Mechanosensing by Peyer's patch stroma regulates lymphocyte migration and mucosal antibody responses.

Fibroblastic reticular cells (FRCs) and their specialized collagen fibers termed 'conduits' form fundamental structural units supporting lymphoid tissues. In lymph nodes, conduits are known to transport interstitial fluid and small molecules from afferent lymphatics into the nodal parenchyma. However, the immunological contributions of conduit function have remained elusive. Here, we report that intestinal Peyer's patches (PPs) contain a specialized conduit system that directs the flow of water absorbed across the intestinal epithelium. Notably, PP FRCs responded to conduit fluid flow via the mechanosensitive ion channel Piezo1. Disruption of fluid flow or genetic deficiency of Piezo1 on CCL19-expressing stroma led to profound structural alterations in perivascular FRCs and associated high endothelial venules. This in turn impaired lymphocyte entry into PPs and initiation of mucosal antibody responses. These results identify a critical role for conduit-mediated fluid flow in the maintenance of PP homeostasis and mucosal immunity.

Stromal infrastructure of the lymph node and coordination of immunity.

The initiation of adaptive immune responses depends upon the careful maneuvering of lymphocytes and antigen into and within strategically placed lymph nodes (LNs). Non-hematopoietic stromal cells form the cellular infrastructure that directs this process. Once regarded as merely structural features of lymphoid tissues, these cells are now appreciated as essential regulators of immune cell trafficking, fluid flow, and LN homeostasis. Recent advances in the identification and in vivo targeting of specific stromal populations have resulted in striking new insights to the function of stromal cells and reveal a level of complexity previously unrealized. We discuss here recent discoveries that highlight the pivotal role that stromal cells play in orchestrating immune cell homeostasis and adaptive immunity.

Intestinal epithelial expression of TNFAIP3 results in microbial invasion of the inner mucus layer and induces colitis in IL-10-deficient mice.

Tumor necrosis factor-induced protein 3 (TNFAIP3; also known as A20) negatively regulates NF-κB and MAPK signals to control inflammatory responses. TNFAIP3 also protects against TNF-induced cell death. Intestinal epithelial cell (IEC) expression of TNFAIP3 improves barrier function and tight junction integrity and prevents dextran sulfate sodium (DSS)-induced IEC death and colitis. We therefore investigated the effects of TNFAIP3 expression in IEC on immune homeostasis in the intestines of immune-compromised mice. Villin-TNFAIP3 (v-TNFAIP3) transgenic mice were interbred with IL-10(-/-) mice (v-TNFAIP3 × IL-10(-/-)) and incidence, onset, and severity of colitis was assessed. v-TNFAIP3 × IL-10(-/-) mice displayed severe, early onset, and highly penetrant colitis that was not observed in IL-10(-/-) or v-TNFAIP3 mice. V-TNFAIP3 mice displayed altered expression of mucosal cytokines, increased numbers of mucosal regulatory T cells, and altered expression of mucosal antimicrobial peptides (AMPs). Microbial colonization of the inner mucus layer of v-TNFAIP3 mice was observed, along with alterations in the microbiome, but this was not sufficient to induce colitis in v-TNFAIP3 mice. The relative sterility of the inner mucus layer observed in wild-type and IL-10(-/-) mice was lost in v-TNFAIP3 × IL-10(-/-) mice. Thus IEC-derived factors, induced by signals that are inhibited by TNFAIP3, suppress the onset of inflammatory bowel disease in IL-10(-/-) mice. Our results indicate that IEC expression of TNFAIP3 alters AMP expression and allows microbial colonization of the inner mucus layer, which activates an IL-10-dependent anti-inflammatory process that is necessary to prevent colitis.

Expression of TNFAIP3 in intestinal epithelial cells protects from DSS- but not TNBS-induced colitis.

Intestinal epithelial cells (IEC) maintain gastrointestinal homeostasis by providing a physical and functional barrier between the intestinal lumen and underlying mucosal immune system. The activation of NF-κB and prevention of apoptosis in IEC are required to maintain the intestinal barrier and prevent colitis. How NF-κB activation in IEC prevents colitis is not fully understood. TNFα-induced protein 3 (TNFAIP3) is a NF-κB-induced gene that acts in a negative-feedback loop to inhibit NF-κB activation and also to inhibit apoptosis; therefore, we investigated whether TNFAIP3 expression in the intestinal epithelium impacts susceptibility of mice to colitis. Transgenic mice expressing TNFAIP3 in IEC (villin-TNFAIP3 Tg mice) were exposed to dextran sodium sulfate (DSS) or 2,4,6-trinitrobenzene sulfonic acid (TNBS), and the severity and characteristics of mucosal inflammation and barrier function were compared with wild-type mice. Villin-TNFAIP3 Tg mice were protected from DSS-induced colitis and displayed reduced production of NF-κB-dependent inflammatory cytokines. Villin-TNFAIP3 Tg mice were also protected from DSS-induced increases in intestinal permeability and induction of IEC death. Villin-TNFAIP3 Tg mice were not protected from colitis induced by TNBS. These results indicate that TNFAIP3 expression in IEC prevents colitis involving DSS-induced IEC death, but not colitis driven by T cell-mediated inflammation. As TNFAIP3 inhibits NF-κB activation and IEC death, expression of TNFAIP3 in IEC may provide an avenue to inhibit IEC NF-κB activation without inducing IEC death and inflammation.

TNFAIP3 maintains intestinal barrier function and supports epithelial cell tight junctions.

Tight junctions between intestinal epithelial cells mediate the permeability of the intestinal barrier, and loss of intestinal barrier function mediated by TNF signaling is associated with the inflammatory pathophysiology observed in Crohn's disease and celiac disease. Thus, factors that modulate intestinal epithelial cell response to TNF may be critical for the maintenance of barrier function. TNF alpha-induced protein 3 (TNFAIP3) is a cytosolic protein that acts in a negative feedback loop to regulate cell signaling induced by Toll-like receptor ligands and TNF, suggesting that TNFAIP3 may play a role in regulating the intestinal barrier. To investigate the specific role of TNFAIP3 in intestinal barrier function we assessed barrier permeability in TNFAIP3(-/-) mice and LPS-treated villin-TNFAIP3 transgenic mice. TNFAIP3(-/-) mice had greater intestinal permeability compared to wild-type littermates, while villin-TNFAIP3 transgenic mice were protected from increases in permeability seen within LPS-treated wild-type littermates, indicating that barrier permeability is controlled by TNFAIP3. In cultured human intestinal epithelial cell lines, TNFAIP3 expression regulated both TNF-induced and myosin light chain kinase-regulated tight junction dynamics but did not affect myosin light chain kinase activity. Immunohistochemistry of mouse intestine revealed that TNFAIP3 expression inhibits LPS-induced loss of the tight junction protein occludin from the apical border of the intestinal epithelium. We also found that TNFAIP3 deubiquitinates polyubiquitinated occludin. These in vivo and in vitro studies support the role of TNFAIP3 in promoting intestinal epithelial barrier integrity and demonstrate its novel ability to maintain intestinal homeostasis through tight junction protein regulation.

hPepT1 transports muramyl dipeptide, activating NF-kappaB and stimulating IL-8 secretion in human colonic Caco2/bbe cells.

BACKGROUND AND AIMS: Bacterial proteoglycan-derived muramyl dipeptide (MDP) activates the intracellular NOD2/CARD15 gene product. How intestinal epithelial cells take up MDP is poorly understood. We hypothesized that the intestinal apical di-/tripeptide transporter, hPepT1, transports MDP, thereby activating downstream pathways similar to nuclear factor kappa B (NF-kappaB). METHODS: Time- and concentration-dependent (3)H-MDP uptakes were studied in Caco2/bbe (C2) cell monolayers where hPepT1 expression was either over- or underexpressed, using an inducible adenovirus system or silencing RNA (siRNA), respectively. NF-kappaB activation and interleukin (IL)-8 and monocyte chemoattractant protein-1 (MCP-1) release were determined by enzyme-linked immunosorbent assay. NOD2/CARD15 expression was inhibited by siRNA. MDP in human duodenal, cecal, and stool samples was measured. RESULTS: MDP, but not its isoforms, inhibited uptake of glycosylsarcosine in C2 cells, indicating stereoselective and competitive inhibition. Approximately 90% of the MDP was cytosolic, showing uptake rather than binding. The K m for MDP uptake was 4.3 mmol/L. Cells overexpressing hPepT1 showed increased Gly-Sar and MDP uptake, whereas decreased uptake was observed after hPepT1 siRNA-inhibition. MDP treatment activated NF-kappaB, resulting in IL-8 release, an effect blocked by siRNA-inhibited expression of NOD2/CARD15. MDP content in cecal and stool samples (in normal subjects) was 20-87 micromol/L, but undetectable in duodenal fluid. CONCLUSIONS: In colonic epithelial cells, MDP is taken up by hPepT1 and activates NF-kappaB and chemokine production. Because hPepT1 expression in chronic colonic inflammation is increased, this may play an important role in promoting colonocyte participation in host defense and pathogen clearance through increased uptake of MDP.