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BI431 LABORATORY PROJECT
Project Title:
Investigating the effect of MSC therapy on the microbiome in acute Graft-versus-Host-Disease
This thesis is submitted in fulfillment of the Biological & Biomedical Sciences Degree.
SUBMITTED BY: Amy O’Callaghan
STUDENT NO: 12381946
SUPERVISOR: Dr. Karen English
DATE: 19th April 2016
1
DECLARATION
I have read and understood the Departmental policy on plagiarism.
I declare that this thesis is my own work and has not been submitted in any form
for another degree or diploma at any university or other institution of tertiary
education.
Information derived from the published or unpublished work of others has been
acknowledged in the text and a list of references is given.
Signature: ………………………………………………
Date: ……………………………………………………
2
Table of ContentsAcknowledgements..............................................................................................................4
Abstract................................................................................................................................ 5
1. Introduction...................................................................................................................... 6
1.1 Acute Graft-versus-host-disease....................................................................................6
1.2 Factors effecting aGvHD severity.................................................................................8
1.3 Humanised mouse models..............................................................................................9
1.4 Mesenchymal Stromal Cells........................................................................................11
1.5 Microbiota.....................................................................................................................13
1.6 Project aims...................................................................................................................15
2. Materials and Methods...................................................................................................16
2.1 Human MSC isolation and culture.............................................................................16
2.2 Establishing a xenogeneic aGvHD model...................................................................16
2.3 Histopathological analysis and scoring.......................................................................17
2.4 Enzyme-Linked Immunosorbent Assay (ELISA)......................................................17
2.5 DNA Isolation................................................................................................................18
2.6 DNA Quantification......................................................................................................18
2.7 16S rRNA Sequencing..................................................................................................18
3. Results............................................................................................................................ 20
3.1 MSC-γ prolonged the survival of aGvHD mice models more significantly than resting MSCs...........................................................................................................20
3.2 Human MSC and MSC-γ reduced pathology and apoptotic damage in the tissues of aGvHD mice........................................................................................................21
3.3 MSC therapies significantly reduced pro-inflammatory cytokines in the tissues of aGvHD mice....................................................................................................................26
3.4 Expected 16S ribosomal RNA sequence results on microbiota diversity following MSC and MSC-γ therapy..................................................................................29
4. Discussion...................................................................................................................... 32
5. Bibliography...................................................................................................................36
3
Acknowledgements
I would like to thank my family and friends for their love and support during my studies at
Maynooth University. I would especially like to thank Jennifer Corbett, an amazing
mentor and advisor throughout this project. Jen has great patience and is an excellent
teacher. I would like to thank Dr. Karen English, my supervisor, for her patience,
encouragement and valuable support. Thank you to the Cellular Immunology Laboratory,
there was always someone to guide and advise me. Finally, I would like to thank Dr. Fiona
Walsh and her team in the Antimicrobial Resistance and Microbiome laboratory for all
their help with the microbiota work, I could not have completed this thesis without you all.
4
Abstract
Graft versus host disease remains a leading cause of immediate morbidity and mortality in
allogeneic human stem cell transplant patients. Division of GvHD into two distinct clinical
entities include acute GvHD and chronic GvHD with a 100 day boundary separating the
classification of both. Following engraftment, the donor (graft) recognises the host
(recipient) as foreign through human leukocyte antigen variance and elicits an exaggerated
inflammatory response influenced by unregulated donor T lymphocyte activation and
proliferation. The corresponding pro-inflammatory cytokines produced create an
unbalanced environment to which target tissues are damaged. The mucosal-epithelium
barrier provides a balanced habitat in the gastrointestinal tract, home to the largest number
of microbial communities in the body. The resultant cytokine storm and synergistic
conditioning regimens can hinder certain aspects of this primary interface, aggravating the
microbiota and their by-products which contribute to the severity of aGvHD. Conventional
and pre-stimulated mesenchymal stromal cell (MSC) therapy alleviates aGvHD symptoms
of target organs in patients and mouse models, to which this study supports these findings.
Since the microbiota contributes to the pathogenesis of aGvHD, very few studies have
outlined the effect of aGvHD therapy, in particular MSC therapies, on the microbiome.
Novel strategies to sustain the microbiota in a rational way to diminish aGvHD while
maintaining host immune functions are examined. Techniques such as 16S rRNA
sequencing seek to delineate these findings, contributing to the output of research in this
field.
5
1. Introduction
1.1 Acute Graft-versus-host-disease
Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is an established
and curative treatment for many threatening haematological malignancies (Shono et al.,
2015), where HSCs are supplied from a healthy donor. Graft-versus-host-disease (GvHD)
is a persistent and at times an unpredictable obstacle of allo-HSCT and can be fatal to
approximately 15% of patients receiving the transplant (Pasquini et al., 2010). The first
report on GvHD was established by Barnes, Loutit and Micklem, which was then
classically described by Billingham as a syndrome recognized by donor immunocompetent
cells, causing an outbreak on specific host tissues (seen as foreign) in allogeneic recipients
who are unable to mount a successful immune response (BARNES et al., 1962,
Billingham, 1966). GvHD can be divided into two distinct pathological processes; acute
and chronic GvHD. Acute GvHD (aGvHD) is a multi-organ disease occurring within 100
days after transplantation and consists of multiple phases:
6
Figure 1.1: The initiation phase of aGvHD involves pre-conditioning regimens damaging
host tissue (the gut is where aGvHD symptoms begin) causing the secretion of pro-
inflammatory cytokines tumour necrosis factor-α (TNF-α) and interleukin-1β (IL-1β).
Increased expression of such cytokines leads to the activation of host antigen presenting
cells (APCs). Such potent regimens systemically exposes microbial bi-products normally
sequestered in the intestinal lumen, contributing to aGvHD manifestation (Mathewson and
Reddy, 2015). During phase 2, host APCs prime mature donor T cells resulting in their
activation, proliferation and expansion into effector T cells. Phase 3 involves migration of
donor-derived effector T-cells to specific target organs where additional pro-inflammatory
secretion, inflammation and necrosis occurs. Figure adapted from Ferrara and Reddy,
(2006).
Cancer patients undergoing allo-HSCT can benefit from aGvHD in that donor T
cells attack and eliminate malignant residual host cells/diverse tumours – Graft versus
Leukaemia effect (GvL) (Kolb, 2008). aGvHD target organs include skin, liver, lung, gut,
spleen, hematopoietic system and endothelium, with organs scored in a grading system
such as the small intestine (SI), liver and skin, as illustrated shortly Filipovich et al. (2005).
During phase 3, alloreactive T cells infiltrate to specific target organs by a complex system
of chemokines and corresponding tissue surface receptors (New et al., 2002). Chemokines
are soluble mediators secreted by various cells and are classified according to the N-
terminal amino acid sequence – C, CC, CXC, and CX3C.
Elevated levels of pro-inflammatory cytokines damage aGvHD target organs in
murine models and patients which are influenced by the conditioning regimen as well as
other factors (Faaij et al., 2006). TNF-α is the main pro-inflammatory cytokine involved in
aGvHD development as it is a key driver of CD4+ effector T cells (Ferrara et al., 2009),
causing tissue damage. Interferon-γ (IFN-γ) upregulates adhesion molecules, chemokines
7
and major histocompatibility complex (MHC), all vital components of antigen presentation
following its secretion from T cells (Goker et al., 2001). IL-2 is involved in T-cell
activation and proliferation, correlating to the development of aGvHD. IL-2 is associated
with other inflammatory conditions (e.g. sepsis) and is used as a general indicator of an
activated immune system (Foley et al., 1998). IL-6 plays a role in differentiating naïve T
cells into Th17 cells that produce IL-17, exacerbating tissue destruction in the gut and liver
in their absence (Yi et al., 2009). IL-1β is secreted in response to the stimulation of the
node-like receptor protein 3 multiprotein inflammasome (Martinon and Tschopp, 2004),
accelerating aGvHD. IL-1β is shown to upregulate TNF-α receptor surface expression on
various cell types/organs and TNF-α production (Aida et al., 2006). Chronic GvHD is a
prevalent long-term complication of allo-HSCT and resembles autoimmune systemic
diseases with prolonged progression (Blazar et al., 2012).
1.2 Factors effecting aGvHD severity
Certain factors are thought to contribute to the development and severity of
aGvHD, including the following; patient age – Weisdorf et al. (1991) stated that older
patients are at a higher risk of aGvHD with Lee et al. (2013) contradicting this. In
complete contrast, some published data revealed no association between age and aGvHD
(Jagasia et al., 2012).
Toxicity of conditioning regimens - combined chemotherapy and radiation provide
space for the donor stem cell engraftment while simultaneously immunosuppressing the
patient in order to prevent rejection. The intensity and toxicity of the non-immunological
insult to tissues was found to be proportional to the severity of aGvHD, with different
forms of conditioning driving different manifestations of the disease (Hill et al., 1997).
Haematopoietic graft source – in order to perform HSCT, a suitable donor must be
available and the best donor for this procedure is a human leukocyte antigen (HLA)-
8
matched sibling or unrelated donor. HLA is very similar to vertebrate MHC class I and
class II. Class I (HLA-A, -B, -C) and II (HLA-DR, -DP, -DQ) are cell surface molecules
present on all nucleated cells (class I), APCs and monocytes (class II), that not only
determine histocompatibility but also regulate T cell recognition and antigen-presentation
(Goker et al., 2001, Chao, 1997). Matched HLA antigens have better engraftment rates
and a reduced rate of aGvHD, unfortunately, less than 30% of patients will have a matched
sibling donor (Ballen et al., 2008). Current alternative donors include a haploidentical
related donor (a maternal donor is preferred due to recognition of foetal antigens during
pregnancy (Stern et al., 2008)), a partially HLA-mismatched unrelated donor in at least one
allele or antigen at HLA-A, -B, -C or -DR or an umbilical cord blood stem cell product
(Kekre and Antin, 2014).
aGvHD prophylaxis approaches – current treatments are limited for this life-
threatening multi-organ complication. Systemic glucocoritcosteroids coupled with
immunosuppressive drugs, such as tacrolimus or cyclosporine A (calcineurin inhibitors),
remains the gold standard primary therapy for aGvHD due to their anti-inflammatory and
immunosuppressive nature (Messina et al., 2008). However, patients with steroid-
refractory aGvHD (patients who do not respond to steroids or do respond but relapse) have
fatal outcomes in 50% of cases (Martínez and Urbano-Ispízua, 2011). Current aGvHD
therapies in clinical trials include Natalizumab, a monoclonal antibody that prevents the
migration of donor T cells to target organs (ClinicalTrials.gov). The most promising and
so far successful therapy are MSCs, as described shortly.
1.3 Humanised mouse models
Shultz et al. (2012) describes an aGvHD murine model as engrafting human cells
and tissues creating a fully functional human immune system. Murine models are mainly
9
utilized due to their feasibility and lack of ethical restraint, enabling researches to create a
comparable system to the disease process seen in patients and develop novel therapies.
The majority of murine models used to study aGvHD are designed for the
transplantation of donor lymphocytes to irradiated hosts, basing the severity of aGvHD
development on the dose of the conditioning regimen, and the amount and type of
transplanted lymphocytes. Approximately 30 years ago, Bosma et al. (1983) identified the
first humanized mouse model (C.B-17-SCID) that went on to develop aGvHD like
symptoms. Since then, many more aGvHD mouse models, such as NOD-SCID
β2microglobulinnull (β2Mnull) (Christianson et al., 1997), NOD-Rag1nullPrf1null (Shultz et al.,
2000), and RAG-2−/−IL2r−/− ᵞ (Traggiai et al., 2004) have been established and utilized.
In this study, the non-obese diabetic (NOD)-severe compromised immunodeficient
(SCID) murine model is used. This xenogeneic humanised mouse model recognises
human immune cells and mouse tissue and engrafts donor-derived peripheral blood
mononuclear cells (PBMCs), causing the onset of aGvHD. Due to the impaired IL-2
receptor chain, this model has a ratified deficiency of mature T and B cells, reduced natural
killer (NK) cell activity, prolonged survival and better engraftment of human leukocytes
(Pearson et al., 2008),
10
Figure 1.2: Development of a humanised mouse model of aGvHD to compare MSC and
MSC-γ therapies and their effects on the microbiota. aGvHD mice were exposed to 2.4
gamma irradiation (2.4 Gy), 8 x 105 PBMC gram-1 or a sham-infusion of phosphate buffer
saline (PBS) was administered intravenously to each mouse through its tail vein. MSC or
MSC-γ (6.4 x104 g) were administered on day 6 intravenously. The development of
aGvHD was monitored closely.
This model is deemed the most commonly used humanised mouse model creating a
suitable system to investigate cellular therapies for the treatment of aGvHD and their effect
on the microbiota (Racki et al., 2010, Ali et al., 2012, Tobin et al., 2013, Healy, 2015).
1.4 Mesenchymal Stromal Cells
Bone marrow-derived mesenchymal stromal cells (MSCs) are a group of
heterogeneous plastic-adherent cells that have the ability to differentiate into an array of
cells in vitro including osteoblasts, adipocytes and chondroblasts (Friedenstein et al., 1966,
Friedenstein et al., 1974). The ability of allogeneic MSCs to influence an immune
response was first depicted by Bartholomew et al. (2002), followed by the revelation of
MSCs capacity to suppress T cell proliferation (English et al., 2007), dendritic cell
maturation, antigen presentation (English et al., 2008) and NK cell function (Lin and
Hogan, 2011).
MSCs display immunoprivileged characteristics; they do not express MHC class II
surface molecules and co-stimulatory ligand (L)/receptors such as CD40, CD40L, B7.1 and
B7.2, avoiding recognition by primed alloreactive CD4+ T-cells (Ryan et al., 2005). Their
immunomodulatory effects, as outlined by Tobin et al. (2013), stem from direct inhibitory
effects on donor-derived T cells instead of MSC-influenced expansion of T-regulatory cell
(Tregs) populations. IFN-ᵞ, alone or in combination with TNF-α, induces MSCs to release
a wide range of soluble factors and enzymes such as transforming growth factor beta
11
(TGF-β) (Di Nicola et al., 2002), IL-10 (Yang et al., 2009), indoleamine 2,3-dioxygenase
(IDO), prostaglandin E2 (PGE2) and cyclooxegenase-2 (COX-2) (Gao et al., 2016),
mediating their immunosuppressive activity. MSCs induce T-cell apoptosis via the FAS-L
dependent FAS pathway, triggering macrophages to produce elevated levels of TGF-β and
stimulating Tregs (Akiyama et al., 2012). FAS-L is a transmembrane protein belonging to
the TNF family. MSCs thrive in an inflamed environment, making these cells ideal
candidates for the treatment of many diseases such as Crohn’s disease, GvHD, amytrophic
lateral sclerosis and cardiac ischemia (Wang et al., 2012).
Le Blanc et al. (2004) outlined successful treatment of a 9 year old patient with
grade IV refractory aGvHD using donor-derived MSCs. Prochymal™ (allogeneic human
MSC therapy) has been shown to effectively treat paediatric patients with aGvHD who
have failed immunosuppressive or steroid therapies (Kurtzberg et al., 2014), however,
Prochymal™ did fail to reach their end-points in phase III clinical trials (Mills, 2009).
MSC therapy in animal models is conflicting; murine studies have reached equivocal
results (Sudres et al., 2006, Tisato et al., 2007, Tobin et al., 2013), while canine studies
have unsuccessfully demonstrated MSC efficacy in preventing aGvHD (Mielcarek et al.,
2011). Kaipe et al. (2014) negatively impacted the large scale use of MSC therapy due to
their potential capacity to become immunogenic, with possible formation of tumours and
rejection. Researchers are now looking at compartments derived from MSCs themselves,
extracellular exosomes, that portray the immunosuppressive, immune dampening and
reparative actions of MSCs (Kordelas et al., 2014).
However, the focus of this project involves the use of conventional MSC and MSC-
γ displaying optimum and consistent results, influencing aGvHD, as described previously
(Prasad et al., 2011, Le Blanc et al., 2008).
12
1.5 Microbiota
The human body is colonized by commensals such as bacteria, fungi and viruses,
collectively referred to as the intestinal microbiota. The human gastrointestinal (GI) – tract
contains trillions of microorganisms, with Sender et al. (2016) revealing that the ratio of
human cells to bacterial cells is closer to 1:1 compared to the previous ratio of 10:1
(Ferreira et al., 2014). 16S ribosomal RNA (rRNA) sequencing carried out by Gangarapu
et al. (2014) on healthy individual stool samples revealed that the human gut is largely
dominated by Firmicutes, Bacteroidetes, Proteobacteria and Actinobacteria, impacting the
host’s biology through production of short-chain fatty acids (SCFA) (Hamer et al., 2008).
Alterations in intestinal microbiota composition are linked to various inflammatory
diseases in humans, including aGvHD.
A role for the microbiome in influencing the intensity of aGvHD was first
identified in 1960s using germ-free recipient mice, with further studies forming the basis
for clinical use of antibiotic treatment prior to transplants, preventing the development of
aGvHD (van BEKKUM and VOS, 1961, van Bekkum et al., 1974). Thus suggesting that
the diversity of the intestinal microbial flora can hinder the patient’s propensity for
aGvHD, with reports illustrating that aGvHD deviates from Clostridiales dominance to the
emergence of Lactobacillales or Enterobacteriales (Holler et al., 2014, Jenq et al., 2012).
Clostridiales are known to induce Tregs (Atarashi et al., 2011), immune cells that possess
a critical role in controlling and regulating intestinal microbial milieu interactions.
Naturally occurring thymus-derived Tregs target T-cells that evoke the development of T-
helper-1 (Th1), Th2 or Th17 cells, dampening the immune response through the secretion
of anti-inflammatory cytokines e.g. IL-10 (Round and Mazmanian, 2010). Administration
of MSCs stimulate the expansion of Tregs enabling them to continue to carry out their
regulatory function, dampening aGvHD (Engela et al., 2012). Clostridiales perform
13
fermentation of consumed non digestible carbohydrates and their by-products are shown to
provide health benefits (Hamer et al., 2008). In the context of a patient with aGvHD, the
lack of such bacterial isolates means they lack the protective effects against aGvHD, as
discussed shortly.
aGvHD and intense conditioning regimens synergistically damages epithelium
surfaces; the primary interface between the gut bacteria and deeper tissues (Hanash et al.,
2012). This leads to imbalanced gut microbial colonies (microbial dysbiosis), resulting in
an immune signalling pathogen recognition receptor (toll like receptor (TLR)) mediated
cytokine storm. The stimulation of such receptors leads to the transcription of
inflammatory genes and upregulation of pro-inflammatory cytokines/costimulatory
molecules, causing local tissue inflammation and migration of leukocytes in aGvHD
(Penack et al., 2010). Generation of pathogen associated molecular patterns (PAMPS;
microbial by-products such as lipopolysaccharide (LPS) and peptidoglycan) and danger
associated molecular patterns (DAMPS; chemokines, cytokines, MHC host-antigens, ATP
from stressed/apoptosing cells) in the systemic circulation activates the inflammasome
complex in myeloid immune cells following conditioning regimens (Gagliani et al., 2014).
Thus contributing to aGvHD through the production of pro-inflammatory cytokines.
aGvHD is regarded as a primary predisposing factor for the development of septicaemia
(Chen et al., 2015), as outlined shortly.
By examining the microbial diversity in the gut on day 0, day 4 and day 9 after
irradiation, administration of PBMCs and MSCs, the capacity of cellular therapeutics to
somewhat modulate intestinal microbiota is examined and delineated, contributing to this
specific research field.
14
1.6 Project aims
The overall aim of this project is to examine the effects of mesenchymal stromal
cell therapy on the microbiome of the gastrointestinal tract in a humanised mouse model of
acute graft-versus-host-disease using up to date techniques such as; Histology and Tunel to
examine architectural changes in specific target organ tissues following cellular therapy
(MSC and MSC-γ), scoring histology images appropriately. ELISA to specifically define
what types of pro-inflammatory cytokines are dampened following cellular therapy. 16S
rRNA sequencing to determine bacterial communities in stool samples on day 0, day 4 and
day 9 following cellular therapy. By confirming what type of MSC therapy has a greater
reduction of aGvHD symptoms, a positive correlation may be noted on the microbiota of
aGvHD mice. The research can be added to the field of aGvHD prophylaxis or MSC
therapy in association with the microbiota in aGvHD mouse models, work that has not
been highlighted to date.
15
2. Materials and Methods
2.1 Human MSC isolation and culture
Human bone marrow MSCs were isolated as described by Barry and Murphy,
(2004), in collaboration with the Regenerative Medicine Institute (REMEDI, NUI Galway,
Ireland). Human MSCs were cultured by my assigned PhD student in complete
Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen-GIBCO, Dublin, Ireland)
supplemented with 10 % (v/v) fetal bovine serum (Gibco-Invitrogen), 50 U/ml penicillin
and 50 µg/ml streptomycin at 37°C in 5% CO2. In some cases, MSCs were stimulated with
recombinant human IFN-γ (500 U/ml) (Peprotech, London, UK) for 24 h and washed
profusely with PBS prior to their use in vivo or in vitro.
2.2 Establishing a xenogeneic aGvHD model
All animal handling procedures were carried out by licensed personnel. A
humanized mouse model of aGvHD was optimized from Pearson et al. (2008) protocol.
NOD.Cg-PrkdcscidIL2tmlWjl/Szj mice (NSG) were exposed to a conditioning dose of 2.4
G-ray of whole-body gamma irradiation day 0. Human PBMC (8 x 105 gram-1) isolated
from buffy coats were administered to NSG mice via the tail vein as previously described
(Tobin et al., 2013). Negative control mice were given PBS alone. Signs of aGvHD
occurred typically between days 10-16 post-PBMC transfusion. In some mice,
conventional human MSC (6.4 x104 g) therapy was administered on day 6 post-PBMC
transfusion. In other groups, IFN-γ stimulated MSC therapy (6.4 x 104g) was administered
on day 6 post-PBMC transfusion. Animals that displayed greater than 15% total body
weight loss or a pathological score of 8 were killed humanely according to local ethical
committee guidelines. Pathological scoring is defined as morphological assessment of the
animals according to a scoring, or grading system. This is used as a tool to derive data
from a biological system (i.e. mouse) for analysis and group comparisons (Gibson-Corley
16
et al., 2013). On day 13, target organs were harvested from all group for histological and
cytokine analysis.
2.3 Histopathological analysis and scoring
Target organs (lung, liver, colon, SI) were recovered from mice and fixed in 10%
(v/v) buffered formalin, prepared for histology and embedded in paraffin wax. Five-µm
tissue sections were stained by haematoxylin and eosin (H&E) and by Tunnel and DAPI.
A semi-quantitative scoring system was used to evaluate abnormalities in the lung, liver,
and GI tract sections in tissues/images stained by H&E.
2.4 Enzyme-Linked Immunosorbent Assay (ELISA)
All ELISA were carried out according to the manufacturer's instructions (R&D
Duoset). 100µl of capture antibodies (42µl IFN-ᵞ, IL-1β, -2, -6, -17, TNF-α in 5mls PBS)
were coated onto the 96 well microtitre plates, covered and incubated at room temperature
overnight. Plates were then washed 3 times in wash buffer (PBS with 0.05 % v/v Tween
20) and incubated in 300µl blocking solution (PBS with 1 % w/v bovine serum albumin
(BSA)) for a minimum of 1 hour. Washing was repeated and incubated with 100 μl/well of
sample supernatant or corresponding cytokine standard for 2 h at room temperature. After
washing, plates were incubated with 100µl of specific detection antibodies for a further 2 h
at room temperature. Plates were washed again and incubated with 100 μl/well of
streptavidin horseradish peroxidase (HRP) (R & D Systems) conjugate diluted 1/200 in
specific reagent diluent (Tris buffered solution (Sigma-Aldrich)) supplemented with BSA
for 20 min, out of direct sunlight. After washing, plates were incubated with 100 μl/well of
tetramethylbenzidine substrate (Sigma-Aldrich) for 20 min at room temperature out of
direct light. The reaction was stopped after 20 min by adding 50 μl/well of 1 M H2SO4.
The absorbance (optical density) of samples and standards were measured at 450 nm for all
17
ELISAs using Biotek plate reader. The cytokine concentration of each sample was
determined by comparison to the standard curve of known cytokine concentrations.
2.5 DNA Isolation
DNA from stool samples of the different treatment groups (PBS, PBMC, MSC and
MSC-γ) were isolated using PowerSoil® DNA Isolation Kit. Each sample followed a step
by step protocol of solutions where cells underwent homogenization, lysation, DNA
purification and eluted in Sterile DNA-Free PCR Grade Water. Gel electrophoresis on a
1.3 % agarose gel was carried out on all samples using GeneRuler 1Kb DNA Ladder (5
µl), to separate and analyse microbial DNA based on their size and charge from the various
treatment groups on different days following their administration (Day 0, Day 4, Day 9).
2.6 DNA Quantification
DNA quantification was carried out using a nanodrop spectrophotometer and gel
electrophoresis. The absorbance of a 1µl sample at a wavelength of 260nm calculated the
amount of DNA in each sample using the equation Abs260OD1=40 ng/µl. The ratio of
absorbance at 260nm and 280nm determined the purity of the sample. All agarose gels
were prepared by adding 1.3g (w/v) agarose (Sigma-Aldrich) to 1X TAE buffer and heated
in the microwave until completely dissolved. 6 µl of Gel Red (Biotium, California, USA)
was added and solution was poured into the gel tray. When solidified, agarose gels were
submerged in TAE buffer and subjected to electrophoresis at 90V for 60 minutes following
administration of each sample (3 µl) and DNA ladder (5 µl).
2.7 16S rRNA Sequencing
The 16S rRNA sequence was amplified from each sample using amplicon
polymerase chain reaction (PCR) (Illumina MiSeq System) using 2 primers that locate that
V3 and V4 regions of the 16S rRNA gene;
16S Amplicon PCR Forward Primer (5 µl) =
18
5'TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG
16S Amplicon PCR Reverse Primer (5 µl) =
5'
GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAAT
CC,
High-Fidelity 2x MasterMix (12.5 µl) (NewEnglands BioLabsinc) and each sample (2.5 µl)
were pipette into individual eppendorfs. The PCR took place in a thermal cycler using the
following program: 95°C for 3 minutes, 25 cycles of: 95°C for 30 seconds, 55°C for 30
seconds, 72°C for 30 seconds, 72°C for 3 minutes, finish at 4°C. Samples were washed
using Clean NA CleanPCR beads (45µl) protocol to clear the product of primers,
nucleotides, salts etc. and eluted in 53µl Elution Buffer. 5 random PCR samples were
tested on a 1.3% agarose gel to confirm that the 16S rRNA gene was amplified. Positive
results were obtained immediately, to which the rest of the samples were completed, stored
in -20°C until being sent to Trinity College Dublin (TCD) for further Index PCR. Index
PCR purifies the 16S V3 and V4 amplicon away from free primers and primer dimer
species. The samples undergo library quantification, normalization, pooling, library
denaturation, MiSeq sample loading and MiSeq reporter metagenomics workflow. The
metagenomics workflow classifies organisms from the specific V3 and V4 amplicon in the
41 samples using a database of 16S rRNA data.
19
3. Results
3.1 MSC-γ prolonged the survival of aGvHD mice models more significantly
than resting MSCs
PBMCs are monocytes and lymphocytes (T, B, NK and NKT cells) isolated from
buffy coats and are administered into irradiated aGvHD mice models in order to replicate
the disease process in patients. aGvHD was established in mice models between day 10-16
and were given two different treatments of MSC (day 6) and MSC-γ (day 6) following
PBMC administration. NSG mice that received PBS alone developed no signs of aGvHD.
NSG mice that were given PBMC acquired aGvHD between days 10 and 16, with a 20%
decrease in weight loss and survived up to day 15.
Figure 3.1: MSC-γ therapy significantly prolonged the survival of aGvHD mice, with both
MSC therapies significantly reducing weight loss. (A) Survival curve and (B) percentage
weight change of aGvHD mice (red circle), MSC treated aGvHD mice (pink circle) and
20
MSC-γ (blue circle). PBS control mice are represented by the green circles. 8 x 105 human
PBMC gram-1 were administered in irradiated NSG mice (2.4 Gy), 6.4 x 104 human gram-1
MSC or MSC-γ were given as cellular therapy on day 6. n=6 mice per group. Statistical
analysis was carried out using Mantel-Cox test and student t-test.
3.2 Human MSC and MSC-γ reduced pathology and apoptotic damage in the tissues
of aGvHD mice
Following aGvHD development between days 10–16, organs were harvested on
day 13 from each treatment group (PBS, PBMC, MSC, and MSC-γ) and tissue sections
were analysed by H&E staining and a scoring system to assess the level of aGvHD
development.
(a) aGvHD in the colons caused an increase in cellular infiltration to the colonic
crypts, with globular abscesses forming and distortion to colonic tissue (denoted by 1a
arrow in figure 3.2a). Hyperplasia is noted. (b) aGvHD of the SI caused an increased
accumulation of cellular infiltration to the lamina propria (denoted by 2a arrow in figure
3.2b), with increased sloughing/blunting of the villi (denoted by 2b arrow in figure 3.2b).
(c) The livers of PBMC mice display an increase in cellular infiltration around the hepatic
ducts and endothelialitis (denoted by 3a and 3b arrows in figure 3.2c). MSC-γ treatment
ameliorated overall liver pathology, given a histological score closer to 2. (d) The lungs of
PBMC mice greatly provoked cellular manifestations/inflammation/infiltration at the
alveolar air spaces (denoted by arrow 4a in figure 3.2d). These results suggest that
conventional and interferon-γ stimulated MSCs reduce clinical signs of aGvHD
development.
21
22
Figure 3.2: MSC therapy significantly reduces
pathology of NOD-SCID IL-2rᵞnull mice suffering
with aGvHD. aGvHD was established in the NSG
mice models and were given two different
treatments of MSC (day 6) and MSC-γ (day 6) following PBMC administration. All
organs were harvested on day 13, fixed in 10% (v/v) buffered formalin, prepared for
histology and embedded in paraffin wax. 5µm sections were cut, stained by H&E and
assessed according to a scoring/grading system where 0 is healthy and 5 is severe aGvHD.
n = 6 mice per group.
23
Tunel is defined as the discovery and quantification of apoptosis (programmed cell-
death) at a single-cell level, based on the physical identification of DNA strand breaks
(‘nicks’). DNA nicks are analysed by terminal deoxynucleotidyl transferase, an enzyme
which catalyses the addition of nucleotides that are secondarily labelled with a marker
(Darzynkiewicz et al., 2008). DAPI (4', 6-diamidino-2-phenylindole) is a nuclear counter
stain accounting for changes in nuclear shape and confirming that the tissue is viable to
work with. aGvHD results in target tissue necrosis and apoptosis which is detected
visually by a fluorescent microscope. Scoring does not take place.
It is clear in figure 3.3 that the blue DAPI images of both the lung and SI indicate
that the tissue samples were viable to work with. The rate of apoptosis is represented by
the green dots in each image. There is a low rate of apoptosis occurring in both PBS
images and a high rate of apoptosis happening in both the PBMC tissue of each organ.
Apoptosis occurs naturally in healthy tissue at a controlled rate. The results suggest that
the rate of apoptosis is reduced in both the lung and SI following MSC and MSC-γ therapy,
24
Figure 3.3: MSCs greatly reduce the rate of apoptosis occurring in NSG murine models.
aGvHD was developed between day 10-16 in NSG mice models following PBMC
administration (day 0) and were given two different treatments of MSC (day 6) and MSC-γ
(day 6). All organs were harvested on day 13 and prepared for analysis. Target organ
tissues were cut into 5µm sections and stained by Tunel and DAPI. Fluorescent
microscopy enables physical identification of the rate of apoptosis and Tunel experimental
data supports data illustrated in H&E images above. n = 6 mice per group.
25
MSCγ
PBS (Healthy)
PBMC (GvHD)
MSC
Lung Small Intestine
DAPI TUNEL DAPI TUNEL
3.3 MSC therapies significantly reduced pro-inflammatory cytokines in the tissues of
aGvHD mice
Typical pro-inflammatory cytokines that are to be expected in great amounts are
TNF-α, IL-1β, -2, -6, -17, and IFN-ᵞ, hallmarks of aGvHD cytokine cascade. They can
amplify graft-versus-host (GvH) reactions and the balance between pro- and anti-
inflammatory cytokines shapes the overall milieu and GvH response.
Specific results are suggested; In the SI, MSC-γ reduces levels of TNF-α and IFN-
γ, two major pro-inflammatory cytokines involved in aGvHD (figure 3.4a). In the colon,
IL-2 is reduced following MSC therapies however, there is no significant difference in both
treatments compared to PBMC IL-2 cytokine levels (figure 3.4b). In the lung, there’s no
statistical significance between MSC and MSC-γ treatment in reducing IL-6. There’s a
similar significant difference between these two therapies regarding every other reduced
cytokine level (figure 3.4c). In the liver, MSC therapy is more significant at reducing IL-
1β in comparison to MSC-γ (figure 3.4d).
26
27
Figure 3.4: Pro-inflammatory cytokines strongly associated with aGvHD are overall
dampened in response to MSC therapy. Following the onset of aGvHD, specific organs
were homogenized and analysed by commercial ELISA assays (R&D duoset) to detect pro-
inflammatory cytokine levels following MSC therapy. The absorbance (optical density) of
samples and standards were measured at 450 nm for all ELISAs using Biotek plate reader.
The cytokine concentration of each sample was determined by comparison to the standard
curve of known cytokine concentrations. Statistical analysis was carried out on each graph
(see tables). Stars with no bar are in comparison to PBMC graphs and the bar between
MSC and MSC-γ tells us if there was significant difference between these two therapies.
3.4 Expected 16S ribosomal RNA sequence results on microbiota diversity following
MSC and MSC-γ therapy
Amplicon polymerase chain reaction (PCR) amplifies a specific gene of interest on
deoxyribonucleic acid (DNA) using varied heat-associated cycles and the sample is
exponentially amplified millions of times. Primers are needed to focus on the area of
interest, binding to the denatured DNA to form new strands and the cycle repeats itself
until desired. The 16S rRNA sequence is a unique, conserved ribosomal subunit that has
played a role in accurately identifying bacterial isolates, leading to the discovery of novel
28
species in metagenomic studies (Woo et al., 2008). 16S rRNA gene is part of the 30S
small subunit of prokaryotic ribosomes and contains nine variable regions interspersed
between conserved regions. Variable regions are frequently used in phylogenetic
classifications such as genus or species in diverse microbial populations. The kit used in
this study focuses on the V3 and V4 regions of the 16S rRNA gene, forming a single
amplicon of 460 base-pairs (amplicon PCR). In order to classify organisms from this
amplicon using a database of 16S rRNA data, index PCR must be done. This generates an
idea of the sort of isolates contained in the stool samples from each treatment group (MSC
and MSC-ᵞ).
DNA was detected in all 41 samples, as indicated through gel electrophoresis
(figure 3.5) and nanodrop confirmation (results not shown). Note there are some wells that
contain little or no DNA smearing. This was overcome by repeating the gel with these
specific samples to rule out human error (figure not shown). 16S rRNA gene was
amplified and confirmed using gel electrophoresis (figure 3.6). Samples were sent to
Trinity College Dublin for Index PCR analysis. Unfortunately, there was an issue with the
16S rRNA machine and so the results were unable to be included due to a delay in
retrieving the results back in time to be analysed correctly.
29
Figure 3.5: Gel electrophoresis was
carried out to physically visualise that
bacterial DNA was detected in the 41 stool samples following DNA isolation technique.
(A) These samples were taken on day 0 following irradiation and PBMC administration.
(B) These stool samples were taken on day 4 and day 9 throughout the 28 day experiment.
Stool samples were homogenized, lysed and purified. The DNA was eluted in Sterile
DNA-Free PCR Grade Water using the PowerSoil® DNA Isolation Kit. Gel
electrophoresis on a 1.3 % agarose gel was carried out on all samples using GeneRuler 1Kb
DNA Ladder (5 µl in first well), enabling physical visualisation of isolated DNA allowing
the continuation of the experiment.
30
Figure 3.6: Following DNA isolation, samples were exposed to varied heat-associated
cycles where the 16S rRNA sequence was exponentially amplified using 16S amplicon
Forward and Reverse Primers and High-Fidelity 2x MasterMix. (a) 5 random samples were
selected to test to see if the PCR worked and that the 16S rRNA sequence was amplified.
Washing of the samples took place using Clean NA Clean PCR beads and after eluting in
50µl of Elution Buffer, 3 µl of sample was placed into the gel for separation. A 1500 kDa
band at 16S rRNA sequence was very clear when compared to the DNA ladder and gave an
indication to decrease the volume of sample used to 1-2 µl. (b) The remaining 36 samples
were tested following PCR procedure. Some bands are faint here as 3 µl of sample was
used; it can be suggested to increase this concentration to 5 µl in order to see very clear
bands.
4. Discussion
An acute graft versus host disease (aGvHD) murine model was employed in this
study to allow reproducible assessment of human cell therapy mechanisms of action on the
microbiota. NOD-SCID IL-2rᵞnull mice were administered with human PBMCs causing the
onset of aGvHD, translating human disease. Throughout the life-span of the aGvHD mice,
a survival curve was created. It is clear from figure 3.1 that PBMC (aGvHD) mice came
down on day 15, with allogenic human MSC and MSC-γ increasing the survival of aGvHD
31
mice. MSC mice came down on day 23. MSC-γ and PBS (healthy) mice survived until
the last day of the experiment, day 28. The weight graph indicates that PBS mice gain
weight, as expected. PBMC mice have a 20% weight loss. There is no significant
difference in terms of weight loss or prolonging survival of aGvHD mice models between
both MSC therapies, however, MSC-γ overall prolonged the survival of aGvHD mice
models at a greater significance than conventional MSCs.
Histological analysis of each target organ occurred after harvest day 13 (figure 3.2).
Visually, there is reduced thickening of the hepatic vein, reduced globular abscess
formation at the colonic crypts, reduced cellular infiltration at alveolar sacs and reduced
sloughing/blunting of the villi. Histology graphs confirm an overall significant reduction
in aGvHD symptoms following cellular therapy. All organs have no significant difference
in histological scoring between both MSC therapies at reducing aGvHD symptoms. The
colon and liver have no significant difference in the histology score given in aGvHD and
conventional MSC therapy. Tunel images mirror the histology images, in that the rate of
apoptosis in the SI and lung is alleviated following both MSC therapies (figure 3.3).
Colonic tissue and SI show similar apoptotic levels (Potten and Grant, 1998) and the rate of
apoptosis is difficult to detect in the liver. Hence, the SI and lung represent systemic and
GI-tract protection at a comparable level following cellular therapy. MSCs migrate to
inflamed lungs first and are trapped there due to them being large cells (Schrepfer et al.,
2007). They exert their immunosuppressive effects into the periphery and foster tissue
repair in other inflamed organs by paracrine effects that dampen inflammation, supporting
tissue regeneration (Caimi et al., 2010). Overall, MSC-γ therapy has a greater significance
at reducing aGvHD symptoms in target organs.
MSC-γ showed enhanced immunosuppressive ability, reducing pro-inflammatory
cytokine levels in aGvHD target organs (figure 3.4), perhaps giving an indication as to why
32
these particular cells have so far been greatly significant at reducing such symptoms. As
aGvHD develops in the murine model, IFN-γ levels increase. This may be an acceptable
amount of IFN-γ needed for the activation of conventional MSCs, hence why MSC
therapies are administered on day 6 (aGvHD progressing). ELISA data reveal a reduction
in TNF-α in specific target organs, indicating MSC mediated immune suppression in vivo.
None of these cytokines are completely diminished, as expected. Chemokines and
cytokines are required to drive the immune response and as the immune system of an
individual matures, the expression of these immune signalling molecules vary
simultaneously (Kleiner et al., 2013). Overall, MSC-γ had a greater significance at
reducing pro-inflammatory cytokines in aGvHD target organs.
To improve the mouse model experiment; perhaps early administration of MSC-γ
(after day 0) or multiple doses of MSC therapies could further reduce TNF-α production
and concomitant activation of T lymphocytes, showing a significant improvement in
aGvHD symptoms. Multiple doses of human MSCs are currently administered to aGvHD
paediatric patients (Kurtzberg et al., 2014), creating the same dosing scheme in the mouse
model as seen in clinical settings.
Distinct differences between mice and humans are obvious but physiology and
anatomical studies are quite similar in these two species, hence why murine models are
widely used in biomedical research (Nguyen et al., 2015). The conserved 16S rRNA gene
was amplified from each sample (figure 3.5). This technique is carried out because a large
number of bacteria are amenable to laboratory culturing techniques or have not yet been
cultured and so their identification is difficult (de Vos and de Vos, 2012). Unfortunately,
the 16S rRNA results were not complete on time to include in this thesis due to machine
malfunctioning in TCD.
33
Published studies reveal an overall loss of Gram positive anaerobic bacteria in
aGvHD mice models such as Clostridials and an outbreak of Gram negative bacteria such
as Escherichia coli (Chen et al., 2015). Eriguchi et al. (2012) revealed that aGvHD and
conditioning regimens damage intestinal epithelium cells (IECs), reducing the production
of α-defensins; major antimicrobial peptides that maintain the GI-tract commensal
microflora. aGvHD reduces butyrate production (SCFA), disturbing the maintenance of
IECs and secretion of α-defensins, causing microbiota dysbiosis and contribution to
aGvHD (Mathewson et al., 2016). This group suggested administering exogenous butyrate
or 17 selected strains of butyrate-producing Clostridia is sufficient for aGvHD protection.
It can be hypothesised that the outgrowth of E.coli is associated with the development of
systemic infection in aGvHD mice, where LPS levels have been significantly higher
compared to controls (corresponding to septicaemia in patients).
In this research project, we are hoping to see a positive shift in the GI-tract
microbiota composition of aGvHD mice following cellular therapy. It will be interesting to
note any differences between both cellular therapies in regards to influencing microbiota
diversity. So far MSC-γ has shown an overall significant reduction in aGvHD target organ
symptoms, specifically the GI-organs and perhaps a similar trend will be noted once the
16S rRNA data is successfully analysed. These results will hopefully be influential in that
patients undergoing allo-HSCT will be closely monitored post-transfusion, allowing
clinicians to note if their microbiota is deviating from normal flora. Thus administration of
cellular therapy could prevent further development of aGvHD. MSC-γ in combination
with butyrate could maintain GI-tract protection and diminish aGvHD. These are all
potential strategies that could originate following a better understanding of the relationship
between cellular therapies, GI-tract microbiota and regulation of the host immune system
during aGvHD. Rapid advances in aGvHD prophylaxis is an ongoing strategy to translate
34
new research findings to clinical implementation, enabling the best possible therapeutic
alternative to potentially prevent aGvHD and protect the GvL effect.
A functional relationship exists amongst an individual and their selective
community of ubiquitous microbes, integrating these vastly diverse colonies as intrinsic
regulators of immune responses. However, intestinal microbial alteration of an individual
in the course of allo-HSCT corresponds to the emergence of aGvHD. It will be interesting
to note any differences in microbiota composition following MSC and MSC-γ therapy in
comparison to aGvHD microbiota. Testing MSCs mechanism of action on the microbiota
in animal models and in future human trials creates hopefulness in the cellular immunology
research community to make MSC therapy a clinical paradigm.
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