FRI0001 NEUTROPHILS IN GRANULOMATOSIS WITH POLYANGIITIS DISPLAY FEATURES OF PYROPTOSIS

Background:Granulomatosis with polyangiitis (GPA) is characterized by extravascular necrotizing granulomatous inflammation and systemic ANCA – associated (AAV) vasculitis with neutrophils as a key player in the pathogenesis (1). We and others have shown that neutrophil-related cell death mechanisms contribute to chronic inflammatory processes in AAV (2, 3). Recently, another form of inflammatory cell death primarily described in monocytes called pyroptosis was also discovered in neutrophils (4). A cardinal feature of pyroptosis is the activation of the NLRP3 inflammasome, a sensor of different pathogen- and damage-associated molecular patterns (PAMP, DAMP), following caspase-1-mediated processing and secretion of IL-1beta (5).Objectives:The aim of this study was to investigate, if neutrophils from GPA patients express pyroptosis-related components NLRP3, active caspase 1 and cleaved IL-1beta.Methods:Polymorphonuclear leukocytes (PMN) were isolated from peripheral blood of GPA patients and healthy controls (HC) (n = 10 each). Expression of NLRP3, inactive/active caspase 1 and active IL-1beta was determined by western blot. In addition, peripheral blood mononuclear cells (PBMC) were isolated from GPA and HC. mRNA expression ofnlrp3andil1bwas determined by qPCR. To exclude false-positive results by contamination with monocytes we performed flow cytometry analysis of whole blood samples with markers CD3, CD14, CD15, CD66b and NLRP3.Results:PMN from GPA patients showed markedly increased expression of NLRP3, active caspase 1 and active IL-1beta compared to HC. In contrast, there was no difference between GPA and HC on the mRNA level of neithernlrp3noril1bin PBMC. In addition, we confirmed by flow cytometry increased expression of NLRP3 in PMN from GPA, but not in monocytes.Conclusion:Here we provide evidence, that neutrophils from GPA undergo pyroptosis, demonstrated by increased NLRP3, active caspase 1 expression as well as IL-1beta processing. Neutrophils are present in high numbers at the site of granulomatous lesions of inflamed tissue in GPA and IL-1beta is increased in GPA sera (2). Therefore, neutrophils represent a potential source of IL-1beta in GPA. Given the fact that GPA-associated features such as massive release of necrosis-related DAMP or microbial agents such asStaphylococcus aureus(6) can activate the NLRP3-inflammasome, we identified here a potential relevant mechanism of neutrophils contributing to chronic inflammation of GPA.References:[1]Jennette, J.C., and Falk, R.J. (2014). Pathogenesis of antineutrophil cytoplasmic autoantibody-mediated disease. Nat. Rev. Rheumatol.10, 463–473.[2]Millet, A., Martin, K.R., Bonnefoy, F., Saas, P., Mocek, J., Alkan, M., Terrier, B.,Kerstein,A., Tamassia, N., Satyanarayanan, S.K., et al. (2015). Proteinase 3 on apoptotic cells disrupts immune silencing in autoimmune vasculitis. J. Clin. Invest. 125, 4107–4121.[3]Schreiber, A., Rousselle, A., Becker, J.U., von Mässenhausen, A., Linkermann, A., and Kettritz, R. (2017). Necroptosis controls NET generation and mediates complement activation, endothelial damage, and autoimmune vasculitis. Proc. Natl. Acad. Sci. 201708247.[4]Tourneur, L., and Witko-Sarsat, V. (2019). Inflammasome activation: Neutrophils go their own way. J. Leukoc. Biol.105, 433–436.[5]Bergsbaken, T., Fink, S.L., and Cookson, B.T. (2009). Pyroptosis: Host cell death and inflammation. Nat. Rev. Microbiol.7, 99–109.[6]Lamprecht, P.,Kerstein, A., Klapa, S., Schinke, S., Karsten, C.M., Yu, X., Ehlers, M., Epplen, J.T., Holl-Ulrich, K., Wiech, T., et al. (2018). Pathogenetic and Clinical Aspects of Anti-Neutrophil Cytoplasmic Autoantibody-Associated Vasculitides. Front. Immunol.9, 1–10.Disclosure of Interests:Anja Kerstein-Staehle: None declared, Nadja Leinung: None declared, Jannik Meyer: None declared, Silke Pitann: None declared, Antje Müller: None declared, Gabriela Riemekasten Consultant of: Cell Trend GmbH, Janssen, Actelion, Boehringer Ingelheim, Speakers bureau: Actelion, Novartis, Janssen, Roche, GlaxoSmithKline, Boehringer Ingelheim, Pfizer, Peter Lamprecht: None declared

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OP0054 NEW ROLE FOR PROTEINASE 3 IN IL-16 BIOACTIVITY CONTROL IN GRANULOMATOSIS WITH POLYANGIITIS

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2021-eular.1314 ◽

2021 ◽

Vol 80 (Suppl 1) ◽

pp. 28.1-29

Author(s):

A. Kerstein-Staehle ◽

C. Alarcin ◽

J. Luo ◽

G. Riemekasten ◽

P. Lamprecht ◽

...

Keyword(s):

Cell Death ◽

Western Blot ◽

Disease Activity ◽

Chronic Inflammation ◽

Immune Cells ◽

Peripheral Blood ◽

Granulomatosis With Polyangiitis ◽

Granulomatous Inflammation ◽

Proteinase 3 ◽

Sds Page

Background:The immunomodulatory cytokine IL-16 is increased in several inflammatory and autoimmune diseases1. IL-16 recruits and activates CD4+ immune cells such as T cells, dendritic cells, or monocytes. IL-16 is produced by various immune and non-immune cells, but synthesis and storage of IL-16 is regulated differentially depending on the cell type and stimulation. For its biological activity, IL-16 cleavage by caspase-3 is required1. Necrotizing granulomatous inflammation is a hallmark of granulomatosis with polyangiitis (GPA) with neutrophil dysregulation as a central driver of chronic inflammation and autoimmunity2. Earlier studies showed a correlation between increased serum IL-16 and disease parameters in AAV, including GPA3, but functional evidence for a direct link between IL-16 and neutrophils in granulomatous inflammation is missing so far.Objectives:In this study we aim to identify a functional link between increased IL-16, neutrophils, and the autoantigen proteinase 3 (PR3) with regard to chronic inflammation and autoimmunity in GPA.Methods:IL-16 was measured in sera of GPA patients (n = 40) and healthy controls (HC, n = 50) by ELISA and correlated with clinical features, such as disease activity (BVAS), creatinine, GFR, VDI and PR3-ANCA status. IL-16 protein expression was analyzed in peripheral blood mononuclear cells (PBMC) and polymorphonuclear cells (PMN) from GPA patients and HC (n = 5, each) by SDS-PAGE and western blot. Binding affinity of recombinant pro-IL-16 to native human PR3 was assessed by microscale thermophoresis. Cleavage of pro-IL-16 by active human PR3 was performed at various time points at 37°C. Cleavage products were analyzed by SDS-PAGE and western blot.Results:Circulating IL-16 was significantly increased in GPA patients compared to HC. Elevated IL-16 positively correlated with BVAS, creatinine, VDI and PR3-ANCA status and negatively correlated with GFR. In PMBC and PMN from GPA and HC we identified different expression patters of precursor and active forms of IL-16. In healthy PBMC we found high amounts of precursor (80kD), pro-IL-16 (55kD) and active IL-16 (17kD). In contrast, PBMC from GPA patients had lower amounts of pro-IL-16 and no active IL-16, indicating activation and secretion of IL-16 due to inflammatory stimulation, as shown earlier5. In GPA PMN we detected no precursor IL-16, but pro-IL-16 and its active form, in contrast to very low amounts of all IL-16 forms in healthy PMN. Processing and release of IL-16 in neutrophils has been linked to apoptosis and secondary necrosis5. By interaction studies we demonstrated direct binding of pro-IL-16 to PR3 with a Kd of 10 nM. In a subsequent cleavage assay we confirmed IL-16 processing by PR3 in a time-dependent manner.Conclusion:Correlation of serum IL-16 with clinical features of GPA suggests that IL-16 is associated with markers of disease activity, tissue damage and autoreactivity. We showed that PBMC and PMN represent a source of IL-16 in GPA. By the identification of PR3 as an additional IL-16-activating enzyme we could demonstrate a potential link between excessive PR3 expression, cell death and IL-16-dependent mechanisms, contributing to chronic granulomatous inflammation and autoimmunity in GPA.References:[1]Glass, W. G. et al. Not-so-sweet sixteen: The role of IL-16 in infectious and immune-mediated inflammatory diseases. J. Interf. Cytokine Res. 26, 511–520 (2006).[2]Millet, A. et al. Proteinase 3 on apoptotic cells disrupts immune silencing in autoimmune vasculitis. J. Clin. Invest. 125, 4107–4121 (2015).[3]Yoon, T. et al. Serum interleukin-16 significantly correlates with the Vasculitis Damage Index in antineutrophil cytoplasmic antibody-associated vasculitis. Arthritis Res. Ther. 22, 1–6 (2020).[4]Elssner, A. et al. IL-16 Is Constitutively Present in Peripheral Blood Monocytes and Spontaneously Released During Apoptosis. J. Immunol. 172, 7721–7725 (2004).[5]Roth, S. et al. Secondary necrotic neutrophils release interleukin-16C and macrophage migration inhibitory factor from stores in the cytosol. Cell Death Discov. 1, 15056 (2015).Disclosure of Interests:None declared

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Z-ligustilide protects BV-2 microglial cells against oxygen-glucose deprivation/reoxygenation-induced injury by inhibiting NLRP3 inflammasome activation and pyroptosis

European Journal of Inflammation ◽

10.1177/2058739220934925 ◽

2020 ◽

Vol 18 ◽

pp. 205873922093492

Author(s):

Jia Hu ◽

Jie Wei ◽

Cheng Zeng ◽

Fengqi Duan ◽

Sijun Liu ◽

...

Keyword(s):

Cell Viability ◽

Nlrp3 Inflammasome ◽

Glucose Deprivation ◽

Microglial Cells ◽

Cell Counting ◽

Oxygen Glucose Deprivation ◽

Inflammasome Activation ◽

Caspase 1 ◽

Nlrp3 Inflammasome Activation ◽

Active Caspase

Z-ligustilide (LIG) is the main bioactive compound of Danggui essential oil, which was reported to exert neuroprotective and anti-inflammatory effects. However, the underlying mechanism remains largely elusive. The present study aims to investigate the effect of LIG on oxygen-glucose deprivation/reoxygenation (OGD/R)-induced injury and whether Nod-like receptor protein 3 (NLRP3) inflammasome and related pyroptosis are targets for the treatment of LIG. The OGD/R model was established in BV-2 microglial cells to investigate the protective effect of LIG. Cell viability and the release of lactate dehydrogenase (LDH) were determined by cell counting assay kit 8 and the LDH release assay kit. Western blot and immunofluorescence staining were carried out to detect NLRP3 inflammasome activation and pyroptosis. Active caspase-1 and TdT-mediated dUTP nick end labeling (TUNEL) double positive cells were defined as pyroptosis population. Statistical comparison among multiple groups was carried out by one-way analysis of variance (ANOVA) followed by least significant difference (LSD) test. Compared with control cells, OGD/R impaired cell viability and induced the release of LDH in BV-2 microglial cells, which were associated with the activation of NLRP3 inflammasome as evidenced by increased expression of NLRP3 and the cleavage of caspase-1 and interleukin-1 beta (IL-1β). In parallel with NLRP3 inflammasome activation, OGD/R induced pyroptotic cell death, manifested by the cleavage of gasdermin D (GSDMD) and increased population of active caspase-1+/TUNEL+ cells. All these events were significantly attenuated by treatment with LIG, indicating that LIG significantly inhibited NLRP3 inflammasome activation and pyroptosis, and ameliorated OGD/R-induced cell injury. In conclusion, LIG protects BV-2 microglial cells against OGD/R-induced injury via inhibition of NLRP3 inflammasome and pyroptosis.

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Bruton’s tyrosine kinase drives neuroinflammation and anxiogenic behavior in mouse models of stress

Journal of Neuroinflammation ◽

10.1186/s12974-021-02322-9 ◽

2021 ◽

Vol 18 (1) ◽

Author(s):

Simantini Ghosh ◽

Zaidan Mohammed ◽

Itender Singh

Keyword(s):

Mouse Models ◽

Peripheral Blood Mononuclear Cells ◽

Nlrp3 Inflammasome ◽

Peripheral Blood ◽

Mononuclear Cells ◽

Specific Inhibitor ◽

Inflammasome Activation ◽

Peripheral Blood Mononuclear ◽

Caspase 1 ◽

Nlrp3 Inflammasome Activation

Abstract Background Current therapies targeting several neurotransmitter systems are only able to partially mitigate the symptoms of stress- and trauma-related disorder. Stress and trauma-related disorders lead to a prominent inflammatory response in humans, and in pre-clinical models. However, mechanisms underlying the induction of neuroinflammatory response in PTSD and anxiety disorders are not clearly understood. The present study investigated the mechanism underlying the activation of proinflammatory NLRP3 inflammasome and IL1β in mouse models of stress. Methods We used two mouse models of stress, i.e., mice subjected to physical restraint stress with brief underwater submersion, and predator odor stress. Mice were injected with MCC950, a small molecule specific inhibitor of NLRP3 activation. To pharmacologically inhibit BTK, a specific inhibitor ibrutinib was used. To validate the observation from ibrutinib studies, a separate group of mice was injected with another BTK-specific inhibitor LFM-A13. Seven days after the induction of stress, mice were examined for anxious behavior using open field test (OFT), light–dark test (LDT), and elevated plus maze test (EPM). Following the behavior tests, hippocampus and amygdale were extracted and analyzed for various components of NLRP3–caspase 1–IL1β pathway. Plasma and peripheral blood mononuclear cells were also used to assess the induction of NLRP3–Caspase 1–IL-1β pathway in stressed mice. Results Using two different pre-clinical models of stress, we demonstrate heightened anxious behavior in female mice as compared to their male counterparts. Stressed animals exhibited upregulation of proinflammatory IL1β, IL-6, Caspase 1 activity and NLRP3 inflammasome activation in brain, which were significantly higher in female mice. Pharmacological inhibition of NLRP3 inflammasome activation led to anxiolysis as well as attenuated neuroinflammatory response. Further, we observed induction of activated Bruton’s tyrosine kinase (BTK), an upstream positive-regulator of NLRP3 inflammasome activation, in hippocampus and amygdala of stressed mice. Next, we conducted proof-of-concept pharmacological BTK inhibitor studies with ibrutinib and LFM-A13. In both sets of experiments, we found BTK inhibition led to anxiolysis and attenuated neuroinflammation, as indicated by significant reduction of NLRP3 inflammasome and proinflammatory IL-1β in hippocampus and amygdala. Analysis of plasma and peripheral blood mononuclear cells indicated peripheral induction of NLRP3–caspase 1–IL1β pathway in stressed mice. Conclusion Our study identified BTK as a key upstream regulator of neuroinflammation, which drives anxiogenic behavior in mouse model of stress. Further, we demonstrated the sexually divergent activation of BTK, providing a clue to heightened neuroinflammation and anxiogenic response to stress in females as compared to their male counterparts. Our data from the pharmacological inhibition studies suggest BTK as a novel target for the development of potential clinical treatment of PTSD and anxiety disorders. Induction of pBTK and NLRP3 in peripheral blood mononuclear cells of stressed mice suggest the potential effect of stress on systemic inflammation.

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Knock Out of Cell Death Pathway Components Results in Differential Caspase Expression in Response to HCV Infection

Proceedings ◽

10.3390/proceedings2020050144 ◽

2020 ◽

Vol 50 (1) ◽

pp. 144

Author(s):

Hannah L. Wallace ◽

Lingyan Wang ◽

Cassandra Davidson ◽

Vipin Chelakkot ◽

Michael Grant ◽

...

Keyword(s):

Flow Cytometry ◽

Cell Death ◽

Cross Talk ◽

Caspase 3 ◽

Caspase Activation ◽

Knock Out ◽

Caspase 1 ◽

Cell Death Pathway ◽

Infected Cells ◽

Active Caspase

Introduction: Pyroptosis (inflammatory programmed cell death) is induced after the activation of an inflammasome, ultimately resulting in pore formation and cell lysis. One factor in the pathology associated with chronic hepatitis C virus (HCV) infection is non-inflammatory caspase-3-mediated apoptosis. Our lab has found both apoptosis and pyroptosis occurring in HCV-infected Huh-7.5 cells. In the context of some viral infections, pyroptosis is beneficial to the virus; for others, pyroptosis is believed to represent an innate antiviral response. This study aimed to test the effects of knocking out components of the inflammasome pathway on caspase activation in HCV-infected cells. Methods: FAM-FLICA (Carboxyfluorescein - Fluorochrome Inhibitor of Caspases) probes or antibodies were used to visualize active caspase-1 and active caspase-3 in vitro. Huh-7.5 cells with components of the pyroptotic or apoptotic pathways knocked out (NLRP3, GSDM-D or caspase-3) were used to determine the effects of their absence on the virus and caspase activation using confocal microscopy and flow cytometry. Results: Increased levels of caspase-1 were consistently observed in HCV-infected cells compared to those in uninfected cells, and these levels increased with subsequent days post-infection. The inhibition of inflammasome activation using knock out cell lines induced the differential activation of caspase-1 and caspase-3, with the inhibition of pyroptosis, resulting in a trend towards greater expression of caspase-3, indicative of apoptosis. The inhibition of NLRP3 did not fully stop caspase-1 activation, but it was decreased. The flow cytometry results revealed a small sub-set of cells positive for both caspase-1 and caspase-3. Conclusions: These data confirm the occurrence of pyroptosis in HCV-infected cells and demonstrate the involvement of the NLRP3 inflammasome, although other inflammasome sensors might be involved. Since the inhibition of one cell death pathway resulted in the increased activation of the other, along with the presence of double-positive cells, there may be cross-talk between apoptotic and pyroptotic pathways; the role of this cross-talk during infection remains to be elucidated.

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Trichomonas vaginalis Induces NLRP3 Inflammasome Activation and Pyroptotic Cell Death in Human Macrophages

Journal of Innate Immunity ◽

10.1159/000493585 ◽

2018 ◽

Vol 11 (1) ◽

pp. 86-98 ◽

Cited By ~ 8

Author(s):

Angelica Montenegro Riestra ◽

J. Andrés Valderrama ◽

Kathryn A. Patras ◽

Sharon D. Booth ◽

Xing Yen Quek ◽

...

Keyword(s):

Cell Death ◽

Host Cell ◽

Nlrp3 Inflammasome ◽

Trichomonas Vaginalis ◽

Inflammasome Activation ◽

Cell Detection ◽

Human Macrophages ◽

Sexually Transmitted ◽

Caspase 1 ◽

Nlrp3 Inflammasome Activation

Trichomonas vaginalis is a sexually transmitted, eukaryotic parasite that causes trichomoniasis, the most common nonviral, sexually transmitted disease in the USA and worldwide. Little is known about the molecular mechanisms involved in the host immune response to this widespread parasite. Here we report that T. vaginalis induces NLRP3 inflammasome activation in human macrophages, leading to caspase-1 activation and the processing of pro-IL-1β to the mature and bioactive form of the cytokine. Using inhibitor-based approaches, we show that NLRP3 activation by T. vaginalis involves host cell detection of extracellular ATP via P2X7 receptors and potassium efflux. In addition, our data reveal that T. vaginalis inflammasome activation induces macrophage inflammatory cell death by pyroptosis, known to occur via caspase-1 cleavage of the gasdermin D protein, which assembles to form pores in the host cell membrane. We found that T. vaginalis-induced cytolysis of macrophages is attenuated in gasdermin D knockout cells. Lastly, in a murine challenge model, we detected IL-1β production in vaginal fluids in response to T. vaginalis infection in vivo. Together, our findings mechanistically dissect how T. vaginalis contributes to the production of the proinflammatory IL-1β cytokine and uncover pyroptosis as a mechanism by which the parasite can trigger host macrophage cell death.

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Using tissue microarray to detect inflammasome signaling components that contribute to the pathogenesis of myelodysplastic syndrome.

Journal of Clinical Oncology ◽

10.1200/jco.2021.39.15_suppl.7044 ◽

2021 ◽

Vol 39 (15_suppl) ◽

pp. 7044-7044

Author(s):

Tony Kurian ◽

Kathy L. McGraw ◽

Sanjay Bridgelall ◽

Rohit Sharma ◽

Jeffrey E. Lancet ◽

...

Keyword(s):

Cell Death ◽

Tissue Microarray ◽

Nlrp3 Inflammasome ◽

Expression Patterns ◽

Low Risk ◽

Inflammasome Activation ◽

Beta Catenin ◽

Spearman Correlation ◽

Caspase 1 ◽

Blast Count

7044 Background: Myelodysplastic syndromes (MDS) are characterized by aberrant maturation, ineffective hematopoiesis, cytopenia, and progression to acute myeloid leukemia. MDS pathogenesis is multifactorial and potentially linked to constitutive innate immune stimulation converging upon the NLRP3 inflammasome to induce pyroptosis, a caspase-1 dependent cell death. Inflammasome assembly is initiated by both cell-extrinsic stimuli including S100A9, the TLR4 and CD33 ligand, and cell-intrinsic danger signals licensing caspase-1 which activates IL1b and beta-catenin resulting in cell death and cellular proliferation leading to maturation and differentiation blocks. Further, EYA2 has been suggested to be an inflammasome activator, whereas cPLA2 has been suggested to be an inhibitor. The purpose of this study is to determine whether immunohistochemistry (IHC) may be utilized to assess expression of inflammasome components. Methods: An IRB protocol was approved prior to initiating this study. We retrospectively identified 43 low risk MDS patients. A tissue microarray (TMA) was constructed utilizing MDS bone marrow biopsy samples (2-3 representative cores per sample). IL-1, S100A9, EYA2, cPLA2, beta-catenin, and TLR4 expression were assessed by IHC after validation of each antibody. IHC expression was scored independently by two hematopathologists by calculating scores (product of staining intensity x percent expression). IHC expression was compared using Spearman correlation estimate. Demographic and clinical data were collected and correlated with IHC expression using Kruskal-Wallis test, Spearman correlation, and Logrank test. Results: Patients were median 72 years of age, 67% men and included 47% MDS-MLD, 35% MDS-RS, 14% MDS-SLD, 2% MDS del5q and 2% MDS-U. IL-1 expression correlated with beta-catenin expression, r = 0.42, 95% CI 0.115 to 0.658 (p = 0.007). There was a trend towards significance between IL-1 and cPLA2, r = 0.30 (p = 0.067); S100A9 and cPLA2, r = 0.31 (p = 0.052); and S100A9 and EYA2, r = 0.31 (p = 0.057). Percentage EYA2 expression correlated with blast count, r = 0.425 (p = 0.008). The IHC expression of these antigens did not correlate with WHO MDS subclassification, IPSS, R-IPSS, disease progression, or survival (p > 0.05). Conclusions: IHC staining of inflammasome activators using TMA may allow better characterization of molecular pathways contributing the MDS pathogenesis. A correlation was seen between expression of antigens known to be increased downstream of NLRP3 inflammasome activation. Furthermore, increased expression of EYA2 correlated with blast count. A future study will compare expression patterns between normal, low risk MDS and high risk MDS samples and correlate these findings with clinical outcome data to further elucidate the pathogenesis of MDS and identify potential targetable markers for novel therapeutic strategies.

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Human and Pathogen Derived Ndpks Act As Novel Damps and PAMPs to Drive Leukemia Cell Survival and Progression through Signaling Via the TLR4-Mediated Alternative NLRP3 Inflammasome Pathway

Blood ◽

10.1182/blood-2019-131236 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2684-2684

Author(s):

Sandro Trova ◽

Matthew Fenton ◽

Bhavini Chauhan ◽

Avani Puri ◽

Santosh Lomada ◽

...

Keyword(s):

Cell Death ◽

Nlrp3 Inflammasome ◽

Peripheral Blood ◽

Active Form ◽

Leukemia Cell ◽

Conditioned Media ◽

Normal Donor ◽

Normal Peripheral Blood ◽

Caspase 1

Elevated plasma levels of the nucleoside diphosphate kinase (NDPK) NM23-H1 are associated with poorer prognosis in acute myeloid leukemia (AML). We previously demonstrated that leukemic blasts release NM23-H1, which binds to more differentiated myeloid cells inducing their secretion of inflammatory cytokines, including IL-1β, that promote survival and proliferation of leukemic blasts1. Both AML and myelodysplastic syndrome (MDS) patients are prone to infections due to impaired hematopoiesis that is worsened by treatment. NDPKs are highly evolutionarily conserved raising the possibility that bacterial/fungal NDPKs could mediate the same survival effect on malignant AML/MDS blasts and exacerbate disease progression. To test this, we generated recombinant NDPKs (rNDPKs) from bacteria and fungi associated with common infections in these patients (E. coli, S. aureus, S. pneumoniae, K. pneumoniae, C. albicans). Cytokine production and survival responses of primary AMLs to these proteins were indistinguishable from their response to rNM23-H1. This activity was independent of NDPK enzyme activity since mutant rNM23-H1 and bacterial and fungal rNDPKs with impaired oligomerization, kinase or exonuclease activity elicited the same cytokine and survival response. Toll like receptors (TLRs) are the major family of human DAMP/PAMP receptors and IL-1β secretion is closely associated with TLR-4 mediated activation of the NLRP3 inflammasome in monocytes. We therefore postulated that NM23-H1 and pathogen derived NDPKs act as novel damage- and pathogen- associated molecular pattern (DAMP, PAMP) molecules. We confirmed that fluorescently labelled rNM23-H1 and S. pneumoniae rNDPK bound selectively to monocytes in peripheral blood. Using in vitro generated monocytes (vitamin D3 differentiated THP-1 cells) we demonstrated that both wild type and mutant rNM23-H1 and bacterial/fungal rNDPKs induced activation of caspase-1 and cleavage of pro-IL-1β into its active form. Secretion of IL-1β was inhibited by antagonists/inhibitors of TLR4, NLRP3 and caspase-1 indicating the involvement of the TLR4-NLRP3 inflammasome axis is mediating the NDPK response. Unlike the canonical NLRP3-inflammasome pathway that leads to monocyte cell death by pyroptosis, rNM23-H1 and rNDPKs did not lead to cell death indicating that rNDPKs are responsible for the activation of the alternative inflammasome. In our earlier studies, and those of others, we demonstrated that not all AML primary samples responsed to NM23-H1 in vitro. We have observed that non responders to NM23-H1 also do not respond to pathogen derived rNDPKs. In contrast, we have observed uniform responses in terms of cytokine release in all normal peripheral blood. We hence hypothesized that the non-rNDPK-responding AML samples may reflect the absence of monocytes in culture. To test this, we generated conditioned media using normal donor leukocytes, in presence or absence of a TLR-4 antagonist to inhibit the IL-1β production. The conditioned media was then used to culture primary AML samples, in parallel with rNDPK in unconditioned media. All the samples analyzed showed increased survival in rNDPK conditioned media even whilst some did not respond to rNDPK in unconditioned media. In summary, our data demonstrate for the first time that NM23-H1 and bacterial/fungal NDPKs are novel DAMPs/PAMPS that signal via TLR4 in monocytes. We further demonstrate that this interaction results in activation of the alternative NLRP3 inflammasome and subsequent cleavage and secretion of IL-1β without death by pyroptosis. Our data showing that bacterial/fungal NDPKs can promote survival of AML blasts indicates that rather than just being a consequence of AML associated immunosuppression, infections may drive the progression and AML. These findings have important implications in the clinical management of AML and its precursor myelodysplastic syndromes (MDS). Lilly AJ, et al.Cancer Res. 2011;71(3):1177-86.Gaidt MM, et al. Immunity. 2016;44(4):833-46 Disclosures Drayson: Abingdon Health: Consultancy, Equity Ownership.

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Dimethyl Fumarate Alleviates NLRP3 Inflammasome Activation in Microglia and Sickness Behavior in LPS-Challenged Mice

Frontiers in Immunology ◽

10.3389/fimmu.2021.737065 ◽

2021 ◽

Vol 12 ◽

Author(s):

Bora Tastan ◽

Burak I. Arioz ◽

Kemal Ugur Tufekci ◽

Emre Tarakcioglu ◽

Ceren Perihan Gonul ◽

...

Keyword(s):

Cell Death ◽

Nlrp3 Inflammasome ◽

Forced Swim Test ◽

Sickness Behavior ◽

Dimethyl Fumarate ◽

Inflammasome Activation ◽

Caspase 1 ◽

Nlrp3 Inflammasome Activation

NLRP3 inflammasome activation contributes to several pathogenic conditions, including lipopolysaccharide (LPS)-induced sickness behavior characterized by reduced mobility and depressive behaviors. Dimethyl fumarate (DMF) is an immunomodulatory and anti-oxidative molecule commonly used for the symptomatic treatment of multiple sclerosis and psoriasis. In this study, we investigated the potential use of DMF against microglial NLRP3 inflammasome activation both in vitro and in vivo. For in vitro studies, LPS- and ATP-stimulated N9 microglial cells were used to induce NLRP3 inflammasome activation. DMF’s effects on inflammasome markers, pyroptotic cell death, ROS formation, and Nrf2/NF-κB pathways were assessed. For in vivo studies, 12–14 weeks-old male BALB/c mice were treated with LPS, DMF + LPS and ML385 + DMF + LPS. Behavioral tests including open field, forced swim test, and tail suspension test were carried out to see changes in lipopolysaccharide-induced sickness behavior. Furthermore, NLRP3 and Caspase-1 expression in isolated microglia were determined by immunostaining. Here we demonstrated that DMF ameliorated LPS and ATP-induced NLRP3 inflammasome activation by reducing IL-1β, IL-18, caspase-1, and NLRP3 levels, reactive oxygen species formation and damage, and inhibiting pyroptotic cell death in N9 murine microglia via Nrf2/NF-κB pathways. DMF also improved LPS-induced sickness behavior in male mice and decreased caspase-1/NLRP3 levels via Nrf2 activation. Additionally, we showed that DMF pretreatment decreased miR-146a and miR-155 both in vivo and in vitro. Our results proved the effectiveness of DMF on the amelioration of microglial NLRP3 inflammasome activation. We anticipate that this study will provide the foundation consideration for further studies aiming to suppress NLRP3 inflammasome activation associated with in many diseases and a better understanding of its underlying mechanisms.

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Vibrio pore-forming leukocidin activates pyroptotic cell death via the NLRP3 inflammasome

10.1101/816553 ◽

2019 ◽

Author(s):

Noam Baram ◽

Hadar Cohen ◽

Liat Edry-Botzer ◽

Dor Salomon ◽

Motti Gerlic

Keyword(s):

Cell Death ◽

Nlrp3 Inflammasome ◽

Host Cells ◽

Inflammasome Activation ◽

Vibrio Proteolyticus ◽

Caspase 1 ◽

Cell Plasma ◽

Target Membrane ◽

Cell Death Mechanisms ◽

Different Diameters

ABSTRACTCell death mechanisms are central to combat infectious microbes and to drive pathological inflammation. One such mechanism, the inflammasome, controls infection through either activation of caspase-1 and the subsequent secretion of the mature pro-inflammatory cytokine, interleukin 1β (IL-1β), or by stopping the dissemination of intracellular pathogens by inducing pyroptotic cell death in infected cells. Hemolysins, which are pore-forming toxins (PFTs), target the host cell plasma membrane by producing pores with different diameters. These pores alter the permeability of the target membrane, often leading to cell death. We previously discovered a functional and potent pore-forming, leukocidin domain-containing hemolysin produced by the Gram-negative marine bacterium Vibrio proteolyticus (V. proteolyticus), termed VPRH. Although leukocidin domains are found in other known PFTs, VPRH constitutes a distinct, understudied class within the leukocidin superfamily. Since PTFs of other pathogens were shown to induce cell death by activating the inflammasome pathway, we hypothesized that VPRH-induced cell death is mediated by direct activation of the inflammasome in mammalian immune host cells. Indeed, we found that VPRH induced a two-step cell death in primary macrophages. The first, a rapid step, was mediated by activating the NLRP3 inflammasome, leading to caspase-1 activation and GSDMD cleavage that resulted in IL-1β secretion and pyroptotic cell death. The second step was independent of the inflammasome; however, its mechanism remains unknown. This study sets the foundation for better understanding the immunological consequences of inflammasome activation by a new leukocidin class of toxins.

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Lycopus lucidus Turcz exerts neuroprotective effect against H2O2‑induced neuroinflammation by inhibiting NLRP3 inflammasome activation in cortical neurons

10.21203/rs.3.rs-149006/v1 ◽

2021 ◽

Author(s):

Hyunseong Kim ◽

Jin Young Hong ◽

Wan-Jin Jeon ◽

Junseon Lee ◽

Seung Ho Baek ◽

...

Keyword(s):

Cell Death ◽

Nlrp3 Inflammasome ◽

Cortical Neurons ◽

Neuronal Damage ◽

Neuroprotective Effect ◽

Synaptic Connectivity ◽

Inflammasome Activation ◽

Caspase 1 ◽

Nlrp3 Inflammasome Activation ◽

Rat Cortical Neurons

Abstract Background Central nervous system (CNS) injuries are a leading cause of permanent functional impairment in humans. Nerve damage can be aggravated by neuroinflammation mediated by protein complexes known as inflammasomes, such as the NLRP3 inflammasome which is a key mediator of caspase-1 and interleukin-1β (IL-1β) /interleukin-18 (IL-18) activation. Lycopus lucidus Turcz (LLT) is a traditional medicinal herb that exerts therapeutic effects against oxidative stress, inflammation, and angiogenesis; however, it remains unclear whether LLT can directly protect neurons against damage, and the underlying molecular mechanisms are poorly understood. Methods We investigated the neuroprotective effect of LLT against hydrogen peroxide (H2O2)-induced neuronal damage in cultured primary rat cortical neurons, as well as the potential underlying mechanisms. Neuronal viability and cell death assays were used to determine the effects of LLT on neuroprotection, while the mode of cell death was confirmed using flow cytometry. Changes in the expression of inflammatory factors involved in activation of the NLRP3 inflammasome were measured using immunocytochemistry (ICC) and confirmed by real-time PCR. And, we analyzed that the effect of LLT on neurotrophic factors secretion and synaptic connectivity using ICC in H2O2-induced neuron at 7 days in vitro. Results LLT effectively protected cultured rat cortical neurons from H2O2-induced injury by significantly inhibiting NLRP3 inflammasome activation. In addition, LLT significantly reduced caspase1 activation, which is known to be induced by inflammasome formation, and consequently regulated the secretion of IL-1β/IL-18. We demonstrated that LLT enhances axonal elongation and synaptic connectivity against H2O2-induced injury of rat primary cortical neuron. Conclusions Together, these results demonstrate that LLT can directly protect cultured cortical neurons from H2O2-induced neuronal damage by inhibiting NLRP3 inflammasome activation and the secretion of caspase-1 and IL-1β/IL-18. Thus, our study provides new insights into the therapeutic mechanisms of LLT and suggests that the NLRP3 inflammasome could be a promising target for treating neurological diseases.

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