ± 0.6

18.8 ± 1.2

3.6 ± 0.3

2.4 ± 0.3

43.8 ± 5.0

10/18 (56%)

0/17 (0%)

Asprv1

16/17 (94%)

13.3 ± 0.4

19.8 ± 1.4

3.1 ± 0.3

2.2 ± 0.2

40.9 ± 3.8

3/17 (18%)

0/16 (0%)

P

0.87

0.82

0.62

0.27

0.82

0.97

0.03

1.00

Asprv1+/+

50/63 (79%)

11.2 ± 0.4

12.9 ± 0.5

2.6 ± 0.1

0.5 ± 0.1

18.2 ± 2.1

2/50 (4%)

3/50 (6%)

Asprv1

32/60 (53%)

12.0 ± 0.5

12.9 ± 0.4

2.1 ± 0.1

0.4 ± 0.1

16.8 ± 2.3

0/32 (0%)

10/32 (31%)

P

0.0003

0.021

0.22

0.0020

0.91

0.78

0.17

0.0040Full recovery

Descriptive statistics

P= 0.009P= 0.22

0 10 20 0 10 20

49

mice; after a recovery phase culminating around day 7 post-onset, wild-type mice experienced a relapse,

whereas Asprv1–/– mice continued to recover, so that 31% of them showed no more clinical signs by the end of

experimentation (Fig. 3.3b, c).

To corroborate and explain these symptomatic differences, we performed flow cytometric analysis on

spinal cords at different phases of bMOG-induced EAE in Asprv1–/– and wild-type mice (Fig. 3.3d). At pre-onset

(day 8 post-immunization), only neutrophils and macrophages had begun to accumulate. At the peak of EAE

(day 13 post-immunization), all types of infiltrating leukocytes reached their maximal numbers; among them,

only macrophages were less numerous in the knockouts (by ~46%) (Fig. 3.3d). More precisely, this reduction

predominantly affected the ICAM1+ subpopulation of macrophages (Fig. 3.3e), which, by analogy with

neutrophils, may be presumed to be extravasated. A similar reduction (43%) in infiltrating macrophages was

estimated by stereology using spinal cord sections stained for F4/80 (P = 0.040, Fig. 3.4). No abnormality was

noticed in the state of activation of either ICAM1+ macrophages or neutrophils, as inferred from their surface

levels of CD45, CD11b, ICAM1, MHCII and CD86 (data not shown). By the chronic phase (day 21 post-

immunization), many leukocytes (neutrophils, macrophages, DCs) had left the spinal cord of both mouse strains,

leaving behind a sizeable population of T cells; in Asprv1–/– mice, even fewer cells remained, and the number of

T cells was only one-sixth that of wild-type mice (Fig. 3.3d).

Fig. 3.4 Stereological investigation of macrophages in Asprv1 knockout mice with bMOG-induced EAE. a, Estimated numbers of F4/80+ macrophages in inflammatory lesions of the spinal cord in wild-type or ASPRV1-deficient mice. At day 13 post immunization with bMOG, frozen sections were cut at 12 µm and every tenth section stained for F4/80 by immunohistochemistry and counterstained with DAPI. Three sections per animal were counted, lesions delineated with DAPI, and the number of macrophages within a constant volume estimated by stereology using the optical fractionator (Gundersen coefficient of error (CE) < 0.07 for all animals with score >0). Sample size: 9 (WT); 6 (KO). b, Variation of macrophage numbers with clinical score in Asprv1+/+ and Asprv1–/– mice. The tendency is best approximated by a quadratic function. c, Representative mosaic images of immunohistochemical staining for the infiltrating macrophage marker F4/80 in Asprv1+/+ spinal cords.

a b

c

DAPI MergeF4/80

50

Zones of infiltration are marked, while the meninges are excluded from counting. False color. Scale bar: 50 µm.

The lower sensitivity of Asprv1 mice to bMOG was not attributable to a defect in neutrophil migration,

because no substantial intergenotype difference was seen either in the number of ICAM1+ neutrophils that had

infiltrated the CNS tissue at the peak of EAE (Fig. 3.3e) or in the number of ICAM1– neutrophils crawling inside

the CNS vasculature (Fig. 3.3e) or circulating in blood (Fig. 3.3f). Nor was this lower sensitivity attributable to a

defect in T cell priming, because the proportion of Th1 and Th17 cells in draining lymph nodes did not differ

between the genotypes (Fig. 3.3g), which is consistent with previous studies showing that neutrophil depletion

does not affect the priming of encephalitogenic T cells (65,62). Finally, this phenotype was only associated to

ASPRV1 and not observed in mice lacking other neutrophil-specific genes (Il1f9 and Mreg). Overall, our results

demonstrate that:

1) ASPRV1 is required both to reach maximum disease severity and to sustain chronic

inflammation in response to bMOG; and

2) ASPRV1 does not significantly affect neutrophil mobilization from bone marrow,

adhesion/crawling on the CNS vasculature, or extravasation into meningeal and submeningeal

compartments, which is consistent with the normal response of Asprv1 mice to MOG35-55.

Therefore, neutrophils exert, most likely in the CNS, a proinflammatory effect via ASPRV1 that is crucial

for perpetuating inflammation specifically in a B-cell-dependent form of EAE.

Methods

Human brain samples: Snap-frozen post-mortem brain samples from MS and control patients were

obtained from the Multiple Sclerosis Society Tissue Bank at the Imperial College London. Additional samples

from a patient with severe rebound MS activity after natalizumab withdrawal (191) was obtained from the

University of Montreal Hospital Research Center. Biopsies were classified as acute active lesion (stage 1 and

2), chronic active lesion (stage 3 and 4), chronic inactive lesion (stage 5) or normal appearing white matter, as

described previously (192). Lesions were scored for severity based on the degree of demyelination and

inflammation, as described previously (192). Frozen sections were cut with a cryostat to obtain ~20 mg of tissue,

which was homogenized in TRI-reagent (Sigma-Aldrich) and processed with the EZ-10 Spin Column Animal

Total RNA Mini-prep Kit (Bio Basic) to extract RNA for RT-qPCR. Additional sections were used for histology.

Our institutional research ethics committee approved this work.

Human blood samples: Healthy volunteers’ blood was layered on top of lymphocyte separation medium

(Wisent Bioproducts) and centrifuged for 20 minutes at 600 ×g. The top cell fraction, enriched in PBMCs, was

51

used directly or processed with the EasySep Human CD4+ T Cell, B Cell or Monocyte Isolation kits; the lower

fraction, enriched with granulocytes, was processed with the EasySep Direct Human Neutrophil Isolation kit,

according to the manufacturer's instructions (StemCell Technologies). RNA was purified from cell fractions using

Aurum Total RNA kit (Bio-Rad Laboratories).

Animals: Asprv1–/– mice (182) were derived from previously established colonies. B1-8 mice (193) with

a homozygous deletion of the Jκ locus (194) were generously provided by Dr. Ann Haberman (Yale University).

bMOG production: The pET-32 MOGtag vector (195) was mutated by PCR using the following primers:

5'-TACCGTCCGCCGTTTTCTCGCGTTGTTCACC-3' and 5'-AAACGGCGGACGGTACCAGCCAACTTCCAT-3'.

The resulting vector was sequenced to confirm the insertion of the S42P mutation, and used to produce bMOG

according to a previously described protocol (196). Final concentration was set to 5 mg/ml with no detectable

impurities, as measured by Bradford assay and SDS-PAGE.

ELISpot: To detect MOG-specific plasma cells in bone marrow, 96-well plates were coated overnight

at 4°C with 0.5 μg mouse MOG1-125. Wells were blocked with 1% (wt/vol) BSA in PBS, then incubated with bone

marrow cells at 37°C with 5% CO2. Spots were detected using a goat alkaline phosphatase-conjugated anti-

mouse IgG antibody (MABTECH) and 5-bromo-4-chloro-3-indolyl-phosphate substrate (Sigma-Aldrich), then

counted under a dissection microscope.

Western blotting: Bone marrow cells were collected by flushing out femurs with 10 mL of HBSS (Wisent

Bioproducts), filtered through 70-μm mesh, resuspended in HBSS containing 45% Percoll (GE Healthcare), and

centrifuged at 1,600 ×g for 30 min over a four-level Percoll density gradient (45%, 55%, 65%, 81%). Cell layers

(mononuclear cells from the top and 45-55% interface; neutrophils from the 65%-81% interface) were retrieved

and lysed by sonication (3 × 5 sec) in 50 μl RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 1% Triton X-100, 0.5%

sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 1× protease and phosphatase inhibitor cocktails

[Sigma-Aldrich, nos. P8340 and P5726], pH 8.0). Protein samples (20 µg) were boiled for 5 min in Laemmli

buffer (50 mM Tris-HCl, 2% SDS, 6% β-mercaptoethanol, 10% glycerol, 12.5 μg/mL bromophenol blue), resolved

on 15% SDS-polyacrylamide gel (Bio-Rad Mini-Protean II), and transferred to a polyvinylidene difluoride

membrane for 45 min at 4°C and 100 V in transfer buffer (25 mM Tris, 200 mM glycine) containing 20% methanol.

The membrane was blocked for 30 min at room temperature in TSM (10 mM Tris, 150 mM NaCl, 5% non-fat dry

milk, 0.1% Tween 20, pH 7.4), and incubated for 1 h at room temperature and then overnight at 4°C in 7 mL of

TSM containing polyclonal rabbit antibody to ASPRV1 C-terminus (182) (1:1000). The membrane was washed

four times in TSM for 5 min and then incubated for 1 h at room temperature with horseradish peroxidase-

conjugated goat anti-rabbit antibody (1:2500; Jackson ImmunoResearch). The membrane was again washed

four times in TSM, then twice in TS (10 mM Tris, 150 mM NaCl, pH 7.4) and once briefly in water, before being

52

incubated for 60 sec with 1 mL Western Lightning chemiluminescence substrate (PerkinElmer), then blotted dry.

The chemiluminescent signal was captured on Carestream Bio-Max Light Film (Kodak), which was scanned at

800 dpi and adjusted for contrast and brightness with Photoshop (Adobe). To control for protein loading, the

membrane was rinsed overnight in TS and blotted for β-actin by 1-h incubations at room temperature first in

mouse anti-β-actin antibody (1:5000; Millipore, clone C4), then in goat anti-mouse antibody (1:2500; Jackson

ImmunoResearch).

Study approval: Studies using mice and human samples were approved, respectively, by the Animal

Protection Committee and the Research Ethics Committee of the University Hospital Center of Quebec – Laval

University.

53

Chapter 4:

Discussion and General Conclusions

In Chapter 1, we detailed the methods neutrophils use to gain access to sites of inflammation,

particularly in the context of demyelinating autoimmune diseases. The basics of neutrophils (their presence in

EAE and in several human demyelinating diseases, as well as the fact that they worsen EAE and correlate to

disease severity in MS) had been established, but beyond this, little had been studied as to the mechanisms of

pathogenesis or of neutrophils’ molecular characteristics in neuroinflammation. In Chapter 2, we have provided

evidence for a distinct neutrophil subpopulation appearing only in EAE. This population of neutrophils infiltrates

the parenchyma of the CNS and is characterized by a high level of surface ICAM1, contrary to the traditional

wisdom on neutrophils in general (197). We demonstrated the idiosyncrasies of ICAM1+ neutrophil

transcriptomes, showing that they share putative functions with macrophages and dendritic cells but also

express some effector genes that are entirely unique. We validated the expression of MHCII and its associated

co-stimulatory molecules by ICAM1+ neutrophils and provided evidence for immunological synapse formation

between these cells and B and T lymphocytes in the EAE spinal cord. In Chapter 3, we also validated a

neutrophil-unique gene, Asprv1, and showed that it contributes to chronic inflammation in bMOG-induced EAE.

Our work represents some of the first investigation of neutrophils in neuroinflammation, yet many questions

about neutrophils’ functions have still not been fully resolved. This discussion chapter will attempt to integrate

our results within the broader scientific literature, discuss points of contention, and highlight some of the future

questions to be addressed.

For the first time, the present study:

1) identifies a surface marker (ICAM1) that distinguishes extravascular from intravascular

neutrophils;

2) identifies a neutrophil-specific marker (ASPRV1) applicable in both mouse and human

within the immune and nervous systems;

3) deciphers the transcriptomic changes undergone by neutrophils after extravasation,

hence revealing plasticity (e.g. acquisition of genes with macrophage-like

immunostimulatory and antigen presentation properties);

4) shows in vivo that neutrophils make intimate physical contacts with T and B lymphocytes

at the site of inflammation;

54

5) specifies the perpetuation of chronic inflammation as one context-dependent function of

neutrophils;

6) ascribes an extra-epithelial role to ASPRV1 as a proinflammatory effector; and

7) introduces a novel humanized myelin antigen (bMOG) that can be used to induce a B cell-

dependent form of EAE and to study neutrophil-B cell interactions.

Catchup

Our study was one of the first to make use of the Catchup model, a reporter mouse for studying

neutrophils. The Catchup mouse has been used for neutrophil-specific gene deletion of Fcgr4 by the inventors.

The authors successfully reduced the surface levels of FCγRIV by around 45% in neutrophils, while leaving

expression levels untouched in monocytes, a closely related cell type (96). In contrast, the LysM-cre system also

reduced expression by around 45%, but also abolished FCγRIV expression in monocytes, decreasing surface

levels by almost 80% of wild-type. For this reason, the Catchup system furnishes clear advantages over previous

neutrophil Cre systems.

We had aimed to use the Catchup Ly6G-cre strain to genetically delete H2-Ab1 in neutrophils, in order

to test the dependence of neutrophils on MHC class II expression in exacerbating EAE. While in Catchup × H2-

Ab1flox/flox mice, the H2-Ab1 gene was excised, as measured by qPCR, and the mRNA was undetectable, surface

MHCII was still present on neutrophils (Fig. 2.S4). While disappointing, this result could stem from differences

in the efficiency of recombination in neutrophils and other cell types, or the persistence of surface MHCII from

earlier stages in development. It is worthy to note that genetic manipulation of neutrophils is still very rare and

in its infancy.

ICAM1

To our knowledge, our study is the first observation of ICAM1-expressing neutrophils in EAE. Yet,

relatively recently, Nourshargh and colleagues reported an ICAM1+ subset of neutrophils in mouse endotoxemia

(198). They found, as did we, low resting levels of ICAM1 on blood neutrophils, which was certainly the prevailing

wisdom of immunologists in general (only a handful of older papers find ICAM1 expression on resting neutrophils,

and these were performed in human cells (199,200)). This could not be increased by leukotriene B4, CXCL1,

formyl-Met-Leu-Phe, or interleukin-1β, but increased drastically upon LPS, TNF, or zymosan ex vivo, by about

an order of magnitude in fluorescence. This effect was not seen in Icam1-null mice (201), ruling out antibody-

staining artifacts. In parallel, the mRNA level increased almost 25-fold in LPS-treated neutrophils ex vivo. As

well, LPS stimulation or ICAM1 cross-linking of wild-type neutrophils but Icam1-null neutrophils enhanced their

ability to phagocytize zymosan particles. Neutralization of Mac-1, the ligand of ICAM1, neutralization of the

55

ICAM1 C-terminal domain, or inhibition of the protein kinase Syk, a downstream effector of ICAM1, was able to

repress phagocytosis in this zymosan assay, but strangely, blocking antibodies to ICAM1 itself did not. Whether

the transcriptomes of these LPS-treated neutrophils would resemble that of our ICAM1+ neutrophils found in

EAE is unknown.

Nourshargh and colleagues also reported increased ICAM1 on “tissue-infiltrated neutrophils”, although,

given that the noxious stimulus used was administered through the blood in this case, they also observed ICAM1

“in blood vascular neutrophils”. These authors contend that “neutrophil transmigration per se is not enough... to

elicit ICAM1 expression... in neutrophils”, since IL-1β induced tissue transmigration of neutrophils but not ICAM1

expression in the absence of a bacterial compound. Yet, another study found that neutrophils transmigrating

through TNF-activated endothelial cells did upregulate ICAM1 (202). In our study, although we determined that

CNS-infiltrating neutrophils express ICAM1 and have an activated phenotype, we have not been able to identify

a factor responsible for this activation process. Thus, the picture is still not clear regarding the stimulus that

causes ICAM1 expression on neutrophils.

As to the functional role of ICAM1—although we have not here demonstrated a direct functional link

between ICAM1 expression and pathogenesis, other studies have shown that ICAM1 increases in expression

on endothelial and glial cells within MS and EAE lesions, helps in lymphocyte transmigration through brain

endothelium, and is a component of signaling pathways leading to pathogenic effector functions in diverse cell

types during EAE (reviewed in (201)). As well, polymorphism in the human ICAM1 gene has been associated

with both protection and susceptibility to MS (203,204). Inhibition of ICAM1, and loss or inhibition of the ICAM1

ligands Mac-1 and LFA-1, generally reduces the severity of disease (201). Antithetically, in the Icam1tm1Bay

mouse, expressing truncated isoforms of ICAM1 only, EAE is more severe, specifically in the chronic phase

(205). If these alternate isoforms are gain-of-function for ICAM1, it is possible they affect EAE severity through

neutrophils, who also act in the chronic phase, via ASPRV1 (Chapter 3). In the Icam1null mouse, EAE is

drastically suppressed, indicating that cell-type-nonspecific ICAM1 expression worsens EAE (201). Further,

adoptive transfer of Icam1null T cells to wild-type mice is incapable of inducing EAE, suggesting that ICAM1 is

required on T lymphocytes as well. However, although the authors noted reduced inflammation in tissue sections,

they did not examine the role of neutrophils in detail in this study. The authors also used evidence from antigen-

presentation assays of mixed genotype, suggesting that ICAM1 is more important on T lymphocytes than on

antigen-presenting cells. This result is seemingly at odds with our hypothesis that ICAM1+ neutrophils could act

as antigen-presenting cells, but the situation may be different if the assay were performed with neutrophils

instead of splenic APCs.

There is another advantage to the identification of ICAM1 as a marker: Previously, when analyzing CNS

samples by flow cytometry, the patrolling and infiltrating neutrophil populations could not be separated. We have

56

shown here that the great majority of extravasated neutrophils in EAE express ICAM1, but not patrolling

neutrophils. By allowing a better adhesion between leukocytes (e.g. via LFA1), ICAM1 facilitates cell-cell

interactions such as the formation of immunological synapses (57). Our results suggest that neutrophils act, at

least in part, in a contact-dependent manner in demyelination, and provide a surface marker for the study of

neutrophil subpopulations.

Transcriptomes

Previous studies over the last decades have consistently reported that neutrophils, rather than being

static, terminally differentiated cells, change their gene expression dramatically upon entering tissues or

encountering inflammatory stimuli (206,207,208,209,210). We have measured the transcriptomes of ICAM1+

and ICAM1− neutrophils sorted from the spinal cord during EAE. Surprisingly, unsupervised hierarchical

clustering revealed that ICAM1+ neutrophils shared more in expression with macrophages and DCs than with

ICAM1-negative neutrophils. We found three differentially regulated modules: those upregulated in ICAM1+,

those downregulated in ICAM1+, and a third class shared between ICAM1+ and ICAM1− but not expressed by

macrophages or DCs. We found that two neutrophil-specific genes, melanoregulin and IL-23α,have no EAE

phenotype, but discovered one, ASPRV1, that is highly important in B-cell-dependent EAE. Our results support

the view that neutrophils are considerably plastic in autoinflammatory disease states.

One recent study (172) by the ImmGen Consortium examined gene expression profiles of mouse

neutrophils in a similar manner to our work. The authors included resting neutrophils as well as neutrophils from

arthritis and peritonitis models. They found uniform upregulation of many genes in the MHCII pathway in their

arthritis-model neutrophil samples, with most (Cd74, Cd80, Ctsb, Ctss, Ctsl, H2-Aa, H2-Ab1, H2-Eb1, Ifi30,

Lgmn) exactly reproducing what we saw in ICAM1+ neutrophils. A principal-component analysis revealed,

consistent with our results, that neutrophils in autoinflammatory environments are far removed both from resting

bone-marrow neutrophils and from other immune cell types (Figure 4.1). It would be interesting to know if

neutrophils from the serum-transfer arthritis mice also express Icam1 or bear surface ICAM1, as the authors did

not mention it in their paper. This paper agrees well with our findings on the plasticity of neutrophils in

inflammatory conditions. The authors hypothesized a weak correlation between transcription and translation in

neutrophils, which presaged our inability to knock down MHCII specifically in neutrophils.

57

Fig. 4.1 Neutrophil phenotypes in other autoimmune diseases. Neutrophils isolated from mice with arthritis induced by thioglycollate, urate or synovial fluid transfer show distinct disparity from neutrophils in bone marrow or other immune cells, according to a principal-component analysis of GeneChip transcriptomic data. From (172).

MHCII

Neutrophils in culture have been made to express MHCII and to present antigen to T lymphocytes

(119,127,130,131,211,212), yet the relevance of these non-classical APCs remains to be shown in vivo.

Neutrophil MHCII expression has also been reported in rheumatoid arthritis, but in this case, no co-stimulatory

molecule expression was noted (213). To our knowledge, our study is the first to:

1) report that EAE neutrophils also transcribe and translate MHCII molecules; and

2) provide evidence for physical cellular interactions between lymphocytes and neutrophils

in the EAE nervous system.

We report here that neutrophils prepare for being APCs at an early stage of development by pre-forming

MHCII mRNA, which can later be translated at the site of inflammation. We also provide the first in vivo evidence

for close contacts between neutrophils and T cells within EAE lesions with a redistribution of cell-surface ICAM1.

Several types of immunological synapses have been described ex vivo (175), but more characterization is

required to determine to which category the synapses we observed in EAE belong. Since current genetic models

cannot specifically delete MHCII in neutrophils, new strategies will be needed to confirm the importance of the

observed immunological synapses. For instance, these synapses could aid neutrophils in enhancing the anti-

MOG Th17 response or modulate epitope spreading by presenting new myelin epitopes to T cells in either a

positive or negative regulatory manner.

Stimuli that can induce MHCII expression on B cells include interleukin-4, cross-linking of the BCR, and

phorbol esters (214). However, LPS results in the downregulation of MHCII; contrary to the induction of ICAM1

by LPS reported by Woodfin et al. (198) Interferon-γ, a Th1 cytokine present in EAE, can also stimulate MHCII

58

expression on a variety of cells that do not normally present antigen (215). Since interferon-γ-induced MHCII

expression is a slow process taking upwards of 24 h (216) and requiring new protein synthesis (217), it would

be important to determine the kinetics of MHCII expression in neutrophils. This in itself would be difficult and

casts doubt on an interferon-γ-mediated mechanism, since neutrophils are short-lived, especially in culture, and

do not have a very high capacity for translation. Consistent with a non-canonical mechanism, certain features of

transcriptional regulation of MHCII expression have been known to differ between APC cell types and

nonlymphoid cell types: for example, the octamer sequence upstream of the TATA box is not required for MHCII

induction in nonlymphoid cells (218,219).

The MHCII transactivator protein (CIITA, Ciita) is a potent stimulator of MHCII expression (220) as well

as expression of the MHCII pathway proteins CD74 and B2M (221), and is expressed by ICAM1+ as well as

ICAM1− neutrophils in our microarray dataset (ICAM1+, hybridization signal 255; ICAM1−, 109; macrophage,

531; DC, 849). B2m was very highly expressed in all cell types in our analysis, and Cd74 was among the

upregulated genes (Figure 2.3) in ICAM1+ cells. This in itself is not enough to tell us whether CIITA is part of

the activation pathway of MHCII in ICAM1+ neutrophils.

The SXY box, another element in the promoter regions of the MHCII locus, is bound by transcription

factors to activate H2-A and H2-E genes as well as many MHCII pathway genes, including Ii, H2-M, and H2-O

(222,223). One SXY-binding transcription factor is RFX5 (Rfx5), which was weakly expressed in both ICAM1+

neutrophils and macrophages (ICAM1+, signal 264; macrophage, 233), but not in ICAM1− neutrophils (signal

117, P = 0.006); this level may be sufficient to bind and activate the SXY box. A very similar trend is seen for

RFXAP (Rfxap) and RFXANK (Rfxank), two other SXY-binding proteins, yet neither is significantly different

between ICAM1+ and ICAM1− neutrophils.

Binding to the Y element of the SXY box requires NF-Y, a dimer of NF-YA and NF-YB (214,222). Both

Nfya and Nfyb are highly expressed in ICAM1+ and ICAM1− neutrophils, at a similar level to macrophages;

therefore, this factor is likely ubiquitous and not the deciding player in the expression of MHCII. Interestingly,

however, CREB (cAMP-response-element-binding protein) is also required to bind, as a dimer with ATF2, the

X2 element of the SXY box. Of all CREB paralogues, only Creb5 (CRE-BPa) showed a strong difference in our

microarrays, rising over 7-fold from ICAM1− to ICAM1+ neutrophils to a level similar to macrophages (P = 0.012).

The cofactor Atf2 is most highly expressed in ICAM1+ neutrophils of all four cell types, suggesting neutrophils

make heavy use of cAMP response elements in fulfilling their functions. In summary, much is still to be elucidated

about the pathways neutrophils use when stimulated to express MHCII, and the details of such a pathway would

work for exciting future work.

59

bMOG

B cells contribute to MS and NMOSD in two major ways:

1) by secreting autoantibodies and

2) by stimulating the development of autoreactive Th cells via antigen presentation and

secretion of cytokines (e.g. IL-6, GM-CSF and IL-15) (224).

Similarly, B cells are essential for the development of EAE when induced with either bMOG, as

demonstrated herein, or human MOG protein (225). However, in the traditional MOG35-55 model, B cells play no

such role, instead acting as beneficial, regulatory cells, since depleting them exacerbates the symptoms (226-

235). This is possibly because DCs and macrophages can only present the immunodominant MOG35-55 epitope

to T cells, whereas B cells can process and present several epitopes, thereby mounting a more diversified

response; by mutating the 35-55 epitope in bMOG, B cells become necessary APCs for T cell activation. The

advantages of using bMOG are twofold: first, compared to the traditional MOG peptide, using B-cell-dependent

antigens such as bMOG and human MOG to investigate B cell responses is important given the recent success

of B cell depletion therapy for MS (224); second, compared to human MOG, bMOG differs from mouse MOG by

only a single amino acid, thus excluding possible confounding effects of multiple mutations present in human

MOG, and when used in mice, is from the same species, avoiding unwanted immunogenicity. Use of bMOG

should help mechanistic studies of how the immune response can drift from foreign to self-epitopes. Also, as

neutrophils are increasingly known to be important communicators and to possess immunoregulatory functions,

the interactions between neutrophils and B cells could be a target for study with bMOG.

ASPRV1

The Ly6G antigen conclusively identifies neutrophils in mice, but no such marker exists in human.

Those that exist (e.g. myeloperoxidase, neutrophil elastase, CD16b and CD66b) can also be expressed by other

myeloid cells and, thus, are not fully specific to neutrophils. This complicates the identification of neutrophils in

human tissues (e.g. CNS), since these cells can adopt a macrophage-like phenotype and since their nucleus

can be more compacted and thus difficult to recognize by conventional microscopy. We propose the use of

ASPRV1 as a specific marker for neutrophils in human and mouse tissue samples, with the added benefit of

facilitating translation of results from animal models.

The substrate and function of ASPRV1 were only known in the context of stratified epithelia (e.g. the

stratum corneum of the skin), where it processes profilaggrin and enhances skin hydration (Chapter 3). We

have now found that ASPRV1 is also expressed in the immune system solely by neutrophils, and promotes

chronic inflammation in EAE. However, the substrate of ASPRV1 in this new context is unknown, as profilaggrin

60

and other members of its family are not expressed in neutrophils (according to our microarray data) and have

not been reported in the immune and nervous systems. Since ASPRV1 was ostensibly acquired from a

retrotransposon sometime around the advent of mammals (184), the selective expression of ASPRV1 in

neutrophils may be associated to a mammal-specific function of neutrophils. By comparison with other aspartic

proteases (e.g. cathepsin D, pepsin, renin, Ddi1p), ASPRV1 may be involved in an intracellular or extracellular

process such as endosomal proteolysis, antigen processing for the MHCII pathway or proteolytic activation of

inflammatory mediators.

The human ASPRV1 isolated by Bernard et al. was able to proteolyze an insulin beta-chain peptide at

Glu13-Ala14. As well, ASPRV1 is able to process itself, and this autoproteolysis is inhibited upon site-directed

mutation of Asp212, providing evidence that ASPRV1 is an active protease. Further studies on the targets of

ASPRV1 are essential to plumbing the depths of its biological functions, especially given that other aspartic

proteases cleave substrates as important as actin and pro-interleukin-1β (181).

Future Directions

A dearth of attention has been given to the contributions of neutrophils, the pervasive innate immune

effector cells, both in EAE and in human demyelinating diseases. Despite direct evidence for neutrophils’

presence in many autoimmune demyelinating diseases, including NMOSD and rare MS variants, only

circumstantial evidence exists for neutrophils contributing to MS. Yet, as we have reviewed (Chapter 1), this

evidence is strong and only growing. Many neutrophil-related factors are correlated to disease activity in MS,

and our study (Chapters 2, 3) has revealed another homologous protein, ASPRV1, that plays a role in EAE and

appears to do the same in human MS. The development of therapies that suppress or modify the activity of

neutrophils would be rational, especially in the case of NMOSD. Current treatments affect neutrophils only in a

non-specific way, yet the success of drugs such as sivelestat show that targeting neutrophil-related factors is

promising.

With the discovery of ICAM1+ neutrophils as a novel subset in Chapter 2 comes the hope for therapies

that selectively target this neutrophil population. While such a concept is still far from implementation, genomics-

and genetics-based therapies (e.g. gene therapy, viral medications, and precision-medicine approaches) are

gaining ground in the broader pharmaceutical sphere. Knowing the transcriptome of neutrophils in EAE as they

compare to healthy states is an important step towards manipulating neutrophils. Further studies could aim to

determine whether a similar population of neutrophils exists in NMOSD or ADEM in humans (which arguably

resemble EAE more than MS). The markers used by an activated neutrophil population may not necessarily be

identical; one well-known example is monocyte subpopulations, which are marked by sequential levels of Ly6C

in mice but by CD16 in humans. The mechanism behind the activation of the MHCII pathway concomitant with

neutrophil stimulation is unclear and not well studied. Future experiments should attempt genetic screens to

61

knock down transcription factors or MHCII pathway proteins under conditions known to stimulate MHCII on

neutrophils, either in culture or in the disease models used by us (EAE) or by other authors (such as experimental

arthritis) (172). Such a forward-genetic approach could identify genes necessary for the induction of the MHCII

pathway in an under-studied cell type and would provide clues as to the molecular mechanism. For example, is

it identical to the pathway utilized by professional antigen-presenting cells such as macrophages, DCs and B

lymphocytes? Furthermore, the use of ChIP-seq methods to compare the transcription-factor occupancy of

promoter regions at the MHCII locus between ICAM1− and ICAM1+ EAE neutrophils could shed light on the

question of ICAM1+ neutrophil MHCII expression, and noncanonical MHCII expression pathways in general.

Finally, the successful use of the Catchup mouse in our study raises hopes for precision study of

neutrophils in the future. While past genetic tools for neutrophil research were unwieldy and imperfect, Catchup

has proven to be a useful system for imaging, cytometry, and possibly also genetic deletion (although the

inherent low transcriptional and translational activity in mature neutrophils still poses a barrier). We are optimistic

about the future of neutrophil research and believe the next ten years will see great strides in our comprehension

of this lonely cell type.

62

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