Spontaneous Colitis In Mice Research Articles

Rap1 prevents colitogenic Th17 cell expansion and facilitates Treg cell differentiation and distal TCR signaling

T-cell-specific Rap1 deletion causes spontaneous colitis in mice. In the present study, we revealed that Rap1 deficiency in T cells impaired the preceding induction of intestinal RORγt+ Treg cells. In the large intestinal lamina propria (LILP) of T-cell-specific Rap1-knockout mice (Rap1KO mice), Th17 cells were found to increase in a microbiota-dependent manner, and the inhibition of IL-17A production prevented the development of colitis. In the LILP of Rap1KO mice, RORγt+ Treg cells were scarcely induced by 4 weeks of age. The expression of CTLA-4 on Rap1-deficient Treg cells was reduced and the expression of CD80 and CD86 on dendritic cells was consequently elevated in Rap1KO mice. When cultured under each polarizing condition, Rap1-deficient naïve CD4+ T cells did not show biased differentiation into Th17 cells; their differentiation into Treg cells as well as Th1 and Th2 cells was lesser than that of wild-type cells. Rap1-deficient naïve CD4+ T cells were found to exhibit the defective nuclear translocation of NFAT and formation of actin foci in response to TCR engagement. These data suggest that Rap1 amplifies the TCR signaling required for Treg-mediated control of intestinal colitogenic Th17 responses.

Microbiota in mesenteric adipose tissue from Crohn\u2019s disease promote colitis in mice

BackgroundMesenteric adipose tissue (mAT) hyperplasia, known as creeping fat is a pathologic characteristic of Crohn’s disease (CD). The reserve of creeping fat in surgery is associated with poor prognosis of CD patients, but the mechanism remains unknown.MethodsMesenteric microbiome, metabolome, and host transcriptome were characterized using a cohort of 48 patients with CD and 16 non-CD controls. Multidimensional data including 16S ribosomal RNA gene sequencing (16S rRNA), host RNA sequencing, and metabolome were integrated to reveal network interaction. Mesenteric resident bacteria were isolated from mAT and functionally investigated both in the dextran sulfate sodium (DSS) model and in the Il10 gene-deficient (Il10−/−) mouse colitis model to validate their pro-inflammatory roles.ResultsMesenteric microbiota contributed to aberrant metabolites production and transcripts in mATs from patients with CD. The presence of mAT resident microbiota was associated with the development of CD. Achromobacter pulmonis (A. pulmonis) isolated from CD mAT could translocate to mAT and exacerbate both DSS-induced and Il10 gene-deficient (Il10−/−) spontaneous colitis in mice. The levels of A. pulmonis in both mAT and mucous layer from CD patients were higher compared to those from the non-CD group.ConclusionsThis study suggests that the mesenteric microbiota from patients with CD sculpt a detrimental microenvironment and promote intestinal inflammation.5k89f_PEiAcxa3djQxTmu9Video abstract

Protective Effects of SIRT6 Overexpression against DSS-Induced Colitis in Mice.

Sirtuin 6 (SIRT6), as a NAD + -dependent deacetylase, plays an indispensable role in the regulation of health and physiology. Loss of SIRT6 causes spontaneous colitis in mice and makes intestinal epithelial cells prone to stress. However, whether SIRT6 overexpression increases resistance to colitis remains unknown. Here, in vivo results demonstrated that SIRT6 overexpression attenuates DSS-induced colitis in terms of clinical manifestations, histopathological damage, loss of tight junction function and imbalanced intestinal microenvironment. Additionally, we also found that the activation of NF-κB and c-Jun induced by DSS is diminished by SIRT6 overexpression. Furthermore, SIRT6 may regulate TAK1 to inhibit NF-κB and c-Jun signaling. Thus, our findings highlight the protective effect of SIRT6 on colon, further supporting the perspective that SIRT6 may be a therapeutic target for intestine injury under stress.

A New Model of Spontaneous Colitis in Mice Induced by Deletion of an RNA m6A Methyltransferase Component METTL14 in T Cells.

Background and aimsMouse models of colitis have been used to study the pathogenesis of inflammatory bowel disease (IBD) and for pre-clinical development of therapeutic agents. Various epigenetic pathways have been shown to play important regulatory roles in IBD. Reversible N6-methyladenosine (m6A) methylation represents a new layer of post-transcriptional gene regulation that affects a variety of biological processes. We aim to study how deletion of a critical component of m6A writer complex, METTL14, in T cells affects the development of colitis.MethodsConditional Mettl14 was lineage specifically deleted with CD4-regulated Cre in T cells. Colitis phenotype was determined by H&E staining, colon weight-to-length ratio and cytokine expression. We additionally utilized T cell transfer model of colitis and adoptive transfer of regulatory T cells. Mice were treated with antibiotics to determine if the colitis could be attenuated.ResultsMETTL14 deficiency in T cells induced spontaneous colitis in mice. This was characterized by increased inflammatory cell infiltration, increased colonic weight-to-length ratio and increased Th1 and Th17 cytokines. The colitis development was due to dysfunctional regulatory T (Treg) cells, as adoptive transfer of WT Treg cells attenuated the colitis phenotype. The METTL14-deficient Treg cells have decreased RORγt expression compared with WT controls. METTL14 deficiency caused impaired induction of naïve T cells into induced Treg cells. Antibiotic treatment notably attenuated the colitis development.ConclusionHere we report a new mouse model of spontaneous colitis based on perturbation of RNA methylation in T cells. The colitis is T cell-mediated and dependent on the microbiome. This model represents a new tool for elucidating pathogenic pathways, studying the contribution of intestinal microbiome and preclinical testing of therapeutic agents for inflammatory bowel disease.

Total CD3 T Cells Are Necessary and Sufficient to Induce Colitis in Immunodeficient Mice With Dendritic Cell-Specific Deletion of TGFbR2: A Novel IBD Model to Study CD4 and CD8 T-Cell Interaction.

Inflammatory bowel disease (IBD) is a multifactorial disorder, with the innate and adaptive immune cells contributing to disease initiation and progression. However, the intricate cross-talk between immune cell lineages remains incompletely understood. The role of CD8+ T cells in IBD pathogenesis has been understudied, largely due to the lack of appropriate models. We previously reported spontaneous colitis in mice with impaired TGFβ signaling due to dendritic cell-specific knockout of TGFbR2 (TGFβR2ΔDC). Here, we demonstrate that crossing TGFβR2ΔDC mice with a Rag1-/- background eliminates all symptoms of colitis and that adoptive transfer of unfractionated CD3+ splenocytes is sufficient to induce progressive colitis in Rag1-/-TGFβR2ΔDC mice. Both CD4+ and CD8+ T cells are required for the induction of colitis accompanied by activation of both T-cell lineages and DCs, increased expression of mucosal IFNγ, TNFα, IL6, IL1β, and IL12, and decreased frequencies of CD4+FoxP3+ regulatory T cells. Development of colitis required CD40L expression in CD4+ T cells, and the disease was partially ameliorated by IFNγ neutralization. This novel model provides an important tool for studying IBD pathogenesis, in particular the complex interactions among innate and adaptive immune cells in a controlled fashion, and represents a valuable tool for preclinical evaluation of novel therapeutics.

Phosphoinositide 3-Kinase P110δ-Signaling Is Critical for Microbiota-Activated IL-10 Production by B Cells that Regulate Intestinal Inflammation.

The phosphoinositide 3-kinase catalytic subunit p110δ (PI3Kδ) gene maps to a human inflammatory bowel diseases (IBD) susceptibility locus, and genetic deletion of PI3Kδ signaling causes spontaneous colitis in mice. However, little is known regarding the role of PI3Kδ on IL-10-producing B cells that help regulate mucosal inflammation in IBD. We investigated the role of PI3Kδ signaling in B cell production of IL-10, following stimulation by resident bacteria and B cell regulatory function against colitis. In vitro, B cells from PI3KδD910A/D910A mice or wild-type B cells treated with PI3K specific inhibitors secreted significantly less IL-10 with greater IL-12p40 following bacterial stimulation. These B cells failed to suppress inflammatory cytokines by co-cultured microbiota-activated macrophages or CD4+ T cells. In vivo, co-transferred wild-type B cells ameliorated T cell-mediated colitis, while PI3KδD910A/D910A B cells did not confer protection from mucosal inflammation. These results indicate that PI3Kδ-signaling mediates regulatory B cell immune differentiation when stimulated with resident microbiota or their components, and is critical for induction and regulatory function of IL-10-producing B cells in intestinal homeostasis and inflammation.

A microbial trigger for colitis.

An intestinal mucus-dwelling bacterial species can trigger spontaneous colitis in mice lacking genes associated with Crohn’s disease.

Core 1- and 3-derived O-glycans collectively maintain the colonic mucus barrier and protect against spontaneous colitis in mice.

Core 1- and core 3-derived mucin-type O-glycans are primary components of the mucus layer in the colon. Reduced mucus thickness and impaired O-glycosylation are observed in human ulcerative colitis. However, how both types of O-glycans maintain mucus barrier function in the colon is unclear. We found that C1galt1 expression, which synthesizes core 1 O-glycans, was detected throughout the colon, whereas C3GnT, which controls core 3 O-glycan formation, was most highly expressed in the proximal colon. Consistent with this, mice lacking intestinal core 1-derived O-glycans (IEC C1galt1−/−) developed spontaneous colitis primarily in the distal colon, whereas mice lacking both intestinal core 1- and core 3-derived O-glycans (DKO) developed spontaneous colitis in both distal and proximal colon. DKO mice showed an early onset and more severe colitis than IEC C1galt1−/− mice. Antibiotic treatment restored the mucus layer and attenuated colitis in DKO mice. Mucins from DKO mice were more susceptible to proteolysis than WT mucins. This study indicates that core 1- and 3-derived O-glycans collectively contribute to the mucus barrier by protecting it from bacterial protease degradation and suggests new therapeutic targets to promote mucus barrier function in colitis patients.

Goblet cell Hypoxia-Inducible Factor (HIF) is necessary for mucin processing and maintenance of mucosal integrity

Abstract Secreted mucus is essential for intestinal homeostasis, separating luminal bacteria from the colonic epithelium and preventing inappropriate inflammation. Perturbations in goblet cell mucus secretion have been shown to lead to spontaneous colitis in mice. Treatment of mice with a prolyl hydroxylase inhibitor (AKB-4924) leads to stabilization of hypoxia inducible factor-1 alpha (HIF-1α) and regulation of hypoxia-responsive genes that promote mucosal protection. Interestingly, goblet cell hyperplasia was observed following in vivo administration of AKB-4924, which may account for the mucosal protection observed. Thus, we hypothesized epithelial stabilization of HIF-1α leads to goblet cell differentiation. Utilizing 3D murine colonic organoid system, treated with a gamma secretase inhibitor (DBZ), AKB-4924 or hypoxia, we analyzed changes in differentiation markers by qPCR. Contrary to our hypothesis, neither AKB-4924 nor hypoxia resulted in the induction of goblet cell differentiation factors. Furthermore, mice lacking HIF-1α in the intestinal epithelium (HIF-1αfl/fl-VillinCRE) exhibited normal colonic goblet cell numbers. However, these goblet cells appeared less organized with diffuse mucin granules. Fluorescent in situ hybridization with a eubacterial probe revealed diminished thickness, with reduced proximity to the microbiome in HIF−/− mice. Finally, profiling the composition of the mucus layer revealed significantly altered expression profile of both secreted and adherent mucins. We conclude that epithelial HIF-1α alters goblet cell function, regulating intestinal mucus, which may contribute to the mucosal-protective role of HIF.

780 Spontaneous Colitis in Mice Carrying GGTase-1 Deficient T-Lymphocytes

780 Spontaneous Colitis in Mice Carrying GGTase-1 Deficient T-Lymphocytes

P-162 Core 1- and Core 3-Derived Mucin-Type O-Glycans Collectively Maintain the Colonic Mucus Barrier and Protect Against Spontaneous Colitis in Mice

P-162 Core 1- and Core 3-Derived Mucin-Type O-Glycans Collectively Maintain the Colonic Mucus Barrier and Protect Against Spontaneous Colitis in Mice

Osteopontin Deficiency Accelerates Spontaneous Colitis in Mice with Disrupted Gut Microbiota and Macrophage Phagocytic Activity.

BackgroundOsteopontin (OPN) is a multifunctional protein expressed in a variety of tissues and cells. Recent studies revealed increased OPN expression in the inflamed intestinal tissues of patients with inflammatory bowel disease (IBD). The role of OPN in the pathophysiology of IBD, however, remains unclear.AimsTo investigate the role of OPN in the development of intestinal inflammation using a murine model of IBD, interleukin-10 knock out (IL-10 KO) mice.MethodsWe compared the development of colitis between IL-10 KO and OPN/IL-10 double KO (DKO) mice. OPN expression in the colonic tissues of IL-10 KO mice was examined by fluorescence in situ hybridization (FISH) analysis. Enteric microbiota were compared between IL-10 KO and OPN/IL-10 DKO mice by terminal restriction fragment length polymorphism analysis. The effect of OPN on macrophage phagocytic function was evaluated by phagocytosis assay.ResultsOPN/IL-10 DKO mice had an accelerated onset of colitis compared to IL-10 KO mice. FISH analysis revealed enhanced OPN synthesis in the colonic epithelial cells of IL-10 KO mice. OPN/IL-10 DKO mice had a distinctly different enteric bacterial profile with a significantly lower abundance of Clostridium subcluster XIVa and a greater abundance of Clostridium cluster XVIII compared to IL-10 KO mice. Intracellular OPN deletion in macrophages impaired phagocytosis of fluorescence particle-conjugated Escherichia coli in vitro. Exogenous OPN enhanced phagocytosis by OPN-deleted macrophages when administered at doses of 1 to 100 ng/ml, but not 1000 ng/ml.ConclusionsOPN deficiency accelerated the spontaneous development of colitis in mice with disrupted gut microbiota and macrophage phagocytic activity.

PHD3 Stabilizes the Tight Junction Protein Occludin and Protects Intestinal Epithelial Barrier Function

Prolyl hydroxylase domain proteins (PHDs) control cellular adaptation to hypoxia. PHDs are found involved in inflammatory bowel disease (IBD); however, the exact role of PHD3, a member of the PHD family, in IBD remains unknown. We show here that PHD3 plays a critical role in maintaining intestinal epithelial barrier function. We found that genetic ablation of Phd3 in intestinal epithelial cells led to spontaneous colitis in mice. Deletion of PHD3 decreases the level of tight junction protein occludin, leading to a failure of intestinal epithelial barrier function. Further studies indicate that PHD3 stabilizes occludin by preventing the interaction between the E3 ligase Itch and occludin, in a hydroxylase-independent manner. Examination of biopsy of human ulcerative colitis patients indicates that PHD3 is decreased with disease severity, indicating that PHD3 down-regulation is associated with progression of this disease. We show that PHD3 protects intestinal epithelial barrier function and reveal a hydroxylase-independent function of PHD3 in stabilizing occludin. These findings may help open avenues for developing a therapeutic strategy for IBD.

The Endoplasmic Reticulum Stress Sensor IRE1α in Intestinal Epithelial Cells Is Essential for Protecting against Colitis

Intestinal epithelial cells (IECs) have critical roles in maintaining homeostasis of intestinal epithelium. Endoplasmic reticulum (ER) stress is implicated in intestinal epithelium homeostasis and inflammatory bowel disease; however, it remains elusive whether IRE1α, a major sensor of ER stress, is directly involved in these processes. We demonstrate here that genetic ablation of Ire1α in IECs leads to spontaneous colitis in mice. Deletion of IRE1α in IECs results in loss of goblet cells and failure of intestinal epithelial barrier function. IRE1α deficiency induces cell apoptosis through induction of CHOP, the pro-apoptotic protein, and sensitizes cells to lipopolysaccharide, an endotoxin from bacteria. IRE1α deficiency confers upon mice higher susceptibility to chemical-induced colitis. These results suggest that IRE1α functions to maintain the intestinal epithelial homeostasis and plays an important role in defending against inflammation bowel diseases.

Nucleotide-Binding Oligomerization Domain 2 Signaling Promotes Hyperresponsive Macrophages and Colitis in IL-10–Deficient Mice

IL-10 contributes to the maintenance of intestinal homeostasis via the regulation of inflammatory responses to enteric bacteria. Loss of IL-10 signaling results in spontaneous colitis in mice and early onset enterocolitis in humans. Nucleotide-binding oligomerization domain (NOD) 2 is an intracellular receptor of bacterial peptidoglycan products, and, although NOD2 mutations are associated with Crohn's disease, the precise role of NOD2 in the development of intestinal inflammation remains undefined. To determine the role of NOD2 in the development of colitis on the clinically relevant genetic background of IL-10-deficient signaling, we generated mice lacking IL-10 and NOD2 (IL-10(-/-)NOD2(-/-)). Loss of NOD2 in IL-10(-/-) mice resulted in significant amelioration of chronic colitis, indicating that NOD2 signaling promotes the development of intestinal inflammation in IL-10(-/-) mice. Contrary to previous reports investigating immune function in NOD2(-/-) mice, T cell proliferative capacity and IL-2 production were not impaired, and immune polarization toward type 1 immunity was not affected. However, loss of NOD2 in IL-10-deficient macrophages reduced IL-6, TNF-α, and IL-12p40 production in response to bacterial stimulation. Further analysis of the intrinsic macrophage response before the onset of inflammation revealed that, in the absence of IL-10, synergistic signaling between various TLRs and NOD2 resulted in hyperresponsive, proinflammatory macrophages, thus providing the appropriate immune environment for the development of colitis. Data presented in this study demonstrate that NOD2 signaling contributes to intestinal inflammation that arises through loss of IL-10 and provides mechanistic insight into the development of colitis in inflammatory bowel disease patients with impaired IL-10 signaling.

Loss of intestinal core 1–derived O-glycans causes spontaneous colitis in mice

Mucin-type O-linked oligosaccharides (O-glycans) are primary components of the intestinal mucins that form the mucus gel layer overlying the gut epithelium. Impaired expression of intestinal O-glycans has been observed in patients with ulcerative colitis (UC), but its role in the etiology of this disease is unknown. Here, we report that mice with intestinal epithelial cell-specific deficiency of core 1-derived O-glycans, the predominant form of O-glycans, developed spontaneous colitis that resembled human UC, including massive myeloid infiltrates and crypt abscesses. The colitis manifested in these mice was also characterized by TNF-producing myeloid infiltrates in colon mucosa in the absence of lymphocytes, supporting an essential role for myeloid cells in colitis initiation. Furthermore, induced deletion of intestinal core 1-derived O-glycans caused spontaneous colitis in adult mice. These data indicate a causal role for the loss of core 1-derived O-glycans in colitis. Finally, we detected a biosynthetic intermediate typically exposed in the absence of core 1 O-glycan, Tn antigen, in the colon epithelium of a subset of UC patients. Somatic mutations in the X-linked gene that encodes core 1 β1,3-galactosyltransferase-specific chaperone 1 (C1GALT1C1, also known as Cosmc), which is essential for core 1 O-glycosylation, were found in Tn-positive epithelia. These data suggest what we believe to be a new molecular mechanism for the pathogenesis of UC.

Deletion of TLR5 results in spontaneous colitis in mice

Activation of TLRs by bacterial products results in rapid activation of genes encoding products designed to protect the host from perturbing microbes. In the intestine, which is colonized by a large and diverse population of commensal bacteria, TLR signaling may not function in a simple on/off mode. Here, we show that the flagellin receptor TLR5 has an essential and nonredundant role in protecting the gut from enteric microbes. Mice lacking TLR5 (TLR5KO mice) developed spontaneous colitis, as assessed by well-defined clinical, serologic, and histopathologic indicators of this disorder. Compared with WT littermates, TLR5KO mice that had not yet developed robust colitis exhibited decreased intestinal expression of TLR5-regulated host defense genes despite having an increased bacterial burden in the colon. In contrast, such TLR5KO mice displayed markedly increased colonic expression of hematopoietic-derived proinflammatory cytokines, suggesting that elevated levels of bacterial products may result in activation of other TLRs that drive colitis in TLR5KO mice. In accordance, deletion of TLR4 rescued the colitis of TLR5KO mice in that mice lacking both TLR4 and TLR5 also had elevated bacterial loads in the colon but lacked immunological, histopathological, and clinical evidence of colitis. That an engineered innate immune deficiency ultimately results in spontaneous intestinal inflammation supports the notion that an innate immune deficiency might underlie some instances of inflammatory bowel disease.