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Background Bacterial flagellin is a major target of innate and adaptive immunity, both of which can promote and/or compensate for deficiencies in each other’s function. Methods To investigate the role of innate immune detection of flagellin irrespective of adaptive immunity, we examined the consequences of loss of Toll-like receptor 5 (T5) and/or Nod-like receptor 4 (N4) upon a Rag1-deficient background. Results Mice lacking Toll-like receptor 5 and Rag1 (T5/Rag-DKO) exhibited frequent lethal Pasteurellaceae-containing abscesses that prevented breeding of these mice. Mice lacking Toll-like receptor 5, Nod-like receptor 4, and Rag1 (T5/N4/Rag-TKO) also resulted in sporadic lethal abdominal abscesses caused by similar Pasteurellaceae. In the absence of such infections, relative to Rag1-KO, T5/N4/Rag-TKO mice exhibited microbiota encroachment, low-grade inflammation, microbiota dysbiosis, and, moreover were highly prone to Citrobacter infection and developed severe colitis when adoptively transferred with colitogenic T cells. Relative proneness of T5/N4/Rag-TKO mice to T-cell colitis was ablated by antibiotics while fecal microbiota transplant from T5/N4/Rag-TKO mice to wild-type mice transferred proneness to Citrobacter infection, indicating that dysbiosis in T5/N4/Rag-TKO mice contributed to these phenotypes. Conclusions These results demonstrate a critical role for innate immune detection of flagellin, especially in the intestinal tract and particularly in hosts deficient in adaptive immunity.

Background & Aims Nourishment of gut microbiota via consumption of fermentable fiber promotes gut health and guards against metabolic syndrome. In contrast, how dietary fiber impacts type 1 diabetes is less clear. Methods To examine impact of dietary fibers on development of type 1 diabetes in the streptozotocin (STZ)-induced and spontaneous non-obese diabetes (NOD) models, mice were fed grain-based chow (GBC) or compositionally defined diets enriched with a fermentable fiber (inulin) or an insoluble fiber (cellulose). Spontaneous (NOD mice) or STZ-induced (wild-type mice) diabetes was monitored. Results Relative to GBC, low-fiber diets exacerbated STZ-induced diabetes, whereas diets enriched with inulin, but not cellulose, strongly protected against or treated it. Inulin’s restoration of glycemic control prevented loss of adipose depots, while reducing food and water consumption. Inulin normalized pancreatic function and markedly enhanced insulin sensitivity. Such amelioration of diabetes was associated with alterations in gut microbiota composition and was eliminated by antibiotic administration. Pharmacologic blockade of fermentation reduced inulin’s beneficial impact on glycemic control, indicating a role for short-chain fatty acids (SCFA). Furthermore, inulin’s microbiota-dependent anti-diabetic effect associated with SCFA-independent restoration of interleukin 22, which was necessary and sufficient to ameliorate STZ-induced diabetes. Inulin-enriched diets significantly delayed diabetes in NOD mice. Conclusions Fermentable fiber confers microbiota-dependent increases in SCFA and interleukin 22 that, together, may have potential to prevent and/or treat type 1 diabetes., Graphical abstract

Bacterial flagellin can elicit production of TLR5-mediated IL-22 and NLRC4-mediated IL-18 cytokines that act in concert to cure and prevent rotavirus (RV) infection. This study investigated the mechanism by which these cyto-kines act to impede RV. Although IL-18 and IL-22 induce each other's expression, we found that IL-18 and IL-22 both impeded RV independently of one another and did so by distinct mechanisms that involved activation of their cognate receptors in intestinal epithelial cells (IEC). IL-22 drove IEC proliferation and migration toward villus tips, which resulted in increased extrusion of highly differentiated IEC that serve as the site of RV replication. In contrast, IL-18 induced cell death of RV-infected IEC thus directly interrupting the RV replication cycle, resulting in spewing of incompetent virus into the intestinal lumen and causing a rapid drop in the number of RV-infected IEC. Together, these actions resulted in rapid and complete expulsion of RV, even in hosts with severely compromised immune systems. These results suggest that a cocktail of IL-18 and IL-22 might be a means of treating viral infections that preferentially target short-lived epithelial cells.

Segmented filamentous bacteria (SFB) are well appreciated for eliciting Th17 cell immune responses. Here, we report the genome sequence of a murine isolate of SFB, which confers strong protection against rotavirus infection independent of acquired immunity.

Fermentable dietary fiber promotes insulin sensitivity thus mitigating type 2 diabetes. Here we report such protection extended to the streptozotocin (STZ)-induced model of type 1 diabetes. Specifically, low-fiber diets exacerbated STZ-induced diabetes while diets enriched with fermentable fiber strongly protected against, or treated, it. Fermentable fiber, inulin, but not insoluble fiber, cellulose, restored glycemic control and prevented loss of adipose depots, while reducing food and water consumption. Inulin normalized pancreatic function and markedly enhanced insulin sensitivity. Such amelioration of diabetes was associated with alterations in gut microbiota composition and was eliminated by antibiotic administration. Pharmacologic blockade of fermentation reduced inulin’s glycemic control benefits indicating a role for short-chain fatty acids in its beneficial action. Furthermore, inulin’s microbiota-dependent anti-diabetic impact correlated with restoration of IL-22, which was necessary and sufficient to ameliorate STZ-induced diabetes. Thus, fermentable fiber, SCFA, and/or IL-22 may have use in prevention and/or treatment of type 1 diabetes.

SummaryAdministration of bacterial flagellin elicits production of TLR5-mediated IL-22 and NLRC4-mediated IL-18 that act in concert to cure and prevent rotavirus (RV) infection. This study investigated the mechanism by which these cytokines act to impede this virus. Although IL-18 and IL-22 induce each other’s expression, we found that IL-18 and IL-22 both impeded RV independently of each other and did so by distinct mechanisms, in both cases via activation of their cognate receptors in intestinal epithelial cells (IEC). IL-22 drove IEC proliferation and migration toward villus tips, which resulted in increased extrusion of highly differentiated IEC that serve as the site of RV replication. In contrast, IL-18 induced pyroptotic death of RV-infected IEC thus directly interrupting the RV replication cycle, resulting in spewing of incompetent virus into the intestinal lumen and causing a rapid drop in levels of RV-infected IEC. Together, these actions resulted in rapid and complete expulsion of RV, even in hosts with severely compromised immune systems. These results suggest that IL-18/22 might be a means of treating viral infections that preferentially target short-lived epithelial cells.

Rotavirus (RV) encounters intestinal epithelial cells amidst diverse microbiota, opening possibilities of microbes influencing RV infection. Although RV clearance typically requires adaptive immunity, we unintentionally generated RV-resistant immunodeficient mice, which, we hypothesized, reflected select microbes protecting against RV. Accordingly, such RV resistance was transferred by co-housing and fecal transplant. RV-protecting microbiota were interrogated by heat, filtration, and antimicrobial agents, followed by limiting dilution transplant to germ-free mice and microbiome analysis. This approach revealed that segmented filamentous bacteria (SFB) were sufficient to protect mice against RV infection and associated diarrhea. Such protection was independent of previously defined RV-impeding factors, including interferon, IL-17, and IL-22. Colonization of the ileum by SFB induced changes in host gene expression and accelerated epithelial cell turnover. Incubation of RV with SFB-containing feces reduced infectivity in vitro, suggesting direct neutralization of RV. Thus, independent of immune cells, SFB confer protection against certain enteric viral infections and associated diarrheal disease.

Most viruses first encounter host cells at mucosal surfaces, which are typically colonized by a complex ecosystem of microbes collectively referred to as the microbiota. Recent studies demonstrate the microbiota plays an important role in mediating host–viral interactions and determining the outcomes of these encounters. This review outlines recently described examples of how bacteria and viruses impact each other particularly during infectious processes. Mechanistically, these effects can be broadly categorized as reflecting direct bacterial–viral interactions and/or involving microbial impacts upon innate and/or adaptive immunity.

The Kir4.1 channel is expressed in the brainstem, retina and kidney where it acts on K(+) transportation and pH-dependent membrane potential regulation. Its heteromerization with Kir5.1 leads to K(+) currents with distinct properties such as single-channel conductance, rectification, pH sensitivity and phosphorylation modulation. Here we show that Kir5.1 also enables S-glutathionylation to the heteromeric channel. Expressed in HEK cells, an exposure to the oxidant H(2)O(2) or diamide produced concentration-dependent inhibitions of the whole-cell Kir4.1-Kir5.1 currents. In inside-out patches, currents were inhibited strongly by a combination of diamide/GSH or H(2)O(2)/GSH but not by either alone. The currents were also suppressed by GSSG and the thiol oxidants pyridine disulfides (PDSs), suggesting S-glutathionylation. In contrast, none of the exposures had significant effects on the homomeric Kir4.1 channel. Cys158 in the TM2 helix of Kir5.1 was critical for the S-glutathionylation, which was accessible to intracellular but not extracellular oxidants. Site-directed mutagenesis of this residue (C158A or C158T) abolished the Kir4.1-Kir5.1 current modulation by oxidants, and eliminated almost completely the biochemical interaction of Kir5.1 with GSH. In tandem Kir4.1-Kir5.1 constructs, the channel with a single Cys158 was inhibited to the same degree as the wild-type channel, suggesting that one glutathione moiety is sufficient to block the channel. Consistent with the location of Cys158, GSSG inhibited the channel only when the channel was open, indicating that the channel inhibition was state dependent. The finding that the heteromeric Kir4.1-Kir5.1 channel but not the homomeric Kir4.1 is subject to the S-glutathionylation thus suggests a novel Kir4.1-Kir5.1 channel modulation mechanism that is likely to occur in oxidative stress.

BACKGROUND AND PURPOSE Rosiglitazone is an anti-diabetic drug acting as an insulin sensitizer. We recently found that rosiglitazone also inhibits the vascular isoform of ATP-sensitive K+ channels and compromises vasodilatory effects of β-adrenoceptor activation and pinacidil. As its potency for the channel inhibition is in the micromolar range, rosiglitazone may be used as an effective KATP channel inhibitor for research and therapeutic purposes. Therefore, we performed experiments to determine whether other isoforms of KATP channels are also sensitive to rosiglitazone and what their sensitivities are. EXPERIMENTAL APPROACH KIR6.1/SUR2B, KIR6.2/SUR1, KIR6.2/SUR2A, KIR6.2/SUR2B and KIR6.2ΔC36 channels were expressed in HEK293 cells and were studied using patch-clamp techniques. KEY RESULTS Rosiglitazone inhibited all isoforms of KATP channels in excised patches and in the whole-cell configuration. Its IC50 was 10 µmol·L−1 for the KIR6.1/SUR2B channel and ∼45 µmol·L−1 for KIR6.2/SURx channels. Rosiglitazone also inhibited KIR6.2ΔC36 channels in the absence of the sulphonylurea receptor (SUR) subunit, with potency (IC50= 45 µmol·L−1) almost identical to that for KIR6.2/SURx channels. Single-channel kinetic analysis showed that the channel inhibition was mediated by augmentation of the long-lasting closures without affecting the channel open state and unitary conductance. In contrast, rosiglitazone had no effect on KIR1.1, KIR2.1 and KIR4.1 channels, suggesting that the channel inhibitory effect is selective for KIR6.x channels. CONCLUSIONS AND IMPLICATIONS These results suggest a novel KATP channel inhibitor that acts on the pore-forming KIR6.x subunit, affecting the channel gating. LINKED ARTICLE This article is commented on by Dart, pp. 23–25 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2012.01990.x

Activators of innate immunity may have the potential to combat a broad range of infectious agents. We report that treatment with bacterial flagellin prevented rotavirus (RV) infection in mice and cured chronically RV-infected mice. Protection was independent of adaptive immunity and interferon (IFN, type I and II) and required flagellin receptors Toll-like receptor 5 (TLR5) and NOD-like receptor C4 (NLRC4). Flagellin-induced activation of TLR5 on dendritic cells elicited production of the cytokine interleukin-22 (IL-22), which induced a protective gene expression program in intestinal epithelial cells. Flagellin also induced NLRC4-dependent production of IL-18 and immediate elimination of RV-infected cells. Administration of IL-22 and IL-18 to mice fully recapitulated the capacity of flagellin to prevent or eliminate RV infection and thus holds promise as a broad-spectrum antiviral agent.

The Kir4.1 channel is expressed in the brainstem, retina and kidney where it acts on K(+) transportation and pH-dependent membrane potential regulation. Its heteromerization with Kir5.1 leads to K(+) currents with distinct properties such as single-channel conductance, rectification, pH sensitivity and phosphorylation modulation. Here we show that Kir5.1 also enables S-glutathionylation to the heteromeric channel. Expressed in HEK cells, an exposure to the oxidant H(2)O(2) or diamide produced concentration-dependent inhibitions of the whole-cell Kir4.1-Kir5.1 currents. In inside-out patches, currents were inhibited strongly by a combination of diamide/GSH or H(2)O(2)/GSH but not by either alone. The currents were also suppressed by GSSG and the thiol oxidants pyridine disulfides (PDSs), suggesting S-glutathionylation. In contrast, none of the exposures had significant effects on the homomeric Kir4.1 channel. Cys158 in the TM2 helix of Kir5.1 was critical for the S-glutathionylation, which was accessible to intracellular but not extracellular oxidants. Site-directed mutagenesis of this residue (C158A or C158T) abolished the Kir4.1-Kir5.1 current modulation by oxidants, and eliminated almost completely the biochemical interaction of Kir5.1 with GSH. In tandem Kir4.1-Kir5.1 constructs, the channel with a single Cys158 was inhibited to the same degree as the wild-type channel, suggesting that one glutathione moiety is sufficient to block the channel. Consistent with the location of Cys158, GSSG inhibited the channel only when the channel was open, indicating that the channel inhibition was state dependent. The finding that the heteromeric Kir4.1-Kir5.1 channel but not the homomeric Kir4.1 is subject to the S-glutathionylation thus suggests a novel Kir4.1-Kir5.1 channel modulation mechanism that is likely to occur in oxidative stress.