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

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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Information derived from the published or unpublished work of others has been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: ……………………………………………………

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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

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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.

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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.

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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:

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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

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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)-

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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

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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),

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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

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(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).

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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

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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.

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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.

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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

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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

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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) =

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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.

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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

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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.

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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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