O008 Type I interferons license caspase-11-dependent NLRP3 inflammasome activation by Gram-negative bacteria

Tight regulation of inflammatory cytokine and interferon (IFN) production in innate immunity is pivotal for optimal control of pathogens and avoidance of immunopathology. The human Nod-like receptor (NLR) NLRP11 has been shown to regulate type I IFN and pro-inflammatory cytokine responses. Here, we identified the ATP-dependent RNA helicase DDX3X as a novel binding partner of NLRP11, using co-immunoprecipitation and LC-MS/MS. DDX3X is known to enhance type I IFN responses and NLRP3 inflammasome activation. We demonstrate that NLRP11 can abolish IKKϵ-mediated phosphorylation of DDX3X, resulting in lower type I IFN induction upon viral infection. These effects were dependent on the LRR domain of NLRP11 that we mapped as the interaction domain for DDX3X. In addition, NLRP11 also suppressed NLRP3-mediated caspase-1 activation in an LRR domain-dependent manner, suggesting that NLRP11 might sequester DDX3X and prevent it from promoting NLRP3-induced inflammasome activation. Taken together, our data revealed DDX3X as a central target of NLRP11, which can mediate the effects of NLRP11 on type I IFN induction as well as NLRP3 inflammasome activation. This expands our knowledge of the molecular mechanisms underlying NLRP11 function in innate immunity and suggests that both NLRP11 and DDX3X might be promising targets for modulation of innate immune responses.

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Agnuside Alleviates Synovitis and Fibrosis in Knee Osteoarthritis through the Inhibition of HIF-1α and NLRP3 Inflammasome

Mediators of Inflammation ◽

10.1155/2021/5534614 ◽

2021 ◽

Vol 2021 ◽

pp. 1-11

Author(s):

Li Zhang ◽

Xiaochen Li ◽

Haosheng Zhang ◽

Zhengquan Huang ◽

Nongshan Zhang ◽

...

Keyword(s):

Knee Osteoarthritis ◽

Nlrp3 Inflammasome ◽

Type I Collagen ◽

Vital Role ◽

Inflammasome Activation ◽

Type I ◽

Aseptic Inflammation ◽

Protein Levels ◽

Nlrp3 Inflammasome Activation ◽

Anti Inflammatory

Increasing evidence has shown that NLRP3 inflammasome activation participates in chronic aseptic inflammation and is related to tissue fibrosis. Our last study also revealed the vital role of NLRP3 inflammasome, highly associated with tissue hypoxia, in the onset and development of knee osteoarthritis (KOA). In this study, we tried to find a possible benign intervention for that pathological process. Agnuside (AGN), a nontoxic, natural small molecule isolated from the extract of Vitex negundo L. (Verbenaceae), has been demonstrated to have antioxidation, anti-inflammatory, analgesia, and many other properties as an iridoid glycoside, although its specific target is still unclear. Therefore, we established MIA-induced KOA model rats and investigated the effects of AGN oral gavage on oxygen-containing state, NLRP3 inflammasome, synovitis, and fibrosis in KOA. Pimonidazole staining and HIF-1α immunohistochemical assay both showed that AGN at the oral dose of 6.25 mg/kg can effectively relieve local hypoxia in synovial tissue. Besides, we observed a decrease of HIF-1α, caspase-1, ASC, and NLRP3 after AGN intervention, both in the mRNA and protein levels. In addition, rats treated with the AGN showed less inflammatory reaction and fibrosis, not only in the expression of NLRP3, inflammasome downstream factors IL-1β and IL-18, and fibrosis markers TGF-β, TIMP1, and VEGF but also in the observation of HE staining, anatomical characteristics, Sirius Red staining, and type I collagen immunohistochemistry. Subsequently, we established LPS-induced models of fibroblast-like synoviocytes (FLSs) mimicking the inflammatory environment of KOA and activating NLRP3 inflammasome. FLSs treated with AGN (3 μM) resulted in a downregulation of HIF-1α and the components required for NLRP3 inflammasome activation. Meanwhile, the content of proinflammatory factors IL-1β and IL-18 in FLS supernatant was also reduced by AGN. In addition, both mRNA and protein levels of the fibrotic markers were significantly decreased after AGN management. To conclude, this study demonstrates that AGN alleviates synovitis and fibrosis in experimental KOA through the inhibition of HIF-1α accumulation and NLRP3 inflammasome activation. Additionally, not only does it reveal some novel targets for anti-inflammatory and antioxidant effects of AGN but also announces its potential value in treating KOA in humans.

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New insights in caspase-11 functions in noncanonical inflammasome signalling

Inflammasome ◽

10.2478/infl-2014-0001 ◽

2014 ◽

Vol 1 (1) ◽

Author(s):

Mélanie Bodnar ◽

Virginie Petrilli

Keyword(s):

Cell Death ◽

Inflammatory Cell ◽

Protein Complexes ◽

Inflammasome Activation ◽

Gram Negative Bacteria ◽

Nucleotide Binding Domain ◽

Gram Negative ◽

Caspase 1 ◽

Nlrp3 Inflammasome Activation ◽

Dependent Pathway

AbstractInflammasomes are multi-protein complexes that play a crucial role in innate immunity. They are assembled by cytosolic sensors of the Nucleotide-binding domain and Leucine-rich repeat containing Receptor (NLR) and PYrin and HIN (PYHIN) domain-containing protein families upon sensing various pathogens and danger signals. Inflammasome formation culminates in caspase-1 activation, which causes the cleavage of pro-IL-1β and pro- IL-18 into active cytokines; this eventually results in the induction of an inflammatory cell death called pyroptosis. Recent data using Gram-negative bacteria suggests a role for caspase-11 not only in NLRP3 inflammasome activation but also in a caspase-1- and inflammasome-independent cell death. This novel caspase-11-dependent pathway is critical to control infection by Gram-negative bacteria and has been named the noncanonical inflammasome.

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Leishmania RNA virus exacerbates Leishmaniasis by subverting innate immunity via TLR3-mediated NLRP3 inflammasome inhibition

Nature Communications ◽

10.1038/s41467-019-13356-2 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 14

Author(s):

Renan V. H. de Carvalho ◽

Djalma S. Lima-Junior ◽

Marcus Vinícius G. da Silva ◽

Marisa Dilucca ◽

Tamara S. Rodrigues ◽

...

Keyword(s):

Nlrp3 Inflammasome ◽

Rna Virus ◽

Severe Form ◽

Inflammasome Activation ◽

Type I ◽

Mucocutaneous Leishmaniasis ◽

Nlrp3 Inflammasome Activation ◽

Parasite Survival ◽

Important Virulence Factor

AbstractLeishmania RNA virus (LRV) is an important virulence factor associated with the development of mucocutaneous Leishmaniasis, a severe form of the disease. LRV-mediated disease exacerbation relies on TLR3 activation, but downstream mechanisms remain largely unexplored. Here, we combine human and mouse data to demonstrate that LRV triggers TLR3 and TRIF to induce type I IFN production, which induces autophagy. This process results in ATG5-mediated degradation of NLRP3 and ASC, thereby limiting NLRP3 inflammasome activation in macrophages. Consistent with the known restricting role of NLRP3 for Leishmania replication, the signaling pathway triggered by LRV results in increased parasite survival and disease progression. In support of this data, we find that lesions in patients infected with LRV+ Leishmania are associated with reduced inflammasome activation and the development of mucocutaneous disease. Our findings reveal the mechanisms triggered by LRV that contribute to the development of the debilitating mucocutaneous form of Leishmaniasis.

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H3 relaxin alleviated vascular injury in rats with type 1 diabetes by inhibiting endoplasmic reticulum stress and inflammation

10.21203/rs.3.rs-435725/v1 ◽

2021 ◽

Author(s):

Xiaohui Zhang ◽

Kelaier Yang ◽

Jinyu Chi ◽

Wenjia Chen ◽

Xiao Ma ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Type 1 Diabetes ◽

Endoplasmic Reticulum Stress ◽

Nlrp3 Inflammasome ◽

Vascular Function ◽

Diabetic Rats ◽

Inflammasome Activation ◽

Type I ◽

Nlrp3 Inflammasome Activation

Abstract Human recombinant relaxin-3 (H3 relaxin ),a small molecule peptide hormone, ameliorated myocardial injury after myocardial infarction or isoprenaline injection by inhibiting apoptosis and fibrosis. However, whether H3 relaxin protects vascular function in rats with type 1 diabetes and its mechanism are unknown. In type 1 diabetes rats model induced by streptozotocin (STZ), rats were subcutaneously injected H3 relaxin (2 µg/kg/d or 0.2 µg/kg/d) for 2 weeks. At 4 or 8 weeks after STZ injection, we detected the expression of fibrosis (type I and III collagen), ERS (endoplasmic reticulum stress) and NLRP3 inflammasome activation in the aortas and inflammation markers in the plasma from rats with diabetes. Compared with the diabetic rats, H3 relaxin treatment exhibited markedly decreased plasma oxidative stress markers (TNF-a and MDA) levels. The protein expression levels of type I and III collagen in the aortas were increased in rats with diabetes, inhibited by H3 relaxin. H3 relaxin treatment inhibited ERS (GRP78 and CHOP) and NLRP3 inflammasome activation in the aortas of diabetic rats. These results suggest that H3 relaxin inhibited fibrosis, ERS and inflammation activation in the aortas of type 1 diabetic rats.

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Genomic-DNA Exposed By Somatic Gene Mutations Engages the cGAS/STING Axis to License the NLRP3 Inflammasome in Myelodysplastic Syndromes

Blood ◽

10.1182/blood-2018-99-116929 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3075-3075 ◽

Cited By ~ 1

Author(s):

Amy F McLemore ◽

Kathy L McGraw ◽

Hsin-An Hou ◽

Grace A Ward ◽

Alexis H Onimus ◽

...

Keyword(s):

Myelodysplastic Syndromes ◽

Nlrp3 Inflammasome ◽

Genomic Dna ◽

Gene Mutations ◽

Interferon Response ◽

Inflammasome Activation ◽

Type I ◽

Caspase 1 ◽

Nlrp3 Inflammasome Activation ◽

Somatic Gene

Abstract Background: The pathogenesis of Myelodysplastic Syndromes (MDS) is linked to constitutive innate immune stimulation that converges upon the NLRP3 inflammasome to induce pyroptosis, a caspase-1 dependent cell death. We have shown that inflammasome assembly is initiated by both cell-extrinsic stimuli such as S100A9 elaborated by Myeloid-Derived Suppressor Cells (MDSC), as well as cell-intrinsic somatic gene mutations (SGM) (Basiorka A, et. al. Blood 2016). SGM of varied classes evoke replication stress caused by transcriptional pauses that can expose genomic DNA to cytosolic sensors through unresolved R-loops or micronuclei formation. The cGMP-AMP Synthase-Stimulator of Interferon Genes (cGAS-STING) is a cell-intrinsic DNA surveillance pathway recognizing both cytosolic pathogenic and autologous DNA, leading to interferon stimulated gene (ISG) transcription and NLRP3 inflammasome activation, key biological features of MDS (Pellagatti A, et. al. Blood 2006; 108:337.). Here, we investigate the contribution of genomic cytosolic DNA engagement by cGAS-STING to NLRP3 inflammasome activation in MDS. Methods: MDS patient and healthy donor bone marrow mononuclear cells (BMMC) were isolated by Ficoll®-Hipaque method from consented participants at the Moffitt Cancer Center or the National Taiwan University Hospital (NTUH). Immortalized murine C57BL/6 Tet2-/- and MX1Cre/SRSF2P95H as well as respective wild type (WT) control BMMCs were used as MDS SGM models. Results: We first assessed cGAS-STING activation in MDS BMMC by measuring ISG response by microarray, demonstrating profoundly increased expression of ISG15, CXCL10, Samd9l, and Ifi27l2 in MDS BMMC (n=213) compared to healthy control BMMC (n=20) (p<0.0001, p=0.013, p=0.0007 and p<0.0001, respectively). Likewise, both the Tet2 and SRSF2 SGM models have increased ISG expression compared to WT. Importantly, treatment with the cGAS inhibitor, RU.521 (0.1-1µM, 24 hours), significantly suppressed ISG expression in the SGM models. Further, we confirmed DNA sensor pathway activation in the SRSF2 SGM model by demonstrating upregulated phosphorylation of the interferon-regulatory factor (IRF)3 and IRF7 transcription factors compared to WT controls, and treatment with RU.521 decreased IRF3 phosphorylation. Caspase-1 cleavage in Tet2 and SRSF2 SGM models confirmed NLRP3 inflammasome activation in mutant cells compared to WT controls, and RU.521 treatment decreased active caspase-1 generation in both SGM models. RU.521 treatment of MDS BMMC harboring DNMT3 and Tet2 mutations induced terminal differentiation as evidenced by increased CD11b expression detected by flow cytometry and morphological assessment of Wright-Giemsa stains. Similarly, treatment with RU.521 promoted cytological differentiation in the Tet2 SGM model. We next investigated cytosolic genomic-DNA sources including micronuclei and unresolved DNA:RNA hybrids (R-loops) in the SGM models and primary MDS BMMC. Using immunofluorescence (IF), we found a significantly increased number of micronuclei in MDS BMMC (n=8) compared to controls (n=4), p=0.0248. Additionally, R-loops were increased in MDS primary BMMC (n=5) harboring varied SGM classes vs. controls (n=5) as well as in the SGM models. Finally, to confirm genomic-DNA engagement by cGAS, we used IF to assess cGAS co-localization. cGAS co-localized with micronuclei at sites of envelope collapse as well as with R-loops in MDS BMMC, thereby demonstrating cGAS/STING engagement of cytosolic self-DNA (n=4, each). Conclusion: These data indicate that cGAS-STING engages redundant sources of cytosolic genomic-DNA in MDS to initiate a Type I interferon response and NLRP3 inflammasome activation. Inhibition of the cGAS-STING axis may represent a novel therapeutic strategy for investigation in MDS. Disclosures List: Celgene: Research Funding.

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