Colitis-induced upregulation of tumor necrosis factor receptor-2 (TNFR2) terminates epithelial regenerative signaling to restore homeostasis.

Colonic epithelial repair is a key determinant of health. Repair involves changes in epithelial differentiation, an extensive proliferative response, and upregulation of regeneration-associated "fetal-like" transcripts, including Ly6a (Sca-1), that represent Yap1 and interferon targets. However, little is known about how this regenerative program terminates and how homeostasis is restored during injury and inflammation. Here we show that, after the initial entry into the regenerative state, the subsequent upregulation of tumor necrosis factor (TNF) receptor 2 (R2, TNFR2, Tnfrsf1b) clears the regenerative signaling and restores homeostatic patterns of epithelial differentiation. Targeted deletion of epithelial TNFR2 in vivo and in colonoid cultures revealed persistent expression of Ly6a, hyperproliferation, and reduced secretory differentiation. Moreover, mice lacking epithelial TNFR2 also failed to complete colon ulcer healing, suggesting that partial resolution of regenerative signaling is essential for the completion of the repair process. These results demonstrate how epithelial cells dynamically leverage a colitis-associated cytokine to choreograph repair.

Epithelial dysfunction is prevented by IL-22 treatment in a Citrobacter rodentium-induced colitis model that shares similarities with inflammatory bowel disease.

Inflammatory bowel disease (IBD) is characterized by a dysregulated intestinal epithelial barrier leading to breach of barrier immunity. Here we identified similar protein expression changes between IBD and Citrobacter rodentium-infected FVB mice with respect to dysregulation of solute transporters as well as components critical for intestinal barrier integrity. We attribute the disease associated changes in the model to the emergence of undifferentiated intermediate intestinal epithelial cells. Prophylactic treatment with IL-22.Fc in C. rodentium-infected FVB mice reduced disease severity and rescued the mice from lethality. Multi-omics and solute analyses revealed that IL-22.Fc treatment prevented disease-associated changes including disruption of the solute transporter machinery and restored proper physiological functions of the intestine, respectively. Taken together, we established the disease relevance of the C. rodentium-induced colitis model to IBD, demonstrated the protective role of IL-22 in amelioration of epithelial dysfunction and elucidated the molecular mechanisms with IL-22's effect on intestinal epithelial cells.

TNF Receptor 1 Promotes Early-Life Immunity and Protects against Colitis in Mice.

Neutralization of tumor necrosis factor (TNF) represents a widely used therapeutic strategy for autoimmune diseases including inflammatory bowel disease (IBD). However, the fact that many patients with IBD are non-responsive to anti-TNF therapies suggests the need for a better understanding of TNF signaling in IBD. Here, we show that co-deletion of TNF receptor 1 (TNFR1, Tnfrsf1a) in the Il10-/- spontaneous colitis model exacerbates disease, resulting in very-early-onset inflammation after weaning. The disease can be interrupted by treatment with antibiotics. The single deletion of TNFR1 induces subclinical colonic epithelial dysfunction and mucosal immune abnormalities, including accumulation of neutrophils and depletion of B cells. During the pre-disease period (before weaning), both Tnfr1-/- and Il10-/-Tnfr1-/- animals exhibit impaired expression of pro-inflammatory cytokines compared with wild-type and Il10-/- controls, respectively. Collectively, these results demonstrate the net anti-inflammatory functions of TNF/TNFR1 signaling through the regulation of colonic immune homeostasis in early life.

ErbB4 signaling stimulates pro-inflammatory macrophage apoptosis and limits colonic inflammation.

Efficient clearance of pro-inflammatory macrophages from tissues after resolution of a challenge is critical to prevent prolonged inflammation. Defects in clearance can contribute to conditions such as inflammatory bowel disease, and thus may be therapeutically targetable. However, the signaling pathways that induce termination of pro-inflammatory macrophages are incompletely defined. We tested whether the ErbB4 receptor tyrosine kinase, previously not known to have role in macrophage biology, is involved in this process. In vitro, pro-inflammatory activation of cultured murine and human macrophages induced ErbB4 expression; in contrast, other ErbB family members were not induced in pro-inflammatory cells, and other innate immune lineages (dendritic cells, neutrophils) did not express detectable ErbB4 levels. Treatment of activated pro-inflammatory macrophages with the ErbB4 ligand neuregulin-4 (NRG4) induced apoptosis. ErbB4 localized to the mitochondria in these cells. Apoptosis was accompanied by loss of mitochondrial membrane potential, and was dependent upon the proteases that generate the cleaved ErbB4 intracellular domain fragment, suggesting a requirement for this fragment and mitochondrial pathway apoptosis. In vivo, ErbB4 was highly expressed on pro-inflammatory macrophages but not neutrophils during experimental DSS colitis in C57Bl/6 mice. Active inflammation in this model suppressed NRG4 expression, which may allow for macrophage persistence and ongoing inflammation. Consistent with this notion, NRG4 levels rebounded during the recovery phase, and administration of exogenous NRG4 during colitis reduced colonic macrophage numbers and ameliorated inflammation. These data define a novel role for ErbB4 in macrophage apoptosis, and outline a mechanism of feedback inhibition that may promote resolution of colitis.

Tumor Necrosis Factor Receptor 2 Restricts the Pathogenicity of CD8(+) T Cells in Mice With Colitis.

BACKGROUND & AIMS: Tumor necrosis factor receptor 2 (TNFR2, Tnfrsf1b) regulates multiple aspects of immune function, but little is known about its role in the immunopathogenesis of inflammatory bowel disease (IBD). We investigated whether TNFR2 restricts the activity of specific immune cell subtypes to protect against the development of colitis in mice. METHODS: Tnfr2(-/-) mice were crossed with interleukin (Il) 10(-/-) mice, which spontaneously develop colitis, to generate Il10(-/-)Tnfr2(-/-) mice. Colonic tissues were collected from Il10(-/-)Tnfr2(-/-) mice along with Il10(-/-) mice (controls) and analyzed by flow cytometry and histology. Bone marrow was transplanted into Il10(-/-) and Il10(-/-)Tnfr2(-/-) mice from Il10(-/-) or Il10(-/-)Tnfr2(-/-) donors by intravenous injection. CD8(+) T cells were neutralized in Il10(-/-)Tnfr2(-/-) mice by intraperitoneal injection of anti-CD8 or isotype control antibodies. Colitis was induced in Rag2(-/-) mice by intravenous injections of naïve CD8(+) T cells isolated from C57BL/6 or Tnfr2(-/-) mice. RESULTS: Il10(-/-)Tnfr2(-/-) mice spontaneously developed more severe colitis compared with Il10(-/-) controls, characterized by selective expansion of colonic CD8(+) T cells. Transplantation of TNFR2-deficient bone marrow resulted in significantly increased incidence and severity of colitis. Transcriptome analyses showed that the expression of genes regulated by TNFR2 were specific to CD8(+) T cells and included genes associated with risk for IBD. Depletion of CD8(+) T cells from Il10(-/-)Tnfr2(-/-) mice prevented colonic inflammation. Adoptive transfer of TNFR2-null naïve CD8(+) T cells compared with CD8(+) T cells from control mice increased the severity of colitis that developed in Rag2(-/-) mice. CONCLUSIONS: TNFR2 protects mice from colitis by inhibiting the expansion of colonic CD8(+) T cells. TNFR2 regulates expression of genes that regulate CD8(+) T cells and have been associated with susceptibility to IBD. Disruption in TNFR2 signaling might therefore be associated with pathogenesis. Strategies to increase levels or activity of TNFR2 and thereby reduce the activity of CD8(+) T cells might be developed to treat IBD patients with CD8(+) T cell dysfunction.

Redeeming an old foe: protective as well as pathophysiological roles for tumor necrosis factor in inflammatory bowel disease.

Tumor necrosis factor (TNF) and its receptors TNFR1 and TNFR2 are major therapeutic targets for inflammatory bowel disease. Research advances have demonstrated that TNF produces pleiotropic responses in the gastrointestinal (GI) tract. Although in excess TNF can contribute to GI pathology, TNF is also a critical protective factor to promote GI homeostasis following injury and inflammation. Genetic studies using candidate and genome-wide association study approaches have identified variants in TNF or its receptors that are associated with Crohn's disease or ulcerative colitis in multiple populations, although the basis for these associations remains unclear. This review considers the efficacy and mechanism of anti-TNF therapies for inflammatory bowel disease to reconcile the many disparate aspects of TNF research and to consider the potential protective effects of TNF signaling in GI health.

Epidermal growth factor receptor inhibits colitis-associated cancer in mice.

Inflammatory bowel disease (IBD) is a chronic illness caused by complex interactions between genetic and environmental factors that propagate inflammation and damage to the gastrointestinal epithelium. This state of chronic inflammation increases the risk for development of colitis-associated cancer in IBD patients. Thus, the development of targeted therapeutics that can disrupt the cycle of inflammation and epithelial injury is highly attractive. However, such biological therapies, including those targeting epidermal growth factor receptor pathways, pose a risk of increasing cancer rates. Using two mouse models of colitis-associated cancer, we found that epidermal growth factor receptor inactivation accelerated the incidence and progression of colorectal tumors. By modulating inflammation and epithelial regeneration, epidermal growth factor receptor optimized the response to chronic inflammation and limited subsequent tumorigenesis. These findings provide important insights into the pathogenesis of colitis-associated cancer and suggest that epidermal growth factor-based therapies for IBD may reduce long-term cancer risk.

Bifidobacterium animalis subsp. lactis fermented milk product reduces inflammation by altering a niche for colitogenic microbes.

Intestinal health requires the coexistence of eukaryotic self with the gut microbiota and dysregulated host-microbial interactions can result in intestinal inflammation. Here, we show that colitis improved in T-bet(-/-)Rag2(-/-) mice that consumed a fermented milk product containing Bifidobacterium animalis subsp. lactis DN-173 010 strain. A decrease in cecal pH and alterations in short chain fatty acid profiles occurred with consumption, and there were concomitant increases in the abundance of select lactate-consuming and butyrate-producing bacteria. These metabolic shifts created a nonpermissive environment for the Enterobacteriaceae recently identified as colitogenic in a T-bet(-/-)Rag2(-/-) ulcerative colitis mouse model. In addition, 16S rRNA-based analysis of the T-bet(-/-)Rag2(-/-) fecal microbiota suggest that the structure of the endogenous gut microbiota played a key role in shaping the host response to the bacterial strains studied herein. We have identified features of the gut microbiota, at the membership and functional level, associated with response to this B. lactis-containing fermented milk product, and therefore this model provides a framework for evaluating and optimizing probiotic-based functional foods.

Enterobacteriaceae act in concert with the gut microbiota to induce spontaneous and maternally transmitted colitis.

Disruption of homeostasis between the host immune system and the intestinal microbiota leads to inflammatory bowel disease (IBD). Whether IBD is instigated by individual species or disruptions of entire microbial communities remains controversial. We characterized the fecal microbial communities in the recently described T-bet(-/-) ×Rag2(-/-) ulcerative colitis (TRUC) model driven by T-bet deficiency in the innate immune system. 16S rRNA-based analysis of TRUC and Rag2(-/-) mice revealed distinctive communities that correlate with host genotype. The presence of Klebsiella pneumoniae and Proteus mirabilis correlates with colitis in TRUC animals, and these TRUC-derived strains can elicit colitis in Rag2(-/-) and WT adults but require a maternally transmitted endogenous microbial community for maximal intestinal inflammation. Cross-fostering experiments indicated a role for these organisms in maternal transmission of disease. Our findings illustrate how gut microbial communities work in concert with specific culturable colitogenic agents to cause IBD.

Colitis-associated colorectal cancer driven by T-bet deficiency in dendritic cells.

We previously described a mouse model of ulcerative colitis linked to T-bet deficiency in the innate immune system. Here, we report that the majority of T-bet(-/-)RAG2(-/-) ulcerative colitis (TRUC) mice spontaneously progress to colonic dysplasia and rectal adenocarcinoma solely as a consequence of MyD88-independent intestinal inflammation. Dendritic cells (DCs) are necessary cellular effectors for a proinflammatory program that is carcinogenic. Whereas these malignancies arise in the setting of a complex inflammatory environment, restoration of T-bet selectively in DCs was sufficient to reduce colonic inflammation and prevent the development of neoplasia. TRUC colitis-associated colorectal cancer resembles the human disease and provides ample opportunity to probe how inflammation drives colorectal cancer development and to test preventative and therapeutic strategies preclinically.

Communicable ulcerative colitis induced by T-bet deficiency in the innate immune system.

Inflammatory bowel disease (IBD) has been attributed to overexuberant host immunity or the emergence of harmful intestinal flora. The transcription factor T-bet orchestrates inflammatory genetic programs in both adaptive and innate immunity. We describe a profound and unexpected function for T-bet in influencing the behavior of host inflammatory activity and commensal bacteria. T-bet deficiency in the innate immune system results in spontaneous and communicable ulcerative colitis in the absence of adaptive immunity and increased susceptibility to colitis in immunologically intact hosts. T-bet controls the response of the mucosal immune system to commensal bacteria by regulating TNF-alpha production in colonic dendritic cells, critical for colonic epithelial barrier maintenance. Loss of T-bet influences bacterial populations to become colitogenic, and this colitis is communicable to genetically intact hosts. These findings reveal a novel function for T-bet as a peacekeeper of host-commensal relationships and provide new perspectives on the pathophysiology of IBD.