Exaggerated IL-15 and Altered Expression of foxp3+ Cell-Derived Cytokines Contribute to Enhanced Colitis in Nlrp3-/- Mice.

The pathogenesis of Crohn's disease (CD) involves defects in the innate immune system, impairing responses to microbes. Studies have revealed that mutations NLRP3 are associated with CD. We reported previously that Nlrp3-/- mice were more susceptible to colitis and exhibited reduced colonic IL-10 expression. In the current study, we sought to determine how the loss of NLRP3 might be altering the function of regulatory T cells, a major source of IL-10. Colitis was induced in wild-type (WT) and Nlrp3-/- mice by treatment with dextran sulphate sodium (DSS). Lamina propria (LP) cells were assessed by flow cytometry and cytokine expression was assessed. DSS-treated Nlrp3-/- mice exhibited increased numbers of colonic foxp3+ T cells that expressed significantly lower levels of IL-10 but increased IL-17. This was associated with increased expression of colonic IL-15 and increased surface expression of IL-15 on LP dendritic cells. Neutralizing IL-15 in Nlrp3-/- mice attenuated the severity of colitis, decreased the number of colonic foxp3+ cells, and reduced the colonic expression of IL-12p40 and IL-17. These data suggest that the NLRP3 inflammasome can regulate intestinal inflammation through noncanonical mechanisms, providing additional insight as to how NLRP3 variants may contribute to the pathogenesis of CD.

The Arabidopsis C2H2 zinc finger INDETERMINATE DOMAIN1/ENHYDROUS promotes the transition to germination by regulating light and hormonal signaling during seed maturation.

Seed development ends with a maturation phase that imparts desiccation tolerance, nutrient reserves, and dormancy degree. Here, we report the functional analysis of an Arabidopsis thaliana C2H2 zinc finger protein INDETERMINATE DOMAIN1 (IDD1)/ENHYDROUS (ENY). Ectopic expression of IDD1/ENY (2x35S:ENY) disrupted seed development, delaying endosperm depletion and testa senescence, resulting in an abbreviated maturation program. Consequently, mature 2x35S:ENY seeds had increased endosperm-specific fatty acids, starch retention, and defective mucilage extrusion. Using RAB18 promoter ENY lines (RAB18:ENY) to confine expression to maturation, when native ENY expression increased and peaked, resulted in mature seed with lower abscisic acid (ABA) content and decreased germination sensitivity to applied ABA. Furthermore, results of far-red and red light treatments of 2x35S:ENY and RAB18:ENY germinating seeds, and of artificial microRNA knockdown lines, suggest that ENY acts to promote germination. After using RAB18:ENY seedlings to induce ENY during ABA application, key genes in gibberellin (GA) metabolism and signaling were differentially regulated in a manner suggesting negative feedback regulation. Furthermore, GA treatment resulted in a skotomorphogenic-like phenotype in light-grown 2x35S:ENY and RAB18:ENY seedlings. The physical interaction of ENY with DELLAs and an ENY-triggered accumulation of DELLA transcripts during maturation support the conclusion that ENY mediates GA effects to balance ABA-promoted maturation during late seed development.

NLRP3 inflammasome plays a key role in the regulation of intestinal homeostasis.

BACKGROUND: Attenuated innate immune responses to the intestinal microbiota have been linked to the pathogenesis of Crohn's disease (CD). Recent genetic studies have revealed that hypofunctional mutations of NLRP3, a member of the NOD-like receptor (NLR) superfamily, are associated with an increased risk of developing CD. NLRP3 is a key component of the inflammasome, an intracellular danger sensor of the innate immune system. When activated, the inflammasome triggers caspase-1-dependent processing of inflammatory mediators, such as IL-1ß and IL-18. METHODS: In the current study we sought to assess the role of the NLRP3 inflammasome in the maintenance of intestinal homeostasis through its regulation of innate protective processes. To investigate this role, Nlrp3(-/-) and wildtype mice were assessed in the dextran sulfate sodium and 2,4,6-trinitrobenzenesulfonic acid models of experimental colitis. RESULTS: Nlrp3(-/-) mice were found to be more susceptible to experimental colitis, an observation that was associated with reduced IL-1ß, reduced antiinflammatory cytokine IL-10, and reduced protective growth factor TGF-ß. Macrophages isolated from Nlrp3(-/-) mice failed to respond to bacterial muramyl dipeptide. Furthermore, Nlrp3-deficient neutrophils exhibited reduced chemotaxis and enhanced spontaneous apoptosis, but no change in oxidative burst. Lastly, Nlrp3(-/-) mice displayed altered colonic ß-defensin expression, reduced colonic antimicrobial secretions, and a unique intestinal microbiota. CONCLUSIONS: Our data confirm an essential role for the NLRP3 inflammasome in the regulation of intestinal homeostasis and provide biological insight into disease mechanisms associated with increased risk of CD in individuals with NLRP3 mutations.

Clostridium difficile toxin-induced inflammation and intestinal injury are mediated by the inflammasome.

BACKGROUND & AIMS: Clostridium difficile-associated disease (CDAD) is the leading cause of nosocomial diarrhea in the United States. C difficile toxins TcdA and TcdB breach the intestinal barrier and trigger mucosal inflammation and intestinal damage. The inflammasome is an intracellular danger sensor of the innate immune system. In the present study, we hypothesize that TcdA and TcdB trigger inflammasome-dependent interleukin (IL)-1beta production, which contributes to the pathogenesis of CDAD. METHODS: Macrophages exposed to TcdA and TcdB were assessed for IL-1beta production, an indication of inflammasome activation. Macrophages deficient in components of the inflammasome were also assessed. Truncated/mutated forms of TcdB were assessed for their ability to activate the inflammasome. The role of inflammasome signaling in vivo was assessed in ASC-deficient and IL-1 receptor antagonist-treated mice. RESULTS: TcdA and TcdB triggered inflammasome activation and IL-1beta secretion in macrophages and human mucosal biopsy specimens. Deletion of Nlrp3 decreased, whereas deletion of ASC completely abolished, toxin-induced IL-1beta release. TcdB-induced IL-1beta release required recognition of the full-length toxin but not its enzymatic function. In vivo, deletion of ASC significantly reduced toxin-induced inflammation and damage, an effect that was mimicked by pretreatment with the IL-1 receptor antagonist anakinra. CONCLUSIONS: TcdA and TcdB trigger IL-1beta release by activating an ASC-containing inflammasome, a response that contributes to toxin-induced inflammation and damage in vivo. Pretreating mice with the IL-1 receptor antagonist anakinra afforded the same level of protection that was observed in ASC-/- mice. These data suggest that targeting inflammasome or IL-1beta signaling may represent new therapeutic targets in the treatment of CDAD.