Reactive oxygen species regulate caspase-11 expression and activation of the non-canonical NLRP3 inflammasome during enteric pathogen infection

Enteropathogenic and enterohemorrhagic bacterial infections in humans are a severe cause of morbidity and mortality. Although NOD-like receptors (NLRs) NOD2 and NLRP3 have important roles in the generation of protective immune responses to enteric pathogens, whether there is crosstalk among NLRs to regulate immune signaling is not known. Here, we show that mice and macrophages deficient in NOD2, or the downstream adaptor RIP2, have enhanced NLRP3-and caspases-11-dependent non-canonical inflammasome activation in a mouse model of enteropathogenic Citrobacter rodentium infection. Mechanistically, NOD2 and RIP2 regulate reactive oxygen species (ROS) production. Increased ROS in Rip2-deficient macrophages subsequently enhances c-Jun N-terminal kinase (JNK) signaling resulting in increased caspase-11 expression and activation, and more non-canonical NLRP3-dependant inflammasome activation. Intriguingly, this leads to protection of the colon epithelium for up to 10 days in Rip2-deficient mice infected with C. rodentium. Our findings designate NOD2 and RIP2 as key regulators of cellular ROS homeostasis and demonstrate for the first time that ROS regulates caspase-11 expression and non-canonical NLRP3 inflammasome activation through the JNK pathway

Inflammasome activation by nucleic acids and nucleosomes in sterile inflammation… or is it sterile?

Inflammasomes are multiprotein complexes that form in the cytoplasm in response to cellular damage and cytosolic pathogen‐associated molecules during infection. These complexes play important roles in initiating innate and adaptive immune responses to infectious disease. In addition, inflammasomes are now recognized as important mediators of sterile inflammation in various autoimmune and autoinflammatory diseases. Interestingly, microbiota and infection play critical roles in the development of ‘sterile inflammation\u27. Herein, we highlight recent advances in our understanding of the role for inflammasomes in nucleic acid‐, nucleosome‐, and histone‐driven sterile inflammation and discuss knowledge gaps and areas of potential future research

Deficiency of the NOD-Like Receptor NLRC5 Results in Decreased CD8+ T Cell Function and Impaired Viral Clearance.

Pathogen recognition receptors are vital components of the immune system. Engagement of these receptors is important not only for instigation of innate immune responses to invading pathogens but also for initiating the adaptive immune response. Members of the NOD-like receptor (NLR) family of pathogen recognition receptors have important roles in orchestrating this response. The NLR family member NLRC5 regulates major histocompatibility complex class I (MHC-I) expression during various types of infections, but its role in immunity to influenza A virus (IAV) is not well studied. Here we show that Nlrc5-/- mice exhibit an altered CD8+ T cell response during IAV infection compared to that of wild-type (WT) mice. Nlrc5-/- mice have decreased MHC-I expression on hematopoietic cells and fewer CD8+ T cells prior to infection. NLRC5 deficiency does not affect the generation of antigen-specific CD8+ T cells following IAV infection; however, a change in epitope dominance is observed in Nlrc5-/- mice. Moreover, IAV-specific CD8+ T cells from Nlrc5-/- mice have impaired effector functions. This change in the adaptive immune response is associated with impaired viral clearance in Nlrc5-/- mice. Collectively, our results demonstrate an important role for NLRC5 in regulation of antiviral immune responses and viral clearance during IAV infection

Enhanced IL-1β production is mediated by a TLR2-MYD88-NLRP3 signaling axis during coinfection with influenza A virus and Streptococcus pneumoniae.

Viral-bacterial coinfections, such as with influenza A virus and Streptococcus pneumoniae (S.p.), are known to cause severe pneumonia. It is well known that the host response has an important role in disease. Interleukin-1β (IL-1β) is an important immune signaling cytokine responsible for inflammation and has been previously shown to contribute to disease severity in numerous infections. Other studies in mice indicate that IL-1β levels are dramatically elevated during IAV-S.p. coinfection. However, the regulation of IL-1β during coinfection is unknown. Here, we report the NLRP3 inflammasome is the major inflammasome regulating IL-1β activation during coinfection. Furthermore, elevated IL-1β mRNA expression is due to enhanced TLR2-MYD88 signaling, which increases the amount of pro-IL-1β substrate for the inflammasome to process. Finally, NLRP3 and high IL-1β levels were associated with increased bacterial load in the brain. Our results show the NLRP3 inflammasome is not protective during IAV-S.p. coinfection

Antifungal Norditerpene Oidiolactones from the Fungus Oidiodendron truncatum, a Potential Biocontrol Agent for White-Nose Syndrome in Bats

White-nose syndrome (WNS) is a devastating disease of hibernating bats caused by the fungus Pseudogymnoascus destructans. We obtained 383 fungal and bacterial isolates from the Soudan Iron Mine, an important bat hibernaculum in Minnesota, then screened this library for antifungal activity to develop biological control treatments for WNS. An extract from the fungus Oidiodendron truncatum was subjected to bioassay-guided fractionation, which led to the isolation of 14 norditerpene and three anthraquinone metabolites. Ten of these compounds were previously described in the literature, and here we present the structures of seven new norditerpene analogues. Additionally, this is the first report of 4-chlorophyscion from a natural source, previously identified as a semisynthetic product. The compounds PR 1388 and LL-Z1271α were the only inhibitors of P. destructans (MIC = 7.5 and 15 μg/mL, respectively). Compounds were tested for cytotoxicity against fibroblast cell cultures obtained from Myotis septentrionalis (northern long eared bat) and M. grisescens (gray bat) using a standard MTT viability assay. The most active antifungal compound, PR 1388, was nontoxic toward cells from both bat species (IC50 \u3e 100 μM). We discuss the implications of these results in the context of the challenges and logistics of developing a substrate treatment or prophylactic for WNS

RIP2 regulates caspases-11 expression through a ROS-JNK pathway.

<p><b>(A)</b> WT and <i>Rip2^−/−</i> BMDM were infected with 20MOI of <i>C. rodentium</i> for 18 h and supernatants collected and examined for type-I interferon using a reporter cell line (U = Units). <b>(B–C)</b> WT or <i>Rip2^−/−</i> BMDM were infected with 20MOI of <i>C. rodentium</i> and mock treated or treated with H₂O₂. Samples were collected at the indicated times and examined for total JNK or phosphorylated JNK by Western blot. <b>(D)</b> WT or <i>Rip2^−/−</i> BMDM were infected with 20MOI of <i>C. rodentium</i> and mock treated or treated with the JNK inhibitor SP600125 (JNKi). Samples were collected at the indicated times and examined for total JNK, phosphorylated JNK and caspase-11 by western blot. <b>(E)</b> Proposed signaling pathway that regulates inflammasome activation. (A) Data are combined from 9 independent experiments and displayed as the mean ± SEM. (B–D) Data are representative of 3 independent experiments. (n.s. = not significant).</p

RIP2 regulates the caspase-11 non-canonical inflammasome.

<p><b>(A–B)</b> WT and <i>Rip2^−/−</i> BMDM were infected with 20MOI of <i>C. rodentium</i> for 18 h and combined supernatant and lysates were examined by Western blot for caspase-11 cleavage (casp-11p30) visually (A) and by densitometry (B). <b>(C–D)</b> WT and <i>Rip2^−/−</i> BMDM were infected with 20MOI of <i>C. rodentium</i> for 2 h and <i>Rip2^−/−</i> cells were subsequently treated with NAC or mock treated as a control. Samples were collected at 18 h and caspase-11 activation was examined as in panels A–B. <b>(E)</b> Densitometry was performed on pro-Caspase-11 p43 band from 3 independent experiments. <b>(F–G)</b> WT and <i>Rip2^−/−</i> BMDM were infected with 20MOI of <i>C. rodentium</i> for 4 h and cells were subsequently treated with 1 µM MG-132 or mock treated as a control. Samples were collected at the indicated time points and pro-caspase-11 levels examined by Western blot and densitometry. <b>(H)</b> WT and <i>Rip2^−/−</i> BMDM were infected with 20MOI of <i>C. rodentium</i> and RNA collected at the indicated time points and analyzed by qRT-PCR for fold induction and normalized to GAPDH. (A–H) Data are representative of three independent experiments with n = 2–3 wells per experiment and displayed as the mean ± SEM. (**, p<0.01; ***, p<0.001).</p

Enhanced inflammasome activation in <i>Nod2^−/−</i> and <i>Rip2^−/−</i> BMDMs.

<p>BMDM were generated from WT, <i>Nod2^−/−</i> and <i>Rip2^−/−</i> mice and infected with 20MOI of <i>Citrobacter rodentium</i> for 18 h. <b>(A–F)</b> Combined supernatant and lysates were examined by Western blot for caspase-1 cleavage (casp-1p20) visually (A,D) and by densitometry (B,E), or (C,F) supernatants were examined for IL-18 secretion by ELISA. <b>(G)</b> BMDM were infected with <i>C. rodentium</i> and assayed for intracellular growth at the indicated times post-infection. (A–F) Data are representative of five independent experiments with n = 2–3 wells per experiment. (G) Data are representative of two independent experiments with n = 2–3 wells per experiment. (B,C,E,F,G) Data are shown as the mean ± SEM. (*, p<0.05; **, p<0.01; ***, p<0.001).</p

NOD2 and RIP2 specifically regulate NLRP3 inflammasome activation.

<p><b>(A–B)</b> WT, <i>Nod2^−/−</i> and <i>Rip2^−/−</i> BMDM were infected with 1MOI <i>Salmonella typhimurium</i> for 4 h and combined supernatant and lysates were examined by Western blot for caspase-1 cleavage (casp-1p20) visually (A) and by densitometry (B). <b>(C)</b> Supernatants were examined 4 h after <i>S. typhimurium</i> infection for IL-18 secretion. <b>(D–F)</b> WT, <i>Nod2^−/−</i> and <i>Rip2^−/−</i> BMDM were infected with 10MOI <i>S typhimurium Δfljb/flic</i> mutant for 18 h. Caspase-1 activation and IL-18 were examined as in panels A–C. <b>(G–I)</b> WT and <i>Rip2^−/−</i> BMDM were infected with 20MOI of <i>C. rodentium</i> for 2 h and <i>Rip2^−/−</i> cells were subsequently treated with the NLRP3 specific inhibitor glyburide or mock treated as a control. Caspase-1 activation and IL-18 were examined as in panels A–C. <b>(J–L)</b> WT and <i>Rip2^−/−</i> BMDM were infected with 1MOI of <i>S. typhimurium</i> and <i>Rip2^−/−</i> cells were simultaneously treated with the NLRP3 specific inhibitor glyburide or mock treated as a control. Caspase-1 activation and IL-18 were examined as in panels A–C. (A–L) Data are representative of three independent experiments with n = 2–3 wells per experiment and displayed as the mean ± SEM. (*, p<0.05; **, p<0.01; ***, p<0.001).</p