Desmoglein 2, but not desmocollin 2, protects intestinal epithelia from injury

Desmosomes are the least understood intercellular junctions in the intestinal epithelia and provide cell–cell adhesion via the cadherins desmoglein (Dsg)2 and desmocollin (Dsc)2. We studied these cadherins in Crohn's disease (CD) patients and in newly generated conditional villin-Cre DSG2 and DSC2 knockout mice (DSG2^ΔIEC; DSC2^ΔIEC). CD patients exhibited altered desmosomes and reduced Dsg2/Dsc2 levels. The intestines of both transgenic animal lines were histopathologically inconspicuous. However, DSG2^ΔIEC, but not DSC2^ΔIEC mice displayed an increased intestinal permeability, a wider desmosomal space as well as alterations in desmosomal and tight junction components. After dextran sodium sulfate (DSS) treatment and Citrobacter rodentium exposure, DSG2^ΔIEC mice developed a more-pronounced colitis, an enhanced intestinal epithelial barrier disruption, leading to a stronger inflammation and activation of epithelial pSTAT3 signaling. No susceptibility to DSS-induced intestinal injury was noted in DSC2^ΔIEC animals. Dsg2 interacted with the cytoprotective chaperone Hsp70. Accordingly, DSG2^ΔIEC mice had lower Hsp70 levels in the plasma membrane compartment, whereas DSC2^ΔIEC mice displayed a compensatory recruitment of galectin 3, a junction-tightening protein. Our results demonstrate that Dsg2, but not Dsc2 is required for the integrity of the intestinal epithelial barrier in vivo.     

IKKβ in intestinal epithelial cells regulates allergen-specific IgA and allergic inflammation at distant mucosal sites

Regulation of allergic responses by intestinal epithelial cells (IECs) remains poorly understood. Using a model of oral allergen sensitization in the presence of cholera toxin as adjuvant and mice with cell-specific deletion of inhibitor-κB kinase (IKKβ) in IECs (IKKβ^ΔIEC), we addressed the contribution of IECs to allergic sensitization to ingested antigens and allergic manifestations at distant mucosal site of the airways. Cholera toxin induced higher pro-inflammatory responses and altered the profile of the gut microbiota in IKKβ^ΔIEC mice. Antigen-specific immunoglobulin E (IgE) responses were unaltered in IKKβ^ΔIEC mice, but their IgA antibodies (Abs), T helper type 1 (Th1) and Th17 responses were enhanced. Upon nasal antigen challenge, these mice developed lower levels of allergic lung inflammation, which correlated with higher levels of IgA Abs in the airways. The IKKβ^ΔIEC mice also recruited a higher number of gut-sensitized T cells in the airways after nasal antigen challenge and developed airway hyper-responsiveness, which were suppressed by treatment with anti-interleukin-17A. Fecal microbiota transplant during allergic sensitization reduced Th17 responses in IKKβ^ΔIEC mice, but did not affect IgA Ab responses. In summary, we show that IKKβ in IECs shapes the gut microbiota and immune responses to ingested antigens and influences allergic responses in the airways via regulation of IgA Ab responses.     

Intestinal epithelial Caspase-8 signaling is essential to prevent necroptosis during Salmonella Typhimurium induced enteritis

Although induction of host cell death is a pivotal step during bacteria-induced gastroenteritis, the molecular regulation remains to be fully characterized. To expand our knowledge, we investigated the role of the central cell death regulator Caspase-8 in response to Salmonella Typhimurium. Here, we uncovered that intestinal salmonellosis was associated with strong upregulation of members of the host cell death machinery in intestinal epithelial cells (IECs) as an early event, suggesting that elimination of infected IECs represents a host defense strategy. Indeed, Casp8^∆IEC mice displayed severe tissue damage and high lethality after infection. Additional deletion of Ripk3 or Mlkl rescued epithelial cell death and lethality of Casp8^∆IEC mice, demonstrating the crucial role of Caspase-8 as a negative regulator of necroptosis. While Casp8^∆IECTnfr1^−/− mice showed improved survival after infection, tissue destruction was similar to Casp8^∆IEC mice, indicating that necroptosis partially depends on TNF-α signaling. Although there was no impairment in antimicrobial peptide secretion during the early phase of infection, functional Caspase-8 seems to be required to control pathogen colonization. Collectively, these results demonstrate that Caspase-8 is essential to prevent Salmonella Typhimurium induced enteritis and to ensure host survival by two different mechanisms: maintenance of intestinal barrier function and restriction of pathogen colonization.     

c-Jun N-terminal kinase 2 promotes enterocyte survival and goblet cell differentiation in the inflamed intestine

c-Jun N-terminal kinases (JNKs) contribute to immune signaling but their functional role during intestinal mucosal inflammation has remained ill defined. Using genetic mouse models, we characterized the role of JNK1 and JNK2 during homeostasis and acute colitis. Epithelial apoptosis, regeneration, differentiation, and barrier function were analyzed in intestinal epithelium-specific (ΔIEC) or complete JNK1 and bone marrow chimeric or complete JNK2 deficient mice as well as double-knockout animals (JNK1^ΔIECJNK2^−/−) during homeostasis and acute dextran sulfate sodium (DSS)-induced colitis. Results were confirmed using human HT-29 cells and wild-type or JNK2-deficient mouse intestinal organoid cultures. We show that nonhematopoietic JNK2 but not JNK1 expression confers protection from DSS-induced intestinal inflammation reducing epithelial barrier dysfunction and enterocyte apoptosis. JNK2 additionally enhanced Atonal homolog 1 expression, goblet cell and enteroendocrine cell differentiation, and mucus production under inflammatory conditions. Our results identify a protective role of epithelial JNK2 signaling to maintain mucosal barrier function, epithelial cell integrity, and mucus layer production in the event of inflammatory tissue damage.     

Epithelial RABGEF1 deficiency promotes intestinal inflammation by dysregulating intrinsic MYD88-dependent innate signaling

Intestinal epithelial cells (IECs) contribute to the regulation of intestinal homeostasis and inflammation through their interactions with the environment and host immune responses. Yet our understanding of IEC-intrinsic regulatory pathways remains incomplete. Here, we identify the guanine nucleotide exchange factor RABGEF1 as a regulator of intestinal homeostasis and innate pathways dependent on IECs. Mice with IEC-specific Rabgef1 deletion (called Rabgef1^IEC-KO mice) developed a delayed spontaneous colitis associated with the local upregulation of IEC chemokine expression. In mouse models of colitis based on Interleukin-10 deficiency or dextran sodium sulfate (DSS) exposure, we found that IEC-intrinsic RABGEF1 deficiency exacerbated development of intestinal pathology and dysregulated IEC innate pathways and chemokine expression. Mechanistically, we showed that RABGEF1 deficiency in mouse IECs in vitro was associated with an impairment of early endocytic events, an increased activation of the p38 mitogen-activated protein kinase (MAPK)-dependent pathway, and increased chemokine secretion. Moreover, we provided evidence that the development of spontaneous colitis was dependent on microbiota-derived signals and intrinsic MYD88-dependent pathways in vivo. Our study identifies mouse RABGEF1 as an important regulator of intestinal inflammation, MYD88-dependent IEC-intrinsic signaling, and chemokine production. This suggests that RABGEF1-dependent pathways represent interesting therapeutic targets for inflammatory conditions in the gut.     

Protein kinase CK2 is a critical regulator of epithelial homeostasis in chronic intestinal inflammation

The molecular mechanisms that restore intestinal epithelial homeostasis during colitis are incompletely understood. Here, we report that during intestinal inflammation, multiple inflammatory cytokines promote the activity of a master regulator of cell proliferation and apoptosis, serine/threonine kinase CK2. Enhanced mucosal CK2 protein expression and activity were observed in animal models of chronic colitis, particularly within intestinal epithelial cells (IECs). The in vitro treatment of intestinal epithelial cell lines with cytokines resulted in increased CK2 expression and nuclear translocation of its catalytic α subunit. Similarly, nuclear translocation of CK2α was a prominent feature observed in colonic crypts from individuals with ulcerative colitis and Crohn’s disease. Further in vitro studies revealed that CK2 activity promotes epithelial restitution, and protects normal IECs from cytokine-induced apoptosis. These observations identify CK2 as a key regulator of homeostatic properties of the intestinal epithelium that serves to promote wound healing, in part through inhibition of apoptosis under conditions of inflammation.     

Chronic intestinal inflammation in mice expressing viral Flip in epithelial cells

Viruses are present in the intestinal microflora and are currently discussed as a potential causative mechanism for the development of inflammatory bowel disease. A number of viruses, such as Human Herpesvirus-8, express homologs to cellular FLIPs, which are major contributors for the regulation of epithelial cell death. In this study we analyzed the consequences of constitutive expression of HHV8-viral FLIP in intestinal epithelial cells (IECs) in mice. Surprisingly, expression of vFlip disrupts tissue homeostasis and induces severe intestinal inflammation. Moreover vFlip^IEC-tg mice showed reduced Paneth cell numbers, associated with excessive necrotic cell death. On a molecular level vFlip expression altered classical and alternative NFκB activation. Blocking of alternative NFκB signaling by deletion of Ikka in vivo largely protected mice from inflammation and Paneth cell loss induced by vFLIP. Collectively, our data provide functional evidence that expression of a single viral protein in IECs can be sufficient to disrupt epithelial homeostasis and to initiate chronic intestinal inflammation.     

Intestinal epithelial cell-specific RARα depletion results in aberrant epithelial cell homeostasis and underdeveloped immune system

Retinoic acid (RA), a dietary vitamin A metabolite, is crucial in maintaining intestinal homeostasis. RA acts on intestinal leukocytes to modulate their lineage commitment and function. Although the role of RA has been characterized in immune cells, whether intestinal epithelial cells (IECs) rely on RA signaling to exert their immune-regulatory function has not been examined. Here we demonstrate that lack of RA receptor α (RARα) signaling in IECs results in deregulated epithelial lineage specification, leading to increased numbers of goblet cells and Paneth cells. Mechanistically, lack of RARα resulted in increased KLF4⁺ goblet cell precursors in the distal bowel, whereas RA treatment inhibited klf4 expression and goblet cell differentiation in zebrafish. These changes in secretory cells are associated with increased Reg3g, reduced luminal bacterial detection, and an underdeveloped intestinal immune system, as evidenced by an almost complete absence of lymphoid follicles and gut resident mononuclear phagocytes. This underdeveloped intestinal immune system shows a decreased ability to clear infection with Citrobacter rodentium. Collectively, our findings indicate that epithelial cell-intrinsic RARα signaling is critical to the global development of the intestinal immune system.     

Regulation of intestinal epithelial cell cytoskeletal remodeling by cellular immunity following gut infection

Gut infections often lead to epithelial cell damage followed by a healing response. We examined changes in the epithelial cell cytoskeleton and the involvement of host adaptive immunity in these events using an in vivo model of parasitic infection. We found that both ezrin and villin, key components of the actin cytoskeleton comprising the brush border (BB) of intestinal epithelial cells (IECs), underwent significant post-translational changes following gut infection and during the recovery phase of gut infection. Intriguingly, using mice lacking either CD4⁺ or CD8⁺ T-cell responses, we demonstrated that the mechanisms by which ezrin and villin are regulated in response to infection are different. Both ezrin and villin undergo proteolysis during the recovery phase of infection. Cleavage of ezrin requires CD4⁺ but not CD8⁺ T cells, whereas cleavage of villin requires both CD4⁺ and CD8⁺ T-cell responses. Both proteins were also regulated by phosphorylation; reduced levels of phosphorylated ezrin and increased levels of villin phosphorylation were observed at the peak of infection and correlated with reduced BB enzyme activity. Finally, we show that infection also leads to enhanced proliferation of IECs in this model. Cytoskeletal remodeling in IECs can have critical roles in the immunopathology and healing responses observed during many infectious and non-infectious intestinal conditions. These data indicate that cellular immune responses can be significant drivers of these processes.     

BVES is required for maintenance of colonic epithelial integrity in experimental colitis by modifying intestinal permeability

Blood vessel epicardial substance (BVES), or POPDC1, is a tight junction-associated transmembrane protein that modulates epithelial-to-mesenchymal transition (EMT) via junctional signaling pathways. There have been no in vivo studies investigating the role of BVES in colitis. We hypothesized that BVES is critical for maintaining colonic epithelial integrity. At baseline, Bves^−/− mouse colons demonstrate increased crypt height, elevated proliferation, decreased apoptosis, altered intestinal lineage allocation, and dysregulation of tight junctions with functional deficits in permeability and altered intestinal immunity. Bves^−/− mice inoculated with Citrobacter rodentium had greater colonic injury, increased colonic and mesenteric lymph node bacterial colonization, and altered immune responses after infection. We propose that increased bacterial colonization and translocation result in amplified immune responses and worsened injury. Similarly, dextran sodium sulfate (DSS) treatment resulted in greater histologic injury in Bves^−/− mice. Two different human cell lines (Caco2 and HEK293Ts) co-cultured with enteropathogenic E. coli showed increased attaching/effacing lesions in the absence of BVES. Finally, BVES mRNA levels were reduced in human ulcerative colitis (UC) biopsy specimens. Collectively, these studies suggest that BVES plays a protective role both in ulcerative and infectious colitis and identify BVES as a critical protector of colonic mucosal integrity.     

Intestinal Epithelium-Specific MyD88 Signaling Impacts Host Susceptibility to Infectious Colitis by Promoting Protective Goblet Cell and Antimicrobial Responses

Intestinal epithelial cells (IECs), including secretory goblet cells, form essential physiochemical barriers that separate luminal bacteria from underlying immune cells in the intestinal mucosa. IECs are common targets for enteric bacterial pathogens, with hosts responding to these microbes through innate toll-like receptors that predominantly signal through the MyD88 adaptor protein. In fact, MyD88 signaling confers protection against several enteric bacterial pathogens, including Salmonella enterica serovar Typhimurium and Citrobacter rodentium. Since IECs are considered innately hyporesponsive, it is unclear whether MyD88 signaling within IECs contributes to this protection. We infected mice lacking MyD88 solely in their IECs (IEC-Myd88^−/−) with S. Typhimurium. Compared to wild-type (WT) mice, infected IEC-Myd88^−/− mice suffered accelerated tissue damage, exaggerated barrier disruption, and impaired goblet cell responses (Muc2 and RELMβ). Immunostaining revealed S. Typhimurium penetrated the IECs of IEC-Myd88^−/− mice, unlike in WT mice, where they were sequestered to the lumen. When isolated crypts were assayed for their antimicrobial actions, crypts from IEC-Myd88^−/− mice were severely impaired in their antimicrobial activity against S. Typhimurium. We also examined whether MyD88 signaling in IECs impacted host defense against C. rodentium, with IEC-Myd88^−/− mice again suffering exaggerated tissue damage, impaired goblet cell responses, and reduced antimicrobial activity against C. rodentium. These results demonstrate that MyD88 signaling within IECs plays an important protective role at early stages of infection, influencing host susceptibility to infection by controlling the ability of the pathogen to reach and survive at the intestinal mucosal surface.     

Hematopoietic plakophilin‐3 regulates acute tissue‐specific and systemic inflammation in mice

Plakophilin‐3 (PKP3) is a member of the armadillo protein family, which is important in cell?cell contacts and signaling during development and tumorigenesis. In conventional facilities, PKP3‐deficient mice (PKP3^?/?) develop spontaneous dermatitis, indicating a possible involvement of PKP3 in inflammatory responses. Here, we show that PKP3 deficiency sensitizes mice to irritant contact dermatitis induced by phorbol myristate acetate (PMA). This sensitization occurred in mice with PKP3 deficiency in the hematopoietic system (PKP3^?/?hem), but not if the deficiency was specific to skin keratinocytes (PKP3^?/?ker). In a model of dextran sulfate sodium induced colitis, ubiquitous PKP3 deletion, but not intestinal epithelial PKP3 deficiency (PKP3^?/?IEC), impaired survival from disease. Interestingly, PKP3^?/?hem mice also displayed increased sensitivity to dextran sulfate sodium induced colitis. Finally, PKP3^?/? mice were more sensitive to the lethality of lipopolysaccharide (LPS) injection than wild‐type (WT) mice, and this phenotype was associated with increased intestinal permeability. PKP3^?/?IEC mice did not reproduce the enhanced endotoxin reactivity of PKP3^?/? mice, in contrast to PKP3^?/?hem mice. Finally, in vitro stimulation of WT neutrophils with LPS or PMA increased Pkp3 expression. In conclusion, our data highlight a novel role for hematopoietic PKP3 in the regulation of both locally and systemically induced immune responses. Nonetheless, further research is needed to unravel the underlying mechanism.     

Targeted deletion of MyD88 in intestinal epithelial cells results in compromised antibacterial immunity associated with downregulation of polymeric immunoglobulin receptor, mucin-2, and antibacterial peptides

Intestinal epithelial cells (IECs) form a physical and immunological barrier that separates the vast gut microbiota from host tissues. MyD88-dependent Toll-like receptor signaling is a key mediator of microbial-host cross-talk. We examined the role of epithelial MyD88 expression by generating mice with an IEC-targeted deletion of the Myd88 gene (MyD88(ΔIEC)). Loss of epithelial MyD88 signaling resulted in increased numbers of mucus-associated bacteria; translocation of bacteria, including the opportunistic pathogen Klebsiella pneumoniae, to mesenteric lymph nodes; reduced transmucosal electrical resistance; impaired mucus-associated antimicrobial activity; and downregulated expression of polymeric immunoglobulin receptor (the epithelial IgA transporter), mucin-2 (the major protein of intestinal mucus), and the antimicrobial peptides RegIIIγ and Defa-rs1. We further observed significant differences in the composition of the gut microbiota between MyD88(ΔIEC) mice and wild-type littermates. These physical, immunological, and microbial defects resulted in increased susceptibility of MyD88(ΔIEC) mice to experimental colitis. We conclude that MyD88 signaling in IECs is crucial for maintenance of gut homeostasis.