IL-1ß blockade prevents cell death and mucosal damage of the small intestine in a model of sterile inflammation.

The intestinal mucosa is covered by a layer of epithelial cells that is constantly challenged by commensal, opportunistic, and pathogenic microorganisms, their components, and harmful compounds. Any inflammatory response to these materials must be tightly controlled to limit tissue damage and restore the integrity of the mucosal barrier. We have shown previously that production of IL-1ß via activation of the inflammasome can lead to mucosal damage in the small intestinal pathology that occurs after intragastric administration of a gluten derived peptide, p31-43. Here we show that specific inhibition of caspase-1 or NLRP3 abolishes the damage induced by p31-43, and that antibody-mediated blocking of IL-1ß inhibits the both the histological changes and the induction of apoptosis and caspase-3 activation driven by p31-43. Understanding the role of IL-1ß in sterile inflammation may help to understand chronic inflammatory pathological processes, and design new intervention strategies.

Arachidonic acid effect on the allosteric gating mechanism of BK (Slo1) channels associated with the ß1 subunit.

Arachidonic acid (AA) is a fatty acid involved in the modulation of several ion channels. Previously, we reported that AA activates the high conductance Ca2+- and voltage-dependent K+ channel (BK) in vascular smooth muscle depending on the expression of the auxiliary ß1 subunit. Here, using the patch-clamp technique on BK channel co-expressed with ß1 subunit in a heterologous cell expression system, we analyzed whether AA modifies the three functional modules involved in the channel gating: the voltage sensor domain (VSD), the pore domain (PD), and the intracellular calcium sensor domain (CSD). We present evidence that AA activates BK channel in a direct way, inducing VSD stabilization on its active configuration observed as a significant left shift in the Q-V curve obtained from gating currents recordings. Moreover, AA facilitates the channel opening transitions when VSD are at rest, and the CSD are unoccupied. Furthermore, the activation was independent of the intracellular Ca2+ concentration and reduced when the BK channel was co-expressed with the Y74A mutant of the ß1 subunit. These results allow us to present new insigths in the mechanism by which AA modulates BK channels co-expressed with its auxiliary ß1 subunit.

Sterile inflammation drives multiple programmed cell death pathways in the gut.

Intestinal epithelial cells have a rapid turnover, being rapidly renewed by newly differentiated enterocytes, balanced by massive and constant removal of damaged cells by programmed cell death (PCD). The main forms of PCD are apoptosis, pyroptosis, and necroptosis, with apoptosis being a noninflammatory process, whereas the others drive innate immune responses. Although apoptosis is thought to be the principal means of cell death in the healthy intestine, which mechanisms are responsible for PCD during inflammation are not fully understood. To address this question, we used an in vivo model of enteropathy in wild-type mice induced by a single intragastric administration of the p31-43 gliadin peptide, which is known to elicit transient MyD88, NLRP3, and caspase-1-dependent mucosal damage and inflammation in the small intestine. Here, we found increased numbers of TUNEL+ cells in the mucosa as early as 2 h after p31-43 administration. Western blot and immunofluorescence analysis showed the presence of caspase-3-mediated apoptosis in the epithelium and lamina propria. In addition, the presence of mature forms of caspase-1, IL-1ß, and gasdermin D showed activation of pyroptosis and inhibition of caspase-1 led to decreased enterocyte death in p31-43-treated mice. There was also up-regulation of RIPK3 in crypt epithelium, suggesting that necroptosis was also occurring. Taken together, these results indicate that the inflammatory response induced by p31-43 can drive multiple PCD pathways in the small intestine.