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Co-regulation of NF-kappaB and inflammasome-mediated inflammatory responses by myxoma virus pyrin domain-containing protein M013.

ABSTRACT: NF-kappaB and inflammasomes both play central roles in orchestrating anti-pathogen responses by rapidly inducing a variety of early-response cytokines and chemokines following infection. Myxoma virus (MYXV), a pathogenic poxvirus of rabbits, encodes a member of the cellular pyrin domain (PYD) superfamily, called M013. The viral M013 protein was previously shown to bind host ASC-1 protein and inhibit the cellular inflammasome complex that regulates the activation and secretion of caspase 1-regulated cytokines such as IL-1beta and IL-18. Here, we report that human THP-1 monocytic cells infected with a MYXV construct deleted for the M013L gene (vMyxM013-KO), in stark contrast to the parental MYXV, rapidly induce high levels of secreted pro-inflammatory cytokines like TNF, IL-6, and MCP-1, all of which are regulated by NF-kappaB. The induction of these NF-kappaB regulated cytokines following infection with vMyxM013-KO was also confirmed in vivo using THP-1 derived xenografts in NOD-SCID mice. vMyxM013-KO virus infection specifically induced the rapid phosphorylation of IKK and degradation of IkappaBalpha, which was followed by nuclear translocation of NF-kappaB/p65. Even in the absence of virus infection, transiently expressed M013 protein alone inhibited cellular NF-kappaB-mediated reporter gene expression and nuclear translocation of NF-kappaB/p65. Using protein/protein interaction analysis, we show that M013 protein also binds directly with cellular NF-kappaB1, suggesting a direct physical and functional linkage between NF-kappaB1 and ASC-1. We further demonstrate that inhibition of the inflammasome with a caspase-1 inhibitor did not prevent the induction of NF-kappaB regulated cytokines following infection with vMyxM013-KO virus, but did block the activation of IL-1beta. Thus, the poxviral M013 inhibitor exerts a dual immuno-subversive role in the simultaneous co-regulation of both the cellular inflammasome complex and NF-kappaB-mediated pro-inflammatory responses.

SUBMITTER: Rahman MM

PROVIDER: S-EPMC2762494 | biostudies-other | 2009 Oct

REPOSITORIES: biostudies-other

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Project description:The NLR family pyrin domain-containing 3 (NLRP3) inflammasome plays an important role in liver fibrosis (LF) development. However, the mechanisms involved in NLRP3-induced fibrosis are unclear. Our aim was to test the hypothesis that the NLRP3 inflammasome in hepatic stellate cells (HSCs) can directly regulate their activation and contribute to LF. Primary HSCs isolated from wild-type (WT), Nlrp3-/- , or Nlrp3L351PneoR knock-in crossed to inducible (estrogen receptor Cre-CreT) mice were incubated with lipopolysaccharide (LPS) and adenosine triphosphate (ATP), or 4OH-tamoxifen, respectively. HSC-specific Nlrp3L351P knock-in mice were generated by crossing transgenic mice expressing lecithin retinol acyltransferase (Lrat)-driven Cre and maintained on standard rodent chow for 6 months. Mice were then sacrificed; liver tissue and serum were harvested. Nlrp3 inflammasome activation along with HSC phenotype and fibrosis were assessed by RT-PCR, western blotting, fluorescence-activated cell sorting (FACS), enzyme-linked immunosorbent assay, immunofluorescence (IF), and immunohistochemistry (IHC). Stimulated WT HSCs displayed increased levels of NLRP3 inflammasome-induced reactive oxygen species (ROS) production and cathepsin B activity, accompanied by an up-regulation of mRNA and protein levels of fibrotic makers, an effect abrogated in Nlrp3-/- HSCs. Nlrp3L351P CreT HSCs also showed elevated mRNA and protein expression of fibrotic markers 24 hours after inflammasome activation induced with 4-hydroxytamoxifen (4OHT). Protein and mRNA expression levels of fibrotic markers were also found to be increased in isolated HSCs and whole liver tissue from Nlrp3L351P Lrat Cre mice compared to WT. Liver sections from 24-week-old NlrpL351P Lrat Cre mice showed fibrotic changes with increased alpha smooth muscle actin (?SMA) and desmin-positive cells and collagen deposition, independent of inflammatory infiltrates; these changes were also observed after LPS challenge in 8-week-old NlrpL351P Lrat Cre mice. Conclusion: Our results highlight a direct role for the NLRP3 inflammasome in the activation of HSCs directly triggering LF.

Project description:AimsThis study hypothesized that activation of endothelial nucleotide oligomerization domain-like receptor protein with pyrin domain containing 3 (Nlrp3) inflammasomes directly produces endothelial dysfunction during hypercholesterolemia, which is distinct from its canonical roles in inflammation.ResultsAcute hypercholesterolemia in mice was induced by intraperitoneal administration of poloxamer 407 (0.5?g/kg) for 24?h. Endothelial dysfunction was assessed by evaluating endothelium-dependent vasodilation in isolated, perfused, and pressurized coronary arteries in response to bradykinin (10(-10)-10(-6) M) and acetylcholine (10(-9)-10(-5) M). Impaired endothelium-dependent vasodilation was observed in Nlrp3(+/+) mice with acute hypercholesterolemia, which was markedly ameliorated in Nlrp3(-/-) mice. Treatment of mice with inhibitors for caspase-1 or high mobility group box 1 (HMGB1) significantly restored endothelium-dependent vasodilation in Nlrp3(+/+) mice with acute hypercholesterolemia. Confocal microscopic analysis demonstrated that hypercholesterolemia markedly increased caspase-1 activity and HMGB1 expression in coronary arterial endothelium of Nlrp3(+/+) mice, which was absent in Nlrp3-deficient mice. Further, recombinant HMGB1 directly induced endothelial dysfunction in normal Nlrp3(+/+) coronary arteries. In vitro, Nlrp3 inflammasome formation and its activity were instigated in cultured endothelial cells by cholesterol crystal, a danger factor associated with hypercholesterolemia. Moreover, cholesterol crystals directly induced endothelial dysfunction in coronary arteries from Nlrp3(+/+) mice, which was attenuated in Nlrp3(-/-) arteries. Such cholesterol crystal-induced impairment was associated with enhanced superoxide production, downregulation of endothelial nitric oxide synthase activity, and pyroptosis.Innovation and conclusionOur data provide the first evidence that activation of endothelial Nlrp3 inflammasome directly impairs endothelial function beyond its canonical inflammatory actions. This novel non-canonical action of Nlrp3 inflammasomes may initiate or exacerbate vascular injury during hypercholesterolemia.

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Co-regulation of NF-kappaB and inflammasome-mediated inflammatory responses by myxoma virus pyrin domain-containing protein M013.

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