NLRP3 regulates a non-canonical platform for caspase-8 activation during epithelial cell apoptosis.

Nod-like receptor, pyrin containing 3 (NLRP3) is characterized primarily as a canonical caspase-1 activating inflammasome in macrophages. NLRP3 is also expressed in the epithelium of the kidney and gut; however, its function remains largely undefined. Primary mouse tubular epithelial cells (TEC) lacking Nlrp3 displayed reduced apoptosis downstream of the tumor necrosis factor (TNF) receptor and CD95. TECs were identified as type II apoptotic cells that activated caspase-8, tBid and mitochondrial apoptosis via caspase-9, responses that were reduced in Nlrp3-/- cells. The activation of caspase-8 during extrinsic apoptosis induced by TNFα/cycloheximide (TNFα/CHX) was dependent on adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC) and completely independent of caspase-1 or caspase-11. TECs and primary human proximal tubular epithelial cells (HPTC) did not activate a canonical inflammasome, caspase-1, or IL-1ß secretion in response to TNFα/CHX or NLRP3-dependent triggers, such as ATP or nigericin. In cell fractionation studies and by confocal microscopy, NLRP3 colocalized with ASC and caspase-8 in speck-like complexes at the mitochondria during apoptosis. The formation of NLRP3/ASC/caspase-8 specks in response to TNFα/CHX was downstream of TNFR signaling and dependent on potassium efflux. Epithelial ASC specks were present in enteroids undergoing apoptosis and in the injured tubules of wild-type but not Nlrp3-/- or ASC-/- mice following ureteric unilateral obstruction in vivo. These data show that NLRP3 and ASC form a conserved non-canonical platform for caspase-8 activation, independent of the inflammasome that regulates apoptosis within epithelial cells.

The goblet cell-derived mediator RELM-ß drives spontaneous colitis in Muc2-deficient mice by promoting commensal microbial dysbiosis.

Intestinal goblet cells are potentially key players in controlling susceptibility to ulcerative colitis (UC). Although impaired mucin (Muc2) production by goblet cells increases microbial stimulation of the colonic mucosa, goblet cells secrete other mediators that may influence or promote UC development. Correspondingly, Muc2-deficient ((-/-)) mice develop spontaneous colitis, concurrent with the dramatic upregulation of the goblet cell mediator, resistin-like molecule-beta (RELM-ß). Testing RELM-ß's role, we generated Muc2(-/-)/Retnlb(-/-) mice, finding that RELM-ß deficiency significantly attenuated colitis development and symptoms compared with Muc2(-/-) mice. RELM-ß expression in Muc2(-/-) mice strongly induced the production/secretion of the antimicrobial lectin RegIIIß, that exerted its microbicidal effect predominantly on Gram-positive Lactobacillus species. Compared with Muc2(-/-)/Retnlb(-/-) mice, this worsened intestinal microbial dysbiosis with a selective loss of colonic Lactobacilli spp. in Muc2(-/-) mice. Orally replenishing Muc2(-/-) mice with murine Lactobacillus spp., but not with a probiotic formulation containing several human Lactobacillus spp. (VSL#3), ameliorated their spontaneous colitis in concert with increased production of short-chain fatty acids. These studies demonstrate that the goblet cell mediator RELM-ß drives colitis in Muc2(-/-) mice by depleting protective commensal microbes. The ability of selective commensal microbial replacement to ameliorate colitis suggests that personalized bacterial therapy may prove beneficial for treatment of UC.

Vasoactive intestinal peptide prevents PKCε-induced intestinal epithelial barrier disruption during EPEC infection.

We previously showed that vasoactive intestinal peptide (VIP) protects against bacterial pathogen-induced epithelial barrier disruption and colitis, although the mechanisms remain poorly defined. The aim of the current study was to identify cellular pathways of VIP-mediated protection with use of pharmacological inhibitors during enteropathogenic Escherichia coli (EPEC) infection of Caco-2 cell monolayers and during Citrobacter rodentium-induced colitis. EPEC-induced epithelial barrier disruption involved the PKC pathway but was independent of functional cAMP, Rho, and NF-κB pathways. VIP mediated its protective effects by inhibiting EPEC-induced PKC activity and increasing expression of the junctional protein claudin-4. Short-term treatment with TPA, which is known to activate PKC, was inhibited by VIP pretreatment, while PKC degradation via long-term treatment with TPA mimicked the protective actions of VIP. Immunostaining for specific PKC isotypes showed upregulated expression of PKCθ and PKCε during EPEC infection. Treatment with specific inhibitors revealed a critical role for PKCε in EPEC-induced barrier disruption. Furthermore, activation of PKCε and loss of barrier integrity correlated with claudin-4 degradation. In contrast, inhibition of PKCε by VIP pretreatment or the PKCε inhibitor maintained membrane-bound claudin-4 levels, along with barrier function. Finally, in vivo treatment with the PKCε inhibitor protected mice from C. rodentium-induced colitis. In conclusion, EPEC infection increases intracellular PKCε levels, leading to decreased claudin-4 levels and compromising epithelial barrier integrity. VIP inhibits PKCε activation, thereby attenuating EPEC-induced barrier disruption.