Inflammasome In Monocytes Research Articles

NLRP3 inflammasome activation and symptom burden in KRAS-mutated CMML patients is reverted by IL-1 blocking therapy

Chronic myelomonocytic leukemia (CMML) is frequently associated with mutations in the rat sarcoma gene (RAS), leading to worse prognosis. RAS mutations result in active RAS-GTP proteins, favoring myeloid cell proliferation and survival and inducing the NLRP3 inflammasome together with the apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), which promote caspase-1 activation and interleukin (IL)-1β release. Here, we report, in a cohort of CMML patients with mutations in KRAS, a constitutive activation of the NLRP3 inflammasome in monocytes, evidenced by ASC oligomerization and IL-1β release, as well as a specific inflammatory cytokine signature. Treatment of a CMML patient with a KRASG12D mutation using the IL-1 receptor blocker anakinra inhibits NLRP3 inflammasome activation, reduces monocyte count, and improves the patient's clinical status, enabling a stem cell transplant. This reveals a basal inflammasome activation in RAS-mutated CMML patients and suggests potential therapeutic applications of NLRP3 and IL-1 blockers.

Cell type-specific roles of NLRP3, inflammasome-dependent and -independent, in host defense, sterile necroinflammation, tissue repair, and fibrosis.

The NLRP3 inflammasome transforms a wide variety of infectious and non-infectious danger signals that activate pro-inflammatory caspases, which promote the secretion of IL-1β and IL-18, and pyroptosis, a pro-inflammatory form of cell necrosis. Most published evidence documents the presence and importance of the NLRP3 inflammasome in monocytes, macrophages, and neutrophils during host defense and sterile forms of inflammation. In contrast, in numerous unbiased data sets, NLRP3 inflammasome-related transcripts are absent in non-immune cells. However, an increasing number of studies report the presence and functionality of the NLRP3 inflammasome in almost every cell type. Here, we take a closer look at the reported cell type-specific expression of the NLRP3 inflammasome components, review the reported inflammasome-dependent and -independent functions, and discuss possible explanations for this discrepancy.

Microbiota-Associated HAF-EVs Regulate Monocytes by Triggering or Inhibiting Inflammasome Activation.

In pregnancy, human amniotic fluid extracellular vesicles (HAF-EVs) exert anti-inflammatory effects on T cells and on monocytes, supporting their immunoregulatory roles. The specific mechanisms are still not completely defined. The aim of this study was to investigate the ability of HAF-EVs, isolated from pregnant women who underwent amniocentesis and purified by gradient ultracentrifugation, to affect inflammasome activation in the human monocytes. Proteomic studies revealed that HAF-EV samples expressed several immunoregulatory molecules as well as small amounts of endotoxin. Surprisingly, metagenomic analysis shows the presence of specific bacterial strain variants associated with HAF-EVs as potential sources of the endotoxin. Remarkably, we showed that a single treatment of THP-1 cells with HAF-EVs triggered inflammasome activation, whereas the same treatment followed by LPS and ATP sensitization prevented inflammasome activation, a pathway resembling monocyte refractories. A bioinformatics analysis of microbiota-HAF-EVs functional pathways confirmed the presence of enzymes for endotoxin biosynthesis as well as others associated with immunoregulatory functions. Overall, these data suggest that HAF-EVs could serve as a source of the isolation of a specific microbiota during early pregnancy. Moreover, HAF-EVs could act as a novel system to balance immune training and tolerance by modulating the inflammasome in monocytes or other cells.

Increased Inflammasome Activation Is Associated with Aging and Chronic Myelomonocytic Leukemia Disease Severity.

Aging causes chronic low-grade inflammation known as inflamm-aging. It is a risk factor for several chronic disorders, including chronic myelomonocytic leukemia (CMML), a hematological malignancy that is most prevalent in older people. Recent studies suggest a critical role for the NLRP3 (NOD-, LRR- and pyrin domain-containing protein 3) inflammasome in inflamm-aging. However, the mechanisms involved in NLRP3 activation in aging and its involvement in CMML progression are not fully understood. In this study, we report that aging increases IL-1β production upon NLRP3 activation in human CD14+ monocytes. Interestingly, we found that the TLR1/2 agonist Pam3CSK4 directly activates the NLRP3 inflammasome in monocytes from older but not from younger healthy donors. Furthermore, we observed a dichotomous response to NLRP3 inflammasome activation in monocytes from a small cohort of CMML patients, and some patients produced high levels of IL-1β and some patients produced low levels of IL-1β compared with older healthy donors. Intriguingly, CMML patients with heightened NLRP3 activation showed increased treatment dependency and disease severity. Collectively, our results suggest that aging causes increased sensitivity to NLRP3 inflammasome activation at a cellular level, which may explain increased inflammation and immune dysregulation in older individuals. Furthermore, NLRP3 inflammasome activation was dysregulated in a small cohort of CMML patients and was positively correlated with disease severity.

A missense variant in the nuclear localization signal of DKC1 causes Hoyeraal-Hreidarsson syndrome

Hoyeraal-Hreidarsson syndrome (HHS) is the most severe form of dyskeratosis congenita (DC) and is caused by mutations in genes involved in telomere maintenance. Here, we identified male siblings from a family with HHS carrying a hemizygous mutation (c.1345C > G, p.R449G), located in the C-terminal nuclear localization signal (NLS) of the DKC1 gene. These patients exhibit progressive cerebellar hypoplasia, recurrent infections, pancytopenia due to bone marrow failure, and short leukocyte telomere lengths. Single-cell RNA sequencing analysis suggested defects in the NLRP3 inflammasome in monocytes and the activation and maturation of NK cells and B cells. In experiments using induced pluripotent stem cells (iPSCs) from patients, DKC1_R449G iPSCs had short telomere lengths due to reduced levels of human telomerase RNA (hTR) and increased cytosolic proportions of DKC1. Treatment with dihydroquinolizinone RG7834 and 3′deoxyanosine cordycepin rescued telomere length in patient-derived iPSCs. Together, our findings not only provide new insights into immunodeficiency in DC patients but also provide treatment options for telomerase insufficiency disorders.

Inflammasome activation in neutrophils of patients with severe COVID-19

AbstractInfection by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) engages the inflammasome in monocytes and macrophages and leads to the cytokine storm in COVID-19. Neutrophils, the most abundant leukocytes, release neutrophil extracellular traps (NETs), which have been implicated in the pathogenesis of COVID-19. Our recent study shows that activation of the NLRP3 inflammasome is important for NET release in sterile inflammation. However, the role of neutrophil inflammasome formation in human disease is unknown. We hypothesized that SARS-CoV-2 infection may induce inflammasome activation in neutrophils. We also aimed to assess the localization of inflammasome formation (ie, apoptosis-associated speck-like protein containing a CARD [ASC] speck assembly) and timing relative to NETosis in stimulated neutrophils by real-time video microscopy. Neutrophils isolated from severe COVID-19 patients demonstrated that ∼2% of neutrophils in both the peripheral blood and tracheal aspirates presented ASC speck. ASC speck was observed in neutrophils with an intact poly-lobulated nucleus, suggesting early formation during neutrophil activation. Additionally, 40% of nuclei were positive for citrullinated histone H3, and there was a significant correlation between speck formation and nuclear histone citrullination. Time-lapse microscopy in lipopolysaccharide -stimulated neutrophils from fluorescent ASC reporter mice showed that ASC speck formed transiently and at the microtubule organizing center long before NET release. Our study shows that ASC speck is present in neutrophils from COVID-19 patients with respiratory failure and that it forms early in NETosis. Our findings suggest that inhibition of neutrophil inflammasomes may be beneficial in COVID-19.

Visualizing P2X7-Dependent Inflammasome Formation in Human Monocytes by Fluorescence Microscopy and Flow Cytometry.

One of the most prominent effects of P2X7 activation in myeloid cells is the induction of the assembly of the NLRP3 inflammasome, a central process controlling the secretion of pro-inflammatory cytokines of the IL-1 family such as IL-1β and IL-18. The ability to visualize inflammasome formation greatly facilitates research into the role of P2X7 in inflammation. In this chapter, a method to monitor the formation of the NLPR3 inflammasome in monocytes and other myeloid cells could be demonstrated. Following priming by lipopolysaccharide (LPS), P2X7 was stimulated by ATP to mediate inflammasome assembly. This causes cytosolically disperse ASC, a central component of the inflammasome, to aggregate into microscopically visible specks due to its recruitment to the inflammasome. Methods to monitor this change in the spatial distribution of ASC in human peripheral blood monocytes by flow cytometry and fluorescence microscopy are presented.

DISULFIRAM EXERTS DOSE-DEPENDENT ACTIVATING AND INHIBITORY EFFECTS ON THE HUMAN INFLAMMASOME

TOPIC: Critical Care TYPE: Original Investigations PURPOSE: Gasdermin-D (GSDMD) is a pore-forming molecule that serves as the final effector of pyroptosis (a form of inflammatory cell death) following activation of the cellular inflammasome in monocytes. Disulfiram (DSM), a drug used in the treatment of alcohol abuse, has recently been shown to inhibit GSDMD via disruption of pore formation after its cleavage by the inflammatory caspases (1, 4, and 5). The aim of this study was to elucidate the inhibitory effects of DSM on GSDMD-mediated pyroptosis and explore its role as a potential therapeutic agent in sepsis. METHODS: A series of experiments was conducted using THP1 cells (a human monocytic cell line) or purified human monocytes from the blood of healthy donors. Samples were pretreated with DSM in various concentrations 30 minutes prior to inflammasome activation with bacterial lipopolysaccharide (LPS). Cells were then assessed under light microscopy for swelling and fluorescent microscopy for the formation of ASC specks (activated oligomers of the adaptor protein required for caspase activation). Cell death was assessed using a lactate dehydrogenase (LDH) cytotoxicity assay. Interleukin-1-beta (IL1s) release was measured by ELISA. GSDMD cleavage, pro-IL1s synthesis, and caspase-1 cleavage were assessed using immunoblots. RESULTS: Unexpectedly, low dose DSM accentuated inflammasome activation. Dramatic inflammasome induction was observed at a concentration of 10uM, a dose associated with near peak inhibition in the prior study. However, higher doses of 50 to 100 uM suppressed pyroptosis as evidenced by loss of cell swelling and ASC speck formation, as well as LDH and IL-1s release. The DSM effect occurred downstream of caspase-1 activation as evidenced by preserved intracellular pro-IL1s synthesis and caspase-1 cleavage in the presence of DSM. Contrary to the prior report, GSDMD cleavage appeared to be inhibited at these higher doses. CONCLUSIONS: DSM unexpectedly exhibits upstream activation of the inflammasome at low doses but downstream inhibition at high doses. CLINICAL IMPLICATIONS: Although DSM's inhibitory effects on inflammasome function make it a promising agent for the future treatment of acute inflammatory conditions such as sepsis and covid-19, our results indicate that further study is needed to better characterize its possible activating effects at lower concentrations. DISCLOSURES: Collaboration via BARDA grant funding relationship with Beckman Coulter, Inc. Please note: 9/19/2019-8/31/2014 Added 04/27/2021 by Elliott Crouser, source=Web Response, value=Grant/Research Support No relevant relationships by Gregory Eisinger, source=Web Response No relevant relationships by Mikhail Gavrilin, source=Web Response No relevant relationships by Wissam Osman, source=Web Response No relevant relationships by Evan Prather, source=Web Response No relevant relationships by Mark Wewers, source=Web Response

NLRP3 and IL-1β Gene Expression Is Elevated in Monocytes From HIV-Treated Patients With Neurocognitive Disorders.

Systemic immune activation and inflammation in chronic HIV infection are driving factors of non-AIDS-related events, including neurocognitive impairment. The role of inflammasome in monocytes from patients with HIV infection has been extensively studied, but its association with the extent of neurocognitive dysfunction has been poorly investigated. We enrolled 79 HIV-positive patients; 44 with varying levels of HIV-associated neurocognitive disorder (HAND) and 35 without and 8 healthy donors. HAND subtypes included asymptomatic neurocognitive impairment (asymptomatic neurocognitive impairment; n = 19), mild neurocognitive disorder (MND; n = 17), and HIV-associated dementia (n = 8). We quantified plasmatic concentrations of proinflammatory cytokines (TNF-α, IL-6, IL-17A, IL-1β, and IFN-γ) for all HIV patients, and the mRNA expression of genes involved in the inflammasome activity (NLRP3, PYCARD, NAIP, AIM2, IL-1β, and IL-18) in monocytes of a subgroup of 28 HIV patients and 8 healthy donors. HIV patients' plasma concentrations of IFN-γ, IL-1β, and IL-17A were undetectable. Levels of TNF-α and IL-6 were similar among the HIV patient groups. A trend toward an increased expression of inflammasome genes according to neurocognitive disorder severity was observed. Of note, the NLRP3 mRNA relative expression was higher in MND compared with other groups, and IL-1β was lower in MND than HIV-associated dementia patients. Changes in inflammasome components in circulating monocytes according to different HAND severity suggest that NLRP3 may be a possible biomarker or target to better understand and treat the link between systemic inflammation and neurocognitive impairment in HIV infection.

Stimulation of Vibratory Urticaria-Associated Adhesion-GPCR, EMR2/ADGRE2, Triggers the NLRP3 Inflammasome Activation Signal in Human Monocytes.

EMR2/ADGRE2 is an adhesion G protein-coupled receptor differentially expressed by human myeloid cells. It modulates diverse cellular functions of innate immune cells and a missense EMR2 variant is directly responsible for vibratory urticaria. Recently, EMR2 was found to activate NLRP3 inflammasome in monocytes via interaction with FHR1, a regulatory protein of complement Factor H. However, the functional involvement of EMR2 activation and its signaling mechanisms in eliciting NLRP3 inflammasome activation remain elusive. In this study, we show that EMR2-mediated signaling plays a critical role in triggering the activation (2nd) signal for the NLRP3 inflammasome in both THP-1 monocytic cell line and primary monocytes. Stimulation of EMR2 by its agonistic 2A1 monoclonal antibody elicits a Gα16-dependent PLC-β activation pathway, inducing the activity of downstream Akt, MAPK, NF-κB, and Ca2+ mobilization, eventually leading to K+ efflux. These results identify EMR2 and its associated signaling intermediates as potential intervention targets of NLRP3 inflammasome activation in inflammatory disorders.

The secretion of cytokines by peripheral blood mononuclear cells of patients with periodontitis and healthy controls when exposed to H2S

ABSTRACT Background: Hydrogen sulfide(H2S) is a bacterial metabolite produced as a result of bacterial growth in subgingival pockets, suggested to partake in the pathogenesis of periodontitis. H2S has previously been shown to induce the secretion of the pro-inflammatory cytokines IL-1β and IL-18 via the NLRP3 inflammasome in monocytes. Objective: To investigate the non-NLRP3 inflammasome-dependent immunological response of human peripheral blood mononuclear cells (PBMCs) of periodontitis patients and healthy controls exposed to H2S in vitro. Methods: PBMCs of periodontitis patients(N = 31) and healthy controls(N = 32) were exposed to 1 mM sodium hydrosulfide (NaHS) at 37°C for 24 h and the secretion of cytokines was compared to resting cells. TNF-α, IFN-γ, IL-6, IL-8, IL-12p40, IL-12p70, IL-17, MCP-1, and IL-1Ra secretions were measured with Bio-Plex Pro™ Human Cytokine Assay. Results: H2S triggered the secretion of the pro-inflammatory IFN-γ, IL-6, IL-17, TNF-α, IL-12p40, and IL-12p70, while the reverse was seen for IL-1Ra. In addition, a higher basal secretion of IFN-γ, IL-6, IL-12p70, IL-17 and MCP-1 was seen from PBMCs of periodontitis patients compared to healthy controls. Conclusion: The bacterial metabolite H2S triggers the secretion of pro-inflammatory cytokines from PBMCs and may thus have a prominent role in the host-bacteria interplay in periodontitis.

Calcium-sensing receptor-mediated NLRP3 inflammasome response to calciprotein particles drives inflammation in rheumatoid arthritis

Increased extracellular Ca2+ concentrations ([Ca2+]ex) trigger activation of the NLRP3 inflammasome in monocytes through calcium-sensing receptor (CaSR). To prevent extraosseous calcification in vivo, the serum protein fetuin-A stabilizes calcium and phosphate into 70-100 nm-sized colloidal calciprotein particles (CPPs). Here we show that monocytes engulf CPPs via macropinocytosis, and this process is strictly dependent on CaSR signaling triggered by increases in [Ca2+]ex. Enhanced macropinocytosis of CPPs results in increased lysosomal activity, NLRP3 inflammasome activation, and IL-1β release. Monocytes in the context of rheumatoid arthritis (RA) exhibit increased CPP uptake and IL-1β release in response to CaSR signaling. CaSR expression in these monocytes and local [Ca2+] in afflicted joints are increased, probably contributing to this enhanced response. We propose that CaSR-mediated NLRP3 inflammasome activation contributes to inflammatory arthritis and systemic inflammation not only in RA, but possibly also in other inflammatory conditions. Inhibition of CaSR-mediated CPP uptake might be a therapeutic approach to treating RA.

Fingolimod (FTY720) Activates Protein Phosphatase‐2A in Human Monocytes and Inhibits Uric Acid Crystal‐Induced Interleukin‐1 Beta Production

IntroductionGout is a chronic inflammatory disease due to monosodium urate (MSU) crystal deposits in articular joints. Phagocytosis of MSU crystals by monocytes/macrophages results in downstream expression and production of interleukin‐1 beta (IL‐1β). The two‐step process of gout inflammation involves 1) priming of monocytes with danger signals e.g. lipopolysaccharide (LPS) or soluble uric acid (UA) and 2) MSU phagocytosis and activation of the NLRP3 inflammasome in monocytes. Protein‐phosphatase‐2A (PP2A) is a serine/threonine phosphatase, and a reduction in intracellular macrophage PP2A activity was shown to aggravate LPS‐induced proinflammatory cytokine production. The prodrug Fingolimod (FTY720) and its phosphorylated active metabolite (p‐FTY720) are efficacious in multiple sclerosis and their mechanism of action involves activation of intracellular PP2A. The objective of this study is to investigate the role of PP2A in regulating monocyte priming and activation by MSU crystals and whether intracellular PP2A activation exerts an anti‐inflammatory effect in MSU‐stimulated monocytes.MethodsHuman THP‐1 monocytes (ATCC; 500,000 cells per well) were treated with UA (50 mg/dL) and LPS (10ng/mL) for 24 hours. Subsequently, cells were treated with MSU crystals (100ug/mL) and MSU phagocytosis was determined at 4 hours using flow cytometry and the percentage of cells displaying side‐scattering above a threshold value, indicative of crystal phagocytosis, were determined. Intracellular PP2A activity was determined at 6 hours following MSU stimulation using the PP2A Immunoprecipitation Phosphatase Assay Kit (Fisher Scientific). IL‐1β expression and protein levels at 6 hours were determined using quantitative polymerase chain reaction and ELISA, respectively. THP‐1 monocytes were treated with FTY720 or p‐FTY720 (2.5mM for both treatments; Cayman Chemicals) and PP2A activity was determined at 1, 3, 6 and 24 hours. THP‐1 monocytes were treated with UA+LPS followed by MSU ± p‐FTY720 (2.5mM for 6 hours) and IL‐1β expression and protein levels were determined. Statistical analyses were performed using analysis of variance (ANOVA) with Tukey’s post‐hoc test.ResultsUA+LPS pre‐treatment increased MSU phagocytosis by THP‐1 monocytes (fig. 1A and 1B; p<0.001) and IL‐1β expression (fig. 1C; p<0.001) and production (fig. 1D; p<0.001). This effect was associated with a reduction in intracellular PP2A activity (fig. 1E; p<0.001). FTY720 and p‐FTY720 increased intracellular PP2A activity in monocytes (fig. 2A; p<0.001) with a higher peak effect achieved at 1 hour for p‐FTY720 (p<0.01). p‐FTY720 treatment reduced IL‐1β expression (fig. 2B; p<0.001) and mature IL‐1β production (fig. 2C; p<0.001) in UA+LPS pre‐treated human monocytes following MSU stimulation.ConclusionUA and LPS enhanced MSU phagocytosis, expression and production of IL‐1β, potentially via a reduction in intracellular PP2A activity. FTY720 and p‐FTY720 activated PP2A in monocytes with an onset of effect of 1 hour and correspondingly reduced IL‐1β expression and production. PP2A is a novel therapeutic target for gout treatment.Impact of soluble uric acid (UA) and lipopolysaccharide (LPS) pre‐treatments on THP‐1 monocyte phagocytic activity towards monosodium urate (MSU) crystals, interleukin‐1 beta (IL‐1β) expression and production and association with intracellular protein phosphatase‐2A (PP2A) activity. A) Representative flow cytometry scatter plots showing enhanced MSU crystal phagocytosis (as shown by more cells with increased side‐scatter in P2 region of interest) by THP‐1 monocytes following UA+LPS pre‐treatment. B) UA and UA+LPS pre‐treatment increased the percentage of THP‐1 monocytes positive for MSU phagocytosis. C) UA+LPS pre‐treatment increased IL‐1β expression by THP‐1 monocytes following MSU challenge. D) UA+LPS pre‐treatment increased IL‐1β production by THP‐1 monocytes following MSU challenge. E) Intracellular PP2A activity was reduced in UA or UA+LPS pre‐treated THP‐1 monocytes. *p<0.001; **p<0.01; n.s.: non significant.Figure 1Impact of FTY720 (Fingolimod; prodrug) and phosphorylated FTY720 (p‐FTY‐720; active metabolite) treatments on protein phosphatase‐2A (PP2A) activity in human THP‐1 monocytes and impact of p‐FTY720 treatment on interleukin‐1 beta (IL‐1β) expression and production by monosodium urate (MSU) crystal stimulated THP‐1 monocytes ± soluble uric acid (UA) and lipopolysaccharide (LPS) pre‐treatment. Treatments were conducted using 2.5μM concentrations. A) FTY720 and p‐FTY720 increased intracellular PP2A activity in THP‐1 monocytes. B) p‐ FTY720 reduced IL‐1β expression in THP‐1 monocytes. C) p‐FTY720 reduced IL‐1β production in THP‐1 monocytes. *p<0.001; **p<0.01; n.s.: non significant.Figure 2

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

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.

Activation of Interleukin-1β Release by the Classical Swine Fever Virus Is Dependent on the NLRP3 Inflammasome, Which Affects Virus Growth in Monocytes.

Classical swine fever virus (CSFV) is a classic Flavivirus that causes the acute, febrile, and highly contagious disease known as classical swine fever (CSF). Inflammasomes are molecular platforms that trigger the maturation of proinflammatory cytokines to engage innate immune defenses that are induced upon cellular infection or stress. However, the relationship between the inflammasome and CSFV infection has not been thoroughly characterized. To understand the function of the inflammasome response to CSFV infection, we infected porcine peripheral blood monocytes (PBMCs) with CSFV. Our results indicated that CSFV infection induced both the generation of pro-interleukin-1β (pro-IL-1β) and its processing in monocytes, leading to the maturation and secretion of IL-1β through the activation of caspase 1. Moreover, CSFV infection in PBMCs induced the production and cleavage of gasdermin D (GSDMD), which is an inducer of pyroptosis. Additional studies showed that CSFV-induced IL-1β secretion was mediated by NLRP3 and that CSFV infection could sufficiently activate the assembly of the NLRP3 inflammasome in monocytes. These results revealed that CSFV infection inhibited the expression of NLRP3, and knockdown of NLRP3 enhanced the replication of CSFV. In conclusion, these findings demonstrate that the NLRP3 inflammasome plays an important role in the innate immune response to CSFV infection.

Reduced NLRP3 Gene Expression Limits the IL-1β Cleavage via Inflammasome in Monocytes from Severely Injured Trauma Patients.

Objective Traumatic injury or severe surgery leads to a profound immune response with a diminished functionality of monocytes and subsequently their IL-1β release. IL-1β plays an important role in host immunity and protection against infections. Its biological activation via IL-1β-precursor processing requires the transcription of inflammasome components and their activation. Deregulated activity of NOD-like receptor inflammasomes (NLR) like NLRP3 that leads to the maturation of IL-1β has been described in various diseases. While the role of other inflammasomes has been studied in monocytes, nothing is known about NLRP3 inflammasome after a traumatic injury. Here, the role of the NLRP3 inflammasome in impaired monocyte functionality after a traumatic injury was analyzed. Measurements and Main Results Ex vivo-in vitro stimulation of isolated CD14+ monocytes with lipopolysaccharide (LPS) showed a significantly higher IL-1β secretion in healthy volunteers (HV) compared to trauma patients (TP) after admission. Reduced IL-1β secretion was paralleled by significantly lowered gene expression of NLRP3 in monocytes from TP compared to those of HV. Transfection of monocytes with NLRP3-encoding plasmid recovered the functionality of monocytes from TP regarding the IL-1β secretion. Conclusions This study demonstrates that CD14+ monocytes from TP are significantly diminished in their function and that the presence of NLRP3 components is necessary in recovering the ability of monocytes to produce active IL-1β. This recovery of the NLRP3 inflammasome in monocytes may imply a new target for treatment and therapy of immune suppression after severe injury.

SAT-067 - Regulation of the NLRP3 inflammasome in monocytes and macrophages from patients with decompensated cirrhosis by low-dose genotoxic stress

SAT-067 - Regulation of the NLRP3 inflammasome in monocytes and macrophages from patients with decompensated cirrhosis by low-dose genotoxic stress

MicroRNA-7 targets Nod-like receptor protein 3 inflammasome to modulate neuroinflammation in the pathogenesis of Parkinson's disease.

Backgroundα-Synuclein (α-Syn), a pathological hallmark of Parkinson’s disease (PD), has been recognized to induce the production of interleukin-1β in a process that depends, at least in vitro, on nod-like receptor protein 3 (NLRP3) inflammasome in monocytes. However, the role of NLRP3 inflammasome activation in the onset of PD has not yet been fully established.ResultsIn this study, we showed that NLRP3 inflammasomes were activated in the serum of PD patients and the midbrain of PD model mice. We further clarified that α-syn activated the NLRP3 inflammasome through microglial endocytosis and subsequent lysosomal cathepsin B release. Deficiency of caspase-1, an important component of NLRP3 inflammasome, significantly inhibited α-syn-induced microglia activation and interleukin-1β production, which in turn alleviated the reduction of mesencephalic dopaminergic neurons treated by microglia medium. Specifically, we demonstrated for the first time that Nlrp3 is a target gene of microRNA-7 (miR-7). Transfection of miR-7 inhibited microglial NLRP3 inflammasome activation whereas anti-miR-7 aggravated inflammasome activation in vitro. Notably, stereotactical injection of miR-7 mimics into mouse striatum attenuated dopaminergic neuron degeneration accompanied by the amelioration of microglial activation in MPTP-induced PD model mice.ConclusionsOur study provides a direct link between miR-7 and NLRP3 inflammasome-mediated neuroinflammation in the pathogenesis of PD. These findings will give us an insight into the potential of miR-7 and NLRP3 inflammasome in terms of opening up novel therapeutic avenues for PD.Electronic supplementary materialThe online version of this article (doi:10.1186/s13024-016-0094-3) contains supplementary material, which is available to authorized users.

Nlrp1 inflammasome is downregulated in trauma patients.

After a major trauma, IL-1β-producing capacity of monocytes is reduced. Generation of IL-1β is important for appropriate immune response after trauma and requires not only synthesis and transcription of inflammasome components but also their activation. Altered IL-1β-processing due to deregulated NLRP inflammasomes assembly is associated with several inflammatory diseases. However, the precise role of NLRP1 inflammasome in monocytes after trauma is unknown. Here, we investigated if NLRP1 inflammasome components are responsible for depressed monocyte function after trauma. We found in ex vivo in vitro assays that LPS-stimulation of CD14(+)-isolated monocytes from healthy volunteers (HV) results in remarkably higher capacity of the IL-1β-release compared to trauma patients (TP). During the 10-day time course, this monocyte depression was highest immediately after admission. Inflammasome activation correlating with this inflammatory response was demonstrated by enhanced protein production of cleaved IL-1β and caspase-1. Furthermore, we found that the gene expression of IL-1β, caspase-1, and ASC was comparable in TP and HV after LPS-stimulation during the 10-day course, while NLRP1 was markedly reduced in TP. We demonstrated that transfected monocytes from TP, which expressed the lacking components, were recovered in their LPS-induced IL-1β-release and that lacking of NLRP1 is responsible for the suppressed monocyte activity after trauma. The restoration of NLRP1 inflammasome suggests new mechanistic target for the recovery of dysbalanced immune reaction after trauma. Suppression in monocyte function occurs early after a major trauma or surgery. Reduced gene expression abrogates NLRP1 inflammasome assembly after trauma. Limited availability of inflammasome components may cause reduced host defense. Restoring NLRP1 in immune-suppressed monocytes recovers NLPR1 activity after trauma. Recovered inflammasome activity may improve the immune response to PAMPs/DAMPs.

Mucosal-associated invariant T cells in inflammatory bowel diseases: bystanders, defenders, or offenders?

Mucosal-associated invariant T cells in inflammatory bowel diseases: bystanders, defenders, or offenders?