The human GI tract has evolved to simultaneously absorb nutrients and be the frontline in
host defence. These seemingly mutually exclusive goals are achieved by a single cell thick
epithelial barrier, and a complex resident immune system which lives in symbiosis with the
intestinal microflora and is also able to rapidly respond to invading pathogens. An
immunological balance is therefore required to permit tolerance to the normal intestinal
microflora, but also prevent the dissemination of pathogenic micro-organisms to the rest of
the host. Inappropriate immune responses in genetically susceptible individuals are the
hallmark of human inflammatory bowel disease (IBD) and are thus targeting effector immune
cells and their cytokines remains the mainstay of treatment. However despite vigorous
efforts to delineate the genetic contribution to IBD disease susceptibility using large
multinational cohorts, the majority of disease heritability remains unknown. Epigenetics
describes heritable changes in chromatin that are not conferred by DNA sequence. These
incorporate changes to histones, chromatin structure and DNA methylation, which confer
changes to gene transcription and thus gene expression and cellular function. Methylbinding
proteins (MBD) have the ability to bind to methylated DNA and recruit large
chromatin remodeling complexes that underpin a variety of epigenetic modifications. Methyl-
CpG-binding domain protein 2 (MBD2) is one such MBD that is required for appropriate
innate (dendritic cell) and adaptive (T cell) immune function, though its role has not been
investigated in the GI tract.
We hypothesized that alterations in chromatin are central to the reprogramming of normal
gene expression that occurs in disease states. By defining the phenotype of immune cells in
the absence of MBDs we hope to understand the mechanisms of chromatin-dysregulation
that lead to immune-mediated diseases such as IBD. We therefore aimed to assess the role
of MBD2 in colon immune cells in the steady state and in murine models of GI tract
inflammation, thereafter identifying the culprit cell types and genes responsible for any
observed changes. We envisaged that investigating heritable, epigenetic changes in gene
expression that are inherently more amenable to environmental manipulation than our DNA
code, may provide novel insight to a poorly understood mechanism of disease
predisposition. In addition identifying the cellular and gene targets of Mbd2 mediated
changes to immune homeostasis that may provide exciting and novel approaches to
therapeutic modulation of pathological inflammatory responses.
In chapter 3 we assessed the expression of Mbd2/MBD2 in the murine/human GI tract.
Consistent with existing mouse data, levels of Mbd2 mRNA increased between anatomical
divisions of small (duodenum, ileum, terminal ileum) and large intestine (caecum, colon,
rectum). In addition MBD2 mRNA was greater in the rectum versus ileum, with active IBD
associated with lower rectal MBD2 mRNA compared to quiescent IBD controls. Thus we
sought to understand the role of Mbd2 in the colon, where mRNA levels were the highest in
the GI tract and where appropriate immune function is central to prevent damaging
inflammation. To address these aims required the development of existing methods of cell
surface marker expression analysis using flow cytometry techniques to simultaneously
identify multiple innate and adaptive immune populations. Using naïve Mbd2 deficient mice
(Mbd2-/-) we observed CD11b+ CD103+ DCs were significantly reduced in number in Mbd2
deficiency.
To understand the role of Mbd2 in colonic inflammation we employed a mouse model of
chemical (DSS) and infectious (T. gondii) colitis comparing Mbd2-/- and littermate controls
(WT). Mbd2-/- were extremely sensitive to DSS and T. gondii mediated colonic inflammation,
characterized by increased symptom score, weight loss and histological score of tissue
inflammation (DSS) and increased antibody specific cytokine responses (T. gondii) in Mbd2
deficient animals. Flow cytometry analysis of colon LP cells in both infectious and chemical
colitis revealed significant accumulation of monocytes and neutrophils in Mbd2-/-. Indeed
monocytes and neutrophils were the principal myeloid sources of IL-1b and TNF in DSS
colitis and the number of IL-1b/TNF+ monocytes/neutrophils was significantly greater in
Mbd2-/-. Lastly we employed our colon LP isolation techniques to analyse immune
populations in active and quiescent IBD and healthy controls, using endoscopically acquired
biopsy samples. Analysis revealed that as in murine colitis, active human IBD is
characterized by the accumulation of CD14High monocyte-like cells, with an associated
increased ratio of macrophage:monocyte-like cells.
In Chapter 4 we sought to understand the cellular sources of Mbd2 that may explain the predisposition
of Mbd2-/- to colitis. Firstly we restricted Mbd2 deficiency to haematopoietic cells
using grafting Mbd2-/- bone marrow (BM) into lethally irradiated WT mice. These animals
treated with DSS displayed increased weight loss, symptom score, neutrophil accumulation
and histopathology score compared to mice irradiated and grafted with WT BM. Given the
accumulation of monocytes in Mbd2-/- DSS treated mice, and existing literature supporting a
pathogenic role in this model, we then investigated the role of Mbd2 in monocyte function.
Colon monocytes sorted from Mbd2-/- and WT DSS treated mice displayed similar
expression for many pro-inflammatory genes (Il6, Il1a, Il1b, Tnf), but demonstrated
significantly dysregulated expression for some others (Regb, Lyz1, Ido1, C4a). To
investigate this in a more refined model, we lethally irradiated WT mice and repopulated
them with a WT:Mbd2-/- BM mix. This enabled the analysis of WT and Mbd2-/- haematopoietic
cells in the same animal. Colon WT and Mbd2-/- monocyte recruitment and cytokine
production in DSS treated mixed BM chimeras was equivalent between genotypes
suggesting that Mbd2 deficiency in monocytes alone did not explain the increased
susceptibility of Mbd2-/- to DSS colitis. We then restricted Mbd2 deficiency to CD11c
expressing cells, given the known role for Mbd2 in their function, and for CD11c+ cells in
DSS, using a CD11cCreMbd2Fl/Fl system. DSS treated mice with Mbd2 deficient CD11c+
cells demonstrated increased weight loss, symptoms score, histolopathology score,
monocyte and neutrophil colon accumulation compared to controls. To further explore the
role of Mbd2 in colon CD11c+ cells, macrophage and DCs from DSS treated WT and Mbd2-/-
mice were purified and their gene expression analysed. Mbd2-/- versus WT macrophages
demonstrated significantly altered expression of both pro- (Il1a, C6, Ido1, Trem2) and antiinflammatory
(Tgfbi, Retnla) pathways that we hypothesized was a method for attempted
host control of excessive colon damage in Mbd2-/- mice. DC gene expression analysis was
hampered by small sample size, but demonstrated a large number of small expression
changes, including IL-12/IL-23 (Jak2) and autophagy (Lrrk2) pathways. Lastly levels of costimualtory
molecules (CD40/CD80) were increased in Mbd2-/- but not CD11cΔMbd2 colon
LP DCs/macrophages suggesting that non-CD11c+ cellular sources of Mbd2 were required
to produce increased activation phenotype in these cells.
Finally in Chapter 5 we explored the role for Mbd2 in non-haematopoietic cells, namely the
colonic epithelium. Here we first developed a novel method for identifying and purifying
these cells using flow cytometry. Mbd2 deficient colonic epithelium demonstrated increased
expression of activation markers MHC II and LY6A/E in the steady state and in DSS / T.
muris mediated colonic inflammation. Indeed FACS purified colon epithelial cells from naive
and DSS treated, Mbd2-/- and WT mice revealed conserved dysregulated gene expression
independent of inflammation: Both naïve and inflamed Mbd2 deficient epithelium displayed
significantly increased expression of genes responsible for antigen processing/presentation
(MHC I, MHC II, immunoproteasome) and decreased expression of genes involved in cell-cell
adhesion (Cldn1, Cldn4). Lastly we investigated whether the observed differences in
Mbd2-/- cell types conferred alterations in the makeup of the intestinal microflora.
Interestingly independent of co-housing of Mbd2-/- and WT animals, Mbd2 deficiency
consistently predicted the microbial composition, with increased levels of Clostridales and
decreased levels of Parabacteroides bacteria.
Collectively we have identified CD11c+ cells, monocytes and colon epithelial cells as key cell
types for Mbd2 mediated changes in gene expression that affect mucosal immune
responses. These data thus identify Mbd2 gene targets within these cell types as exciting
new areas for investigation and therapeutic modulation to limit damaging GI tract
inflammation