Proliferation in the developing intestine is regulated by the endosomal protein Endotubin.

During postnatal intestinal development, the intestinal epithelium is highly proliferative, and this proliferation is regulated by signaling in the intervillous and crypt regions. This signaling is primarily mediated by Wnt, and requires membrane trafficking. However, the mechanisms by which membrane trafficking regulates signaling during this developmental phase are largely unknown. Endotubin (EDTB, MAMDC4) is an endosomal protein that is highly expressed in the apical endocytic complex (AEC) of villus enterocytes during fetal and postnatal development, and knockout of EDTB results in defective formation of the AEC and giant lysosome. Further, knockout of EDTB in cell lines results in decreased proliferation. However, the role of EDTB in proliferation during the development of the intestine is unknown. Using Villin-CreERT2 in EDTBfl/fl mice, we deleted EDTB in the intestine in the early postnatal period, or in enteroids in vitro after isolation of intervillous cells. Loss of EDTB results in decreased proliferation in the developing intestinal epithelium and decreased ability to form enteroids. EDTB is present in cells that contain the stem cell markers LGR5 and OLFM4, indicating that it is expressed in the proliferative compartment. Further, using immunoblot analysis and TCF/LEF-GFP mice as a reporter of Wnt activity, we find that knockout of EDTB results in decreased Wnt signaling. Our results show that EDTB is essential for normal proliferation during the early stages of intestinal development and suggest that this effect is through modulation of Wnt signaling.

Intestinal Regulatory T Cells.

Mucosal surfaces are distinctive sites exposed to environmental, dietary, and microbial antigens. Particularly in the gut, the host continuously actively adapts via complex interactions between the microbiota and dietary compounds and immune and other tissue cells. Regulatory T cells (Tregs) are critical for tuning the intestinal immune response to self- and non-self-antigens in the intestine. Its importance in intestinal homeostasis is illustrated by the onset of overt inflammation caused by deficiency in Treg generation, function, or stability in the gut. A substantial imbalance in Tregs has been observed in intestinal tissue during pathogenic conditions, when a tightly regulated and equilibrated system becomes dysregulated and leads to unimpeded and chronic immune responses. In this chapter, we compile and critically discuss the current knowledge on the key factors that promote Treg-mediated tolerance in the gut, such as those involved in intestinal Treg differentiation, specificity and suppressive function, and their immunophenotype during health and disease. We also discuss the current state of knowledge on Treg dysregulation in human intestine during pathological states such as inflammatory bowel disease (IBD), necrotizing enterocolitis (NEC), graft-versus-host disease (GVHD), and colorectal cancer (CRC), and how that knowledge is guiding development of Treg-targeted therapies to treat or prevent intestinal disorders.

Emerging Roles of Disabled Homolog 2 (DAB2) in Immune Regulation.

Disabled-2 (DAB2) is a clathrin and cargo binding endocytic adaptor protein recognized for its multifaceted roles in signaling pathways involved in cellular differentiation, proliferation, migration, tumor suppression, and other fundamental homeostatic cellular mechanisms. The requirement for DAB2 in the canonical TGFß signaling in fibroblasts suggested that a similar mechanism may exist in immune cells and that DAB2 may contribute to immunological tolerance and suppression of inflammatory responses. In this review, we synthesize the current state of knowledge on the roles of DAB2 in the cells of the innate and adaptive immune system, with particular focus on antigen presenting cells (APCs; macrophages and dendritic cells) and regulatory T cells (Tregs). The emerging role of DAB2 in the immune system is that of an immunoregulatory molecule with significant roles in Treg-mediated immunosuppression, and suppression of TLR signaling in APC. DAB2 itself is downregulated by inflammatory stimuli, an event that likely contributes to the immunogenic function of APC. However, contrary findings have been described in neuroinflammatory disorders, thus suggesting a highly context-specific roles for DAB2 in immune cell regulation. There is need for better understanding of DAB2 regulation and its roles in different immune cells, their specialized sub-populations, and their responses under specific inflammatory conditions.

An indisputable role of NHE8 in mucosal protection.

The loss of the intestinal Na+/H+ exchanger isoform 8 (NHE8) results in an ulcerative colitis-like condition with reduction of mucin production and dysbiosis, indicating that NHE8 plays an important role in intestinal mucosal protection. The aim of this study was to investigate the potential rebalance of the altered microbiota community of NHE8-deficient mice via fecal microbiota transplantation (FMT) and feeding probiotic VSL#3. We also aimed to stimulate mucin production by sodium butyrate administration via enema. Data from 16S rRNA sequencing showed that loss of NHE8 contributes to colonic microbial dysbiosis with reduction of butyrate-producing bacteria. FMT increased bacterial adhesion in the colon in NHE8 knockout (NHE8KO) mice. Periodic-acid Schiff reagent (PAS) stain and quantitative PCR showed no changes in mucin production during FMT. In mice treated with the probiotic VSL#3, a reduction of Lactobacillus and segmented filamentous bacteria (SFB) in NHE8KO mouse colon was detected and an increase in goblet cell theca was observed. In NHE8KO mice receiving sodium butyrate (NaB), 1 mM NaB stimulated Muc2 expression without changing goblet cell theca, but 10 mM NaB induced a significant reduction of goblet cell theca without altering Muc2 expression. Furthermore, 5 mM and 10 mM NaB-treated HT29-MTX cells displayed increased apoptosis, while 0.5 mM NaB stimulated Muc2 gene expression. These data showed that loss of NHE8 leads to dysbiosis with reduction of butyrate-producing bacteria and FMT and VSL#3 failed to rebalance the microbiota in NHE8KO mice. Therefore, FMT, VSL#3, and NaB are not able to restore mucin production in the absence of NHE8 in the intestine.NEW & NOTEWORTHY Loss of Na+/H+ exchanger isoform 8 (NHE8), a Slc9 family of exchanger that contributes to sodium uptake, cell volume regulation, and intracellular pH homeostasis, resulted in dysbiosis with reduction of butyrate-producing bacteria and decrease of Muc2 production in the intestine in mice. Introducing fecal microbiota transplantation (FMT) and VSL#3 in NHE8 knockout (NHE8KO) mice failed to rebalance the microbiota in these mice. Furthermore, administration of FMT, VSL#3, and sodium butyrate was unable to restore mucin production in the absence of NHE8 in the intestine.

Intestinal Epithelial Expression of MHCII Determines Severity of Chemical, T-Cell-Induced, and Infectious Colitis in Mice.

BACKGROUND & AIMS: Intestinal epithelial cells (IECs) provide a barrier that separates the mucosal immune system from the luminal microbiota. IECs constitutively express low levels of major histocompatibility complex (MHC) class II proteins, which are upregulated upon exposure to interferon gamma. We investigated the effects of deleting MHCII proteins specifically in mice with infectious, dextran sodium sulfate (DSS)-, and T-cell-induced colitis. METHODS: We disrupted the histocompatibility 2, class II antigen A, beta 1 gene (H2-Ab1) in IECs of C57BL/6 mice (I-AbΔIEC) or Rag1-/- mice (Rag1-/-I-AbΔIEC); we used I-AbWT mice as controls. Colitis was induced by administration of DSS, transfer of CD4+CD45RBhi T cells, or infection with Citrobacter rodentium. Colon tissues were collected and analyzed by histology, immunofluorescence, xMAP, and reverse-transcription polymerase chain reaction and organoids were generated. Microbiota (total and immunoglobulin [Ig]A-coated) in intestinal samples were analyzed by16S amplicon profiling. IgA+CD138+ plasma cells from Peyer's patches and lamina propria were analyzed by flow cytometry and IgA repertoire was determined by next-generation sequencing. RESULTS: Mice with IEC-specific loss of MHCII (I-AbΔIEC mice) developed less severe DSS- or T-cell transfer-induced colitis than control mice. Intestinal tissues from I-AbΔIEC mice had a lower proportion of IgA-coated bacteria compared with control mice, and a reduced luminal concentration of secretory IgA (SIgA) following infection with C rodentium. There was no significant difference in the mucosal IgA repertoire of I-AbΔIEC vs control mice, but opsonization of cultured C rodentium by SIgA isolated from I-AbΔIEC mice was 50% lower than that of SIgA from mAbWT mice. Fifty percent of I-AbΔIEC mice died after infection with C rodentium, compared with none of the control mice. We observed a transient but significant expansion of the pathogen in the feces of I-AbΔIEC mice compared with I-AbWT mice. CONCLUSIONS: In mice with DSS or T-cell-induced colitis, loss of MHCII from IECs reduces but does not eliminate mucosal inflammation. However, in mice with C rodentium-induced colitis, loss of MHCII reduces bacterial clearance by decreasing binding of IgA to commensal and pathogenic bacteria.

Rapid Downregulation of DAB2 by Toll-Like Receptor Activation Contributes to a Pro-Inflammatory Switch in Activated Dendritic Cells.

Dendritic cells (DCs) are pivotal in regulating tolerogenic as well as immunogenic responses against microorganisms by directing both the innate and adaptive immune response. In health, phenotypically different DC subsets found in the gut mucosa are maintained in their tolerogenic state but switch to a pro-inflammatory phenotype during infection or chronic autoinflammatory conditions such as inflammatory bowel disease (IBD). The mechanisms that promote the switch among the mucosal DCs from a tolerogenic to an immunogenic, pro-inflammatory phenotype are incompletely understood. We hypothesized that disabled homolog 2 (DAB2), recently described as a negative regulator of DC immunogenicity during their development, is regulated during intestinal inflammation and modulates mucosal DC function. We show that DAB2 is highly expressed in colonic CD11b+CD103- DCs, a subset known for its capacity to induce inflammatory Th1/Th17 responses in the colon, and is downregulated predominantly in this DC subset during adoptive T cell transfer colitis. Administration of Dab2-deficient DCs (DC2.4 Dab2-/- cells) modulated the course of DSS colitis in wild-type mice, enhanced mucosal expression of Tnfa, Il6, and Il17a, and promoted neutrophil recruitment. In bone-marrow derived dendritic cells (BMDC), DAB2 expression correlated with CD11b levels and DAB2 was rapidly and profoundly inhibited by TLR ligands in a TRIF- and MyD88-dependent manner. The negative modulation of DAB2 was biphasic, initiated with a quick drop in DAB2 protein, followed by a sustained reduction in Dab2 mRNA. DAB2 downregulation promoted a more functional and activated DC phenotype, reduced phagocytosis, and increased CD40 expression after TLR activation. Furthermore, Dab2 knockout in DCs inhibited autophagy and promoted apoptotic cell death. Collectively, our results highlight the immunoregulatory role for DAB2 in the intestinal dendritic cells and suggest that DAB2 downregulation after microbial exposure promotes their switch to an inflammatory phenotype.

Interaction and involvement of cellular adhesion molecules in the pathogenesis of Schistosomiasis mansoni.

Parasites from genus Schistosoma currently infect more than 200 million people worldwide. Infection with Schistosoma mansoni causes intestinal schistosomiasis with geographical distribution across Africa, Middle East, Caribbean, Brazil, Venezuela and Suriname. People with Schistosomiasis mansoni suffer from a chronic disease as result of an exacerbated immune response to the eggs deposited in hepatic tissue. The presence of eggs in the tissue triggers the recruitment and activation of immune cells to wall off and isolate them from the rest of the organism. In this context, immune cells turn activated and increase the expression of cellular adhesion molecules (CAM), such as l-selectin and LFA-1, and DC-SIGN which through interaction with CAM expressed on activated endothelial vessels, help moving leukocytes quickly to the sites of infection (inflammation around the eggs), as a strategy to defend the organism from foreign invaders. Since the vertebrate host is not able to eliminate the foreign invader a granuloma formation take place in the tissue where the eggs are trapped, originating granulomas. Patients and mice with chronic schistosomiasis have increased levels of CAM in their circulation and egg-trapped tissue, which may contribute to the inflammatory process, granuloma formation and pathology aggravation. Here we systematically reviewed the findings raised over the last two decades that addressed the involvement of cellular adhesion molecules in the intestinal and hepatic inflammatory response and liver granuloma formation during Schistosomiasis mansoni. This review intends to contribute to the understanding of Schistosomiasis mansoni pathogenesis by discussing alterations and interactions in cellular adhesion molecules during the disease.