Rajaraman Eri

Aberrant Mucin Assembly in Mice Causes Endoplasmic Reticulum Stress and Spontaneous Inflammation Resembling Ulcerative Colitis

Background MUC2 mucin produced by intestinal goblet cells is the major component of the intestinal mucus barrier. The inflammatory bowel disease ulcerative colitis is characterized by depleted goblet cells and a reduced mucus layer, but the aetiology remains obscure. In this study we used random mutagenesis to produce two murine models of inflammatory bowel disease, characterised the basis and nature of the inflammation in these mice, and compared the pathology with human ulcerative colitis. Methods and Findings By murine N-ethyl-N-nitrosourea mutagenesis we identified two distinct noncomplementing missense mutations in Muc2 causing an ulcerative colitis-like phenotype. 100% of mice of both strains developed mild spontaneous distal intestinal inflammation by 6 wk (histological colitis scores versus wild-type mice, p < 0.01) and chronic diarrhoea. Monitoring over 300 mice of each strain demonstrated that 25% and 40% of each strain, respectively, developed severe clinical signs of colitis by age 1 y. Mutant mice showed aberrant Muc2 biosynthesis, less stored mucin in goblet cells, a diminished mucus barrier, and increased susceptibility to colitis induced by a luminal toxin. Enhanced local production of IL-1β, TNF-α, and IFN-γ was seen in the distal colon, and intestinal permeability increased 2-fold. The number of leukocytes within mesenteric lymph nodes increased 5-fold and leukocytes cultured in vitro produced more Th1 and Th2 cytokines (IFN-γ, TNF-α, and IL-13). This pathology was accompanied by accumulation of the Muc2 precursor and ultrastructural and biochemical evidence of endoplasmic reticulum (ER) stress in goblet cells, activation of the unfolded protein response, and altered intestinal expression of genes involved in ER stress, inflammation, apoptosis, and wound repair. Expression of mutated Muc2 oligomerisation domains in vitro demonstrated that aberrant Muc2 oligomerisation underlies the ER stress. In human ulcerative colitis we demonstrate similar accumulation of nonglycosylated MUC2 precursor in goblet cells together with ultrastructural and biochemical evidence of ER stress even in noninflamed intestinal tissue. Although our study demonstrates that mucin misfolding and ER stress initiate colitis in mice, it does not ascertain the genetic or environmental drivers of ER stress in human colitis. Conclusions Characterisation of the mouse models we created and comparison with human disease suggest that ER stress-related mucin depletion could be a fundamental component of the pathogenesis of human colitis and that clinical studies combining genetics, ER stress-related pathology and relevant environmental epidemiology are warranted.

Intestinal barrier dysfunction in inflammatory bowel diseases.

The etiology of human inflammatory bowel diseases (IBDs) is believed to involve inappropriate host responses to the complex commensal microbial flora in the gut, although an altered commensal flora is not completely excluded. A multifunctional cellular and secreted barrier separates the microbial flora from host tissues. Altered function of this barrier remains a major largely unexplored pathway to IBD. Although there is evidence of barrier dysfunction in IBD, it remains unclear whether this is a primary contributor to disease or a consequence of mucosal inflammation. Recent evidence from animal models demonstrating that genetic defects restricted to the epithelium can initiate intestinal inflammation in the presence of normal underlying immunity has refocused attention on epithelial dysfunction in IBD. We review the components of the secreted and cellular barrier, their regulation, including interactions with underlying innate and adaptive immunity, evidence from animal models of the barriers role in preventing intestinal inflammation, and evidence of barrier dysfunction in both Crohns disease and ulcerative colitis.

ER stress and the unfolded protein response in intestinal inflammation

Endoplasmic reticulum (ER) stress is a phenomenon that occurs when excessive protein misfolding occurs during biosynthesis. ER stress triggers a series of signaling and transcriptional events known as the unfolded protein response (UPR). The UPR attempts to restore homeostasis in the ER but if unsuccessful can trigger apoptosis in the stressed cells and local inflammation. Intestinal secretory cells are susceptible to ER stress because they produce large amounts of complex proteins for secretion, most of which are involved in mucosal defense. This review focuses on ER stress in intestinal secretory cells and describes how increased protein misfolding could occur in these cells, the process of degradation of misfolded proteins, the major molecular elements of the UPR pathway, and links between the UPR and inflammation. Evidence is reviewed from mouse models and human inflammatory bowel diseases that ties ER stress and activation of the UPR with intestinal inflammation, and possible therapeutic approaches to ameliorate ER stress are discussed.

An intestinal epithelial defect conferring ER stress results in inflammation involving both innate and adaptive immunity

We recently characterized Winnie mice carrying a missense mutation in Muc2, leading to severe endoplasmic reticulum stress in intestinal goblet cells and spontaneous colitis. In this study, we characterized the immune responses due to this intestinal epithelial dysfunction. In Winnie, there was a fourfold increase in activated dendritic cells (DCs; CD11c+ major histocompatibility complex (MHC) class IIhi) in the colonic lamina propria accompanied by decreased colonic secretion of an inhibitor of DC activation, thymic stromal lymphopoietin (TSLP). Winnie also displayed a significant increase in mRNA expression of the mucosal TH17 signature genes Il17a, IL17f, Tgfb, and Ccr6, particularly in the distal colon. Winnie mesenteric lymph node leukocytes secreted multiple TH1, TH2, and TH17 cytokines on activation, with a large increase in interleukin-17A (IL-17A) progressively with age. A major source of mucosal IL-17A in Winnie was CD4+ T lymphocytes. Loss of T and B lymphocytes in Rag1-/- × Winnie (RaW) crosses did not prevent spontaneous inflammation but did prevent progression with age in the colon but not the cecum. Adoptive transfer of naive T cells into RaW mice caused more rapid and severe colitis than in Rag1-/-, indicating that the epithelial defect results in an intestinal microenvironment conducive to T-cell activation. Thus, the Winnie primary epithelial defect results in complex multicytokine-mediated colitis involving both innate and adaptive immune components with a prominent IL-23/TH17 response, similar to that of human ulcerative colitis.

CCR5-Delta32 mutation is strongly associated with primary sclerosing cholangitis.

CCR5 plays a key role in the distribution of CD45RO+ T cells and contributes to generation of a T helper 1 immune response. CCR5-Δ32 is a 32-bp deletion associated with significant reduction in cell surface expression of the receptor. We investigated the role of CCR5-Δ32 on susceptibility to ulcerative colitis (UC), Crohns disease (CD) and primary sclerosing cholangitis (PSC). Genotype and allelic association analyses were performed in 162 patients with UC, 131 with CD, 71 with PSC and 419 matched controls. There was a significant difference in CCR5 genotype (OR 2.27, P=0.003) between patients with sclerosing cholangitis and controls. Similarly, CCR5-Δ32 allele frequency was significantly higher in sclerosing cholangitis (17.6%) compared to controls (9.9%, OR 2.47, P=0.007) and inflammatory bowel disease patients without sclerosing cholangitis (11.3%, OR 1.9, P=0.027). There were no significant differences in CCR5 genotype or allele frequency between those with either UC or CD and controls. Genotypes with the CCR5-Δ32 variant were increased in patients with severe liver disease defined by portal hypertension and/or transplantation (45%) compared to those with mild liver disease (21%, OR 3.17, P=0.03). The CCR5-Δ32 mutation may influence disease susceptibility and severity in patients with PSC.

TNFα and IL10 SNPs act together to predict disease behaviour in Crohn’s disease

Background: The cytokines tumour necrosis factor (TNF)α and interleukin (IL)10 have been implicated in the pathogenesis of Crohn’s disease (CD), with increased concentrations reported in patients with active disease. However, limited data exist on their effects on disease phenotype in the same population. Certain single nucleotide polymorphisms (SNPs) within the promoter region of the IL10 (-1082G/A, -592C/A) and TNFα (-308G/A, -857C/T) genes have been associated with altered levels of circulating IL10 and TNFα. Methods: We conducted an Australian based case–control study (304 CD patients; 231 healthy controls) of these four SNPs. Further investigation of two SNPs was conducted using a logistic regression analysis. Results: We identified a possible association of both IL10 SNPs and TNFα-857 with CD. Further investigation of a relationship with disease severity showed a significant association of higher producing IL10-1082G and TNFα-857C alleles with stricturing behaviour, which was strongest when these alleles were combined and persisted after multivariate analysis (p = 0.007; odds ratio (OR) 2.37, 95% CI 1.26 to 4.43). In addition, the TNFα-857CC genotype was independently associated with familial CD (p = 0.03; OR 3.12; 95% CI 1.15 to 8.46). Conclusion: These two SNPs may help to predict disease behaviour in CD patients, which may be clinically useful in shaping treatment of the disease at an earlier stage.

CC Chemokine Ligand 20 and Its Cognate Receptor CCR6 in Mucosal T Cell Immunology and Inflammatory Bowel Disease: Odd Couple or Axis of Evil?

Chemokines and their cognate receptors have been identified as major factors initiating and governing cell movement and interaction. These ligands and their receptors are expressed on a wide variety of cells and act during steady-state migration as well as inflammatory recruitment. CCR6 is a non-promiscuous chemokine receptor that has only one known chemokine ligand, CCL20, and is present on B and T cells as well as dendritic cells (DCs). Two CD4+ T cell populations with opposing functions present in the intestines and the mesenteric lymph nodes express CCR6: the pro-inflammatory TH17 and regulatory Treg cells. CCL20 is also present in the intestine and is strongly up-regulated after an inflammatory stimulus. Interestingly, this ligand is also expressed by TH17 cells, which opens up the possibility of autocrine/paracrine signaling and, consequently, a self-perpetuating cycle of recruitment, thereby promoting inflammation. Recently, CCR6 has been implicated in inflammatory bowel disease (IBD) by genome wide association studies which showed an association between SNPs in the genomic region of the CCR6 gene and the inflammation. Furthermore, recent research targeting the biological function of CCR6 indicates a significant role for this chemokine receptor in the development of chronic IBD. It is therefore possible that IBD is facilitated by a disordered regulation of TH17 and Treg cells due to a disruption in the CCL20-CCR6 axis and consequently disturbed mucosal homeostasis. This review will summarize the literature on CCL20-CCR6 in mucosal immunology and will analyze the role this receptor-ligand axis has in chronic IBD.

Intestinal Epithelium and Autophagy: Partners in Gut Homeostasis

One of the most significant challenges of cell biology is to understand how each type of cell copes with its specific workload without suffering damage. Among the most intriguing questions concerns intestinal epithelial cells in mammals; these cells act as a barrier between the internally protected region and the external environment that is exposed constantly to food and microbes. A major process involved in the processing of microbes is autophagy. In the intestine, through multiple, complex signaling pathways, autophagy including macroautophagy and xenophagy is pivotal in mounting appropriate intestinal immune responses and anti-microbial protection. Dysfunctional autophagy mechanism leads to chronic intestinal inflammation, such as inflammatory bowel disease (IBD). Studies involving a number of in vitro and in vivo mouse models in addition to human clinical studies have revealed a detailed role for autophagy in the generation of chronic intestinal inflammation. A number of genome-wide association studies identified roles for numerous autophagy genes in IBD, especially in Crohn’s disease. In this review, we will explore in detail the latest research linking autophagy to intestinal homeostasis and how alterations in autophagy pathways lead to intestinal inflammation.

Angiotensinogen and transforming growth factor beta1: novel genes in the pathogenesis of Crohn's disease.

Background: Angiotensin peptides may act locally as cytokines in several organ systems with elevated mucosal levels present in Crohn’s disease. A variant in the angiotensinogen gene promoter results in increased peptide production, while transforming growth factor β1 (TGFβ1) codon 25 variants demonstrate variable peptide production, predisposing to fibrosis in several organs. Aims: Conduct an Australian-based analysis of the angiotensinogen-6 variant in two independent inflammatory bowel disease (IBD) cohorts, and examine the role of angiotensinogen-6 and TGFβ1 codon 25 variants in shaping Crohn’s disease phenotype. Methods: IBD Patients (Crohn’s disease = 347, ulcerative colitis = 147) and CD families (n = 148) from two cohorts, together with 185 healthy controls were genotyped for angiotensinogen-6. Genotype-phenotype analyses were performed for both angiotensinogen-6 and TGFβ1 codon 25. Results: Angiotensinogen-6 AA genotype was significantly associated with Crohns disease (p = 0.007, OR = 2.38, CI = 1.32–4.32) in cohort 1, but not in the smaller cohort 2 (p = 0.19). The association remained significant when the two cohorts were combined (p = 0.008), and in a TDT family analysis (p = 0.03). TGF 1 codon 25 was associated with stricturing Crohn’s disease (p = 0.01, OR = 2.63, CI = 1.16–5.88) and a shorter time to intestinal resection (p = 0.06). Conclusions: The association of the angiotensinogen-6 variant with Crohn’s disease supports a potential role for angiotensin-converting enzyme inhibitors and angiotensin II receptor antagonists in disease treatment.

Intestinal secretory cell ER stress and inflammation

Data from animal models and human inflammatory bowel diseases have implicated the ER (endoplasmic reticulum) stress pathway in intestinal inflammation. We have characterized the development of inflammation in Winnie mice in which ER stress arises due to a single missense mutation in the MUC2 mucin produced by intestinal goblet cells. This model has allowed us to explore the genesis of inflammation ensuing from a single gene polymorphism affecting secretory cells. In these mice, a proportion of MUC2 misfolds during biosynthesis, leading to ER stress and activation of the unfolded protein response. Winnie mice develop spontaneous complex progressive inflammation that is most severe in the distal colon. Inflammation involves TH1, TH2 and TH17 T-cells, with a progressive development of a TH17-dominated response, but also involves innate immunity, in a pattern not dissimilar to human colitis. Experimental inhibition of tolerance in this model severely exacerbates colitis, demonstrating active effective suppression of inflammation. Even though the misfolding of MUC2 is a consequence of an inherited mutation, as inflammation develops, the molecular markers of ER stress increase further and goblet cell pathology becomes worse, suggesting that inflammation itself exacerbates ER stress.