Steady-state neutrophil homeostasis is dependent on TLR4/TRIF signaling

doi:10.1182/blood-2012-05-429589Prepublished online December 5, 2012;

Hans-Georg Rammensee, Lothar Kanz and Hans-Georg KoppR. Müller, Elke Malenke, Tina Wiesner, Melanie Märklin, Julia-Stefanie Frick, Rupert Handgretinger, Stefanie Bugl, Stefan Wirths, Markus P. Radsak, Hansjörg Schild, Pamela Stein, Maya C. André, Martin Steady-state neutrophil homeostasis is dependent on TLR4/TRIF signaling

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Steady-state neutrophil homeostasis is dependent on TLR4/TRIF signaling

Stefanie Bugl*, Stefan Wirths*, Markus P. Radsak1, Hansjörg Schild2, Pamela Stein2,

Maya C. André3, Martin R. Müller, Elke Malenke, Tina Wiesner, Melanie Märklin, Julia-

Stefanie Frick4, Rupert Handgretinger3, Hans-Georg Rammensee5, Lothar Kanz, and

Hans-Georg Kopp

Department of Medical Oncology, Hematology, Immunology, Rheumatology and

Pulmonology, Medical Center II, South West German Comprehensive Cancer Center,

University Hospital of Tuebingen, 72076 Tuebingen, Germany 1Dept. of Internal Medicine III (Hematology, Oncology, Pneumology), Johannes

Gutenberg University Medical Center, 55131 Mainz, Germany 2Institute for Immunology, Johannes Gutenberg University Medical Center, 55131

Mainz, Germany 3Department of Pediatric Hematology and Oncology, University Children’s Hospital,

72076 Tuebingen, Germany 4Institute of Medical Microbiology and Hygiene, Eberhard Karls University Tuebingen,

72076 Tuebingen, Germany 5Department of Immunology, Institute for Cell Biology, Eberhard Karls University

Tuebingen, 72076 Tuebingen, Germany.

*authors contributed equally to this work

Corresponding author:

Hans-Georg Kopp, MD

Dept. Hematology/Oncology, Eberhard-Karls University

Otfried-Mueller-Str. 10

D-72076 Tuebingen, Germany

Email: [email protected]

Phone: ++49-7071-29 8 7289

Fax: ++49-7071-29 5689

Running Title: TLR4/TRIF are essential in neutrophil homeostasis

Blood First Edition Paper, prepublished online December 5, 2012; DOI 10.1182/blood-2012-05-429589

Copyright © 2012 American Society of Hematology




2

Abstract

Polymorphonuclear neutrophil granulocytes (neutrophils) are tightly controlled by an

incompletely understood homeostatic feedback loop adjusting the marrow’s supply to

peripheral needs. While it has long been known that marrow cellularity is inversely

correlated with G-CSF levels, the mechanism linking peripheral clearance to production

remains unknown.

Herein, the feedback response to antibody induced neutropenia is characterized to

consist of G-CSF dependent shifts of marrow hematopoietic progenitor populations

including expansion of the lin-/Sca-1+/c-kit+ (LSK) and granulocyte macrophage

progenitor (GMP) compartments at the expense of thrombopoietic and red cell

precursors. Evidence is provided that positive feedback regulation is independent from

commensal germs as well as T-, B- and NK-cells. However, in vivo feedback is impaired

in TLR4-/- and TRIF-/-, but not MyD88-/- animals.

In conclusion, steady-state neutrophil homeostasis is G-CSF dependent and regulated

through pattern-recognition receptors, thereby directly linking TLR-triggering to

granulopoiesis.




3

Introduction

Neutrophils are indispensable in generating the early inborn immunologic response to

invading bacteria and fungi. Because both the lack of neutrophils and their increased or

misguided activity contribute to human disease, neutrophil homeostasis is tightly

regulated 1. The discovery and cloning of granulocyte colony stimulating factor (G-CSF),

the principal cytokine stimulating neutrophil production and egress from the bone

marrow, has opened up the door for an understanding of neutrophil homeostasis 2.

Pancytopenia due to bone marrow aplasia after myeloablative therapy and G-CSF

levels have been described to be inversely correlated 3. The regulatory circuits

determining plasma G-CSF in wild type mice, however, have not been well determined.

While the physiologic response of granulopoiesis to infection, termed emergency

granulopoiesis, has been characterized in more detail, steady-state granulopoiesis

remains incompletely understood. Indeed, current evidence suggests that it may be

regulated in a completely different manner 4. We therefore examined the effects of

neutrophil depletion in an established mouse model of neutropenia. Our results show

that there is specific sensing of neutropenia in the absence of inflammation. The

positive feedback phenomena are characterized by typical quantitative shifts of

hematopoietic marrow progenitors, dependence on upregulated G-CSF, and

downregulated marrow CXCL12. Analyses of the underlying mechanisms suggest the

existence of several, redundant pathways regulating G-CSF dependent granulopoiesis,

including IL-23 and IL-17, as previously described 5.

While TLR4-signaling has been implicated in emergency granulopoiesis 6-8, this highly

conserved pathway may also be suitable for tailoring neutrophil production to prevailing

needs in the steady-state 9. Our results suggest that TLR4-signaling represents a

conditio sine qua non for the sensing of peripheral blood neutropenia in the steady-

state.




4

Material and Methods

Mice

C57BL/6, NOD.Cg-Prkdcscid IL2rgtmWjl/Sz (NSG), B6.129P2(SJL)-Myd88tm1.1Defr/J

(MyD88-/-), C3H/HeJ and C3H/N mice were obtained from Jackson Laboratories (Bar

Harbor, Maine, USA) and maintained under specific pathogen-free conditions.

Germ-free (GF) C57BL/6 mice were maintained as previously described 10 (University of

Ulm, Germany). C3H/HeJ/TLR2-/- mice were provided by H.-G. Rammensee. TRIF-/- and

TLR4-/- mice by M. Radsak. All animals received amoxycillin in their drinking water.

Animal experiments were performed with the authorization of the Institutional Animal

Care and Use Committee of the University of Tuebingen according to German federal

and state regulations.

Antibody-induced neutropenia

To induce neutropenia, anti-Gr-1 clone RB6-8C5 (BioXCell, West Lebanon, NH, USA)

was injected at a dose of 500 µg every other day. Alternatively, anti-mLy6G clone 1A8

(BioXCell, USA) was injected at 1 mg every 36 hours. Antibodies were injected i.p. in

500 µl PBS for 8 days. Control groups received 500 µl PBS i.p..

Antibody-induced depletion NK cells

To deplete NK cells, 0.3 mg anti-NK1.1, clone PK 136 (BioXCell, USA) in 500 µl PBS

was injected i.p. daily for 7 days. 500 µl PBS was injected as control.

Flow cytometry

Antibodies were purchased from eBioscience (Natutec, Frankfurt am Main, Germany):

CD34 (Pacific Blue); Sca-1, Gr-1 and IgG2a (PE); CD11b and c-kit (APC); streptavidin

(PECy7); CD16/32 (PerCP-Cy5), CD127 (biotin). CD3, CD11b, B220, Gr-1, Ter119




5

(biotinylated) were used as mouse lineage panel (BD Pharmingen, Heidelberg,

Germany).

Peripheral blood analysis

Retro-orbital blood was collected and differential blood counts were obtained using an

automated Bayer Advia 120 MultiSpecies Analyzer (Bayer HealthCare, Leverkusen,

Germany). For flow cytometric analysis (FACS-Canto II; BD Bioscience, Heidelberg,

Germany), red blood cells (RBC) were lysed with ammonium chloride buffer (0.150 mM

NH4Cl, 0.1 mM EDTA, 0.150 mM KHCO3) for 10 minutes on ice. Cells were stained to

determine myeloid cells (CD11b, Gr-1). Isotype controls were used as indicated.

Bone marrow flow cytometry

After 8 days of continuous neutropenia, bones were harvested and flushed. Additionally

the vertebral column was harvested and pestled to obtain a maximum of marrow cells.

Red blood cells were lysed with ammonium chloride buffer for 10 minutes on ice. Then

cells were washed with PBS and stained for flow cytometric analysis of progenitor cells.

Cytokine ELISA

Plasma levels of G-CSF, M-CSF, IL-17 and IL-23 were measured using Quantikine

ELISA kits (RnD Systems, Wiesbaden, Germany) according to the recommendations of

the manufacturer.

Quantitative Real Time PCR

Whole marrow mRNA was isolated by RNeasy mini kit (Qiagen, Hilden, Germany). After

reverse transcription (SuperScriptII, Invitrogen, Darmstadt, Germany), quantitative PCR

was performed in a LC480 (Roche, Mannheim, Germany). Primers for G-CSF and β-




6

Actin were purchased from Applied Biosystems, Taqman gene expression assay

(Foster City, USA). The ratio was calculated to β-Actin.

Hematoxylin-Eosine staining of femora

Femora were harvested, fixed with 2% PFA, and embedded in paraffin after

decalcification (Richard Allan Scientific, Kalamazoo, MI, USA). Sections were stained

with H&E.

Statistics

Data are shown as mean ± standard error of the mean (SEM). Were indicated,

statistical significance of results in paired t-test analysis is given as p-values (Microsoft

Excel). P < 0.05 was considered statistically significant.




7

Results

Effective and durable antibody mediated peripheral blood neutropenia

C57BL/6 mice received either RB6-8C5 or 1A8 antibody versus PBS i.p.. Within twelve

hours, the Gr-1+/CD11b+ population was completely eradicated in the antibody treated,

but not in the placebo injected mice (Figure S1A). Continuous peripheral blood

neutropenia could be induced in all animals (Figure S1B). Forward vs. side scatter

FACS analysis of peripheral blood cells after red blood cell lysis revealed a relative

increase of Gr-1-/CD11b+ cells after antibody administration (Figure S1A). This

population showed immature myelocytic morphology (Figure S1 D) 11.

Mature neutrophils express Ly6G, which was previously defined as myeloid

differentiation antigen (Gr-1), and anti-Ly6G antibody clone RB6-8C5 depletes these

cells 12. Numerous publications on neutrophil depleted states in murine models of

infection have been published. However, RB6-8C5 antibody has been shown to

additionally deplete dendritic cells and subsets of macrophages, lymphocytes,

monocytes, and may be functionally active in stimulating Gr-1 positive myeloid

precursors 13. We, therefore, additionally utilized the more selective anti-Ly6G antibody

(clone 1A8) to deplete neutrophils in vivo 14.

Automated as well as manual microscopic peripheral blood analysis revealed that both

antibodies resulted in a highly significant reduction of neutrophils (Figure S1 B, C, D).

Peripheral blood counts showed that neutropenic mice had a concomitant decrease in

absolute white cell numbers on day 8 (Figure S1 C) (control = 7,330/µl, RB6-8C5 =

2,260/µl, 1A8 = 2,800/µl).

Antibody-induced neutropenia has previously been shown to induce hematopoietic stem

and progenitor cell (HSPC) proliferation in the marrow independent from complement or




8

Fc receptor γ 15, which strongly argues against inflammation mediated by complement

fixation or Fc receptor signaling as a mediator of Gr-1 antibody induced effects.

Nevertheless, in order to prove the specificity of the observed changes after antibody-

dependent neutrophil depletion, C57BL/6 mice received anti NK cell antibody (NK1.1,

clone PK136) at a dose of 0.3 mg once daily. After 7 days, mice underwent peripheral

blood and bone marrow analysis. In contrast to neutrophil depletion, NK depletion did

not cause LSK or GMP expansion. Moreover, G-CSF levels were unchanged (data not

shown). Direct effects of 1A8-antibody on stromal G-CSF production as previously

described for anti-Sca-1 antibodies 16 could be ruled out: C57BL/6 mesenchymal

stromal cells did not upregulate G-CSF RNA upon addition of 1A8 (data not shown).

Additionally, we analyzed whether residual endotoxin in the antibody was responsible

for the changes. To this end, wild type mice received 2 EU/LAL LPS every 36 hours.

After 8 days, animals were analyzed, and no changes of marrow progenitors were

detected (Figure S2 A). We conclude that the observed changes after neutrophil

depletion represent specific results.

Neutropenia induces expansion of lineage negative and myeloid lineage

committed stem and progenitor cells in the bone marrow

In order to establish the effects of neutropenia on hematopoietic marrow, mice

underwent extensive analyses of their femoral marrows. For optimum comparability,

analyses were done on day 8 of neutropenia. Histology demonstrated normocellularity

with an increased granulopoiesis/erythropoiesis ratio (Figure 1 A). Interestingly,

progenitor frequencies were affected at all levels of hematopoietic differentiation in

neutropenic animals: there were both relative and absolute increases in LSK cells

(Figure 1 B, C). Myeloid progenitors displayed a shift from lin- CD127- Sca-1- c-kit+

CD16/32- CD34- megakaryocyte/erythrocyte lineage restricted progenitor (MEP)




9

towards the lin- CD127- Sca-1- c-kit+ CD16/32+ CD34+ granulocyte/macrophage lineage-

restricted progenitor (GMP) phenotype (Figure 1 B, C). Lin- CD127- Sca-1- c-kit+

CD16/32- CD34+ common myeloid progenitors (CMP) were not significantly affected.

In summary, antibody induced neutropenia specifically stimulated CMP and GMP

expansion at the cost of MEP. Moreover, there was a strong effect on the LSK

population, which increased by 3-fold. Importantly, these changes were dependent on

G-CSF: concomitant administration of anti-G-CSF completely abrogated the above-

described feedback phenomena in neutropenic mice (Figure S2 B, C).

Positive feedback regulation of G-CSF in neutropenic mice

In adhesion molecule deficient mice, neutrophil homeostasis has been shown to be

regulated through a feedback loop involving IL-23, IL-17, and G-CSF 5. IL-17 has been

described to mediate G-CSF induced granulopoiesis 17. We therefore analyzed plasma

concentrations of known modulators of granulopoiesis including G-CSF, M-CSF, IL-17,

and IL-23. Within 8 days of neutrophil depletion with RB6-8C5, G-CSF concentration

increased to a mean concentration of 317 pg/ml, corresponding to a 4.5-fold increase

over baseline levels (Figure 1 D). 1A8 induced neutropenia resulted in a 2.5-fold

increase over baseline levels to 165 pg/ml. Interestingly, although RB6-8C5 eliminated

monocytes in addition to neutrophils, M-CSF plasma levels remained unchanged

(Figure 1 D). Further studies will be necessary to elucidate the underlying regulatory

mechanisms.

Interestingly, effects on IL-23 and IL-17 levels were not pronounced and dependent on

the anti-Ly6G antibody used. In agreement with recent publications, IL-17 levels

increased from 21 pg/ml to 67 pg/ml at day 8 of RB6-8C5 induced neutropenia (p =

0.05) and from 21 pg/ml to 43 pg/ml with 1A8 (p = 0.05) (Figure 1E).




10

In contrast to control mice, IL-23 plasma levels increased from levels below the ELISA

detection threshold to 40 pg/ml with 1A8 (p = 0.03) (Figure 1E). RB6-8C5 treated mice

did not display an increase in IL-23. IL-23 is assumed to be a monocyte/macrophage

and dendritic cell secreted cytokine. Nonspecific depletion of these cells by RB6-8C5

may explain the absent increase of IL-23 in these animals.

In summary, induction of peripheral blood neutropenia in vivo induced strong increases

of G-CSF, IL-17, and IL-23. These data are in line with results in leukocyte adhesion

molecule deficient mice, where a regulatory loop including TH17 cells was suggested 5.

T-/B-/NK-deficient mice display steady-state neutropenia accompanied by an

increase of non-committed hematopoietic stem cells

We next hypothesized that deficiency in IL-17 producing T-cells would result in steady-

state neutropenia due to reduced G-CSF production. Comparative analysis of absolute

neutrophil numbers and their degree of maturation in the peripheral blood of wild type

and lymphocytopenic mice should therefore reveal potential redundancy of neutrophil

regulatory loops. NSG mice have been reported to be devoid of T, B, and NK cells due

to a deficiency in common IL-2 receptor γ-chain 18.

Steady-state analysis of the peripheral blood showed significantly decreased white

blood counts in NSG mice as compared with C57BL/6 mice (1,920/µl in NSG; 7,330/µl

in C57BL/6). Of note, leukocytopenia in these mice was not only attributable to the total

lack of lymphocytes in NSG mice, but also to a reduction of Gr-1+/CD11b+ mature

polymorphonuclear and band neutrophils as well as Gr-1low/CD11b+ monocytes (Figure

S3 A, B).




11

Analysis of bone marrows showed increased absolute numbers of LSK cells. In

contrast, CMP as well as GMP were significantly reduced in NSG mice (Figure S3 C,

D). These findings are in line with reduced stimulation of myelopoiesis by loss of TH17

cells. Accordingly, a significant reduction of plasma G-CSF was detected in NSG mice

(Figure S3 E).

In summary, differences at the marrow and peripheral blood level consistent with

lymphocyte-dependent regulation of granulopoiesis can be observed in NSG. However,

almost normal neutrophil counts and plasma G-CSF levels are maintained in NSG mice.

These data confirm the contribution of lymphocytes to steady-state neutrophil

granulopoiesis, but indicate the existence of additional, redundant pathways.

Lymphocytes are dispensable in neutropenia-induced G-CSF mediated feedback

granulopoiesis

In order to gain further insight into lymphocyte-independent granulopoiesis, NSG mice

were made neutropenic. Analysis of the marrow revealed a pattern of changes over

baseline identical to the findings described above in C57BL/6 mice, including a

massively increased LSK population in both RB6-8C5 and 1A8 treated animals as well

as significantly increased GMP at the expense of MEP in RB6-8C5 treated mice (Figure

2 A, B). A possible explanation for the more pronounced effect on LSK and GMP as

compared to CMP-levels may be the faster transit time of the smaller, more short-lived

CMP-subpopulation 19. Plasma G-CSF increased from a mean of 35 pg/ml to 370 pg/ml,

corresponding to a 10.5-fold increase with RB6-8C5 antibody and nearly 30 fold up to

1,064 pg/ml in NSG mice depleted with 1A8 antibody (Figure 2 C). Plasma IL-17 as well

as IL-23 levels remained below detection threshold (4.37 pg/ml for IL-17, 2.28 pg/ml for

IL-23) in control and experimental groups throughout the experiment (data not shown).




12

These data support the notion that neutropenia can be sensed and translated into

profound changes in the composition of hematopoietic marrow in T-/NK-/B-cell deficient

mice as efficiently as in wild type mice.

Transcriptional regulation of G-CSF is dependent on neutrophil mass

Peripheral blood neutrophils bear the highest expression of G-CSF receptor (colony

stimulating factor 3 receptor, CSF3R) among hematopoietic cells 20. We therefore

hypothesized that plasma G-CSF may be a function of total neutrophil mass, i.e. G-CSF

could be regulated by binding to its receptor on the neutrophil surface with consecutive

effects on G-CSF plasma levels. In analogy to megakaryopoiesis 21, homeostatic

granulopoiesis could thus be the result of an indirect regulation of G-CSF through the

prevailing neutrophil mass. Alternatively, G-CSF may be regulated at the transcriptional

level.

To test the direct effect of neutrophil mass on G-CSF protein levels, granulocyte

transfusions were conducted. C57BL/6 mice received 1A8 antibody to deplete

neutrophils for 8 days and subsequently received granulocyte transfusions, which

resulted in measurable increases of peripheral blood neutrophils (Figure S4 A). Plasma

G-CSF in acceptor mice determined immediately before and 36 hours after transfusion

showed no significant changes over baseline (Figure S4 B). We conclude that Gr-1+

neutrophil cell mass may not be a direct regulator of plasma G-CSF. Total marrow cell

derived G-CSF RNA showed an insignificant increase over baseline (Figure 3C).

To examine transcriptional regulation of G-CSF in response to neutrophil leukocytosis,

C57BL/6 mice received daily injections of rh-G-CSF 22. On day 5, peripheral blood was

obtained and animals were sacrificed. Bone marrow flow cytometry (Figure 3 A, B)




13

revealed changes indistinguishable from mice with antibody induced neutropenia.

Specifically, marrow hematopoietic precursors showed a 4-fold increase of LSK cells

and a 3-fold increase of GMP at the expense of MEP (Figure 3 A, B). Although

hematopoiesis in neutropenic and rhG-CSF treated mice appeared phenotypically

indistinguishable, whole bone marrow-derived G-CSF mRNA in G-CSF treated mice

plummeted (Figure 3C). Thus, there is a negative feedback loop, whereby G-CSF

induced neutrophil leukocytosis induces downregulation of marrow G-CSF transcription.

In summary, G-CSF is downregulated upon rh-G-CSF-induced neutrophilia, showing

negative feedback on the RNA level. On the other hand, G-CSF plasma levels are not

affected by neutrophil transfusions. We therefore conclude that transcriptional regulation

of G-CSF is indeed dependent on neutrophil mass. However, in contrast to TPO, G-

CSF receptor scavenging does not seem to play a role in G-CSF regulation. In

lymphocyte deficient mice, this pathway is robust and therefore independent of IL-17

secreting NK-, NK-like- and T-cells.

Reduced steady-state granulopoiesis with conserved feedback homeostatic

regulation in germ-free mice

Bacterial colonization has been established to influence development of both innate and

adaptive immunity 23. Granulopoiesis is hypothesized to be highly dependent on

peripheral needs. While results obtained in G-CSF and CSF3R deficient mice, who

display strongly reduced neutrophil numbers have revealed the importance of G-CSF

signaling in steady-state neutrophil homeostasis 24;25, “emergency granulopoiesis” is

mediated by microbial compounds binding to pattern recognition receptors 26. Bacterial

flora was also shown to enhance the ability of neutrophils to kill pathogenic bacteria 26.




14

We hypothesized that steady-state granulopoiesis could indirectly be influenced by

microbial colonization and therefore analyzed GF mice in the steady-state.

Steady-state comparative analyses of C57BL/6 mice kept under SPF conditions vs. GF

C57BL/6 mice revealed reduced total white blood counts (7,300/µl vs 3,400/µl) and a

significantly smaller proportion of Gr-1+/CD11b+ neutrophils in GF mice (1,424/µl vs.

374/µl). Indeed, neutrophil counts in GF mice were lower than described in G-CSF or

CSF3R deficient animals 24;25. Manual differential blood counts confirmed reduced

relative proportions of band and polymorphonuclear neutrophils. These results may

indicate that steady-state granulopoiesis could be at least partly dependent on pattern

recognition receptors.

Toll-like receptors (TLR) have been found to be expressed on hematopoietic stem cells

and myeloid progenitors of the granulocyte lineage, and microbial compounds may

provide cues for hematopoiesis including neutrophil production 27. Since neutropenia

may feedback stimulate hematopoiesis via commensal bacteria invading mucosal

membranes unopposed by neutrophils thereby triggering TLR-signaling, we utilized GF

mice to study the influence of commensal microbiotes on neutrophil homeostasis. To

this end, GF mice either received 1A8 antibody or PBS and effects on hematopoiesis

were analyzed as described above (Figure 4 A). Comparable to SPF mice, neutropenia

induced absolute and relative increases of the LSK and GMP populations: the number

of LSK cells in neutropenic GF mice went up from 0.31x105 ± 0.14x105 to 1.72x105 ±

0.54x105. Myeloid progenitors were enriched in the marrows of neutropenic GF mice,

GMP went up from 3.18x105 ± 0.28x105 to 9.25x105 ± 0.49x105 (Figure 4B). Moreover,

analysis of cytokine plasma levels revealed significant increases of plasma G-CSF in

the antibody-treated group from 34 pg/ml to 293 pg/ml (Figure 4 C).




15

Taken together, analysis of GF C57BL/6 mice shows that a total lack of microbial

colonization is associated with extraordinarily decreased neutrophil numbers in the

steady-state as well as decreased baseline G-CSF levels. However, upon antibody

induced neutropenia, GF mice generate responses both at the marrow and peripheral

blood level indistinguishable from wild type or NSG animals.

Neutropenia induced feedback regulation is dependent on TLR4 and TRIF, but not

TLR2 and MyD88

GF maintained mice may receive TLR stimuli through LPS in their food and drinking

water 28. We therefore analyzed, whether interference with downstream signaling

pathways might abrogate feedback neutrophil granulopoiesis in mice resistant to

endotoxin (Tlr4Lps-d) by a spontaneous mutation 29 and mice deficient in both TLR-2 and

TLR-4 30.

When C3H/HeJ(TLR4mut) /TLR2-/- mice, C3H/HeJ (TLR4mut) mice, and C3H/N wild type

control mice were challenged with 1A8 antibody, we found only insignificant changes

from baseline in the knock-out mice (Figure 5 A, B, C). Indeed, while wild type mice

showed an increase of G-CSF by 8.5-fold from 156 pg/ml to 1,318 pg/ml, there was a

non-significant increase in the knock-out animals (Figure 5 C). Moreover, all

neutropenia induced changes at the hematopoietic progenitor cell level were to be

observed in control C3H/N, but not in knock-out mice (Figure 5 A, B, D, E). Numbers of

marrow hematopoietic progenitors were not significantly different in neutropenic

C3H/HeJ(TLR4mut) /TLR2-/- mice versus control mice, and LSK cells remained

unchanged (Figure 5 A). Interestingly, steady-state G-CSF levels differed between

mouse strains, but were identical within a given background, i.e. C57BL/6 or C3H/N.




16

We therefore additionally analyzed TLR4 knockout mice on the C57BL/6 background.

As expected from the results obtained in C3H/HeJ mice, TLR4-deficient animals

showed no significant changes in plasma G-CSF levels (78 pg/ml in control mice vs. 77

pg/ml in 1A8 treated mice) (Figure 6 A). Moreover, analysis of the marrows showed that

treatment with 1A8 resulted in minor, non-significant changes at the progenitor level:

LSK cells increased insignificantly from 1.26x105 (control) to 1.75x105 cells (1A8). GMP

increased from 2.49x105 to 3.0x105 cells, and there was a slight decrease of MEP from

2.32x105 in control mice to 1.87x105 in 1A8 treated mice (Figure 6 B). We conclude

from these findings that TLR4 is essential in homeostatic, G-CSF dependent feedback

regulation of neutrophil granulopoiesis.

TLR-signaling involves the recruitment of one or several TIR-domain-containing adaptor

proteins like MyD88, TIRAP, TRIF or TRAM 31. Therefore, MyD88-/- 32 and TRIF-/- mice

33 were analyzed in addition. MyD88-/- and C57BL/6 control mice received 1A8 antibody

as described above. Analysis of MyD88-/- peripheral blood, plasma, and marrow

displayed changes indistinguishable from results obtained in C57BL/6 animals (Figure 6

A, C). Moreover, plasma G-CSF increased significantly up to 6- fold in neutropenic

MyD88-/- mice from 56 pg/ml to 347 pg/ml. Analysis of neutropenic vs. control TRIF-/-

mice showed a non-significant increase of plasma G-CSF from 184 pg/ml (PBS) to 422

pg/ml (1A8) (Figure 6 A). Moreover, LSK expansion was detectable with a significant

increase of LSK cells from 1.06x105 in control to 2.54x105 in antibody treated animals.

GMP numbers as well as MEP levels, however, remained unchanged (Figure 6 D).

Expressional analysis of TLR4, MyD88, and TRIF RNA in the marrow of all tested

mouse types revealed unchanged expression levels (data not shown).




17

Taken together, TLR-4 is essential for feedback regulation in neutropenia. Interestingly,

absence of TRIF partially abrogates feedback G-CSF upregulation and respective

changes in the marrow, while TLR-2 and MyD88 are completely dispensable. We

conclude that undisturbed signaling via TLR-4 represents necessary elements of the

signaling pathway sensing neutropenia and initiating the appropriate response.

Neutropenia inhibits bone marrow CXCL12 transcription

Neutrophil supply is not only dependent on production, but also on release from the

marrow. Downregulation of CXCL12 within the marrow milieu has been described to be

associated with facilitated neutrophil egress 34. We therefore analyzed marrow CXCL12

under various experimental conditions.

C57BL/6 mice received rh-G-CSF at a dose of 300 µg/kg body weight for five

consecutive days 22. Peripheral blood revealed leukocytosis with 82% neutrophils

(13,260/µl ± 2,180/µl leukocytes, 10,864/µl ± 1,810/µl neutrophils). As previously

described, whole marrow CXCL12 RNA levels were significantly reduced by about 10-

fold (Figure S5 A). Interestingly, neutropenia downregulated CXCL12 RNA at a

comparable magnitude both in spf-kept and in GF wild type mice (Figure S5 B).

Additionally, flow cytometric surface expression levels of CXCR4 were determined in

LSK, GMP, CMP, MEP, and Gr1low/CD11b+ cells. CXCR4 was slightly downregulated

upon granulocyte depletion in all examined populations except from MEPs (data not

shown). Thus, antibody induced neutropenia may result in facilitated egress through

suppression of marrow CXCL12.

Analysis of marrow CXCL12 in NSG mice in the steady-state demonstrated a significant

increase by approximately 3-fold as compared with C57BL/6 mice (Figure S5 A).

Therefore, steady-state neutropenia in NSG mice may also be due to reduced egress of




18

neutrophils. Upon neutropenia, however, downregulation of CXCL12 in NSG mice was

similar to wild type mice (Figure S5 C).

To establish the importance of TLR-signaling, TLR4-/-, C3H/HeJ, C3H/HeJ/TLR2-/-, and

C3H/N control mice received PBS or 1A8, and CXCL12 RNA was analyzed.

Neutropenic C3H/HeJ and C3H/N mice displayed significantly decreased CXCL12 RNA.

However, in C3H/HeJ/TLR2-/-and C57BL/6 TLR4-/- mice, the CXCL12 decrease did not

reach a significant extent (Figure S5 D).

In conclusion, increased marrow granulopoiesis, whether driven by exogenous rh-G-

CSF or neutropenia, results in decreased CXCL12 levels. In addition, peripheral blood

neutropenia in NSG mice is not only due to loss of the IL-17 signaling axis, but may also

be ascribed to CXCL12 mediated retention of neutrophils.

Discussion

In contrast to feedback regulation of erythropoiesis and megakaryopoiesis, knowledge

on mechanisms of neutrophil granulopoiesis is fragmentary. Early observations of

upregulated G-CSF levels in neutropenic patients after myelosuppressive

chemotherapy and in cyclic neutropenia have led to the conclusion that either a

“neutrostat” would sense peripheral neutrophil levels or a neutrophil turnstile located at

the marrow-blood interface would enumerate released neutrophils in order to provide

feedback regulation of G-CSF and adapt marrow neutrophil production to peripheral

needs 35.

In order to elucidate feedback mechanisms, we utilized a model of neutropenia that

includes minimum inflammation 15. Animals were kept SPF and received amoxycillin in

their drinking water. During prolonged neutropenia, mice were closely monitored for




19

signs of infection. Therefore, infection induced changes, also known as “emergency

granulopoiesis” did not play a role in our study of neutrophil homeostasis.

Our findings show that two different anti Ly6G-antibodies effectively and specifically

induced neutropenia in all studied mice (Figure S6, S7). Neutropenia resulted in

identical changes of hematopoietic marrow composition and G-CSF feedback

stimulation independent from the binding epitope.

G-CSF is the principal neutrophil regulating cytokine, and the absence of G-CSF in

humans or in knock-out mice results in severe neutropenia 1;24. Based on observations

in adhesion-molecule deficient mice, IL-23 and IL-17, secreted by dendritic

cells/macrophages and TH17-CD4- T-cells, respectively, have been described as

upstream regulators of G-CSF 5;36. While G-CSF seems to be necessary for the

maintenance of neutrophils in steady-state mice, interference with IL-17 did not reduce

neutrophil numbers 37. In our model of antibody-induced neutropenia, IL-17 increased

significantly, confirming previous results 5. However, neutropenia in lymphocytopenic

NSG-mice displaying baseline IL-17 levels without any variation resulted in positive

feedback regulation of marrow hematopoiesis and increased G-CSF expression at the

transcriptional level. We, therefore, conclude that the IL-17 dependent pathway is

redundant.

Because feedback regulation of G-CSF was efficient in NSG-mice, we hypothesized

that highly conserved sensing mechanisms of neutropenia may perceive indirect

sequelae of neutropenia such as mucosal membrane invasion by commensal germs,

which have been shown to regulate systemic immunity 38. Sensing receptors of

pathogen-associated molecules include TLR2, TLR4, NOD1, and NOD2. TLR4 is

known as the major LPS receptor, TLR2 is a mediator of responses to gram-positive

bacteria. TLR2- and 4-agonists regulate neutrophil functions and contribute to inhibition




20

of neutrophil apoptosis in states of antimicrobial response 39;40. Furthermore, several

molecules including hyaluronan, surfactant protein-A, β-defensin and byglycan

represent endogenous TLR2 and TLR4 ligands 41. Moreover, heat shock proteins like

Gp96 may represent endogenous potentiating factors allowing minute concentrations of

TLR-ligands to generate a TLR-dependent signal 42. To study the influence of the

mucosal bacterial flora on feedback granulopoiesis, we took advantage of germ-free

C57BL/6 mice 38. These animals do not harbor commensal microbiotes, but they have

also been shown to lack TH17- cells in the colonic bowel wall. When germ-free mice

were made neutropenic, however, we found that their ability to mount the typical

response was preserved in the absence of commensal germs. Therefore, intestinal

microflora is not a necessary prerequisite for the observed feedback regulation of

neutrophil granulopoiesis. A potential disturbing factor in this model are effects of

lipopolysaccharide (LPS) contained in the experimental animals’ autoclaved food 28

which exert TLR2 and TLR4 agonist activity 39, enabling these mice to create feedback

granulopoiesis upon neutropenia. Thus, we examined TLR4-deficient, TLR4-mutated,

and TLR2-deficient/TLR4-mutated animals. All of these genetically modified mice

displayed markedly disturbed feedback upregulation of G-CSF, LSK and GMP

expansion 9. Indeed, absence of TLR4 resulted in a complete loss of feedback to

neutropenia with a marrow composition and peripheral G-CSF levels identical to control

treated mice. We, therefore, conclude that TLR4 is required for positive feedback

signaling in antibody-induced neutropenia. Moreover, TRIF, but not MyD88 signaling is

involved in G-CSF upregulation in vivo, since MyD88-/- display an undisturbed feedback

both at the G-CSF level and the marrow progenitor cell level. In contrast, TRIF-/- mice

showed an insignificant increase of G-CSF over baseline and no GMP expansion.




21

While binding of G-CSF to its cognate receptor (CSF3R) expressed on committed

hematopoietic progenitors of the granulocyte lineage mediates proliferative and

differentiative effects 35, G-CSF mediated mobilization of hematopoietic stem and

progenitor cells as well as neutrophils from the marrow has been elegantly shown to be

mediated indirectly by CSF3R+ monocytes 43;44. Our data clearly show that neutropenia

per se upregulates plasma G-CSF levels. Moreover, we demonstrate for the first time

that administration of exogenous rh-G-CSF downregulates marrow G-CSF expression.

While negative feedback mechanisms of G-CSF stimulated neutrophilia on

granulopoiesis have been described to be in part dependent on enzymatic cleavage of

G-CSF by neutrophil elastase 45, our results demonstrate that there is feedback on the

transcriptional level. Interestingly, infusion of CSF3R+ neutrophils did not significantly

influence plasma G-CSF. The latter finding provides evidence against a model of

indirect regulation of plasma G-CSF by neutrophil pool size.

Neutrophil numbers in peripheral blood are determined by their production rate, half-life

and their positioning (margination and marrow release); only 1-2% of neutrophils

circulate in the periphery in the steady-state 46. Emergency granulopoiesis results in

both increased neutrophil production and release to circulation 47. There is evidence that

G-CSF releases neutrophils from the marrow by disrupting their anchoring to a

CXCL12-positive marrow niche through CXCR4 expressed on the neutrophil surface 48.

Our results are in line with previous observations that marrow CXCL12 expression is

reduced during neutrophil depletion 46. Similar to emergency granulopoiesis,

neutropenia-induced feedback granulopoiesis in our model is regulated by production

and release. G-CSF may however be responsible for both effects.

Regulation of myelopoiesis has been demonstrated to be dependent on its own

downstream cellular components as well as on stromal cells. Loss or depletion of both




22

conventional dendritic cells and macrophages was shown to increase myelopoiesis 49.

Both cell types may function as negative regulators of myelopoiesis. Thus, neutrophil

mass may either be sensed by macrophages as previously suggested 5 or function as a

negative regulator of myelopoiesis similar to macrophages and dendritic cells. Our

findings suggest that, indeed, there is a “neutrostat”, which may be located on the

marrow level. In fact, emergency granulopoiesis has been shown to be dependent on

TLR4 expressed on the non-hematopoietic marrow compartment 7. Preliminary results

show that marrow-derived mesenchymal stromal cells potently upregulate G-CSF

expression upon TLR4 triggering and that upregulation of G-CSF RNA upon

neutropenia is to be detected in the CD45- marrow population (data not shown).

Our results challenge the current dogmatic distinction of steady-state versus emergency

granulopoiesis: we suggest a mechanism relying on highly conserved signalling

pathways that constantly adapt neutrophil production to environmental needs. The

importance of exogenous vs. endogenous TLR-ligands as well as cell type dependent

mechanisms are still to be determined, but the fact that antibiotic treatment reduces the

efficiency of stem cell mobilization by G-CSF in humans 50 underscores the potential

clinical importance of our findings.




23

Acknowledgments

HGK was supported by grants from the German Research Foundation (SFB685, project

A7) and Deutsche Krebshilfe (Max Eder Program, project 109833). MR is supported by

the German Research Foundation KFO183 (Ra 988/4-2) and by the federal Ministry of

Education and Research (BMBF 01EO1003). MCA was supported by grants from the

Deutsche Forschungsgemeinschaft (KFO 183/TP 4). The authors thank Nicole

Kosgalwis, University of Ulm, for expert gnotobiotic animal breeding facilities.

Authorship Contributions

SB performed the majority of the experiments and contributed to the writing of the

paper; SW designed some of the experiments, analyzed and interpreted data and

contributed to the writing of the paper; MPR, HS, JSF, HGR, MCA and RH provided

mice; PS, EM, TW and MM contributed some of the experiments;

MRM, LK analyzed and interpreted data; HGK designed the project and experiments,

analyzed and interpreted data and wrote the manuscript.

Disclosure of Conflicts of Interest

The authors declare no competing financial interests.




24

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Figure Legends

Figure 1: Neutropenia induces expansion of myeloid hematopoietic progenitors

and myelopoietic cytokines

A) H&E stained femora of neutropenic and control C57BL/6 mice on day 8 of treatment

with granulocyte/erythrocyte ratio. (microscope: Apotome, Zeiss, Esslingen,

Germany; acquisition software: Axiovision (Zeiss); magnification (63 x and 20x)).

Note that marrow cellularity remains high in neutropenic animals and the increased

granulocyte/erythrocyte ratio in antibody-treated mice.

B) Marrow flow cytometry including gating strategy in C57BL/6 wild type mice. Myeloid

progenitor cells (lin- CD127- Sca-1- c-kit+) were further differentiated into GMP

(CD16/32+ CD34+), CMP (CD16/32- CD34+), and MEP (CD16/32- CD34-). Note

expansion of the LSK population and increase of GMP at the cost of MEP.

C) Absolute marrow cell numbers calculated to reflect total cell counts in both hindlimbs

(n = 3). RB6-8C5: p(LSK) < 0.001; p(GMP) = 0.003; p(MEP) = 0.02; p(CMP) = n.s.

1A8: p(LSK) = 0.01; p(GMP) = 0.03; p(MEP) = n.s.; p(CMP) = n.s..

D) Plasma G-CSF and M-CSF levels in control and neutropenic C57BL/6 mice. Note

the significant increase of G-CSF (n = 3). G-CSF: p(RB6-8C5) = 0.002; p(1A8) <

0.001. M-CSF: p(1A8 and RB6-8C5) = n.s..

E) Plasma IL-17 and IL-23 levels in control and neutropenic C57BL/6 mice (n = 3 each

group). IL-17: p(RB6-8C5) = 0.05; p(1A8) = 0.05. IL-23: p(RB6-8C5) = n.s., p(1A8) =

0.03.

Figure 2: Neutropenia induced feedback in NSG mice

A) NSG mice received 1A8, RB6-8C5 or PBS. Flow cytometric analyses of marrows are

shown. Note the significant changes of LSK, GMP and MEP.




29

B) Absolute cell numbers in both femora and tibiae of NSG mice (n = 5). RB6-8C5:

p(LSK) = 0.04; p(GMP) = 0.05; p(CMP) < 0.001; p(MEP) = 0.02; 1A8: p(LSK) <

0.001; p(GMP) = n.s.; p(MEP) = n.s.; p(CMP) = n.s.

C) Plasma G-CSF and M-CSF levels in control and antibody-treated NSG mice. Note

significant change of G-CSF (n(control) = 4; n(RB6-8C5) = 3; n(1A8) = 3). G-CSF:

p(RB6-8C5) = 0.003; p(1A8) < 0.001. M-CSF: p(1A8 and RB6-8C5) = n.s.

Figure 3: Exogenous G-CSF yields changes similar to neutropenia and reveals

transcriptional regulation of G-CSF in the marrow

A) Marrow flow cytometry in control and rh-G-CSF treated C57BL/6 mice (n = 5).

B) Absolute marrow cell numbers in both hindlimbs of C57BL/6 mice after rh-G-CSF (n

= 5) vs. PBS (n = 3). rh-G-CSF: p(LSK) < 0.001; p(GMP) = 0.01; p(CMP) = 0.02;

p(MEP) < 0.001.

C) Transcriptional G-CSF levels in the marrow of control, rh-G-CSF, RB6-8C5 and 1A8-

treated C57BL/6 mice. Note the increase of G-CSF upon neutropenia and the

negative feedback at the RNA level after application of rh-G-CSF. p(rh-G-CSF) =

0.045; p(RB6-8C5) = n.s.; p(1A8) = n.s..

Figure 4: Commensal germs and MyD88 are dispensable in G-CSF mediated

feedback

A) Flow cytometry of peripheral blood from neutropenic GF C57BL/6 mice and controls

(n = 3).

B) Absolute marrow cell numbers in both hindlimbs of GF C57BL/6 mice. Note the

similarity of changes in GF mice as compared with SPF-kept C57BL/6 (n = 3).

p(LSK) = 0.04; p(GMP) = 0.05; p(CMP) = n.s.; p(MEP) = n.s..




30

C) Plasma G-CSF levels in C57BL/6 and GF mice after neutrophil depletion with 1A8

vs. controls. Note the significant increase of G-CSF in neutropenic mice and

decreased baseline G-CSF levels in GF mice compared with animals maintained

under SPF conditions (n(GF) = 3, n(C57BL/6) = 3). p(C57BL/6) = 0.04; p(GF

C57BL/6) = 0.02.

Figure 5: Neutropenia induced feedback regulation is TLR-dependent

A) Absolute cell numbers in hindlimb marrows of C3H/HeJ/TLR2-KO mice. Note that

control and neutropenic mice (n = 5, each) are identical. p(LSK, GMP, CMP and

MEP) = n.s.

B) Absolute cell numbers in hindlimb marrows of C3H/HeJ mice after 8 days of 1A8-

induced neutropenia. There are no significant differences in control vs. neutropenic

mice (n = 5). p(LSK, GMP, CMP and MEP) = n.s.

C) Plasma G-CSF levels in control and neutropenic C3H/N, C3H/HeJ/TLR2-KO and

C3H/HeJ mice. Note the significant increase of G-CSF levels in C3H/N and the

insignificant differences in C3H/HeJ mice (n = 5). p(C3H/N) = 0.002;

p(C3H/HeJ/TLR2-KO) = n.s.; p(C3H/HeJ) = n.s..

D) Absolute cell numbers calculated to reflect total cell counts in both femora and tibiae

of n = 3 C3H/N wild type mice after treatment with 1A8 for 8 days. Note the

significant changes of LSK and GMP. p(LSK) = 0.05, p(GMP) = 0.02, p(CMP) = n.s.,

p(MEP) = n.s..

E) Flow cytometric analyses of marrow cells in control and 1A8-treated C3H/HeJ mice.

Note equal numbers in control and neutropenic mice.




31

Figure 6: TLR4 and TRIF are necessary for feedback granulopoiesis

A) Plasma G-CSF levels in control and antibody treated C57BL/6 (n = 3, each), TLR4-/-

(n = 3, each), MyD88-/- (n = 5, each) and TRIF-/- mice (n(1A8) = 5, n(PBS) = 4). Note

the significant increase of G-CSF in C57BL/6 and MyD88-/- and non-significant

changes in the TLR4-/- and TRIF-/- mice. p(C57BL/6) = 0.04; p(TLR4-/-) = n.s.;

p(MyD88-/-) < 0.001; p(TRIF-/-) = n.s..

B) Absolute marrow cell numbers in TLR4-/- hindlimbs after 8 days of 1A8-induced

neutropenia. Note the insignificant changes of LSK, GMP, and MEP (n = 3). p(LSK,

GMP, CMP and MEP) = n.s..

C) Absolute cell numbers in hindlimbs of n = 5 MyD88-/- mice. LSK and GMP are

significantly increased in neutropenia. p(LSK) = 0.03, p(GMP) = 0.02, p(CMP) = n.s.,

p(MEP) = 0.03.

D) Hindlimb marrow cell numbers of n = 5 TRIF-/- mice after 8 days of 1A8-induced

neutropenia vs. PBS (n = 4). While there is an increase of LSK, changes at

progenitor level are non-significant. p(LSK) = 0.02, p(GMP) = n.s., p(CMP) = n.s.,

p(MEP) = n.s..




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Documents

Steady-state neutrophil homeostasis is dependent on TLR4/TRIF signaling