Prolonged ethanol exposure causes central nervous system hyperexcitability that involves a loss of GABAergic inhibition. We previously demonstrated that long-term ethanol exposure enhances the internalization of synaptic GABA A receptors composed of ␣1␤2/3␥2 subunits. However, the mechanisms of ethanol-mediated internalization are unknown. This study explored the effect of ethanol on surface expression of GABA A ␣1 subunit-containing receptors in cultured cerebral cortical neurons and the role of protein kinase C (PKC) ␤, ␥, and isoforms in their trafficking. Cultured neurons were prepared from rat pups on postnatal day 1 and maintained for 18 days. Cells were exposed to ethanol, and surface receptors were isolated by biotinylation and P2 fractionation, whereas functional analysis was conducted by whole-cell patch-clamp recording of GABAand zolpidem-evoked responses. Ethanol exposure for 4 h decreased biotinylated surface expression of GABA A receptor ␣1 subunits and reduced zolpidem (100 nM) enhancement of GABA-evoked currents. The PKC activator phorbol-12,13-dibutyrate mimicked the effect of ethanol, and the selective PKC inhibitor calphostin C prevented ethanol-induced internalization of these receptors. Ethanol exposure for 4 h also increased the colocalization and coimmunoprecipitation of PKC␥ with ␣1 subunits, whereas PKC␤/␣1 association and PKC/␣1 colocalization were not altered by ethanol exposure. Selective PKC␥ inhibition by transfection of selective PKC␥ small interfering RNAs blocked ethanol-induced internalization of GABA A receptor ␣1 subunits, whereas PKC␤ inhibition using pseudo-PKC␤ had no effect. These findings suggest that ethanol exposure selectively alters PKC␥ translocation to GABA A receptors and PKC␥ regulates GABA A ␣1 receptor trafficking after ethanol exposure.

Flagellin is a potent immunogen that activates the innate immune system via TLR5 and Naip5/6, and generates strong T and B cell responses. The adaptor protein MyD88 is critical for signaling by TLR5, as well as IL-1 and IL-18 receptors, major downstream mediators of the Naip5/6 Nlrc4-inflammasome. Herein we define roles of known flagellin receptors and MyD88 in antibody responses generated towards flagellin. We used mice genetically deficient in flagellin recognition pathways to characterize innate immune components that regulate isotype specific antibody responses. Using purified flagellin from Salmonella, we dissected the contribution of innate flagellin recognition pathways to promote antibody responses towards flagellin and co-administered ovalbumin in C57BL/6 mice. We demonstrate IgG2c responses towards flagellin were TLR5- and inflammasome-dependent; IgG1 was the dominant isotype and partially TLR5- and inflammasome-dependent. Our data indicates a substantial flagellin-specific IgG1 response was induced through a TLR5-, inflammasome-, and MyD88-independent pathway. IgA anti-FliC responses were TLR5- & MyD88-dependent and caspase-1-independent. Unlike C57BL/6 mice, flagellin immunized A/J mice induced co-dominant IgG1 and IgG2a responses. Furthermore, MyD88-independent flagellin-induced antibody responses were even more pronounced in A/J MyD88−/− mice, and IgA anti-FliC responses were suppressed by MyD88. Flagellin also worked as an adjuvant toward co-administered ovalbumin, but it only promoted IgG1 anti-OVA responses. Our results demonstrate that a novel pathway for flagellin recognition contributes to antibody production. Characterization of this pathway will be useful for understanding immunity to flagellin and the rationale design of flagellin-based vaccines.

Background: Acyl-CoA synthetase 1 (ACSL1) promotes inflammatory effects in macrophages, but its regulation and biological role remain largely unknown. Results: Multiple inflammatory pathways contribute to ACSL1 induction, and this induction allows for phospholipid turnover in activated macrophages. Conclusion:The regulation and function of ACSL1 differ substantially in macrophages and insulin target tissues. Significance: These findings indicate a novel role for ACSL1 in innate immunity.

Salmonella enterica serovar Typhimurium is a flagellated bacterium and one of the leading causes of gastroenteritis in humans. Bacterial flagellin is required for motility and also a prime target of the innate immune system. Innate immune recognition of flagellin is mediated by at least two independent pathways, TLR5 and Naip5-Naip6/NlrC4/Caspase-1. The functional significance of each of the two independent flagellin recognition systems for host defense against wild type Salmonella infection is complex, and innate immune detection of flagellin contributes to both protection and susceptibility. We hypothesized that efficient modulation of flagellin expression in vivo permits Salmonella to evade innate immune detection and limit the functional role of flagellin-specific host innate defenses. To test this hypothesis, we used Salmonella deficient in the anti-sigma factor flgM, which overproduce flagella and are attenuated in vivo. In this study we demonstrate that flagellin recognition by the innate immune system is responsible for the attenuation of flgM− S. Typhimurium, and dissect the contribution of each flagellin recognition pathway to bacterial clearance and inflammation. We demonstrate that caspase-1 controls mucosal and systemic infection of flgM− S. Typhimurium, and also limits intestinal inflammation and injury. In contrast, TLR5 paradoxically promotes bacterial colonization in the cecum and systemic infection, but attenuates intestinal inflammation. Our results indicate that Salmonella evasion of caspase-1 dependent flagellin recognition is critical for establishing infection and that evasion of TLR5 and caspase-1 dependent flagellin recognition helps Salmonella induce intestinal inflammation and establish a niche in the inflamed gut.

Salmonella enterica Typhimurium is a flagellated bacterium and one of the leading causes of gastroenteritis in humans. Bacterial flagellin is critical for motility and also a prime target of the innate immune system. Innate immune recognition of flagellin is mediated by at least two independent pathways, TLR5 and Naip5-Naip6/NlrC4/Caspase-1. The functional significance of each of the two independent flagellin recognition systems in innate immunity to wild type Salmonella infection is complex, and contribute to both protection and susceptibility. We hypothesized that this was due to efficient modulation of flagellin expression in vivo and evasion of innate immune detection. Salmonella deficient in the anti-sigma factor flgM overexpress flagellin, are attenuated in vivo, and this attenuation is dependent on flagellin expression. In this study, we used flgM- Salmonella to determine if flagellin recognition by the innate immune system is responsible for the attenuation of flgM- S. typhimurium, and to dissect the contribution of each flagellin recognition pathway to bacterial clearance, inflammation and infection. We demonstrate that caspase-1 controls systemic infection of flgM- S. Typhimurium, and also limits intestinal inflammation and injury. In contrast, TLR5 paradoxically promotes bacterial colonization in the cecum, and attenuates intestinal inflammation. Our results indicate that Salmonella evasion of caspase-1 dependent flagellin recognition is critical for establishing systemic infection and that evasion of TLR5 and caspase-1 dependent flagellin recognition help Salmonella induce intestinal inflammation and establish a niche in the inflamed gut.