1

Establishment of a mouse model of colitis and its use to evaluate the anti-inflammatory effects of two

ghrelin peptides

Samia Taufiq Bachelor of Applied Science (Honours)

School of Life Sciences, Faculty of Science

Masters of Applied Science 2009

Supervisors:

A/Prof Mike McGuckin - Mater Medical Research Institute

(MMRI)

Dr. Lisa Chopin - Queensland University of Technology (QUT)

Dr. Penny Jeffery - Mater Medical Research Institute (MMRI)

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Keywords

Ghrelin, GHSR, Δ4 peptide, DSS, IBD

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Abstract Ghrelin is a gut-brain peptide hormone that induces appetite, stimulates the

release of growth hormone, and has recently been shown to ameliorate inflammation.

Recent studies have suggested that ghrelin may play a potential role in inflammation-

related diseases such as inflammatory bowel diseases (IBD). A previous study with

ghrelin in the TNBS mouse model of colitis demonstrated that ghrelin treatment

decreased the clinical severity of colitis and inflammation and prevented the

recurrence of disease. Ghrelin may be acting at the immunological and epithelial

level as the ghrelin receptor (GHSR) is expressed by immune cells and intestinal

epithelial cells. The current project investigated the effect of ghrelin in a different

mouse model of colitis using dextran sodium sulphate (DSS) – a luminal toxin. Two

molecular weight forms of DSS were used as they give differing effects (5kDa and

40kDa). Ghrelin treatment significantly improved clinical colitis scores (p=0.012) in

the C57BL/6 mouse strain with colitis induced by 2% DSS (5kDa). Treatment with

ghrelin suppressed colitis in the proximal colon as indicated by reduced

accumulative histopathology scores (p=0.03). Whilst there was a trend toward

reduced scores in the mid and distal colon in these mice this did not reach

significance. Ghrelin did not affect histopathology scores in the 40kDa model. There

was no significant effect on the number of regulatory T cells or TNF-α secretion

from cultured lymph node cells from these mice.

The discovery of C-terminal ghrelin peptides, for example, obestatin and the

peptide derived from exon 4 deleted proghrelin (Δ4 preproghrelin peptide) have

raised questions regarding their potential role in biological functions. The current

project investigated the effect of Δ4 peptide in the DSS model of colitis however no

significant suppression of colitis was observed. In vitro epithelial wound healing

assays were also undertaken to determine the effect of ghrelin on intestinal epithelial

cell migration. Ghrelin did not significantly improve wound healing in these assays.

In conclusion, ghrelin treatment displays a mild anti-inflammatory effect in the 5kDa

DSS model. The potential mechanisms behind this effect and the disparity between

these results and those published previously will be discussed.

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Table of Contents 1. Chapter One: Introduction............................................11

2. Chapter Two: Literature Review...................................14 2.1. Inflammatory Bowel Disease

2.1.1. Genetic factors associated with IBD

2.1.2. The intestinal barrier in IBD

2.1.3. Aberrant immune response in IBD

2.2. Animal models of IBD 2.2.1. Chemically-induced models

2.2.2. Genetically engineered models

2.3. Current treatments for IBD 2.3.1. New treatments for IBD need to be evaluated

2.4. Ghrelin 2.4.1. Ghrelin gene derived peptides

2.4.2. Ghrelin O-Acyltransferase (GOAT)

2.4.3. The role of Ghrelin in inflammation and IBD

2.4.4. Ghrelin in mouse models of colitis

2.5. Relevance of project

2.6. Conclusion

3. Chapter Three: Materials and Methods.......................36 3.1. Mice

3.2. Induction of colitis and experimental design

3.3. Assessment of inflammation: symptoms and inflammatory score

3.4. Haematological analysis

3.5. Tissue collection

3.6. Histological assessment of colitis

3.7. Isolation and culture of Mesenteric lymph node (MLN) cells

3.8. Enzyme-Linked ImmunoSorbent Assay (ELISA) for TNF-α

3.9. Mesenteric lymph nodes (MLN) T Regulatory Cells counting using Flow

Cytometry

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3.10. Cell Culture

3.11. Wound assay

3.12. Statistical Data analysis

3.13. Presentation of Results

4. Chapter Four: Results..................................................43 4.1. Effect of ghrelin peptide treatment on clinical colitis scores in C57BL/6 and

BALB/c mice administered with dextran sodium sulphate (DSS)

4.2. Treatment with ghrelin peptides does not affect bodyweight change in mice

with DSS-induced colitis

4.3. Effect of ghrelin peptides on colon shortening in C57BL/6 and BALB/c

mice given DSS

4.4. Blood analyses of BALB/c and C57BL/6 mice

4.5. Treatment with ghrelin peptides suppressed histolopathological colitis in

C57BL/6 mice

4.6. Measurement of TNF-α from mesenteric lymph node lymphocyte cultures

4.7. Enumeration of CD4+CD25+Foxp3+ T regulatory cells in mesenteric lymph

nodes

4.8. Treatment with ghrelin did not improve wound healing in the HT29 cell line

5. Chapter Five: Discussion..............................................74

Appendix............................................................................80 1.1 Score sheet for mice undergoing DSS Treatment

1.2 Assessment of DSS colitis

1.3 Table A.1

Figure A.1

References...........................................................................84

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List of Illustrations and Diagrams

A. Chapter Two: Literature Review 1. Figure 1. Mucosal immune responses in IBD.

2. Figure 2. Alternative splicing of mouse ghrelin gene generates multiple

ghrelin peptides.

3. Figure 3. A potential mechanism of ghrelin peptides in inflammation.

B. Chapter Four: Results 1. Figure 1. Clinical evidence of colitis in BALB/c mice treated with DSS and

ghrelin peptides.

2. Figure 2. Clinical evidence of colitis in C57BL/6 mice treated with DSS and

ghrelin peptides.

3. Figure 3. Changes in body weight in BALB/c mice treated with DSS and

ghrelin peptides.

4. Figure 4. Changes in body weight in C57BL/6 mice treated with DSS and

ghrelin peptides.

5. Figure 5. Colon lengths of mice treated with DSS for 8 days.

6. Figure 6. Two-Way ANOVA comparison of colon lengths of BALB/c and

C57BL/6 mice treated with ghrelin peptides with 5kDa DSS.

7. Figure 7. Haematological parameters in BALB/c mice treated with DSS and

ghrelin peptides for 8 days.

8. Figure 8. Haematological parameters in C57BL/6 mice treated with DSS and

ghrelin peptides for 8 days.

9. Figure 9. Histological colitis scores in BALB/c mice treated with DSS and

ghrelin peptides.

10. Figure 10. Histological colitis scores in C57BL/6 mice treated with DSS and

ghrelin peptides.

11. Figure 11. Representative H&E sections of colons dissected from 6 week old

Naive C57BL/6 mice.

12. Figure 12. Representative histology images of C57BL/6 mice treated with

5kDa DSS.

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13. Figure 13. Representative histology images of C57BL/6 mice treated with

40kDa DSS

14. Figure 14. Histological assessment of inflammation in the Proximal colon of

BALB/c and C57BL/6 mice.

15. Figure 15. Histological assessment of inflammation in the Distal colon of

BALB/c and C57BL/6 mice.

16. Figure 16. TNF-α concentration in mesenteric lymph node cultures from

mice treated with DSS and ghrelin peptides

17. Figure 17. Representative flow cytometry plots of T regulatory cell

populations in mesenteric lymph nodes in the BALB/c mice.

18. Figure 18. Representative flow cytometry plots of T regulatory cell

populations in mesenteric lymph nodes in the C57BL/6 mice.

19. Figure 19. T regulatory cell numbers in mesenteric lymph nodes in BALB/c

(A) and C57BL/6 (B) mice.

20. Figure 20. Analysis of wound assay on HT29 cells to measure the effect of

various ghrelin concentrations in wound healing.

21. Figure 21. Pre-treatment of HT29 cells with various concentrations of ghrelin

to measure its effect in wound healing.

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Abbreviations

IBD Inflammatory Bowel Disease

GHS-R1a Growth Hormone Secretagogue Receptor 1a

DC Dendritic Cell

CD Crohn’s disease

UC Ulcerative Colitis

Treg Regulatory T cells

IEC Intestinal epithelial cells

IL-1β Interleukin-1β

Il-12 Interleukin-12

IFN-γ Interferon- γ

TNF-α Tumour Necrosis Factor- α

IL-10 Interleukin-10

TGFβ Transforming growth factor beta

NF-κB Nuclear Factor-κB

DSS dextran sodium sulphate

IL-10 -/- Interleukin-10 knock out

TNBS 2,4,6-Trinitrobenzene sulfonic acid

MLNs Mesenteric lymph nodes

Th T helper cells

CD4 Cluster of Differentiation 4

CD25 Cluster of Differentiation 25

Foxp3 Forkhead box P3

TLR Toll-like Receptors

TCR T-cell receptor

NEMO NF-κB essential modulator

GOAT Ghrelin O-Acyltransferase

PPAR-γ Peroxisome proliferator-activated receptor- γ

LPS lipopolysaccharides

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Statement of Original Authorship

“The work contained in this thesis has not been previously submitted to

meet requirements for an award at this or any other higher education

institution. To the best of my knowledge and belief, the thesis contains

no material previously published or written by another person except

where due reference is made.”

Samia Taufiq

Signature Date

10

Acknowledgement

This study was performed at the Mater Medical Research Institute

(MMRI) – Mucosal Diseases Program, under team leader, Associate

Professor Michael McGuckin. I would specially like to thank my MMRI

supervisor- Dr. Penny Jeffery and my Queensland University of

Technology (QUT) principal supervisor - Dr. Lisa Chopin for their right

guidance and help. I am grateful to Dr. Rajaraman Eri, Deborah Roche,

Sharyn B. Tauro, Patricia Lusby, and the rest of the team from the

Mucin and IBD lab for their valuable contribution. Lastly, I would like

to thank my parents, sister, husband, and friends for supporting me

throughout my degree.

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Chapter One: Introduction

Inflammatory bowel disease (IBD) is characterized as a chronic

inflammatory condition which predominantly affects the gastrointestinal tract. IBD

can be further classified into two chronic disorders: Crohn’s disease (CD) and

ulcerative colitis (UC). CD is characterised by patchy transmural inflammation

affecting any area of the gastrointestinal tract, whereas UC is limited to the colon,

causing continuous mucosal inflammation also involving the rectum. The etiology of

IBD is not completely known, but it is thought to arise from an abnormal

immunological response to antigens present in the gut lumen (Fiocchi, 1998). IBD is

affected by a combination of environmental, genetic, and immunological

abnormalities which influences the immune response within the intestinal mucosa

leading to inflammation (Hanaeur et al., 1996; Podolsky et al., 2002). An immune

response is generated when immune cells, including monocytes, macrophages, T

cells, and dendritic cells (DC) detect microbes (Guillot et al., 2004). Once an antigen

is detected DCs migrate to the mesenteric lymph nodes and stimulate naive T cells,

which are primarily dominated by mucosal CD4+ lymphocytes (Uhlig et al., 2006).

The CD4+ T cells differentiation is controlled by cytokines which determine the type

of inflammatory response to occur. Pro-inflammatory cytokines such as interleukin

(IL-12), interferon- γ (IFN-γ) and tumour necrosis factor (TNF-α) play a major role

in colonic tissue destruction, and are released upon failure to regulate T cell

responses in the intestinal or colonic mucosa and in response to infection (Weinstein

et al., 1997; Gonzalez-Rey et al., 2006). Animal studies suggest that the presence of

regulatory T cells (Tregs), which produce the anti-inflammatory cytokines, IL-10

and transforming growth factor beta (TGFβ), supports the immune balance in the

normal gut by restoring mucosal tolerance (Mowat, 2003; Kelsen et al., 2005).

Much of our knowledge of the pathogenesis of IBD comes from numerous

animal models, which are designed to mimic the clinical features seen in human

IBD. Two such types of experimental animal models are discussed in this review, the

chemically induced colitis models and the genetically induced colitis models. Both

types of models can reflect immune cell and epithelial cell dysfunction. Even though

these animal models do not completely exhibit the complexity of the human

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diseases, they are useful tools to investigate mechanisms of colitis and to evaluate

new therapies for IBD.

Current therapies used in IBD have significant side effects and many are

ineffective for severe and relapsing IBD. We, therefore, require therapeutic agents

which will suppress the aberrant immune response seen in IBD (to downregulate

pro-inflammatory cytokine release) and also repair the damaged epithelium. A

potential anti-inflammatory agent is ghrelin, a 28 amino acid gut peptide hormone.

Ghrelin inhibits the production of pro-inflammatory cytokines in vitro and exerts

strong protective actions on the gastric mucosa thereby helping to accelerate the

healing of lesions (Dixit et al., 2004; Li et al., 2004b; Konturek et al., 2006). Ghrelin

treatment is also effective in ameliorating experimental colitis in mice, however,

studies by Zhao et al., (2006) have shown that the activation of the ghrelin receptor

can trigger a pro-inflammatory response and can also activate the production of NF-

κB, a pro-inflammatory transcription factor, in human colonocytes in vitro. These

authors suggest that the release of pro-inflammatory cytokines during colonic

inflammation could directly trigger the upregulation of ghrelin during colitis through

the activation of NF-κB. Due to these differing theories, it is important to further

investigate the role of ghrelin in inflammation and IBD, in order to understand the

complete mechanism of its actions.

It is now known that multiple ghrelin peptides are produced from the

preproghrelin pro-hormone and these include the mature ghrelin hormone and

obestatin, the C terminal peptide of proghrelin which is thought to be active (Zhu et

al., 2006 ). A splice variant of the ghrelin gene results in an exon 4-deleted

proghrelin isoform (Jeffery et al., 2003). As of yet, no study has clearly shown

whether the unique C-terminal peptide of the exon 4-deleted proghrelin isoform is

functionally active or not. Its mRNA expression in the colon or mucosal epithelial

cells has also not been investigated. The human orthologue, exon 3-deleted

proghrelin, is expressed in a wide range of human tissues including human prostate

and breast cancer cell lines, and is upregulated in cancer tissue as compared to

normal tissue (Jeffery et al., 2002; Jeffery et al., 2005a, b). It is important to examine

the functional significance of the exon 4-deleted proghrelin isoform in animal

models of colitis to determine if it has similar anti-inflammatory properties to

ghrelin.

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This study will investigate whether the administration of ghrelin and Δ4

proghrelin peptide can ameliorate intestinal inflammation in the DSS model of

colitis. We hypothesise that ghrelin peptides will reduce colitis and rescue epithelial

damage by stimulating proliferation and migration.

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Chapter Two: Literature Review

Inflammatory Bowel Disease Inflammatory bowel diseases (IBD) are chronic inflammatory conditions of

the gastrointestinal tract present in two forms: Crohn’s disease (CD) and ulcerative

colitis (UC). IBD affects over 61,000 people in Australia, out of which 28,000

people are affected with CD and 33,000 with UC (ACCA report). There are around

776 new cases of CD and 846 cases of UC diagnosed each year (ACCA). Even

though IBD can be diagnosed at any age it is more prevalent between the ages 15 to

40. Disease morbidity can be significantly higher in younger patients, with the risk

of lifelong problems in relation to emotional well being, physiological growth,

reproductive health issues, education, and employment (Van Limbergen et al.,

2007).

The unique pathophysiological feature of IBD is the close apposition of the intestinal

immune system to high concentrations of intraluminal bacteria. There has been

strong evidence which supports that the dysregulation of the normally controlled

immune response to commensal bacteria in a genetically susceptible individual

causes IBD (Cho, 2008). IBD is thought to be characterised by dysfunction of

mucosal T cells, altered cytokine production, and cellular inflammation which

ultimately leads to damaging the distal small intestine and the colonic mucosa

(Fiocchi, 1998; Hanauer & Present, 2003). Whilst it is difficult to diagnose a patient

with IBD in the early stages, clinical symptoms including abdominal pain, rectal

bleeding, malabsorption and weight loss aid in the diagnosis. (Fiocchi, 1998;

Dieleman & Heizer, 1999). However, in some cases extra-intestinal manifestations

are found to affect skin, joints and eyes in both CD and UC patients (Russel et al.,

2004).

Although the pathogenesis of IBD is still unclear it is thought to result from a

combination of genetic, environmental, and immunologic abnormalities in which an

uncontrolled immune response within the intestinal lumen leads to inflammation in

genetically predisposed individuals (Yu et al., 2004). Environmental factors such as

drinking, and variations in food intake affect the pathophysiology of IBD (Sakamoto

et al., 2005). The risk of smoking has been found to be increased in patients with

CD, but decreased in patients with UC (Podolsky, 2002). The luminal environment,

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in particular the luminal flora, play a crucial role in IBD (Korzenik & Podolsky,

2006), for instance, an active balance between the presence of commensal flora and

the dampening of protective host mechanisms is essential to maintain non-inflamed

mucosa. Even though intestinal bacteria play a pivotal role in the development of

IBD, their contribution to inflammation remains unclear (Ott et al., 2004). Studies

about the relationship of microbes-host are limited due to lack of knowledge of the

diversity and complexity of the microbial flora (MacDonald & Monteleone, 2000).

Genetic factors associated with IBD

Genetic factors also play a significant role in the pathogenesis of IBD in CD

and UC. NOD2 has been identified (also known as CARD15/IBD1) as a

susceptibility gene in CD using positional cloning and other gene approaches (Hugot

et al., 2001; Ogura et al., 2001). Since then several other genes have also been

implicated in CD, such as IBD5, IL23R, and ATG16L1 (Peltekova et al., 2004; Duerr

et al., 2006; Hampe et al., 2007; Silverberg et al., 2007). NOD2 is a pattern-

recognition receptor which functions as an intracellular sensor for bacterial

peptidoglycan and can also be activated by a small bioactive component of

peptidoglycans, muramyl dipeptide (MDP) (Giardin et al., 2003; Inohara et al.,

2003). The activation of NOD2 by the MDPs results in the activation of NF-κB and

mitogen-activated protein (MAP) kinase signalling pathways (Kobayashi et al.,

2005). ATG16L1, an autophagy-related gene, is involved in the degradation of

intracellular pathogens, regulation of cell signalling and of T-cell homeostasis

(Levine & Deretic, 2007). Since autophagy results in restricting the growth of certain

microorganisms, a mutation in the gene would therefore result in the reduction of

clearance of pathogens and allow a more permissive growth to the intracellular

bacterial pathogens (Amano et al., 2006). The association of CD with

polymorphisms in NOD2 and the ATG16L1 suggests that alterations in the

recognition and intracellular processing of bacterial components may have a role in

the immunopathogenesis of the disease (Cho, 2008). However, studies with NOD2-

deficient mice, which showed the absence of intestinal inflammation, have

highlighted that the dysregulation of the NOD2 pathway alone is insufficient to

induce CD (Pauleau & Murray 2003; Kobayashi et al, 2005). Therefore, being a

carrier of any of these genes does not necessarily lead to developing colitis leading to

IBD. The susceptibility of developing IBD results from the change in microbial

16

factors, which is a consequence from the influence of environmental factors. Genetic

factors affecting the barrier function and the innate and adaptive immunity in IBD

are also affected by the alterations between commensal microbes and the mucosa

leading to intestinal inflammation (Van Limbergen et al., 2007).

The intestinal barrier in IBD

The intestinal barrier is primarily made up of biofilm with commensal

bacteria, the mucous layer and the epithelium and innate immune defences, including

macrophages, dendritic cells, and neutrophils. Any damage caused to this barrier can

subsequently result in the persistent activation of the immune system (Figure 1).

Immune cells including monocytes, macrophages, T cells and DCs participate in the

detection of microbes, as there is constant communication between the luminal flora

and the underlying dense network of innate and adaptive immune cells in the

epithelium (Guillot et al., 2004). The intestinal epithelium consists of intestinal

epithelial cells (IEC) which are important for the absorption and transportation of

nutrients and the formation of a protective mucosal barrier in the gut which is

normally rapidly regenerated after damage (Han et al., 2003; Okamoto & Watanabe,

2004; Cario & Podolsky, 2005). IECs express Toll-like receptors (TLRs), which are

a family of innate immune recognition receptors that control adaptive immune

responses and induce antimicrobial effector pathways, which, therefore, lead to the

elimination of host-threatening pathogens (Guillot et al., 2004; Cario, 2005).

Aberrant immune response in IBD

The mucosal immune response is initiated by the sensing of microbes, which

later activates the adaptive immune response (Xavier & Podolsky, 2007). The

activated immune response, the characteristic of IBD, is mostly dominated by the

mucosal CD4+ T lymphocytes (Uhlig et al., 2006). The differentiation of CD4+ T

cells is tightly controlled by cytokines which determine the type of inflammatory

response that occurs in the host. Failure to regulate T cell responses in the intestinal

or colonic mucosa causes the release of pro-inflammatory cytokines which leads to

death of enterocytes and inflammation of the tissue (Weinstein et al., 1997).

Recruited macrophages cause damage to epithelial barrier by producing reactive

oxygen radicals, nitric oxide radicals, and pro-inflammatory cytokines such as

interleukin-12 (IL-12), interleukin 1β (IL-1β), interferon-γ (IFN-γ) and tumour

17

necrosis factor (TNF-α) which play a major role in colonic tissue destruction

(Podolsky, 2002).

Regulatory T cells (Tregs), are characterised as being CD4+,CD25+ Foxp3+

and produce IL-10 and transforming growth factor beta (TGFβ) which have anti-

inflammatory effects in the gut (Mowat, 2003; Uhlig et al., 2006). Without the

presence of any inflammatory mediators, TGFβ results in the development of Foxp3+

Treg cells, which then suppress inflammatory responses (Kim & Rudensky 2006).

Interestingly, if TGFβ cooperates with IL-6 (a pro-inflammatory cytokine) it results

in the generation of Th17 cells, which are involved in numerous inflammatory

diseases, including colitis (Fantini et al., 2007). This suggests that TGFβ can be both

anti-inflammatory and pro-inflammatory depending on the expression of other

cytokines. Thus the combination of TGFβ and IL-6 results in the inhibition of Treg

cell development and the differentiation of naive CD4+ T cells into Th17 T cells

(Bettelli et al., 2006). Theoretically, the potential role for Tregs to suppress IBD in

humans via the production of TGFβ is also limited. Monteleone et al., (2004) have

shown high levels of Smad7, which prevents TGFβ signalling and also down-

regulates the immune response, in inflammatory cells in IBD lesions. In contrast, Yu

et al., (2006) have claimed that Treg cells in the lamina propria have evolved to

suppress immune responses to the resident commensal bacteria, and play a pivotal

role in modulating the clinical range of UC. The true role of Treg cells in the IBD

immune responses in both humans and mouse models of colitis requires further

examination.

Dendritic cells are the dominant antigen presenting cell (APC) type in the

lamina propria. They form an extensive network beneath the intestinal epithelium

and can sample luminal antigens via long processes which project through the

interstices of epithelial cells (Chieppa et al., 2006). The sampling of bacteria by

resident DCs is enabled by direct dendritic cell-microbial contact (Niess et al., 2005;

Chieppa et al., 2006). Once stimulated, DCs then migrate to mesenteric lymph nodes

(MLN) where they promote the differentiation of naive T cells into effector and

regulatory T cells. The cytokines secreted by DCs, then stimulates the differentiation

of naive CD4+ T cells into a Th1, Th2, Th17 or regulatory T cells subsets. Aberrant

activity of lamina propria DCs has been suggested to be a vital component of the

immune response in IBD patients (Niess, 2008).

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The recruitment of activated neutrophils, DCs, and macrophages in the

lamina propria initiates the local immune response, which leads to inflammation

(Fort et al., 1998). The master regulator of cytokine expression is the transcription

factor NF-κB, which resides in the cytoplasm and translocates to the nucleus upon

activation. NF-κB is a pro-inflammatory transcription factor that plays an important

role in immune, inflammatory, and stress responses where it induces the expression

of target genes to promote cell cycle progression, cell survival, adhesion, invasion,

and angiogenesis. However, the inappropriate activation of NF-κB promotes

uncontrolled inflammation (Finco et al., 1997; Bharti & Aggarwal, 2002).

Animal models of IBD A significant portion of our understanding of IBD comes from studies in

animal models of intestinal inflammation (Blumberg et al., 1999; Strober et al.,

2002; Pizarro et al., 2003). These animal models have been designed to imitate

different forms of IBD, but they still fail to represent the exact mechanisms of

IEC

Lamina propria

Macrophages

IL-12 TNF-α IFN-γ

CD4+ cells

Regulatory T cells

Inflammation DC

DC

DC

Lumen

Figure 1. Mucosal immune responses in IBD. An immune response is generated when foreign pathogens gain entry through the intestinal epithelial cells (IECs) which act as a protective barrier. These foreign antigens then activate dendritic cells (DCs), and macrophages in the lamina propria. CD4+ cells release an over production of pro-inflammatory cytokines, such as- IL-12, TNF-α, IFN-γ which results in severe damage of the colonic tissue. Regulatory T cells, which normally suppress inflammation, maybe impaired in IBD.

19

human diseases. However, they are still useful for investigating important aspects

such as the pathogenic mechanisms during the initiation of colitis, and can also act as

important tools in studying the therapeutic effects of drugs in IBD.

There are numerous animal models used to study the inflammatory

mechanisms in IBD, but the two most commonly used are chemically-induced colitis

and genetically-engineered colitis (Table 1). This review will primarily focus on

these two models used in IBD.

Intestinal inflammation- epithelial integrity

Chemically-induced models

The intestinal epithelium not only acts as a physical but also as an

immunological barrier which prevents the direct contact of the intestinal mucosa

with the luminal bacteria. Therefore, any damage to the intestinal barrier can be

crucial for the development of IBD, as luminal antigens and microorganisms can

easily gain entry into the mucosa resulting in the initiation of inflammatory

responses (Wirtz & Neurath, 2000). Two models which highlight the intestinal

epithelial cell function in IBD are the dextran sodium sulphate (DSS)-induced model

and 2,4,6-Trinitrobenzene sulfonic acid (TNBS) induced colitis model.

The TNBS model is very useful in studying aspects of gut inflammation,

including cytokine secretion patterns, mechanisms of oral tolerance and

immunotherapy (Wirtz et al., 2007). In this model the TNBS solution has to remain

in the colon lumen to create reproducible results. Therefore, this model requires

constant observation when inducing colitis. Higher doses of these compounds can

lead to substantial death rates resulting from colon damage (Wirtz et al., 2007).

There is a high variability within experimental groups, and with the reproducibility

of the injury and inflammation, which are either strain-specific or specific to the lot

of TNBS (Fedorak & Madsen, 2000). For example, C57BL/6 mice are relatively

resistant to TNBS, whereas BALB/c mice demonstrate higher severity of colitis.

Significant care needs to be executed during the intrarectal step to make sure that the

gut is not damaged or punctured.

The DSS model in comparison to the TNBS model is a simple oral

administration of these polymers. DSS is a heparin-like polysaccharide which

contains three sulphate groups per molecule. DSS in drinking water can induce both

acute and chronic colitis in mice, depending on the time course of the oral

20

administration of DSS (Vowinkel et al., 2004). DSS-induced colitis exhibits

morphological and pathophysiological features that resemble human UC (Hamilton

et al., 1983; Okayasu et al., 1990; Cooper et al., 1993). Features such as superficial

ulcerations, mucosal damage, production of cytokines and other inflammatory

mediators, and leukocyte infiltration are similar to the human UC (Okayasu et al.,

1990; Cooper et al., 1993; Elson et al., 1995). The direct toxic effect of these

polymers on the colonic epithelium affect on the integrity of the mucosal barrier

(Fedorak & Madsen, 2000), stimulate macrophage activation, and the alteration of

colon microflora have all been implicated in the pathogenesis of DSS-induced colitis

(Okayasu et al., 1990; Cooper et al., 1993). Kitajima et al., (2000) have shown that

colitis in mice treated with 40kDa DSS was more severe when compared to mice

treated with 5kDa DSS, and there was also a difference in location of colitis in the

colon.

The severity of DSS-induced colitis depends on the molecular weight,

sulphate percentage (of DSS), and the dosage and duration of the DSS administration

(Okayasu et al., 1990; Yamada, et al., 1992; Cooper et al., 1993; Axelsson et al.,

1996). Therefore, these factors have to be considered when the DSS-colitis is used in

mice. As is the case with TNBS model, DSS induced colitis is also affected by the

mouse strain, the age of the mice, individual differences in the intestinal microflora

between the animal groups, the dosage applied to these animals and the duration of

DSS induction (Wirtz et al., 2007). BALB/c mice develop mild to severe colitis with

5% DSS, whereas C57BL/6 mice develop mild to severe colitis with 2% DSS.

However, in comparison to other colitis models, this model can produce acute,

chronic or relapsing forms of colitis by changing the concentration and cycle of

administration of DSS (Okayasu et al., 1990). This model is widely used in the

exploration of immunological mediated aspects of chronic mucosal inflammation,

and also for evaluating new potential therapeutic agents.

Intestinal inflammation- cells of the adaptive immune system

Genetically engineered models

The genetically engineered models of IBD have allowed the exploration of

the interaction between genetic and environmental factors (Fedorak & Madsen,

2000). Although, these models do not necessarily duplicate all the histologic and

clinical characteristics associated with IBD, they do exhibit features which are

21

similar to human disease, such as the development of colitis and acute inflammation

(Fedorak & Madsen, 2000; Wirtz et al., 2000). There are several genetically

engineered models, but one of the most commonly used is the IL-10 knock-out,

mouse which causes a disease with similar features to CD in humans (Van Deventer

et al., 1997; Schreiber et al., 2000). Other transgenic models such as T-cell receptor

(TCR) mutant mice and NEMO (NF-κB essential modulator) are also briefly

discussed in this review.

IL-10 is a potent anti-inflammatory cytokine which down-regulates activation

of Th1 cells, and also inhibits macrophage inflammatory cytokine production

including TNF-α, IL-1, and IL-2, and other T-cell associated macrophage activity

(Schreiber et al., 2000). In this model, the IL-10 gene is inactivated by targeted

mutation (Fedorak et al., 2000). Furthermore, IL-10-/- mice experience a

“spontaneous” enterocolitis which is dependent on the presence of luminal flora

(Kuhn et al., 1993). IL-10-/- mouse studies have shown that a Th1 cell response is

generated, which in normal mice is suppressed by Th2 cell production of IL-10

(Berg et al., 1996). These increased levels of pro-inflammatory cytokines in IL-10 -/-

mice imply that the uncontrolled cytokine production by the macrophages

contributes to the pathogenic response (Rennick et al., 1997; Fedorak & Madsen,

2000). However, the disease onset of this model is prolonged, and the expression of

colitis can take several months (Kuhn et al., 1993). The severity of the disease in this

spontaneous gene targeted model can be inconsistent and depends on environmental

factors such as commensal flora (Wirtz et al., 2007). However, there are variations in

the IL-10-/- mice such as the differences in the strain of mice, and also in the

phenotype of colitis. Nevertheless, this model of IBD has similar features as CD in

humans, which helps to develop our understanding of the pathogenesis of the

disease.

Models such as TCR deficient mouse colitis and NEMO -/- have similar

features to UC (Mombaerts et al., 1993; Schmidt-Supprian et al., 2000). The TCR

deficient model and NEMO -/-, like IL-10 -/- mice, also take a long time to develop

colitis, but the colitis develops differently. TCR mice develop chronic colitis

spontaneously with an increase in T and B cell production. In comparison, NEMO -/-

mice develop colitis by extensive epithelial destruction, suggesting that the epithelial

integrity is damaged by the invasion of bacteria from the lumen into the mucosa

(Nenci et al., 2007; Zaph et al., 2007). Zaph et al., (2007) showed that NEMO -/-

22

mice are unable to eliminate infection by pathogens, suggesting that NF-κB plays an

important role in IECs against pathogens through protective immune responses.

Table 1. Different mouse models used to induce colitis and their

mechanisms. Mouse models Mode of

administration Mechanisms

underlying colitis References

Chemical induction TNBS Intrarectal Breakage of mucosal

barrier resulting in Th1 response (similar to CD) with increased production of inflammatory cytokines (IL-12, IFN-γ, and TNF-α) and chemokines from infiltrating cells and isolated macrophages

Morris et al., 1989; Neurath et al, 1995; Elson et al., 1996; Strober et al., 2002; Wirtz et al., 2007

DSS Oral Acute colitis is stimulated through the direct toxic effect of DSS on colonic epithelial cells affecting the integrity of the mucosal barrier

Fedorak & Madsen, 2000

Genetically engineered IL-10 -/- Genetically

transferred Lack of anti-inflammatory cytokine IL-10, causes spontaneous colitis due to suppressed immune tolerance

Kuhn et al., 1993

TCR mutant mice Genetically transferred

Spontaneous colitis; Lack majority of CD4+ and CD8+ T cells. Suggested to be associated with an increase in Th2 response similar to UC

Mombaerts et al., 1993

NEMO -/- Genetically transferred

Blocks NF-κB by pro-inflammatory cytokines and interferes with the generation of lymphocytes. Similar to UC

Schmidt-Supprian et al., 2000

23

Each mouse model has its advantages and disadvantages, and this highlights

the importance of utilising a number of different models to trial drugs or helps

understand specific features of IBD.

Current treatments for IBD Over the years, there has been intense study regarding the treatment of IBD.

However, new treatments which suppress inflammation and restore damaged mucosa

are required. Table 2 summarises the commonly used drugs and their mechanisms of

action.

5-ASA or mesalamine used to treat patients with IBD in its active disease

state is known to maintain patients in remission (Sutherland & Shaffer, 1993).

Although it is considered to be a safe drug, its efficacy rates are not very high.

Nevertheless, past studies have shown that there seems to be a dose–response

relationship in the efficacy for the treatment of both UC and CD (Khan et al., 1977;

Khan et al., 1980; Singleton et al., 1993). However, long-term treatment with

mesalamine in CD is not as effective as it is for UC.

In comparison, corticosteroids reduce inflammation in the intestine of

patients with UC and CD. They have anti-inflammatory actions by suppressing the

immune system, however, this results in patients being more susceptible to

infections. Corticosteroids are unable to keep UC or CD disease in remission and

also cannot prevent the re-occurrence of the disease after surgery. The side-effects

related to corticosteroids are dependent on the dose and the duration of the therapy

(Hanauer et al., 1991; Lindmark, 1993). Therefore, long-term uses of corticosteroids

are discouraged, due to the risk of long-lasting side effects. Patients who take these

drugs are at a risk of developing osteoporosis and long-term use in children can

result in delayed growth. Immunomodulators such as AZA and 6-MP have a slow

onset of action compared to corticosteroids (Dieleman et al., 1991). Since these

drugs suppress the immune system there are greater chances of infection. 6-MPs also

cause severe hepatotoxicity in a significant number of patients (Dieleman et al.,

1991).

24

Table 2. Therapeutic treatments and their mechanisms in IBD. Therapeutic drugs Disease Mechanisms of

Action Side effects References

5-ASA/Mesalamine CD & mild-moderate UC

Inhibits IL-1, NF-κB, TNF-α activation, and B cells and oxygen radical production

Kidney related problems, when administered in high doses

Jarnerot, 1994; Egan et al., 1999; Kaiser et al., 1999; Nikolaus et al., 2000; Weber et al., 2000; Sutherland et al., 2002 & 2003

Corticosteroids Moderate-severe CD & UC

Down-regulates the activation of NF-κB, and also decreases cytokine production by inducing cytokine inhibition. Decreases IL-1 and IL-2 production

Dyspepsia, hyperglycemia, alteration of fat distribution, high risk of infection, and adrenal suppression

Lindmark, 1993; Auphan et al., 1995; Brattsand & Linden, 1996; Hanauer & Kane, 2000

Immunomodulators- AZA & 6-MP

CD Inhibits cell proliferation in DNA synthesis phase of the cell cycle

Nausea, fever, rash, joint pain, diarrhea, renal dysfunction, and high risk of infection

Sandborn, 1996; Hanauer & Kane, 2000

Infliximab CD Blocks the action of TNF-α by binding to it and prevents its signalling to the TNF-α receptors on the surface of leukocytes and epithelial cells

Allergic reaction or a delayed hypersensitivity reaction, high blood pressure, chest pain, difficulty in breathing, and high risk of infection (i.e. respiratory)

Knight et al., 1993; D’Haens et al., 1999; Hanauer et al., 2002

Probiotics CD & UC Unknown, but has been suggested to suppress growth or the epithelial binding by pathogenic bacteria. Increases production of protective cytokines, IL-10, and suppress production of pro-inflammatory cytokines, TNF-α

Not many known, but may cause an allergic reaction if taken with another medication or supplement

Hart et al., 2003; Mach, 2006; Ebtissam, 2007

*5-ASA (5-Aminosalicylates); AZA (Azathioprine); 6-MP (6-Mercaptopurine)

25

The monoclonal antibody infliximab is a mouse-human chimeric antibody to

TNF-α (Knight et al., 1993). TNF-α is one of the key cytokines that triggers and

sustains the inflammatory response (D’Haens et al., 1999; Hanauer et al., 2002). Due

to immunogenity, patients being treated with infliximab can form human anti-

chimeric antibodies (HACA), but co-treatment with immunomodulators can reduce

the risk of HACA formation (Kuhbachler & Foisch, 2007). There is also a risk of

decreased white and red blood cells, as well as decreased platelet count. Even though

they are an efficacious method for treating CD the disadvantage with this therapy is

that it is relatively expensive and infection is a serious side-effect. There are other

antibodies in the pipeline for treatment of CD. IL-23R gene is associated with CD,

but has also been reported in patients with UC (Duerr et al., 2006). IL-23 is

expressed at high levels by activated macrophages and DCs and the functional IL-23

cytokine is comprised of p19 and p40. Blocking antibodies specific for p40 inhibits

IL-23 and IL-12 induced signaling. This administration of the p40-specific

antibodies has proved to be a promising approach for the treatment of CD (Mannon

et al., 2004).

A contentious class of therapeutic agents for treatment of IBD are probiotics.

Probiotics are living microorganisms which are present in the intestine as normal

flora and are important to the health and well-being of the host (Campieri et al.,

2001). Increasing evidence suggests that gastrointestinal microflora are involved in

the pathogenesis of IBD in genetically susceptible subjects with immunological

dysregulation (Dotan & Rachmilewitz, 2005). Supporting this hypothesis is the

observation that there is an increase in the number of microorganisms and a change

in various populations of normal flora in IBD patients (Ebtissam, 2007). The

interactions between the commensal microflora and the intestinal mucosa stimulate

inflammatory activity (Dotan & Rachmilewitz, 2005; Bai & Quyang, 2006). A recent

study by Fedorak (2008) has identified the mechanism of action of probiotics to have

direct effect on epithelial cell function and intestinal health, including enhancing

epithelial barrier function, modulating epithelial cytokine secretion into an anti-

inflammatory dominant profile, altering mucus production, changing bacterial

luminal flora, modifying the innate and systemic immune system, and inducing

regulatory T-cell effects. However, for probiotics to have a therapeutic effect on

IBD, their therapeutic mechanism of action has to be associated with the pathogenic

mechanism of action of the disease (Fedorak 2008). Furthermore, recent studies have

26

shown that the administration of probiotics ameliorates inflammation by exerting

positive effects on epithelial cell and mucosal immune system dysfunction which

form the basis of the inflammation (Boirivant & Strober, 2007). Nevertheless, the

use of probiotics for therapy requires both in vitro and in vivo study to prove their

effectiveness in IBD.

The use of antibiotics such as metronidazole and ciprofloxacin seem to have

a scientific rationale in treating IBD especially in the long-term treatment of CD and

in the management of pouchitis (Guslandi, 2005). However, the use of antibiotics in

IBD is still debated as they are administered for long periods of time resulting in

tolerability problems as well as adverse side effects (Guslandi, 2005; Perencevich &

Burakoff, 2006).

New treatments for IBD need to be evaluated

Peptides isolated from the gut have shown promising effects in animal

models of IBD. Gut peptides such as ghrelin, vasoactive intestinal peptide (VIP),

cortistatin, and somatostatin have been shown to exhibit anti-inflammatory effects

(van Bergeijk & Wilson, 1997; Delgado et al., 1999; Dixit et al., 2004; Li et al.,

2004; Gonzalez-Rey et al., 2006; Szliter et al., 2007). Ghrelin and VIP suppress

inflammation through both the innate and adaptive immune systems (Delgado et al.,

1999; Dixit et al., 2004; Szliter et al., 2007). In a toxin-induced colitis study, it was

found that ghrelin had anti-inflammatory effects and decreased the severity of colitis

and inflammation and prevented the recurrence of the disease (Gonzalez-Rey et al.,

2006). It significantly reduced the clinical symptoms and pathology by down-

regulating both pro-inflammatory and Th-1 mediated immune responses (Gonzalez-

Rey et al., 2006). Ghrelin has been shown to affect cell proliferation in colon cancer

cell lines (Taufiq et al., in preparation). Our work demonstrates that nanomolar

concentration of ghrelin significantly increases the proliferation of human

colonocytes. Novel treatments are required which will not only suppress

inflammation by acting directly on the immune system, but also repair and restore

the injured epithelium to prevent further damage. Therefore, ghrelin peptides are

promising therapeutic candidates for treating IBD and other inflammatory diseases.

27

Ghrelin Ghrelin is a 28-amino acid peptide which is octanoylated at its third amino

acid and is the endogenous ligand for the ghrelin receptor, the growth hormone

secretagogue receptor (GHS-R1a) (Kojima et al., 1999). Ghrelin is mainly produced

by stomach tissue and it has an unusual acyl modification on its critical serine-3

residue (Kojima et al., 1999). The acyl modification is important for the activation of

the GHS-R1a (Kojima et al., 1999). As well as stimulating growth hormone (GH)

release (Kojima et al., 1999), ghrelin also influences appetite (Nakazato et al., 2001),

energy balance (Inui, 2001), and gastric motility (Sibilia et al.,2002). Ghrelin

circulates in the plasma in two forms: acylated (octanoylated) and non-acylated (des-

octanoyl or des-acyl) ghrelin. Ghrelin is predominantly expressed in the entero-

endocrine cells of the stomach although co-expression of ghrelin and the GHS-R1a

have been observed widely throughout human and mouse tissues at the mRNA or

protein levels, albeit at low levels (Date et al., 2000). Ghrelin expressing cells, also

known as X/A-like cells, are found in the stomach, where they are not in contact

with the lumen but are close to the capillaries (Korbontis et al., 2004). Ghrelin is

found in the fundus of the stomach and also in a smaller number of immunopositive

cells in the small and large intestine (Korbontis et al., 2004). Lee et al., (2002) have

shown the presence of ghrelin within the mucosa of both rat stomach and colon, with

the highest levels being in the stomach. It has also been demonstrated in RT-PCR

analyses that ghrelin receptor mRNA expression is present in many peripheral

organs, such as heart, lung, liver, kidney, pancreas, stomach, small and large

intestines, adipose tissues, and immune cells which indicate that ghrelin has multiple

functions in these tissues (Guan et al., 1997; Hattori et al., 2001; Gnanapavan et al.,

2002).

In our study we also observed mRNA and protein expression of ghrelin and

its receptor, GHS-R1a, in colon epithelial cell lines (Taufiq et al., in preparation). In

particular ghrelin and its receptor are expressed in the Caco-2 cell line, which is

similar in biochemical and morphological aspects to normal intestinal enterocytes.

As summarised in Table 3, ghrelin has been shown to affect various factors including

cell proliferation and inflammation. The expression of ghrelin and its receptor have

been identified in T cells, which suggest that ghrelin levels found in IBD patients can

28

clarify the possible involvement of ghrelin in intestinal inflammation (Hattori et al.,

2001; Dixit et al., 2003; Dixit et al., 2004).

Ghrelin also affects epithelial cell proliferation in several different cell types

(Pettersson et al., 2002; Jeffery et al., 2003; Kim et al., 2004; Mazzocchi et al.,

2004; Zhang et al., 2004; Jeffery et al., 2005 a, b; Maccarinelli et al., 2005;

DeVriese et al., 2005). We have observed in our previous study that ghrelin

increased cell proliferation in Caco-2 (colon epithelial) cell lines by nearly 50% with

the administration of nanomolar concentration of ghrelin (Taufiq et al., in

preparation). However some studies have shown that ghrelin inhibits proliferation

(Xia et al., 2004). The effect of ghrelin on cell proliferation has been shown to have

conflicting results (Nanzer et al., 2004), however, these apparent discrepancies may

be due to different cells types and receptor expression.

Table 3. Multiple Effects of Ghrelin function. Parameter Effects References

GH release Kojima et al., 1999

Appetite Nakazato et al., 2001

Obesity Tschop et al., 2000

Inflammation Dixit et al., 2004; Dixit et al., 2009; Li et al., 2004; De Smet et al., 2009

Cardiovascular functions Cardiac output Blood pressure

Nagaya et al., 2001; Nagaya & Kangawa, 2003

Gastric functions Gastric motility

Sibilia et al.,2002

Cell Proliferation Jeffery et al., 2002; 2003; 2005a, b; DeVriese et al., 2006

Ghrelin gene derived peptides

The 28 amino acid peptide hormone, ghrelin is derived proteolytically from a

precursor of 117 amino acids (Kojima et al., 1999). Zhu et al (2006) have shown that

cleavage of the 23 amino acid signal sequence yields pro-ghrelin, which has a

glycine as its N terminal residue. However, the C terminus of the mature ghrelin is

?

?

29

generated via the prohormone convertase 1/3 (PC1/3), which cleaves after the

arginine-28 of pro-ghrelin, creating the mature 28 amino acid peptide (Zhu et al.,

2006). Furthermore, several potentially functional peptide hormones can be derived

from alternative splicing of the ghrelin gene, as can be seen in Figure 2. These

include the mature ghrelin peptide, obestatin, and the novel Δ4 C-terminal proghrelin

peptide (Jeffery et al., 2003; Jeffery et al., 2005a,b). It has been predicted that the

exon 4-deleted variant is produced potentially by an alternative splicing mechanism

such as exon skipping (Jeffery et al., 2005a,b). The exclusion of exon 4 from mouse

preproghrelin mRNA transcript results in a cDNA frameshift that, when translated,

encodes a unique 16 amino acid C-terminal peptide sequence (Δ4 proghrelin

peptide) which is highly conserved (Jeffery et al., 2003; Jeffery et al., 2005a,b).

However, whether this unique C-terminal peptide of the Δ4 proghrelin peptide is

functionally active or not has not been determined.

Ghrelin O-Acyltransferase (GOAT)

Ghrelin is the only protein in animals that is known to be modified by O-

acylation with octanoate, an eight-carbon fatty acid, which is necessary for the

central action of ghrelin (Kojima & Kangawa, 2005), however, the enzyme which

catalyses this modification has only recently been discovered (Yang et al., 2008).

Figure 2. Alternative splicing of mouse ghrelin gene generates multiple ghrelin peptides.

1 2 3 4 5

2 3 4 5 2 3 5

2 3 4 2 3 5

3’ 5’

Cleaved and post-translationally modified

ghrelin Δ4 peptide obestatin ghrelin

Exon

Transcription, translation, and alternative splicing

30

The polytopic membrane-bound enzyme which attaches octanoate to serine-3 of

ghrelin has been named Ghrelin O-Acyltransferase (GOAT). GOAT belongs to the

family of Membrane-Bound O-Acyltransferases (MBOATs), which attach fatty acids

to lipids and proteins (Hofmann, 2000). GOAT is a unique enzyme since it is the

first family member which transfers a medium-chain fatty acid such as octanoate,

whereas previously studied enzymes transferred long-chain fatty acids of at least 16

carbons (Yang et al., 2008). Yang et al, (2008) further discuss that since ghrelin is

the only known octanoylated protein in animals, it is perhaps the sole substrate for

GOAT. The relative distribution of GOAT mRNA in different mouse tissue reveals

that like ghrelin, GOAT is highly expressed in the stomach (Yang et al., 2008).

Gutierrez et al., (2008) have identified and characterised human GOAT, and have

also demonstrated the occurrence of ghrelin and GOAT in stomach and pancreas

tissue implicating a crucial role of GOAT in the acylation of ghrelin in pancreatic

function. Since ghrelin is primarily localised to a minor population of X/A cells it

will be interesting to determine whether GOAT is also restricted to these cells (Yang

et al., 2008). There was a moderate level of expression of GOAT mRNA in mouse

tissue of the colon suggesting that is does play some role in the acylation of ghrelin

in the colon. Since we have demonstrated the expression of ghrelin in some colonic

epithelial cells, it would therefore be interesting to identify the role of GOAT and its

acylation of ghrelin in both human and mouse colon. GOAT is now being studied as

a critical molecular target in developing novel therapy for obesity, type-2 diabetes,

and perhaps in inflammatory-mediated diseases (Gutierrez et al., 2008).

The role of Ghrelin in inflammation and IBD

Ghrelin has been described as a potent anti-inflammatory factor which

inhibits the production of pro-inflammatory cytokines through activated monocytes

and endothelial cells, and results in protection from endotoxic shock (Dixit et al.,

2004; Li et al., 2004). An inhibitory effect of ghrelin on cytokine mRNA expression

might be responsible for the inhibition of splenic T cell proliferation by ghrelin in

vitro (Xia et al., 2004). Ghrelin also modulates an immune response in several

disease processes such as: arthritis (Granado et al., 2005) pancreatitis (Dembinski et

al., 2003), stomach inflammation (Ariyasu et al., 2004; Konturek et al., 2006) and

intestinal colitis (Gonzalez-Rey et al., 2006). In a study by Konturek et al., (2006) it

was found that due to its anti-inflammatory properties, ghrelin exerts strong

31

protective actions on the gastric mucosa and also accelerates healing of lesions.

Ghrelin and its receptor have been identified in T cells, and can inhibit the activation

of cytokines including, IL-1β, IL-6, TNF-α, and also leptin (Dixit et al., 2003).

Ghrelin also reduces Th1 (IL-2 and IFN-γ) cytokine mRNA expression in activated

lymphocytes, and almost completely inhibits Th2 cytokine mRNA expression (Xia et

al., 2004). What is also interesting is that serum levels of ghrelin are increased in

patients with IBD (Karmiris et al., 2006). Therefore, elevated serum levels are a

feature of active IBD, suggesting a role for endogenous ghrelin in active human IBD

(Peracchi et al., 2006).

NF-κB is considered to play an important role in inflammation and IBD

pathogenesis (Finco et al., 1997; Bharti & Aggarwal, 2002). Ghrelin has strong anti-

inflammatory effects in human endothelial cells, potentially mediated by the

inhibition of NF-κB activation. Li et al., (2004) have also shown that ghrelin inhibits

TNF-α-induced NF-κB activation, whereas Zhao et al., (2006) claim that the up-

regulation of ghrelin during colitis is through NF-κB activation. Nevertheless, Zhao

et al., (2006) have not addressed the functional importance of the overexpression of

the ghrelin receptor in pathogenesis of colitis. Due to the differences in results,

further research into the role of ghrelin in inflammation and IBD is required to

understand the complete mechanism of its actions.

Ghrelin in mouse models of colitis

A study by Gonzalez-Rey et al., (2006) used a TNBS-induced colitis model

which, like CD, causes an archetypal CD4+ Th1 cell-mediated colitis, to investigate

the effect of ghrelin treatment on IBD. In this study the anti-inflammatory effect of

ghrelin decreased the severity of colitis and inflammation and prevented the

recurrence of the disease (Gonzalez-Rey et al., 2006). The therapeutic effect was

linked with the down-regulation of both pro-inflammatory cytokines (IL-12, IFN-γ,

and TNF-α) and the Th1-driven autoimmune response and increased levels of the

anti-inflammatory cytokine IL-10 (Gonzalez-Rey et al., 2006). Ghrelin treatment

reduced colonic infiltration of neutrophils, and macrophages due to the down-

regulation of multiple chemokines and cytokines in the lamina propria. Interestingly,

lamina propria mononuclear cells and mesenteric lymph node (MLN) T cells, which

were isolated from ghrelin treated mice, secreted lower levels of pro-inflammatory

cytokines in vitro upon activation (Dixit et al., 2004; Gonzalez-Rey et al., 2006).

32

The study did not investigate the effects of ghrelin on epithelial barrier damage and

did not look at the expression of ghrelin and its receptor, GHSR, in the colonic

specimens from the mice. The histological sections did not specify from what region

of the colon the histopathology scores were derived. This is important especially in

the 5kDa DSS model, where the location of damage induced by DSS is dependent

upon the molecular weight of DSS and the mouse strain. Also the cytokine and

chemokine contents from the protein extracts were measured from only the TNBS

model and not the DSS model. Since these two models induce different kinds and

degree of colitis, it is important to measure the levels of cytokine/chemokines from

each model.

Relevance of Project The advancement in the understanding of mucosal immunology in IBD in

recent years has generated a robust variety of potential therapeutics. These potential

therapeutic agents may be useful in treating CD and UC however; thorough pre-

clinical studies need to be undertaken. The recent discovery of the anti-inflammatory

actions of ghrelin in colitis, have raised many questions related to the role of ghrelin

in the colonic epithelial barrier and the mucosal immune system. According to

current studies it is evident that ghrelin does influence production of inflammatory

cytokines and transcription factors by both antigen presenting cells and immune

effector cells, and is an element of co-regulation between the endocrine and the

immune system. The recent discovery of exon 4-deletd preproghrelin and its high

expression in the stomach creates a new aspect in the field of ghrelin research.

A significant knowledge gap is whether ghrelin acts as a pro- or anti-

inflammatory mediator during immune responses. This research project will

investigate the mechanism of ghrelin peptides by treating mice with colitis with

different concentrations to determine whether the ghrelin peptides will ameliorate

colitis. This study will also compare the disease severity of acute colitis induced by

DSS in two different mouse strains. It has been identified that C57BL/6 mice are

more susceptible than BALB/c mice to DSS-induced colitis (Sasaki et al., 2008). In

a study by Kitajima et al., (2000) the molecular weight of the DSS polymers used to

induce colitis made a huge impact on the severity of colitis and epithelial damage of

the mucosa. They showed that the severity and primary location of colitis differ with

33

administration of DSS at different molecular weights for 7 days in mice (Kitajima et

al., 2000). Their study concluded that colitis induced by 40kDa DSS was more

severe than that induced by 5kDa DSS in BALB/c mice. The differences in genetic

background are also a major determinant of the inflammatory response. While the

inflammatory response in C57BL/6 is driven by Th1, the response in BALB/c mice

is Th2-driven. This reflects the fact that BALB/c mice have a defective Th1 response

and are more susceptible to bacterial infections such as Leishmania major. C57BL/6

infected mice, in comparison, effectively mount a Th1 response, leading to the

clearance of the infection followed by healing (Sacks & Noben-Trauth, 2002). This

leads us to predict that the induction of colitis in these two strains of mice will be

relatively different both histopathologically and immunologically. Therefore, our

project will use 5kDa and 40kDa of DSS to develop colitis in BALB/c and C57BL/6

mice, predicting that mice treated with 40kDa will develop more severe colitis

compared to mice treated with 5kDa.

The first aim of this project is: To determine whether the administration of

ghrelin and Δ4 proghrelin peptide will ameliorate intestinal inflammation in two

different mouse strains with DSS-induced colitis. We hypothesise that ghrelin

peptides will reduce colitis and rescue epithelial damage through proliferative

mechanisms. Since IBD is a multifactorial disease which is caused by both

environmental and immunological factors, we will examine the influence of the

ghrelin peptides in two different animal strains which will be treated with different

molecular weights of DSS to induce colitis. This will be the first study to investigate

the functional significance, if any, of Δ4 proghrelin peptide in DSS-induced colitis

mouse models. Mice in the DSS model will develop acute colitis, and will be treated

with the ghrelin peptides. The inflammatory activity will be measured by scoring

histopathology in the colon and measuring the expression of pro-inflammatory

cytokines and the relative abundance of Treg cells from the mesenteric lymph nodes.

Another knowledge gap is whether ghrelin is working at the epithelial level.

It has been previously demonstrated by our group that ghrelin and its receptor are

expressed in some colon cancer cells, but whether the protective mechanism in

colitis occurs at an epithelial level is still not clear. Previous studies have revealed

that both exogenous and endogenously produced ghrelin increases proliferation in

some in vitro cell systems (Waseem et al., 2008). Therefore the second aim of this

project is: To investigate whether exogenous ghrelin modulates intestinal

34

proliferation/migration using colon cancer cell lines, as in vitro models of

intestinal epithelium. We hypothesise that ghrelin peptides improve epithelial would

healing in colon cancer cell line- HT29. In this model, the cells are grown to

confluence and then insulted by creating a “wound”. The cells are then treated with

different concentrations of ghrelin peptides over 48 hours, photographed at regular

intervals. This study would act as a model for intestinal epithelial growth and/or

recovery after treatment with ghrelin.

In a study by Konturek et al., (2006) it was found that due to its anti-

inflammatory properties, ghrelin exerts strong protective actions on the gastric

mucosa and also accelerates healing of lesions. Therefore, it would be interesting to

identify whether ghrelin will cause wound healing in the colonic epithelium. The

exact mechanism through which ghrelin may mediate proliferation and migration are

not clear.

Conclusion The current therapies for treating patients with severe IBD are often

ineffective and have been associated with numerous side effects. We thus require

ideal treatments which will not only suppress inflammation by acting directly on the

immune system but will also repair the injured colonic mucosa to prevent any further

damage. The recent discoveries of the anti-inflammatory actions of the ghrelin

peptides in the immune system are promising. With the help of experimental animal

models, which have clinical manifestations similar to those observed in IBD, we can

investigate the immunological, pathological, and physiological features of intestinal

inflammation in IBD. These models can help us to elucidate the influence of ghrelin

on the immune system including on T cells, dendritic cells, and regulatory T cells.

Figure 3 summarises a hypothesised role of ghrelin peptides in the immune system,

where they potentially target DCs which may affect T cells directly and cause the

production of anti-inflammatory cytokines to suppress inflammation caused in

colitis. Therefore, future studies in the role and underlying mechanisms of novel

ghrelin peptides in IBD will contribute to the development of new targets for

medical therapies.

35

Ghrelin peptides

IL-12 TNF-α IFN-γ

Lamina propria

Lumen

CD4+ cells Macrophages

IL-10 TGFβ Foxp3

Regulatory T cells

DC

IL-10

DC

Figure 3. A potential mechanism of ghrelin peptides in inflammation. We hypothesise that ghrelin peptides can alter the functions of DC and cause them to tolerise and induce the differentiation of Foxp3 expressing Treg cells. Treg cells will then secrete the production of anti-inflammatory cytokines, such as IL-10 and TGFβ, and suppress the production of pro-inflammatory cytokines such as IL-12, TNF-α, IFN-γ in response to IEC barrier injury. Ghrelin could also work directly on mucosal epithelial cells and T cells to repress inflammation.

IEC

36

Chapter Three: Materials and Methods

3.1 Mice

Female BALB/c and C57BL/6 mice, six weeks old, were purchased from

Animal Resources Centre (WA, Australia) and housed in individual filtered air cages

within the MMRI animal handling facility under 12:12-h light-dark cycles. The mice

were fed a standard rodent pellet diet and tap water and were acclimatized for a

minimum of one week before the start of the study. All animal experiments were

approved by the University of Queensland animal ethics committee (AEC No.

MMRI/397/08).

3.2 Induction of colitis and experimental design

Experimental colitis was induced by the administration of dextran sodium

sulphate (DSS) dissolved in drinking water. Two molecular weights of DSS were

used: 40kDa (ICN Biomedicals, Costa Mesa, CA) and 5kDa (Wako Pure Chemicals,

Tokyo, Japan). BALB/c mice were given five percent DSS and C57BL/6 mice

received two percent DSS as they are more susceptible to DSS-induced colitis (Wirtz

et al., 2007). Mice were divided into groups of six per cage/treatment and the

experiment was performed twice (N=10-12 per treatment; four animals died

spontaneously during two separate experiments). DSS was administered for eight

days and mice were sacrificed on day eight. Octanoylated ghrelin

(1nmol/mouse/day), ghrelin Δ4 peptide (1nmol/mouse/day) or sterile phosphate-

buffered saline (PBS,vehicle control) were injected intraperitoneally on days four

and six. The time period for DSS treatment and timing of injections were optimised

by the lab previously, and is also a published protocol (Gonzalez-Rey et al., 2006).

Control mice (naives) received tap water only. Ghrelin (Auspep, Parkville, Victoria)

and ghrelin Δ4 peptide (Mimotopes, Clayton, Victoria) were diluted in sterile PBS

immediately before injection.

3.3 Assessment of inflammation: symptoms and inflammatory score

The development of colitis was assessed daily by measurement of body

weight and evaluation of stool consistency, faecal bleeding, and diarrhoea. Clinical

symptoms, including shivering, hunching and ruffling of the coat were evaluated

37

daily using clinical score indices as described more fully in the score sheet

(Appendix 1.1). Body weight on day zero was taken as 100% weight and change in

weight each day was measured as a deviation from day zero weight. The

presence/severity of diarrhea was scored on a scale of 0 to 3 (0= normal; 1= slightly

soft but well formed; 2=soft and not well formed; 3=watery faeces with mucus).

Similarly, rectal bleeding was defined as faeces containing visible blood or gross

rectal bleeding scored on a scale of 0 to 3 (0= no traces of blood; 1= specks of blood

in the faeces; 2= blood present in faeces; 3= prominent blood in the faeces and/or

sticking to fur around the rectum). The cumulative score for diarrhea and rectal

bleeding was calculated by adding the scores for each day.

3.4 Haematological analysis

Blood samples were collected on the final day of the experiment (day eight).

Blood was collected from the mice either by tail vein bleed, cardiac puncture (in

extreme cases where mice were too sick to bleed) or submandibular puncture,

following which the mice were sacrificed by either asphyxiation or cervical

dislocation. For tail vein bleeds the animals were placed in containers and heated for

5-10 minutes with an infrared heat lamp to promote vasodilation. Each animal was

then placed in a plastic restrainer which only exposed the tail. A sterile scalpel blade

was used to cut one of the lateral veins in the tail to collect blood into eppendorf

tubes containing 2µl heparin (BD Biosciences). Haematocrit, total leukocyte counts

and differential white cell counts were measured in the blood samples (50µl) using

the Sysmex SF3000 Haematology Analyser (Sysmex Corp, Kobe, Japan).

3.5 Tissue collection

Mice were euthanized by either asphyxiation or cervical dislocation on day 8.

Post-mortem, the colons were excised, opened longitudinally and washed gently in

PBS to remove faecal debris and their lengths were measured. The colons were then

dissected in half with one half rolled in the “swiss roll” formation and fixed in

neutral buffered 10% formalin solution. From the remaining colon, 5mm sections

from distal and proximal colon were dissected for RNA and protein extraction. For

RNA extraction, the colon tissues were crushed immediately in 0.5ml Trizol

(Invitrogen, Carlsbad, CA) and snap frozen on dry ice. For protein extraction, the

colon samples were snap frozen in liquid nitrogen and stored at -80°C until the full

38

protein extraction protocol could be performed. The mesenteric lymph nodes (MLN)

were also dissected from each mouse and placed into complete RPMI (RPMI-1640

supplemented with 100U/mL penicillin/streptomycin, 2nmol/L L-glutamine,

50µmol/L β-mercaptoethanol (Invitrogen), and 10% heat-inactivated foetal calf

serum (Invitrogen) and kept on ice until processing.

3.6 Histological assessment of colitis

Colon swiss rolls collected in 10% formalin were transferred to 70% ethanol

24h later. These were then sent to the histology service at the Queensland Institute of

Medical Research (QIMR) and were dehydrated, embedded in paraffin and 5 micron

sections were cut for immunohistochemical analysis or for haematoxylin and eosin

(H&E) staining. H&E stained sections were examined to assess the degree of colitis

in each mouse. Sections were examined at various magnifications using an Olympus

BX50 microscope and representative digital photographs were taken. Histological

colitis severity was evaluated based on microscopic features including crypt length,

ulceration, goblet cell loss and inflammatory cell infiltration (full details in scoring

sheet, Appendix 1.2). The grading system used for this project is well established in

our laboratory (Heazlewood et al., 2008) and the scoring was done by two blinded

investigators.

3.7 Isolation and culture of Mesenteric lymph node (MLN) cells

Whole mesenteric lymph nodes, adjacent to the proximal colon were

dissected from the mice. The lymph nodes were then gently crushed between sterile

glass slides to disaggregate cells and then passed through a 40µm cell strainer

(Falcon, BD Biosciences, Franklin Lakes, NJ) to remove fat and connective tissue.

Lymph node cells were then washed in fresh RPMI, counted using a

haemocytometer, obtained in a single-cell suspension and plated into 24 well plates

at a concentration of 2 X 106 cells/mL, in the presence of 50ng/ml phorbol 12-

myristyl 13-acetate (PMA, Sigma) and 750ng/ml ionomycin (Sigma) to stimulate

immune cells. The culture supernatants were collected at 48h time point and

centrifuged at 400g for 5min to remove cells and debris and were then aliquoted and

stored at -80°C. TNF-α production in culture supernatants was determined after 48h

culture as described below.

39

3.8 Enzyme-Linked ImmunoSorbent Assay (ELISA) for TNF-α

ELISA was performed to measure the level of mouse TNF-α in stimulated

mesenteric lymph node cultures from mice treated with DSS and mice co-treated

with ghrelin peptides using BD ELISA kits (BD Biosciences). Capture antibody for

TNF-α was diluted 1:250 in coating buffer (1M sodium carbonate) and was plated

100μl/well in a 96 well microplate (Nunc, Roskilde, Denmark). The plate was

sealed and left overnight at 4°C. The following day the capture antibody solutions

were aspirated and washed 3 times with 300μl/well wash buffer (0.05% Tween in

PBS) in an automatic microplate washer (BioTek ELx405, Winooski, Vermont).

After the plate was washed it was inverted and blotted on an absorbent paper to

remove any residual debris. The plates were then blocked with assay diluent (3%

Bovine Serum Albumin in PBS, 200μl/well) and incubated at room temperature (RT)

for 1h. At this point the lymph node culture supernatants were thawed on ice. The

plate was washed in the plate washer 3 times as described above. The standard for

the cytokine, TNF-α, and the supernatants were diluted 1:10 – 1:20 in the assay

diluent. The supernatants and the standard (100μl/well) were pipetted into the

appropriate wells in the plates were sealed and incubated at RT for 2h. The plates

were then washed 5 times. The detection antibody (1:250) was diluted in the assay

diluent and then 100μl diluted detection antibody was added to each well. The plate

was once again sealed and incubated at RT for 1h. This was followed by another 5

washes in the plate washer. Next, the enzyme reagent (Sav-HRP) 1:250 was diluted

in assay diluent and 100μl was added to each well. The plate was sealed and

incubated for 30 mins at RT. The plate was then washed 7 times, which was

followed by adding 100μl/well of tetramethylbenzidine (TMB) Substrate Solution

(BD Bioscience) and incubation at RT for 30 mins in the dark. This was followed by

the addition of 50μl of stop solution (1M H2SO4) to each well. The plate was then

analysed in a Sunrise microplate reader (Tecan, Zurich, Switzerland) at an

absorbance of 450nm.

3.9 Mesenteric lymph nodes (MLN) T Regulatory Cells counting using Flow

Cytometry

Mesenteric lymph nodes were crushed, using sterile glass slides, after

dissection and filtered to make a single cell suspension (as described in section 3.7).

Lymph node cells (106 cells) were then washed in ice-cold RPMI complete medium

40

and then centrifuged at 400g for 5 mins (at 4°C). Cell pellets were resuspended in

flow cytometry staining buffer (0.05% BSA, 0.09% NaN3 in PBS), vortexed and

centrifuged again. All antibodies were purchased from eBioscience. Supernatant was

aspirated, and the cells were then co-stained for the T regulatory cell surface

molecules CD4 and CD25 (0.125µg/106 cells of CD4-FITC and 0.06µg/test of

CD25-PE antibodies in 100µl flow staining buffer). Samples were incubated at 4°C

for 30 min in the dark, and then washed with cold PBS (1ml). Cells were centrifuged

at 400g for 5 min, and cell pellets were resuspended in 1ml

Fixation/Permeabilization solution (eBioscience) and vortexed. The samples were

incubated at 4°C for 18h in the dark. The cells were then washed twice with

permeabilization buffer (1ml, eBioscience), centrifuged, and supernatant aspirated.

The samples were then blocked with 1-2µg/test Fc Block (anti-mouse CD16/32

clone, eBioscience) in permeabilization buffer (100µl) at 4°C for 15 min to minimise

non-specific binding of the Foxp3 antibody to macrophage and lymphocyte Fc

receptor. After the blocking step, 0.5µg/test anti-mouse/rat Foxp3 (clone FJK-16s)

antibody or isotype control in permeabilization buffer was added to the samples and

incubated for a minimum of 30 mins in dark at 4°C. Control samples included auto-

fluorescent controls (staining buffer only), CD4-FITC single stained control, CD25-

PE single stain control, Foxp3-APC single stain control and APC isotype control.

The cells were then washed in permeabilization buffer (1ml), centrifuged and

supernatant was aspirated. Cells were resuspended in cold PBS (400µl) and analysed

on a LSRII flow cytometer (BD). Gates were drawn to include the lymphocyte

population and exclude dead cells, erythrocytes and debris and were based on

forward and side scatter profiles. Gating strategy and compensation data, including

PMT voltages (included in Appendix 1.3). Data were analysed using FlowJo

software (Tree Star, Harvard).

3.10 Cell Culture

The HT29 colon cancer cell line obtained from ATCC was cultured in

complete RPMI 1640 medium (Invitrogen, Carlsbad, CA) containing 10% heat-

inactivated foetal calf serum (FCS), 50 units/ml penicillin G and 50µl/ml

streptomycin sulphate and 5mmol/ml L-glutamine (Invitrogen). The cells were

cultured in 80 cm2 cell culture flasks (Nunc) at 37°C in a Sony incubator with 5%

41

CO2. The cells were tested routinely by the Scientific Support Staff at the MMRI and

were found to be free from Mycoplasma.

3.11 Wound assay

HT29 cells (1x106cells/well) were plated into 6 well plates (Nunc) and

cultured until they reached 100% confluence. Once confluent the cells were then

washed twice with 37ºC PBS and then serum starved overnight in 0.1% FCS in

RPMI. Prior to creating the wounds, the cells were treated with Mitomycin C

(10µg/ml, Sigma, St Louis, MO) for two hours to inhibit cell proliferation. This

concentration of Mitomycin C was determined empirically and does not induce

apoptosis in colon cells. A sterile p200 pipette tip was used to create a “wound” (3

scratches per well). The cells were washed again once with warm PBS (1ml/well) to

remove cell debris and to smooth the edge of the scratch and then replaced with 2ml

of media with different concentrations (0-10nM) of ghrelin (Auspep) and EGF

(100ng/ml, Sigma) as a positive control. The cells were photographed (Canon EOS

40D, Australia) under phase-contrast microscopy (Olympus CKX41, USA), at 0, 12,

24, and 48h time points. Wound closure was determined quantitatively by using

ImageJ (National Institutes of Health). The percentage of wound closure was

calculated as (initial area-final area)/initial x 100. Experiments were performed once,

with two wells per treatment, where each well had three scratches.

3.12 Statistical Data analysis

All data were expressed as mean + S.D. Data were analysed using GraphPad

Prism (v5.01), statistics analysis software program (San Diego, CA). Parametric tests

such as One-way ANOVA followed with Tukey’s post hoc comparison was used to

determine statistically significant differences in the mean values between the groups.

Where necessary, Two-way ANOVA was used to analyse two independent factors.

Non-parametric analysis using Mann-Whitney U-test was used when comparing two

groups. The statistical test used and the sample sizes for each analyses are provided

within the figure legend. Probability values p<0.05 were considered statistically

significant.

42

3.13 Presentation of Results

The data presented in the results section examines two different variables (mouse

strains and DSS molecular weight) as well the efficacy of ghrelin in colitis.

Therefore, the results are presented in a comparative format. The total clinical scores

and body weight change are shown over a period of time to indicate the start of

colitis and the effect of ghrelin. These data were further split into 5kDa DSS +/-

ghrelin peptides treated mice vs. 40kDa DSS +/- ghrelin peptide treated mice.

However, the rest of the data presented evaluates the efficacy of the ghrelin peptides

in comparison to PBS in the different molecular weights of DSS treated mice in each

mouse strain.

43

Chapter Four: Results 4.1 Effect of ghrelin peptide treatment on clinical colitis scores in C57BL/6 and

BALB/c mice administered with dextran sodium sulphate (DSS)

Eight weeks old female BALB/c and C57BL/6 mice were induced with colitis by

treating them with 5% and 2% DSS, respectively over 8 days. The DSS treatment

was conducted with two molecular weights- 5kDa and 40kDa which differentially

affect the proximal and distal large intestine. The mice were injected with ghrelin

peptides on day 4 and day 6 via i.p. injections. A pilot study was conducted with the

administration of ghrelin peptides prior to day 4, however no significant benefit was

observed over the two day injection protocol. The total clinical scores were

evaluated daily on clinical symptoms such as diarrhoea, rectal bleeding, shivering,

hunching and ruffling (described in more detail in the score sheet, Appendix 1.1).

Changes in bodyweight change were not included in the clinical scores because

ghrelin is a known appetite stimulant. In BALB/c mice clinical scores were higher

when treated with the larger molecular weight of DSS (40kDa) (Figure 1B) than the

5kDa DSS group (Mann-Whitney U-test, p=0.0001; Figure 1A) on day 7. In

contrast, in the C57BL/6 mice total clinical scores were higher when treated with the

5kDa DSS (Figure 2A) as compared to the 40kDa DSS (Figure 2B), but this did not

reach statistical significance (Mann-Whitney U-test, p=0.309). Treatment with

ghrelin or ∆4 peptide did not suppress clinical colitis in BALB/c mice whether they

were given 5kDa or 40kDa DSS (Figures 1A & B). However, C57BL/6 mice

administered 5kDa DSS (Figure 2A) did display significantly less clinical signs of

colitis when they were treated with ghrelin (p= 0.012) by day 8. There were no

clinical scores recorded for the BALB/c mice on day 8 due to an oversight.

Therefore, a comparison between the two strains of mice on that day could not be

measured.

4.2 Treatment with ghrelin peptides does not affect bodyweight change in mice

with DSS-induced colitis

There was no significant effect on body weight in mice with DSS-induced colitis

when treated with ghrelin peptides. A decrease in body weight was observed from

day 5, in both the BALB/c (Figure 3) and C57BL/6 mice DSS-treated mice (Figure

44

4). However, the main difference observed was that each strain of mice responded

differently towards the different molecular weights of DSS. There was a significant

difference in bodyweight in the BALB/c mice between the 5kDa DSS + PBS group

(Figure 3A) when compared to the 40kDa DSS + PBS group (Figure 3B). There was

a significant drop in bodyweight in from day 6, p=0.002 in the 40kDa DSS + PBS

group mice in comparison to the naive mice, and also on day 7, p<0.0001 and day 8,

p<0.0001, using Mann-Whitney U-test. In the BALB/c mice when the 5kDa DSS +

PBS group was compared to the 5kDa DSS + ghrelin group on days 6, p=0.77; day

7, p=0.71 and day 8, p=0.98, there was no significant difference observed. No

significance was observed with the Δ4 peptide treatment in the BALB/c mice, on day

6, p=0.51, day 7, p=0.97 and day 8, p=0.79, using a Mann-Whitney U-test. In

contrast, the reverse was found in the C57BL/6 group of mice where the 5kDa DSS

+ PBS group (Figure 4A) progressively lost weight from day 6 when compared to

the 40kDa DSS + PBS group (Figure 4B). The BALB/c mice did not lose weight

when treated with the smaller molecular weight of DSS (5kDa) whereas when

treated with 40kDa DSS there was a significant drop in weight. Conversely, the

C57BL/6 mice were resistant to the 40kDa DSS treatment with no significant weight

change. However, in comparison to the BALB/c, we did notice a significant decrease

of initial body weight loss in the C57BL/6 5kDa DSS treated mice. The C57BL/6

5kDa DSS + PBS group lost significant weight from day 6, p=0.0002 and on day 7,

p=0.0001 and day 8, p=0.002, in comparison to the naive mice; Mann-Whitney U-

test. There was no significant difference with ghrelin treatment in the 5kDa DSS

group, on day 6, p=0.69, day 7, p=0.31 or day 8, p=1.00, using a Mann-Whitney U

test. Similarly, there was no significant effect of ghrelin and its peptide, Δ4, on body

weight in C57Bl/6 mice treated with DSS (5kDa or 40kDa).

4.3 Effect of ghrelin peptides on colon shortening in C57BL/6 and BALB/c mice

given DSS

Colon lengths were measured in all the groups to examine whether ghrelin peptides

were able to rescue colitis. Mice treated with DSS had shrunken and swollen colons

as compared to the naive/healthy group of mice due to apoptosis (Maki et al., 2005).

The colon lengths in the 40kDa DSS groups of BALB/c mice (Figure 5A) were

much shorter in length as compared to the 5kDa DSS group, Mann-Whitney U-test:

p<0.0005. This observation is in agreement with the greater loss of body weight, in

45

the 40kDa DSS. In C57BL/6 mice (Figure 5B) there was no significant difference in

colon lengths between the two molecular weights of DSS, in contrast to the BALB/c

mice. Colitis was induced because the colons of the 5kDa DSS + PBS group were

significantly shorter in length when compared to the naive C57BL/6 mice, p=0.0001,

Mann-Whitney U-test. There was also a significant difference in colon length

between the naive C57BL/6 mice and the 40kDa DSS + PBS group, p=0.002, using a

Mann-Whitney U-test. However, a Two-Way ANOVA (Figure 6A and B) test did

not show any significant difference between the colon lengths of the ghrelin peptides

treated groups when compared to the DSS + PBS groups in either strain of mice in

both the 5 and 40kDa DSS groups.

46

Naives5kDa DSS + PBS5kDa DSS + 1nmol Ghrelin5kDa DSS + 1nmol ∆4 peptide40kDa DSS + PBS40kDa DSS + 1nmol Ghrelin40kDa DSS + 1nmol ∆4 peptide

Figure 1. Clinical evidence of colitis in BALB/c mice treated with DSS and ghrelin peptides. BALB/c mice were assessed on the severity of colitis by calculating total clinical scores daily with evaluation of stool consistency, faecal bleeding and diarrhoea. Clinical symptoms such as shivering, hunching and ruffling were also evaluated daily using clinical score indices as described in complete detail in the score sheet (Appendix 1.1). Black arrows indicate administration of ghrelin peptides or PBS on Days 4 and 6. Clinical scores of BALB/c mice treated with 40kDa DSS (B) were higher when compared to 5kDa DSS (A) on day 7, (Mann-Whitney U-test, p=0.0001). (N=11-12 mice/group, Mean ± S.D.) The data is representative of 2 individual studies.

Total Clinical scores BALB/c

0 1 2 3 4 5 6 70

1

2

3

4

5

6

7

8

Days

Tota

l Clin

ical

Sco

res

B

Total Clinical scores BALB/c

0 1 2 3 4 5 6 70

1

2

3

4

5

6

7

8

Days

Tota

l Clin

ical

Sco

res

A

47

Naives5kDa DSS + PBS5kDa DSS + 1nmol Ghrelin5kDa DSS + 1nmol ∆4 peptide40kDa DSS + PBS40kDa DSS + 1nmol Ghrelin40kDa DSS + 1nmol ∆4 peptide

Figure 2. Clinical evidence of colitis in C57BL/6 mice treated with DSS and ghrelin peptides. C57BL/6 mice were assessed on severity of colitis by calculating total clinical scores daily with evaluation of stool consistency, faecal bleeding and diarrhoea. Clinical symptoms such as shivering, hunching and ruffling were also evaluated daily using clinical score indices as described in complete detail in the score sheet (Appendix 1.1). Black arrows indicate administration of ghrelin peptides or PBS on Days 4 and 6. There was a significant improvement in colitis in mice treated with 5kDa DSS with ghrelin by day 8 (A). Mann-Whitney, U-test, *p=0.012. However, there was no such improvement in the 40kDa DSS group (B). (N=11-12 mice/group, Mean ± S.D). The data is representative of 2 individual studies.

Total Clinical scores C57BL/6

0 1 2 3 4 5 6 7 80

1

2

3

4

5

6

7

8

Days

Tota

l Clin

ical

Sco

res *p=0.012

A

Total Clinical scores C57BL/6

0 1 2 3 4 5 6 7 80

1

2

3

4

5

6

7

8

Days

Tota

l Clin

ical

Sco

res

B

48

Naives5kDa DSS + PBS5kDa DSS + 1nmol Ghrelin5kDa DSS + 1nmol ∆4 peptide40kDa DSS + PBS40kDa DSS + 1nmol Ghrelin40kDa DSS + 1nmol ∆4 peptide

Figure 3. Changes in body weight in BALB/c mice treated with DSS and ghrelin peptides. Body weight of mice was expressed as percentage of initial weight. BALB/c mice were given 5% (5kDa DSS/40kDaDSS) with or without the presence of ghrelin peptides. Black arrows indicate administration of ghrelin peptides or PBS on Days 4 and 6. There was a significant difference in body weight in the BALB/c group, ***p=0.0009, between the 5kDa DSS + PBS (A) and 40kDa DSS + PBS (B) using Mann-Whitney, U-test on day 6, day 7, ***p<0.0001, and day 8, ***p<0.0001. Colitis was induced from day 6, **p=0.002, to day 7, ***p<0.0001, and on day 8, ***p<0.0001 in BALB/c mice treated with 40kDa DSS using Mann-Whitney, U-test, when compared to the Naïve mice. (N=11-12 mice/group, Mean ± SD).

Body weight change BALB/c

0 1 2 3 4 5 6 7 890

100

110

120

Days

Perc

enta

ge o

f ini

tial w

eigh

t cha

nge

A

Body weight change BALB/c

0 1 2 3 4 5 6 7 870

80

90

100

110

120

Days

Perc

enta

ge o

f ini

tial w

eigh

t cha

nge

B

*** p<0.0001

** p=0.002

49

A

Figure 4. Changes in body weight in C57BL/6 mice treated with DSS and ghrelin peptides. Body weight of mice was expressed as percentage of initial weight. C57BL/6 mice were given 2% DSS (5kDa DSS / 40kDa DSS) with or without the presence of ghrelin peptides. Black arrows indicate administration of ghrelin peptides or PBS on days 4 and 6. There was a significant difference in body weight in the C57Bl/6 group, **p=0.0046, between the 5kDa DSS + PBS (A) and 40kDa DSS + PBS(B) using Mann-Whitney, U-test on day 6, ***p=0.0003, day 7, ***p<0.0001, day 8, ***p<0.0001. Colitis was induced with 5kDa DSS from day 6, p=0.0002; day 7, p=0.0001 and till day 8, p=0.002, Mann-Whitney, U-test when compared to the naïve mice. (N=11-12 per group, Mean ± SD).

Naives5kDa DSS + PBS5kDa DSS + 1nmol Ghrelin5kDa DSS + 1nmol ∆4 peptide40kDa DSS + PBS40kDa DSS + 1nmol Ghrelin40kDa DSS + 1nmol ∆4 peptide

Body weight change C57BL/6

0 1 2 3 4 5 6 7 870

80

90

100

110

120

Days

Perc

enta

ge o

f ini

tial w

eigh

t cha

nge

A

Body weight change C57BL/6

0 1 2 3 4 5 6 7 870

80

90

100

110

120

Days

Perc

enta

ge o

f ini

tial w

eigh

t cha

nge

B

50

Colon length C57BL/6

Naives

5kDa D

SS + PBS

5kDa D

SS + 1nmol G

hrelin

4 pep

tide

∆

5kDa D

SS + 1nmol

40kD

a DSS + PBS

40kD

a + 1n

mol Ghrel

in

4 pep

tide

∆

40kD

a DSS + 1n

mol 1n

mol Ghrel

in

4 pep

tide

∆

1nmol

0102030405060708090

100110120

Treatment Groups

Colo

n le

ngth

(mm

)

***p=0.0001 **p=0.002

B

Figure 5. Colon lengths of mice treated with DSS for 8 days. There was a significant difference (Mann-Whitney U-test: ***p<0.0005) in colon lengths between the 5kDa DSS + PBS group and the 40kda DSS + PBS group in the BALB/c mice (A). In the C57BL/6 group (B) there was no significant difference in the DSS groups. However, the naïve C57BL/6 mice had a significant difference in colon length when compared to the 5kDa DSS + PBS mice, ***p=0.0001 and with the 40kDa DSS + PBS mice, **p=0.002 using a Mann-Whitney, U-test. (N=11-12 mice/group, Mean ± S.D).

Colon length BALB/c

Naives

5kDa D

SS + PBS

5kDa D

SS + 1nmol G

hrelin

4 pep

tide

∆

5kDa D

SS + 1nmol

40kD

a DSS + PBS

40kD

a DSS+ 1n

mol Ghrel

in

4 pep

tide

∆

40kD

a DSS + 1n

mol 1n

mol Ghrel

in

4 pep

tide

∆

1nmol

0102030405060708090

100110120

Treatment Groups

Col

on le

ngth

(mm

)

A

***p<0.0005

51

Figure 6. Two-Way ANOVA comparison of colon lengths of BALB/c and C57BL/6 mice treated with ghrelin peptides with 5kDa DSS. There was a significant difference between the strain of mice (Two-way ANOVA, ***p<0.0005) in colon lengths between the 5kDa DSS + PBS group in the BALB/c mice and C57BL/6 group. However, there was no significant difference in the DSS + PBS group compared to the DSS + Ghrelin group (A) nor the DSS + Δ4 peptide (B) in either strain of mice (N=11-12 mice/ group, Mean ± S.D).

Colon length

BALB/c

C57BL/6

0

20

40

60

80

100

1205kDa DSS + PBS5kDa DSS + 1nmol Ghrelin

Col

on le

ngth

(m

m)

A

***

Colon length

BALB/c

C57BL/6

0

20

40

60

80

100

1205kDa DSS + PBS5kDa DSS + 1nmol ∆4 peptide

Col

on le

ngth

(m

m)

B

***

52

4.4 Blood analyses of BALB/c and C57BL/6 mice

The mice were bled before they were culled on Day 8. Around 50µl of blood was

collected from each mouse; however due to severity of colitis this was not always

possible. When mice were dehydrated, the method of blood collection was cardiac-

puncture. The blood was then run through a Sysmex SF3000 Haematology Analyser

(Sysmex Corp, Kobe, Japan). Figures 7 and 8, show a summary of the blood analysis on

BALB/c and C57BL/6 mice. There was no significant difference in the haematocrit

(HCT) count between the different DSS-treated groups in the BALB/c strain (Figure

7A). The HCT of the 40kDa DSS + PBS, mean 48%, were lower when compared to the

40kDa DSS + Ghrelin group, 55% but this was not found to be statistically significant

(p=0.5 Mann-Whitney, U-test). When comparing between the strains of mice it was

found that the C57BL/6 mice HCT (Figure 8A) were much lower following DSS when

compared to the BALB/c strain of mice. In contrast, the leukocyte levels in the BALB/c

mice treated with 40kDa DSS (Figure 7B) were relatively higher than the C57BL/6

group (Figure 8B). The 40kDa DSS treated BALB/c mice had a slightly increased

leukocyte count when compared to the 5kDa DSS treated group. There was no

significant difference observed between the various DSS groups of the C57BL/6 mice

treated with or without the ghrelin peptides. Similar results observed in the leukocyte

count were found with the lymphocyte count in the BALB/c (Figure 7C) and C57BL/6

mice (Figure 8C). The levels of leukocyte and lymphocytes in the 40kDa DSS group in

the BALB/c mice were elevated but this may have been due to technical error. In some

cases, when the mice were too severely dehydrated, it was difficult to obtain enough

blood, therefore the collected blood samples were then diluted in PBS to obtain enough

sample to run through the Sysmex machine. This reason may have accounted for the

elevated levels of the leukocyte and lymphocyte count.

53

Figure 7. Haematological parameters in BALB/c mice treated with DSS and ghrelin peptides for 8 days. Mice were bled on day 8 (final day of experiment) and 50µl of blood samples were collected for measuring haematocrit (A), leukocyte (B) and lymphocyte (C) counts using the Sysmex SF3000 Haematology Analyser. There was no significant difference observed between the DSS and ghrelin treated groups or in comparison to the naive mice. The levels of leukocyte and lymphocyte in the 40kDa DSS group were elevated but this may have been due to technical error. (N= 11-12 mice/group, Mean ± S.D).

A Haematocrit BALB/c

Naives

5kDa D

SS + PBS

5kDa D

SS + 1n

mol Ghr

elin

4 pep

tide

∆

5kDa D

SS + 1n

mol

40kD

a DSS +

PBS

40kD

a DSS +

1nmol G

hrelin

4 pep

tide

∆

40kD

a DSS +

1nmol 1n

mol Ghrel

in

4 pep

tide

∆

1nmol

0

10

20

30

40

50

60

70

80

Treatment Groups

Hae

mat

ocri

t %

Leukocyte BALB/c

Naives

5kDa D

SS + PBS

5kDa D

SS + 1n

mol Ghr

elin

4 pep

tide

∆

5kDa D

SS + 1n

mol

40kD

a DSS +

PBS

40kD

a DSS +

1nmol

Ghreli

n

4 pep

tide

∆

40kD

a DSS +

1nmol

d1n

mol Ghr

elin

4 pep

tide

∆

1nmol

0

10

20

30

40

50

60

70

80

Treatment Groups

Leuk

ocyt

e (x

10^9

/L)

B

Lymphocyte BALB/c

Naives

5kDa D

SS + PBS

5kDa D

SS + 1n

mol Ghrel

in

4 pep

tide

∆

5kDa D

SS + 1n

mol

40kD

a DSS +

PBS

40kD

a DSS +

1nmol G

hrelin

4 pep

tide

∆

40kD

a DSS +

1nmol

1nmol G

hrelin

4 pep

tide

∆

1nmol

0

10

20

30

40

50

60

Treatment Groups

Lym

phoc

yte

(x10

^9/L

)

C

54

Figure 8. Haematological parameters in C57BL/6 mice treated with DSS and ghrelin peptides for 8 days. Mice were bled on day 8 (final day of experiment) and 50µl of blood samples were collected for measuring haematocrit (A), leukocyte (B) and lymphocyte (C) counts using the Sysmex SF3000 Haematology Analyser. There was no significant difference observed between the DSS and ghrelin treated groups or in comparison to the naive mice. (N= 11-12 mice/group, Mean ± S.D).

Haematocrit C57BL/6

Naives

5kDa D

SS + PBS

5kDa D

SS + 1n

mol Gheli

n

4 pep

tide

∆

5kDa D

SS + 1n

mol

40kD

a DSS +

PBS

40kD

a + 1n

mol Ghrel

in

4 pep

tide

∆

40kD

a DSS +

1nmol

1nmol G

hrelin

4 pep

tide

∆

1nmol

0

10

20

30

40

50

60

70

80

Treatment Groups

Hae

mat

ocrit

%A Leukocyte C57BL/6

Naives

5kDa D

SS + PBS

5kDa D

SS + 1n

mol Ghrel

in

4 pep

tide

∆

5kDa D

SS + 1n

mol

40kD

a DSS +

PBS

40kD

a + 1n

mol Ghrel

in

4 pep

tide

∆

40kD

a DSS +

1nmol

1nmol G

hrelin

4 pep

tide

∆

1nmol

0

10

20

30

40

50

60

70

80

Treatment GroupsLe

ukoc

yte

(x10

^9/L

)

B

Lymphocyte C57BL/6

Naives

5kDa D

SS + PBS

5kDa D

SS + 1nmol G

hrelin

4 pep

tide

∆

5kDa D

SS + 1nmol

40kD

a DSS + PBS

40kD

a + 1n

mol Ghrel

in

4 pep

tide

∆

40kD

a DSS + 1n

mol 1n

mol Ghrel

in

4 pep

tide

∆

1nmol

0

10

20

30

40

50

60

Treatment Groups

Lym

phoc

yte

(x10

^9/L

)

C

55

4.5 Treatment with ghrelin peptides suppressed histolopathological colitis in C57BL/6

mice

Figures 9 and 10 show the summary of total histology scores, which were scored by two

blinded individuals. The colons were fixed in formalin in a “swiss roll” manner, which

were then sent for paraffin embedding followed by sectioning and finally H&E stained

(Figure 11). The scores were based on inflammation severity, epithelial damage (i.e.

crypt loss, goblet cell loss, crypt abscesses, and ulceration), infiltration extent; whether

there was cell infiltration around the base of the crypt, or more in the muscularis

mucosa resulting in oedema, and finally on a overall percentage involving crypt

abscesses, loss of crypt and ulcers (more details of scoring system in Appendix 1.2).

Figures 9 and 10 show a comparison in the colon sections between all of the groups in

the BALB/c and C57BL/6 mice. The colon sections observed for scoring were:

proximal colon, mid colon, and distal colon. The sections were observed and

photographed at various magnifications.

Ghrelin treatment in the 5kDa DSS induced colitis group in the C57BL/6 mice

was more effective as compared to the 40kDa DSS. Ghrelin was able to suppress

inflammation to some extent in the proximal colon of the C57BL/6 mice (Figure 10A)

as compared to BALB/c mice in Figure 9A. There was less goblet cell loss, fewer crypt

abscesses and less cell infiltration in the proximal colon when the C57BL/6 mice were

treated with ghrelin (Figure 12D-F). The crypt architecture was intact with no

formations of ulcers in the distal colon when compared to the PBS treated group (Figure

12A-C). Two-way ANOVA analysis showed that the total histological scores of the

5kDa DSS treated group in the proximal colon were decreased when treated with

ghrelin. A statistical significance (p=0.03) was found between the 5kDa DSS + PBS

group and the 5kDa DSS + Ghrelin group in the C57BL/6 mice (Figure 14A). There

was also a significant difference (p<0.0001) in the total histology scores between the

two strains of mice, when treated with the same molecular weight of DSS (5kDa). This

is consistent with the previous results where treatment with 5kDa DSS was more severe

in the C57BL/6 mice as compared to the BALB/c strain of mice. Interestingly, there

also seemed to be an effect with the Δ4 peptide which caused a slight decrease in the

histology scores in the proximal colon (Figure 14B) in the C57BL/6 mice. Less goblet

cell loss was observed however there was some infiltration present in the mid and distal

colon (Figure 12H and I). Even though, this result was not statistically significant

(p=0.06) it indicates that there was a slight difference in the total histology score

56

between the 5kDa DSS + PBS and the 5kDa DSS + Δ4 peptide groups in the C57BL/6

mice.

There was not any significant difference between the PBS and ghrelin peptides

treated groups in the mid colon both in the BALB/c (Figure 9B) and the C57BL/6

(Figure 10B) strain of mice. However, there was a very slight improvement with ghrelin

treatment in the 5kDa DSS group in the C57BL/6 mice in the total histology score. In

Figure 12E, presence of goblet cells can be seen when compared to the mid colon in the

5kDa DSS + PBS group (Figure 12B). Figure 10A, and B indicate that the C57BL/6

group of mice had a higher total histology score when compared to the respective

treatment groups in the BALB/c mice.

Figure 9C and 10C shows the summary of histological scores in the distal colon.

Interestingly, there was a slight improvement discovered in the C57BL/6 mice (Figure

10C) with the treatment of ghrelin in the 5kDa DSS treated group. However, a Two-

way ANOVA indicate that these trends were not significant p=0.28 (Figure 15A). In

Figure 12F, the crypt architecture and goblet cells are present; however some

infiltration can also be noticed. There was less histological damage in the C57BL/6

mice treated with 40kDa DSS when compared to the 5kDa DSS. There was less damage

in the proximal colon with mice treated with 40kDa DSS + PBS (Figure 13A). Ghrelin

treatment did not have any effect in the proximal and mid colon in the C57BL/6 mice

treated with 40kDa DSS. There was small improvement noticed in the distal colon of

the mice treated with ghrelin (Figure 13F), as there were less crypt abscesses and less

crypt loss seen in comparison with mice treated with PBS (Figure 13C). In contrast, the

Δ4 peptide did not have any effect in the C57BL/6 mice in the 40kDa DSS group

(Figure 13G-I).

Unlike its effect in C57BL/6 mice, ghrelin peptides were unable to repress

colitis in the 5 and 40kDa DSS treated mice in the BALB/c group. It was also observed

that the naive mice in the BALB/c group were unexpectedly exhibiting mild colitis in

the proximal, mid and distal colons (Figures 9). Furthermore, some mice treated with

ghrelin and Δ4 peptide only, without DSS also exhibited mild inflammation. However,

it was interesting to observe that BALB/c mice treated with Δ4 peptide only, showed no

sign of inflammation in the distal colon when compared to the naive mice. Regardless

of the naive mice scoring, there was no difference in histological colitis scores between

the 5kDa DSS and the 40kDa DSS. Even though, the 40kDa DSS group seemed to have

worse colitis as compared to the 5kDa DSS group in terms of body weight loss and

57

clinical scores, no such difference was observed at the histology level. Since the

BALB/c mice unexpectedly showed low grade inflammation in the naive group, this

potentially skewed the results for the other treatment groups making clear

interpretations difficult.

58

Figure 9. Histological colitis scores in BALB/c mice treated with DSS and ghrelin peptides. There was no significant difference between the naive and 5kDa DSS or 40kDa DSS group, or with treatment of ghrelin and Δ4 peptide in the proximal colon (A). In the Mid colon (B) there was no significant difference between the 5kDa DSS + PBS group and the ghrelin peptide treated groups. There was also no significant improvement in colitis with ghrelin and Δ4 peptide treatment in the distal colon (C). Histological colitis severity was evaluated based on microscopic features including crypt length, ulceration, goblet cell loss and inflammatory cell infiltration (full details in the scoring sheet in Appendix 1.2). (N= 11-12 mice/group, Mean ± S.D).

Proximal colon BALB/c

Naives

5kDa D

SS + PBS

5kDa D

SS + 1n

mol Ghrel

in

4 pep

tide

∆

5kDa D

SS + 1n

mol

40kD

a DSS +P

BS

40kD

a DSS +

1nmol G

hrelin

4 pep

tide

∆

40kD

a DSS +

1nmol

1nmol G

hrelin

4 pep

tide

∆

1nmol

0

5

10

15

20

Treatment Groups

His

tolo

gy s

core

A Mid colon BALB/c

Naives

5kDa D

SS + PBS

5kDa D

SS + 1n

mol Ghrel

in

4 pep

tide

∆

5kDa D

SS + 1n

mol

40kD

a DSS +P

BS

40kD

a DSS +

1nmol G

hrelin

4 pep

tide

∆

40kD

a DSS +

1nmol

1nmol G

hrelin

4 pep

tide

∆

1nmol

0

5

10

15

20

Treatment GroupsH

isto

logy

sco

re

B

Distal colon BALB/c

Naives

5kDa D

SS + PBS

5kDa D

SS + 1nmol G

hrelin

4 pep

tide

∆

5kDa D

SS + 1nmol

40kD

a DSS +PBS

40kD

a DSS + 1n

mol Ghrel

in

4 pep

tide

∆

40kD

a DSS + 1n

mol 1n

mol Ghrel

in

4 pep

tide

∆

1nmol

0

5

10

15

20

Treatment Groups

Hist

olog

y sc

ore

C

59

Mid colon C57BL/6

Naives

5kDa D

SS + PBS

5kDa D

SS + 1nmol G

hrelin

4 pep

tide

∆

5kDa D

SS + 1nmol

40kD

a DSS + PBS

40kD

a DSS + 1n

mol Ghrel

in

4 pep

tide

∆

40kD

a DSS + 1n

mol 1n

mol Ghrel

in

4 pep

tide

∆

1nmol

0

5

10

15

20

Treatment GroupsH

isto

logy

sco

re

B

Distal colon C57BL/6

Naives

5kDa D

SS + PBS

5kDa D

SS + 1nmol G

hrelin

4 pep

tide

∆

5kDa D

SS + 1nmol

40kD

a DSS + PBS

40kD

a DSS + 1n

mol Ghrel

in

4 pep

tide

∆

40kD

a DSS + 1n

mol 1n

mol Ghrel

in

4 pep

tide

∆

1nmol

0

5

10

15

20

Treatment Groups

Hist

olog

y sc

ore

C

Figure 10. Histological colitis scores in C57BL/6 mice treated with DSS and ghrelin peptides. There was a statistical difference in the proximal colon (A) of mice treated with 5kDa DSS + PBS in comparison to 5kDa DSS + Ghrelin, *p=0.03, using a Two-Way ANOVA. There was a slight improvement in the proximal colon with 5kDa DSS + Δ4 peptide, however this was not significant, p=0.06, using a Two-Way ANOVA. There was no difference observed in the mid colon (B) within the treatment groups. There again was a slight improvement observed in the 5kDa DSS + Ghrelin group when compared to the 5kDa DSS + PBS group in the distal colon (C), p=0.28, but this was not significant. No significant difference was noticed in the 40kDa DSS group. Histological colitis severity was evaluated based on microscopic features including crypt length, ulceration, goblet cell loss and inflammatory cell infiltration (full details in the scoring sheet in Appendix 1.2). (N= 11-12 mice/group, Mean ± S.D).

A Proximal colon C57BL/6

Naives

5kDa D

SS + PBS

5kDa D

SS + 1n

mol Ghrel

in

4 pep

tide

∆

5kDa D

SS + 1n

mol

40kD

a DSS +

PBS

40kD

a DSS +

1nmol G

hrelin

4 pep

tide

∆

40kD

a DSS +

1nmol

1nmol G

hrelin

4 pep

tide

∆

1nmol

0

5

10

15

20

Treatment Groups

His

tolo

gy s

core

*p=0.03

60

Figure 11. Representative H&E sections of colons dissected from 6 week old Naive C57BL/6 mice. Histology pictures (A) show an overall “swiss roll” formation of the colon (X40 magnification). Histological severity was assessed as per the scoring system attached in the Appendix. The circles in histology pictures represent goblet cells in the (B) proximal region of the colon, (C) mid colon and (D) distal colon (all X200 magnification). Representative digital photographs were taken using an Olympus BX50 microscope. The arrows from picture A (X40 magnification) indicate which section of the colon was observed for histology in X200 magnification.

A

B C

D

61

Figure 12. Representative histology images of C57BL/6 mice treated with 5kDa DSS. Histological severity was assessed as per the scoring system attached in the Appendix 1.2. In mice treated with 5kDa DSS + PBS (A-C) goblet cell loss (black circle), loss of crypt architecture, crypt abscesses (black arrow), infiltration (red arrow), and ulceration were observed. In comparison, mice treated with 1nmol ghrelin (D-F) and 1nmol delta 4 peptide (G-I) showed less goblet cell loss and infiltration.

A B C

D E F

G H I

Prox 5kDa DSS + PBS Mid 5kDa DSS + PBS Distal 5kDa DSS + PBS

Prox 5kDa DSS + 1nmol Ghrelin Mid 5kDa DSS + 1nmol Ghrelin Distal 5kDa DSS + 1nmol Ghrelin

Prox 5kDa DSS + 1nmol Δ4 peptide

Mid 5kDa DSS + 1nmol Δ4 peptide

Distal 5kDa DSS + 1nmol Δ peptide

62

Figure 13. Representative histology images of C57BL/6 mice treated with 40kDa DSS. Histological severity was assessed as per the scoring system attached in the Appendix 1.2. In mice treated with 40kDa DSS + PBS (A-C) goblet cell loss, loss of crypt architecture, crypt abscesses and infiltration (red arrow), were starting to be observed. Mice treated with 1nmol ghrelin (D-F) and 1nmol delta 4 peptide (G-I) also showed goblet cell loss and infiltration.

Prox 40kDa DSS + PBS Mid 40kDa DSS + PBS Distal 40kDa DSS + PBS

Prox 40kDa DSS + 1nmol Ghrelin Mid 40kDa DSS + 1nmol

Ghrelin Distal 40kDa DSS + 1nmol

Ghrelin

Prox 40kDa DSS + 1nmol Δ4 peptide

Mid 40kDa DSS + 1nmol Δ4 peptide

Distal 40kDa DSS + 1nmol Δ4 peptide

A B C

D E F

G H I

63

Proximal colon

BALB/c

C57BL/6

0

5

10

15

205kDa DSS + PBS5kDa DSS + 1nmol Ghrelin

Tot

al H

isto

logy

sco

res *

***

A

Figure 14. Histological assessment of inflammation in the Proximal colon of BALB/c and C57BL/6 mice. There was a significant difference (A) between the 5kDa DSS + PBS vs. 5kDa DSS + Ghrelin (Two-way ANOVA, *p=0.03), where treatment with ghrelin reduced colitis. A significant difference between the severity of colitis was also found between the two strains of mice (***p<0.0001). Treatment with Δ4 peptide (B) was unable to reach statistical significance in suppressing colitis in the C57BL/6 mice (Two-way ANOVA, p=0.06). (N=11-12 mice/group, ± Mean S.D). The data is representative of 2 individual studies.

B Proximal colon

BALB/c

C57BL/6

0

5

10

15

205kDa DSS + PBS5kDa DSS + 1nmol ∆4 peptide

Tot

al H

isto

logy

sco

res

64

Figure 15. Histological assessment of inflammation in the Distal colon of BALB/c and C57BL/6 mice. There was no significant difference between the 5kDa DSS + PBS vs. 5kDa DSS + Ghrelin (A) and 5kDa DSS + PBS vs. 5kDa DSS + Δ4 peptide (B). There was a slight, but not significant, improvement in treatment with ghrelin and Δ4 peptide in the C57BL/6 mice. No difference was seen in the BALB/c mice treated with PBS compared to Ghrelin or the Δ4 peptide. Colitis severity was assessed on the scoring system of histology scores attached in the Appendix 1.2. (N=11-12 mice group, Mean ± S.D). The data is representative of 2 individual studies.

Distal colon

BALB/c

C57BL/6

0

5

10

15

205kDa DSS + PBS5kDa DSS + 1nmol Ghrelin

Tot

al H

isto

logy

sco

res

A

Distal colon

BALB/c

C57BL/6

0

5

10

15

205kDa DSS + PBS5kDa DSS + 1nmol ∆4 peptide

Tot

al H

isto

logy

sco

res

B

65

4.6 Measurement of TNF-α from mesenteric lymph node lymphocyte cultures

The levels of Th1 pro-inflammatory cytokine, TNF-α, in stimulated mesenteric lymph

node (MLN) cultures from mice treated with DSS and mice co-treated with ghrelin

peptides was measured using an ELISA. MLN leukocytes were treated with 50ng/ml

PMA and 750ng/ml ionomycin to stimulate immune cells for 48h. Even though there

was no effect of treatment of ghrelin in either strain of mice for both the 5kDa DSS or

the 40kDa DSS, the naïve mice in both the strains had unexpectedly high levels of

TNF-α in comparison to what had been established by our lab (TNF-α levels in cultures

from naive C57BL/6 mice: 250pg/ml; Heazlewood et al., 2008). Figure 16A

demonstrates that the naïve BALB/c mice had similar levels of TNF- α, when compared

to the other groups. Ghrelin did slightly decrease the levels of TNF- α in the 5kDa DSS

group in the BALB/c mice (Figure 16A), this result is not significant and a conclusion

cannot be made since there is not any difference in TNF-α levels between the naives

and the 5kDa DSS + Ghrelin. These elevated levels of TNF-α are in conjunction with

the histology results where the naïve BALB/c mice were scoring for low grade

inflammation. However, similar TNF-α levels were also seen in the C57BL/6 naïve

mice (Figure 16B). These mice were healthy and had no inflammation in the

histological assessment. Similarly to the BALB/c mice, there was no difference in the

TNF-α levels in the C57BL/6 mice following DSS treatment. C57BL/6 mice treated

with 40kDa DSS + Ghrelin did slightly decrease the levels of TNF-α when compared to

40kDa DSS + PBS, however, this did not reach significance (Mann-Whitney, p=0.25).

66

Figure 16. TNF-α concentration in mesenteric lymph node cultures from mice treated with DSS and ghrelin peptides. To determine the cytokine profile between each treatment group, mesenteric lymph nodes, adjacent to proximal colons were dissected from the mice, on day 8. The lymph nodes were then obtained in a single-cell suspension, plated, and then stimulated with 50ng/ml PMA and 750ng/ml ionomycin. The culture supernatants were collected after 48h time points. Cytokine levels of TNF-α in BALB/c (A) and C57BL/6 (B) mice were determined using an ELISA. There was so statistical significance observed between the different treatment groups and the naive mice (N=3-6 mice/group, Mean ± S.D).

BALB/c TNF-α levels 48h

Naives

5kDa D

SS + PBS

5kDa D

SS + 1nmol G

hrelin

40kD

a DSS + PBS

40kD

a DSS + 1n

mol Ghrel

in

4 pep

tide

∆

40kD

a DSS + 1n

mol

0

1000

2000

3000

4000

5000

Treatment Groups

pg/m

l

A

C57BL/6 TNF-α levels 48h

Naives

5kDa D

SS + PBS

5kDa D

SS + 1nmol G

hrelin

4 pep

tide

∆

5kDa D

SS + 1nmol

40kD

a DSS + PBS

40kD

a DSS + 1

nmol Ghrel

in

4 pep

tide

∆

40kD

a DSS + 1n

mol

0

1000

2000

3000

4000

5000

Treatment Groups

pg/m

L

B

67

4.7 Enumeration of CD4+CD25+Foxp3+ T regulatory cells in mesenteric lymph

nodes

Figures 17 and 18 demonstrate flow cytometric analysis of T regulatory (Treg) cells

from BALB/c and C57BL/6 mice respectively. The x-axis represents CD25 staining in

cells CD4+ gated against, y-axis staining for the Foxp3 transcription factor. Figure 19A

shows that there was not any significant difference in the Treg cells between the PBS

treated and the ghrelin treated group in both the 5kDa and 40kDa DSS in the BALB/c

mice. There was a slight increase in the 40kDa DSS + Δ 4 peptide as compared to the

40kDa DSS + PBS; however this did not reach significance, Mann-Whitney U-test,

p=0.06. There was a significant increase in Treg cells in C57BL/6 mice treated with

5kDa DSS + Ghrelin when compared to the naïve mice, Mann-Whitney, U-test

p=0.0009. There was also a slight increase in the percentage of Treg cells in the

C57BL/6 mice (Figure 19B) in the 5kDa DSS + Ghrelin group as compared to the 5kDa

DSS + PBS, however using a Mann-Whitney U-test failed to show significance, p=0.2.

There was no statistical difference observed between each treatment group in both the

strains of mice (Figure 19). Ghrelin did not seem to have a substantial effect in

significantly increasing the proportion of CD4+CD25+Foxp3+ cells in MLNs from

BALB/c and C57BL/6 mice.

68

5kDa DSS + PBS 5kDa DSS + 1nmol Ghrelin 5kDa DSS + 1nmol Δ4 peptide

40kDa DSS + PBS 40kDa DSS + 1nmol Ghrelin 40kDa DSS + 1nmol Δ4 peptide

CD25 – gated on CD4+

Foxp

3

Isotype control

Figure 17. Representative flow cytometry plots of T regulatory cell populations in mesenteric lymph nodes in BALB/c mice. Cells were stained with CD4-FTIC, CD25-PE, Foxp3-APC. The percentage of CD4+ CD25+Foxp3+ cells was determined using LSRII Flow Cytometer and shown within the gate on each graph. Data were analysed using FlowJo software (Tree Star, Harvard). N=11-12 mice/group.

69

CD25 – gated on CD4+

Naives 5kDa DSS + PBS 5kDa DSS + 1nmol Ghrelin

Figure 18. Representative flow cytometry plots of T regulatory cell populations in mesenteric lymph nodes in C57BL/6 mice. Cells were stained with CD4-FTIC, CD25-PE, Foxp3-APC. The percentage of CD4+ CD25+Foxp3+ cells was determined using LSRII Flow Cytometer and shown within the gate on each graph. Data were analysed using FlowJo software (Tree Star, Harvard). N=11-12 mice/group.

5kDa DSS + 1nmol Δ4 peptide 40kDa DSS + PBS 40kDa DSS + 1nmol Ghrelin

40kDa DSS + 1nmol Δ4 peptide

Foxp

3

70

Figure 19. Regulatory T cell numbers in mesenteric lymph nodes in BALB/c (A) and C57BL/6 (B) mice. The percentage of CD4+ CD25+Foxp3+ cells was determined using LSRII Flow Cytometer. Data were analysed using FlowJo software. There was no significant difference observed between the different DSS groups and with ghrelin treatment. However, there was significant increase in Treg cells in C57BL/6 (B) mice when comparing the naive mice to the 5kDa DSS + Ghrelin treated group, **p=0.0009, using a Mann-Whitney U-test. (N=11-12 mice/group, Mean ± S.D).

A BALB/c MLN regulatory T cells

Naives

5kDa D

SS + PBS

5kDa D

SS + 1n

mol Ghrel

in

4 pep

tide

∆

5kDa D

SS + 1n

mol

40kD

a DSS +

PBS

40kD

a DSS +

1nmol G

hrelin

4 pep

tide

∆

40kD

a DSS +

1nmol

0

10

20

30

Treatment Groups

CD

4+C

D25

+Fox

p3+

C57BL/6 MLN regulatory T cells

Naives

5kDa D

SS + PBS

5kDa D

SS + 1n

mol Ghrel

in

4 pep

tide

∆

5kDa D

SS + 1n

mol

40kD

a DSS +

PBS

40kD

a DSS +

1nmol G

hrelin

4 pep

tide

∆

40kD

a DSS +

1nmol

1nmol G

hrelin

4 pep

tide

∆

1nmol

0

10

20

30

Treatment Groups

CD

4+C

D25

+Fox

p3+

B

**

71

4.8 Treatment with ghrelin did not improve wound healing in the HT29 cell line

Wound healing assay was used as a model for intestinal epithelial growth and recovery.

To measure the effect of ghrelin in cell migration/wound healing, the colon cancer cell

line HT29 was obtained. HT29 cells were grown to confluence and were treated with

Mitomycin C (to inhibit proliferation) for two hours prior to creating the “wound”.

Once a scratch/wound was created the cells were immediately treated with various

concentrations of ghrelin (0.01, 0.1, 1, 10nM), media only as negative control and EGF

(100ng/ml) as positive control and were photographed at different time points (0, 10,

24, 48h). Images taken were analysed using the ImageJ program to quantify wound

closure in the cells. As can be seen from Figure 20A there was not any significant effect

in wound closure after 10 h of treatment with ghrelin. There was no difference between

the different concentrations of ghrelin in the wound closure of HT29 cells. Treatment

with EGF caused a significant increase in the cells after 10h (One-way ANOVA,

Tukey’s post hoc: p<0.001) and 24h (p<0.05) treatment. However, there was not any

difference observed between the media only (negative control) and different

concentrations of ghrelin treated cells. Figure 21, shows HT29 cells were pre-treated

with ghrelin for one hour before the “wound” was created. After 10hr (Figure 21A) of

treatment with ghrelin there was not any difference observed between any group. Even

the EGF did not cause any significant increase in wound closure after 10 and 24 h.

There was a slight increase in wound closure after 24h in the HT29 cells treated with

0.1nM ghrelin. However, results from One-way ANOVA, with Tukey’s post hoc tests

revealed that this was not statistically significant.

72

Figure 20. Analysis of wound assay on HT29 cells to measure the effect of various ghrelin concentrations in wound healing. Cells were photographed at different time points (0, 10, 24, 48h). Graph represents the percentage of wound closure of HT29 cells over a period of 48 h. * indicates p<0.05; **p<0.001 (one-way ANOVA with Tukey’s post hoc comparisons). Data represent 1 experiment. (N=6 per treatment group, Mean ± S.D).

HT29 24h wound

Neg co

ntrol

EGF

0.01n

M G

0.1nM G

1nM G

10nM G

0

10

20

30

40

Treatment Groups

Perc

ent o

f wou

nd c

losu

re

*

B

HT29 10h wound

Neg co

ntrol

EGF

0.01n

M G

0.1nM G

1nM G

10nM G

0

10

20

30

40

Treatment Groups

Perc

ent o

f wou

nd c

losu

re**

A

73

Figure 21. Pre-treatment of HT29 cells with various concentrations of ghrelin to measure its effect in wound healing. The cells were pre-treated with various ghrelin concentrations before creating the “wound”. Cells were then photographed at different time points (0, 10, 24, 48h). Graph represents the percentage of wound closure of HT29 cells over a period 48h. Data represent 1 experiment. (N=3 per treatment group, Mean ± S.D).

HT29 10h woundpre-treated with Ghrelin

Neg co

ntrol

EGF

0.01n

M G

0.1nM

G1n

M G

10nM G

0

10

20

30

40

Treatment Groups

Per

cent

of w

ound

clo

sure

A

HT29 24h woundpre-treated with Ghrelin

Neg co

ntrol

EGF

0.01n

M G

0.1nM G

1nM G

10nM G

0

10

20

30

40

Treatment Groups

Perc

ent o

f wou

nd c

losu

re

B

74

Chapter Five: Discussion

This study demonstrates that ghrelin treatment suppresses clinical colitis in

C57BL/6 mice treated with DSS (5kDa) and this is reflected in the reduction in

accumulative histopathological scores in the colon. Significant reduction of

inflammation was found in the proximal colon of these mice, although there also was a

trend towards less inflammation in the mid and distal colons of ghrelin treated mice.

This may have been due to expression of the ghrelin receptor or other receptors which

mediate ghrelin’s actions, being most highly expressed in the proximal colon region.

Peroxisome proliferator-activated receptors (PPAR-γ) are a group of nuclear receptor

proteins, which have been recently shown to interact with ghrelin receptor signalling

(Demers et al., 2008). A study by Avallone et al., (2006) has shown that scavenger

receptor, CD36, and/or GHS-R1a receptor may signal to enhance PPAR-γ activity in

macrophages. PPAR-γ has a major role in mediating anti-inflammatory processes and

the ability from its ligands to inhibit the expression of inflammatory genes in

macrophages and other vascular cells (Lee et al., 2003; Castrillo & Tontonoz, 2004).

Thus, Avallone et al, (2006) speculate that the activation of CD36 and/or GHS-R1a

may impact the inflammatory response in macrophages through PPAR-γ. Therefore it

can be potentially hypothesized that PPAR-γ could play a major role in inflammatory

responses mediated via ghrelin. PPAR-γ is expressed throughout the colon, mostly

occurring in the luminal epithelial cells of the proximal colon (Su et al., 2007).

Therefore this may explain why there was an effect of ghrelin primarily in the proximal

colon and this hypothesis is currently being investigated in our laboratory. Su et al.,

(2007) further showed that PPAR-γ was lowly expressed in the distal colon in the

C57BL/6 mice, which supports our findings that ghrelin showing only slight

suppression of colitis in the distal colon.

Our results showed mild action of ghrelin in the DSS model in comparison to

the TNBS model used by the Gonzalez-Rey group. The difference in colitis models

could be one of the reasons why a difference in ghrelin’s effect was noticed. Different

mouse strains and animal housing conditions could also result in the variance of results.

The previous study also used the BALB/c mouse strain with 5kDa DSS, however, their

study focused on the TNBS model, and they only showed difference in colon lengths,

clinical scores, and neutrophil activation activity results from their DSS model. Since

75

ghrelin is des-acylated very easily due to instability of the esterification, we made every

effort to preserve the activity of ghrelin however, this could be responsible for the

difference in results. Perhaps in the future the use of more stable analogues of ghrelin

such as the growth hormone secretagogues (Merck Research Laboratories) would be

efficacious.

A previous study by Melgar et al., (2005) demonstrated that the severity of

acute colitis induced by DSS was strain specific, where the colitis was more chronic in

C57BL/6 but not in the BALB/c mice. Our results showed that colitis in C57BL/6 mice

was more severe with 5kDa DSS treatment and colitis in BALB/c mice was more

severe with 40kDa DSS. Moreover, many studies have shown that the severity of colitis

induced by DSS is dependent on inbred mouse strain (Maehler et al., 1998), the

concentration of DSS (Egger et al., 2000), the molecular weight of DSS (Axelsson et

al., 1996; Kitajima et al., 2000), and the duration of DSS (Cooper et al., 1993). To

further investigate the pathophysiological effect of ghrelin in intestinal inflammation,

we studied the anti-inflammatory effect of ghrelin peptides using different molecular

weights of DSS in two different mouse strains. Our results indicate that treatment with

ghrelin was more effective in the C57BL/6 mouse strain treated with 5kDa DSS as

compared to the 40kDa DSS. C57BL/6 mice treated with 40kDa DSS showed no

beneficial effects of ghrelin, possibly because the severity of colitis in the 40kDa DSS

is more prominently seen in the lower colon (Axelsson et al., 1996). Kitajima et al.,

(2000) examined the effect of different molecular weights of DSS and found that the

severity of disease in the 5kDa DSS was mostly in the caecum and the upper colon. Our

results show that the C57BL/6 mice had severe inflammation in their proximal and

distal colons after being treated with 5kDa DSS in comparison to 40kDa DSS. This

inflammation was improved significantly after treatment with ghrelin peptides in the

proximal colon. BALB/c mice however, had more inflammation in the distal colon with

40kDa DSS treatment. Kitajima et al., (2000) also showed that the severity of colitis in

the 40kDa DSS was more prominently seen in the lower colon of BALB/c Cr Slc mice.

Axelsson et al., (1996), discuss that perhaps the difference in the severity of colitis

caused by the 5kDa and 40kDa DSS may be due to the sulphur content per molecule,

which may play an important role in the induction of colitis. It is suggested that the

larger molecular weight of DSS may be more prone to induce severe colitis in the

region of the colon which is more permeable where large amount of DSS can pass

through (Axelsson et al., 1996). Some in vitro studies of the large intestine with test

76

markers have shown that the proximal colon is more permeable than the distal colon

(Hosoya et al., 1993; Lange et al., 1994; van Meeteren et al., 1998). This therefore

suggests that there is a strong relationship between the colitis induced by different

molecular size forms of DSS and the region of permeability in the mucosal barrier

(Kitajima et al., 2000).

It is still unknown whether the Δ4 proghrelin peptide is functionally active.

Other prohormone fragments of ghrelin, such as obestatin, have been shown to be

bioactive. Obestatin has been reported to have inhibitory effects on feeding and

digestive motility and therefore has been postulated to antagonize ghrelin actions on

energy homeostasis and gastrointestinal functions (Zhang et al., 2005). However, these

findings have been questioned and further studies are required to determine the

physiological function of obestatin (Gourcerol et al., 2006; Lauwers et al., 2006; Bassil

et al., 2007). We therefore investigated whether the Δ4 peptide had similar or opposing

effects in our colitis models as ghrelin. Our results indicate that whilst there appears to

be mild benefit this is not significant or as potent as ghrelin, but it is mostly working in

the proximal colon. This effect was only seen in the C57BL/6 mice and not in the

BALB/c mice. Therefore, we may need to optimise the delivery of this peptide in mice

and further study of its administration and dosage is being investigated in our

laboratory. We would also need to determine the expression of Δ4 proghrelin peptide in

the both normal and inflamed colon and also in the epithelial cells.

Regulatory T cells (Treg) play a central role in IBD. They are characterised by

CD4+CD25+Foxp3+ which produce anti-inflammatory cytokines, IL-10 and TGFβ

(Mowat, 2003; Uhlig et al., 2006). In our study we did not find any significant

difference in Treg cells isolated from mesenteric lymph nodes (MLNs) with ghrelin

treatment. There seemed to be a slight increase in the Treg cells with ghrelin treatment

in the C57B/6 mice treated with 5kDa DSS. This may have been because these mice

had severe colitis in comparison to 40kDa DSS treatment. This study however did not

look at the function of Treg cells in these mice. Gonzalez-Rey et al., (2006) found

increased levels of IL-10 and TGFβ in MLNs and the lamina propria mesenteric cells of

their BALB/c mice. Our study failed to show any such difference in the number of Treg

cells in our BALB/c mice treated with ghrelin.

We also investigated the levels of the Th-1 pro-inflammatory cytokine, TNF-α

in this study. However, we could not determine a conclusion because cultures MLNs

from our naive mice had unexpectedly high levels of TNF-α than normally identified in

77

our lab. This perhaps could be a technical issue, since we had elevated levels of TNF-α

isolated from the MLNs. Other studies have measured pro-inflammatory cytokine levels

using protein extracts from the colon or colon implants to determine the effect of

ghrelin in colitis (Gonzalez-Rey et al., 2006; Heazlewood et al., 2008). There can be

numerous reasons explaining the increased cytokine levels. The MLN cells may have

been over-stimulated with PMA/ionomycin after 48 h. Gonzalez-Rey et al. (2006) used

a low concentration of PMA (10ng/ml) whereas we used a fairly higher concentration

(50ng/ml). Since naïve mice are healthy, at times it is very difficult to distinguish the

lymph nodes from fat. Therefore, there could be some possibility that fat cells were also

incubated along with the MLN cells. If this were the case, then fat cells could have

caused an increase in various cytokine production causing the MLN cells from the

naïve mice to have elevated TNF-α levels. Leptin, a nonglycosylated protein is

produced by adipocytes, it induces activation of cytokine towards a Th1 response and

has been found to enhance mesenteric TNF-α expression (Zhang et al., 1994; Barbier et

al., 2003; Fain et al., 2004). Explants from the lamina propria could also be beneficial

in investigating the effect of ghrelin in colitis in these DSS-induced mice. The

recruitment of immune cells, dendritic cells and macrophages into the lamina propria

could help in determining the mechanism of ghrelin’s action in inflammation. Since it

has been found that ghrelin receptors are expressed in macrophages, B cells, and

dendritic cells, it would be important to investigate the role of ghrelin in these cells

(Dixit et al., 2004). It would be interesting to perform in vitro studies with mouse

intestinal epithelial cells and by insulting the cells with TNF-α or lipopolysaccharides

(LPS) first and then treating the cells with ghrelin to determine the anti-inflammatory

effects of ghrelin.

We also found that the naïve BALB/c mice had a consistent presence of perhaps

a parasitic infection primarily occurring in the caecum and the proximal colon in the

histology images. It was present in either the luminal region or in the surface

epithelium, but was very rarely seen in the crypt or in the crypt base. It is suspected that

there may be an underlying parasitic infection and a senior parasite expert, Dr. Damien

Stark (Division of Microbiology, SydPath St. Vincent's Hospital) has suggested it to be

Dientamoeba fragilis, after observing histological images taken from the colons of the

BALB/c mice. Perhaps this underlying parasite could be the cause of the low-grade

inflammation occurring in the naïve and ghrelin and delta 4 only mice. D. fragilis is a

trichomonad parasite which has been implicated in causing gastrointestinal disease in

78

humans (Stark et al., 2007). It has been found that D. fragilis infection may be

symptomatic with both acute and chronic infections being reported in children and

adults causing symptoms such as diarrhoea and abdominal pain (Stark et al., 2005).

However, no such infections have been observed in rodents so far. Therefore, it would

be too early to predict the reason of low grade infection in the BALB/c mice. Currently,

there are further investigations taking place to determine if D. fragilis is indeed present

or whether there is another cause of the background inflammation.

It is not known if ghrelin affects wound healing in the intestinal epithelial cells

of mice so we investigated an in vitro wound assay to determine if it could help repair

the epithelial barrier. Previous study with ghrelin has shown it can increase migration in

astrocytoma cells (Dixit et al., 2006). However, we did not see an improvement in

wound healing in intestinal epithelial cells- HT29. This could have been because we

needed to insult the cells with TNF-α first, so it would interfere with the inherent

restitutive potential of mucosal epithelial cells through inhibition of proliferation, cell

migration and induce cellular apoptosis (Waseem et al., 2008). A study by Waseem et

al., (2008) showed that in the presence of TNF-α, ghrelin promoted the migration of

intestinal epithelial Caco-2 cells. We are confident our assay worked because treatment

with the positive control, EGF, did cause some significant increase in wound closure

after 10 and 24h. However there was not any effect on wound healing in the HT29 cells

with various concentrations of ghrelin. A complete closure with EGF after 24h

treatment could have been a better control in this assay. In the future, it would be useful

to study would closure in HT29 cells using different concentrations of EGF. It would

also be beneficial to study the effects of EGF + ghrelin in HT29 cells and other colon

cancer cells line. This would address whether there are potential synergistic or additive

effects of ghrelin on these cell lines. For future studies it would be useful to examine

the effect of ghrelin in wound healing in mouse epithelial cell lines to confirm the effect

of ghrelin in suppressing colitis in C57BL/6 mice in the proximal colon. Further

investigation would be required to study the role of ghrelin in other intestinal epithelial

cell lines using an siRNA approach and to determine if the down-regulation of ghrelin

and its receptor mRNA decreases the rate of proliferation and migration in these cell

lines. In order to establish the clinical relevance of ghrelin expression, future studies are

required in human colon (histopathological) tissue and this should be performed using

RT-PCR, Western analysis and immunohistochemistry.

79

In conclusion, we demonstrate that ghrelin was able to partially suppress colitis

in the C57BL/6 mice in the proximal colon and very mildly in the mid and distal colon.

However, these effects were only in the 5kDa DSS model and not in the 40kDa DSS

model. Although these results confirm the anti-inflammatory actions of ghrelin, they are

not in complete agreement with the findings of the Gonzalez-Rey et al., (2006). Our

study only showed improvement with ghrelin in the proximal colon of C57BL/6 mice

treated with a smaller molecular weight of DSS. This study also demonstrates that the

severity of colitis depends very highly on the molecular weight of DSS along with the

strain of mice. Our study showed that ghrelin was able to improve colitis in the

proximal colon, therefore it may be applicable to use ghrelin treatment in IBD in the

future. Patients with Crohn’s Disease of the terminal ileum could benefit with ghrelin

treatment as it is more effective in that region. Future studies examining the expression

of ghrelin and its receptor in human colons would be able to guide us in treatment with

ghrelin.

80

Appendix 1.1 SCORE SHEET FOR MICE UNDERGOING DSS TREATMENT

DAILY OBSERVATIONS

Experiment number: Mouse number:

Date and time of challenge:

Dose: Pre-challenge weight (g):

DATE

Time post-challenge Days post-DSS Day

1 Day

2 Day

3 Day

4 Day

5 Day

6 Day

7 Day

8 Day

9 Observations from a distance

Inactive Hunched posture Ruffled fur Rate of breathing Crusty Eyes Shivering Diarrhoea Rectal bleeding

On handling

Not inquisitive or alert Bodyweight (% change from start/score)*

Any other abnormal behaviour or signs noted

ACTION TAKEN^

NOTES

TOTAL SCORE Scoring Details: 0 = normal, 1 = equivocal symptoms, 2 = mild symptoms, 4 = severe symptoms. Total of 4 or over = mouse culled. 0= no weight loss, 2 = 5-15% weight loss, 4 = >15% weight loss

♀/♂

81

1.2 Assessment of DSS colitis – scoring system adapted from Obermeier Clin Exp Immunol. 1999; 116(2): 238–245 and Tamaki et al. Gastroenterology 2006; 131:1110-1121. Inflammation Severity (IS) 0 = none 1 = mild 2 = moderate 3 = severe

Infiltration Extent (IE) 0 = no infiltrate 1 = infiltrate around crypt base 2 = infiltrate reaching to muscularis mucosae 3 = extensive infiltration reaching the muscularis mucosae and thickening of the mucosa with abundant oedema 4 = infiltration of the submucosa.

Epithelial Damage (ED) 0 = normal morphology 1 = some loss of goblet cells /some crypt abscesses or damage 2 = loss of goblet cells in large areas /extensive crypt abscesses or damage 3 = loss of crypts <5 crypt widths 4 = loss of crypts > 5 crypt widths, < 20% ulceration 5 = > 20% ulceration

Percentage Involvement of Epithelial Damage (PD) (crypt abscessed, crypt loss or ulceration) 0 = 0% 1 = 1-25% 2 = 26-50% 3 = 51-75% 4 = 76-100%

Experiment …………………………………….. Read by…………………… Blind Date / / No. CA PC MC DC/REC

IS IE ED PD IS IE ED PD IS IE ED PD IS IE ED PD

82

1.3

Parameter PMT Voltages C57BL/6 5kDa Figure 18

PMT Voltages C57BL/6 40kDa Figure 18

PMT Voltages BALB/c 5kDa Figure 19

PMT Voltages BALB/c 40kDa Figure 19

FSC-A 549 599 599 599 FSC-W 549 599 599 599 FSC-H 549 599 599 599 SSC-A 308 374 368 324 SSC-W 308 374 368 324 SSC-H 308 374 368 324 FITC 484 500 484 451 PE 533 516 522 495 APC 632 604 622 593

TABLE A.1. Photomultiplier (PMT) voltages used for flow cytometric enumeration of T regulatory cells in mesenteric lymph nodes.

83

CD4 CD25

Foxp

3

Figure A.1. A representative example of the gating strategies employed to enumerate T regulatory cells in mesenteric lymph nodes. Erythrocytes, adipocytes, granulocytes and cellular debris were eliminated from analysis by the use of forward and side scatter profiles. Lymphocytes were further gated on size and side-scatter profile. CD4+ T lymphocytes were determined by CD4-FITC staining and only these cells were included in the T regulatory cell analysis. T regulatory cells were characterised as CD4+CD25+Foxp3+

(all antibodies from eBioscience).

84

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