Cytokine Maturation Research Articles

Shiga Toxin/Lipopolysaccharide Activates Caspase-4 and Gasdermin D to Trigger Mitochondrial Reactive Oxygen Species Upstream of the NLRP3 Inflammasome

The non-canonical caspase-4 and canonical NLRP3 inflammasomes are both activated by intracellular lipopolysaccharide (LPS), but the crosstalk between these two pathways remains unclear. Shiga toxin 2 (Stx2)/LPS complex, from pathogenic enterohemorrhagic Escherichia coli, activates caspase-4, gasdermin D (GSDMD), and the NLRP3 inflammasome in human THP-1 macrophages, but not mouse macrophages that lack the Stx receptor CD77. Stx2/LPS-mediated IL-1β secretion and pyroptosis are dependent on mitochondrial reactive oxygen species (ROS) downstream of the non-canonical caspase-4 inflammasome and cleaved GSDMD, which is enriched at the mitochondria. Blockade of caspase-4 activation and ROS generation as well as GSDMD deficiency significantly reduces Stx2/LPS-induced IL-1β production and pyroptosis. The NLRP3 inflammasome plays a significant role in amplifying Stx2/LPS-induced GSDMD cleavage and pyroptosis, with significant reduction of these responses in NLRP3-deficientTHP-1 cells. Together, these data show that Stx2/LPS complex activates the non-canonical inflammasome and mitochondrial ROS upstream of the NLRP3 inflammasome to promote cytokine maturation and pyroptosis.

The Salmonella pathogenicity island-2 subverts human NLRP3 and NLRC4 inflammasome responses.

Inflammasomes are signaling hubs that activate inflammatory caspases to drive cytokine maturation and cell lysis. Inflammasome activation by Salmonella Typhimurium infection or Salmonella-derived molecules is extensively studied in murine myeloid cells. Salmonella-induced inflammasome signaling in human innate immune cells, is however, poorly characterized. Here, we show that Salmonella mutation to inactivate the Salmonella pathogenicity island-2 type III secretion system (SPI2 T3SS) potentiates S. Typhimurium-induced inflammasome responses from primary human macrophages, resulting in strong IL-1β production and macrophage death. Inactivation of the SPI1 T3SS diminished human macrophage responses to WT and ΔSPI2 Salmonella. Salmonella ΔSPI2 elicited a mixed inflammasome response from human myeloid cells, in which NLR family CARD-domain containing protein 4 (NLRC4) and NLR family PYRIN-domain containing protein 3 (NLRP3) perform somewhat redundant functions in generating IL-1β and inducing pyroptosis. Our data suggest that Salmonella employs the SPI2 T3SS to subvert SPI1-induced NLRP3 and NLRC4 inflammasome responses in human primary macrophages, in a species-specific immune evasion mechanism.

NLRP3/Caspase-1 Pathway-Induced Pyroptosis Mediated Cognitive Deficits in a Mouse Model of Sepsis-Associated Encephalopathy

Sepsis-associated encephalopathy (SAE) is a common complication that leads to long-term cognitive impairments and increased mortality in sepsis survivors. The mechanisms underlying this complication remain unclear and an effective intervention is lacking. Accumulating evidence suggests the nucleotide-binding domain-like receptor protein3 (NLRP3)/caspase-1 pathway is involved in several neurodegenerative diseases. Thus, we hypothesized that the NLRP3/caspase-1 pathway is involved in NLRP3-mediated pyroptosis, maturation and release of inflammatory cytokines, and cognitive deficits in SAE. We used the NLRP3 inhibitor MCC950 and the caspase-1 inhibitor Ac-YVAD-CMK to study the role of the NLRP3/caspase-1 pathway in pyroptosis and cognitive deficits in a mouse model of SAE. Mice were randomly assigned to one of six groups: sham+saline, sham+MCC950, sham+Ac-YVAD-CMK, cecal ligation and puncture (CLP)+saline, CLP+MCC950, and CLP+Ac-YVAD-CMK. Surviving mice underwent behavioral tests or had hippocampal tissues collected for histochemical analysis and biochemical assays. Our results show that CLP-induced hippocampus-dependent memory deficits are accompanied by increased NLRP3 and caspase-1 positive cells, and augmented protein levels of NLRP3, caspase-1, gasdermin-D, and pro-inflammatory cytokines in the hippocampus. In addition, administration of MCC950 or Ac-YVAD-CMK rescues cognitive deficits and ameliorates increased hippocampal NLRP3-mediated neuronal pyroptosis and pro-inflammatory cytokines. Our results suggest that the NLRP3/caspase-1 pathway-induced pyroptosis mediates cognitive deficits in a mouse model of SAE.

Sesame seeds essential oil and Sesamol modulate the pro-inflammatory function of macrophages and dendritic cells and promote Th2 response.

Background: Herbal medicine is becoming progressively accepted treatment for management of different diseases worldwide. Recognition of the active ingredients and mechanisms of herbal medicine against the immune system and related anomalies is highly favorable. This experimental study aimed to investigate the effects of Sesame (Sesamum indicum L.) essential oil and sesamol as effective components on mouse splenocytes subsets, macrophages and dendritic cells (DCs). Methods: Effective components of sesame were extracted and used to treat splenocytes, PHA (5μg/ml) and LPS (10 μg/ml) stimulated splenocytes, macrophages and DCs in different concentration (0.01–100 μg/ml). The cell proliferation/viability was measured using the MTT assay and nitrite levels were measured by the diazotization method. Moreover, TNF-α and IL-1β cytokines concentration were assayed by ELISA. Treated DCs also analysed for maturation marker levels and cytokine production. Results: Analysis of the results indicated that sesame components suppress PHA-stimulated splenocytes with no effect on LPS-stimulated subsets. Furthermore, the sesame ingredients reduced the release of IFN-γ and increased secretion of IL-4 from lymphocytes. Macrophages viability was not affected and production of NO, TNF-α, and IL-1β were inhibited using sesame essential oil and sesamol. DCs phenotype skewed to immature and release of TNF-α and IL-1β were abrogated form DCs. Conclusion: These results indicate that sesame essential oil and its effective component as sesamol may capable of suppressing the response of cellular immunity with the domination of Th2 responses and also could modulate macrophages and the dendritic cells proinflammatory functions.

Mercury and arsenic attenuate canonical and non-canonical NLRP3 inflammasome activation

Exposure to heavy metals can cause several diseases associated with the immune system. Although the effects of heavy metals on production of inflammatory cytokines have been previously studied, the role of heavy metals in inflammasome activation remains poorly studied. The inflammasome is an intracellular multi-protein complex that detects intracellular danger signals, resulting in inflammatory responses such as cytokine maturation and pyroptosis. In this study, we elucidated the effects of four heavy metals, including cadmium (Cd), mercury (Hg), arsenic (As), and lead (Pb), on the activation of NLRP3, NLRC4, and AIM2 inflammasomes. In our results, mercury and arsenic inhibited interleukin (IL)-1β and IL-18 secretion resulting from canonical and non-canonical NLRP3 inflammasome activation in macrophages and attenuated elevation of serum IL-1β in response to LPS treatment in mice. In the mechanical studies, mercury interrupted production of mitochondrial reactive oxygen species, release of mitochondrial DNA, and activity of recombinant caspase-1, whereas arsenic down-regulated expression of promyelocytic leukemia protein. Both mercury and arsenic inhibited Asc pyroptosome formation and gasdermin D cleavage. Thus, we suggest that exposure to mercury and/or arsenic could disrupt inflammasome-mediated inflammatory responses, which might cause unexpected side effects.

Increased Binding of Specificity Protein 1 to the IL21R Promoter in B Cells Results in Enhanced B Cell Responses in Rheumatoid Arthritis.

B cells are implicated in rheumatoid arthritis (RA) based on the presence of autoantibodies and the therapeutic response to B cell depletion. IL-21 has a significant role in B cell development and function. Here we assess B cell responses to IL-21 and the mechanisms responsible for altered IL-21R expression in RA. Flow cytometry of PBMC and cultured B cells was used to quantify protein and mRNA levels of IL-21R, IL-21 signaling through pSTAT3, specificity protein 1 (SP1) and to determine cytokine production (IL-6) and maturation status of B cells in RA and healthy control subjects. SP1 binding to the IL21R promoter region in B cells was assessed with ChIP-qPCR. We demonstrate an increase in IL-21R expression in total and memory B cells from RA subjects, which correlated with responsiveness to IL-21 stimulation. Stimulation of naïve RA B cells with IL-21 and CD40L resulted in an increase in differentiation into plasmablasts and an increase in IL-6 production in comparison to healthy controls, which was dose dependent on IL-21 stimulation. IL-21R expression on memory B cells in RA synovial fluid was comparable to peripheral blood making our study pertinent to understanding B cell responses in the joint and site of inflammation. We identified an increase in SP1 protein and mRNA in RA B cells and demonstrate an increase in binding of SP1 to the IL21R promoter region, which suggests a mechanism by which IL-21R expression is enhanced on B cells in RA. Taken together, our results indicate a mechanism by which IL-21 enhances B cell development and function in RA through an SP1 mediated increase in IL-21R expression on B cells.

LncRNA Malat1 modulates the maturation process, cytokine secretion and apoptosis in airway epithelial cell-conditioned dendritic cells.

Airway epithelial cells (AECs) are the first point of contact with airborne antigens and are able to instruct resident immune cells to appropriate immune responses. Previous studies have shown that the abnormal expression of metastasis-associated lung adenocarcinoma transcript 1 (Malat1) was associated with tumorigenesis, progression, metastasis, and apoptosis in many cancer types. However, little is known about its functional involvement in the cross-talk of AECs with dendritic cells (DCs). The aim of the present study was to identify Malat1 as a novel epithelial cell-derived immune-modulating factor that contributes to the specific inflammatory-immune airway microenvironment. By using an in vitro co-culture model, where layers of AECs can interact with DCs, and transfecting Malat1 siRNA in AECs, AEC-conditioned DCs were harvested for further analysis of the celluar phenotype, secretion of inflammatory chemokines, and expression of apoptotic markers. The present study clearly demonstrated that Malat1 modulates the maturation process, pro-inflammatory cytokine secretion and apoptosis in AECs-conditioned DCs.

Spotlight on the NLRP3 inflammasome pathway.

Inflammation is triggered by a repertoire of receptors detecting infections and damages. Some of these receptors directly bind microbial ligands, while others recognize endogenous molecules exposed under stress conditions, including infections. Most of these receptors can be engaged by a relatively limited number of stimuli. Differently, NLRP3 acts as a broad sensor of cell homeostasis rupture and can be activated downstream of a plethora of stimuli. NLRP3 then assembles a multiprotein platform resulting in caspase-1 activation, which controls, by direct cleavage, the maturation of cytosolic pro-cytokines including pro-interleukin-1β. In addition, caspase-1 processes cytosolic gasdermin-D and unleashes its pore-forming N-terminal domain, leading to the release of mature cytosolic cytokines and alarmins, as well as pyroptotic cell lysis. Accumulating evidences of the aggravating role of NLRP3-mediated inflammation in various highly prevalent human conditions, including diabetes, neurodegenerative and cardiovascular diseases, raises a huge clinical interest. Nevertheless, the molecular mechanism governing NLRP3 activation remains insufficiently understood. In line with the detrimental consequences of NLRP3 activation illustrated by the aforementioned pathologies, this process is tightly regulated. In this review, we address the current understanding of the control of NLRP3 activity which can be divided into two coordinated processes referred to as priming and activation. In particular, we detail the emerging role of NLRP3 post-translational modifications critical in inflammasome assembly regulation.

Long Noncoding RNA Signatures Induced by Toll-Like Receptor 7 and Type I Interferon Signaling in Activated Human Plasmacytoid Dendritic Cells.

Long noncoding RNAs (lncRNAs) exhibit highly lineage-specific expression and act through diverse mechanisms to exert control over a wide range of cellular processes. lncRNAs can function as potent modulators of innate immune responses through control of transcriptional and posttranscriptional regulation of mRNA expression and processing. Recent studies have demonstrated that lncRNAs participate in the regulation of antiviral responses and autoimmune disease. Despite their emerging role as immune mediators, the mechanisms that govern lncRNA expression and function have only begun to be characterized. In this study, we explore the role of lncRNAs in human plasmacytoid dendritic cells (pDCs), which are critical sentinel sensors of viral infection and contribute to the development of autoimmune disease. Using genome-wide sequencing approaches, we dissect the contributions of Toll-like receptor 7 (TLR7) and type I interferon (IFN-I) in the regulation of coding and noncoding RNA expression in CAL-1 pDCs treated with R848 or IFNβ. Functional enrichment analysis reveals both the unique and synergistic roles of TLR7 and IFN-I signaling in the orchestration of pDC function. These observations were consistent with primary cell immune responses elicited by detection of viral infection. We identified and characterized the conditional TLR7- and IFN-I-dependent regulation of 588 lncRNAs. Dysregulation of these lncRNAs could significantly alter pDC maturation, IFN-I and inflammatory cytokine production, antigen presentation, costimulation or tolerance cues, turnover, or localization, all consequential events during viral infection or IFN-I-driven autoimmune diseases such as systemic lupus erythematosus. These findings demonstrate the differential responsiveness of lncRNAs to unique immune stimuli, uncover regulatory mechanisms of lncRNA expression, and reveal a novel and tractable platform for the study of lncRNA expression and function.

Cardiomyocyte Inflammasome Signaling in Cardiomyopathies and Atrial Fibrillation: Mechanisms and Potential Therapeutic Implications.

Inflammasomes are high molecular weight protein complexes in the cytosol of immune and other cells that play a critical role in the innate immune system in response to cellular stress. NLRP3 inflammasome, the best-understood inflammasome, is known to mediate the maturation (activation) of caspase-1 from pro-caspase-1, causing the maturation and release of cytokines (e.g., interleukin-1β) and potentially leading to a form of inflammatory programmed cell death called pyroptosis. Previous work has shown that the NLRP3 components are expressed in cardiomyocytes and cardiac fibroblasts and recent studies have identified the NLRP3 inflammasome as a key nodal point in the pathogenesis of cardiomyopathies and atrial fibrillation, which may create an opportunity for the development of new therapeutic agents. Here we review the recent evidence for a role of NLRP3 inflammasome in the cardiomyocytes and discuss its potential role in the evolution of cardiac remodeling and arrhythmias and new opportunities created by these very recent developments.

The immunoregulatory role of corneal epithelium-derived thrombospondin-1 in dry eye disease

PurposeIn this study, we examine the expression of corneal epithelium-derived thrombospondin-1 (TSP-1) and its immunomodulatory functions in a validated murine model of dry eye disease (DED). MethodsDED was induced in female C57BL/6 using a controlled environment chamber (CEC) for 14 days. mRNA and protein expression of TSP-1 by corneal epithelial cells was quantified using real-time PCR and flow cytometry. Corneal epithelial cells from either naïve or DED mice were cultured with bone marrow derived dendritic cells (BMDCs) in the presence of IFNγ for 48 h, and BMDC expression of MHC-II and CD86 was determined using flow cytometry. Next, either recombinant TSP-1 or anti-TSP-1 antibody was added to the co-culture, and BMDC expression of above activation markers was evaluated. Finally, either DED mice were topically treated with either recombinant TSP-1 or human serum albumin (HSA), and maturation of corneal DCs, expression of inflammatory cytokines, and DED severity were investigated. ResultsmRNA expression of TSP-1 by the corneal epithelium was upregulated in DED. Corneal epithelial cells derived from mice with DED demonstrated an enhanced capacity in suppressing BMDC expression of MHC-II and CD86 relative to wild type mice, and this effect was abrogated by TSP-1 blockade and potentiated by recombinant TSP-1. Finally, topical application of recombinant TSP-1 significantly suppressed corneal DC maturation and mRNA expression of pro-inflammatory cytokines, and ameliorated disease severity in mice with DED. ConclusionsOur study elucidates the function of epithelium-derived TSP-1 in inhibiting DC maturation and shows its translational potential to limit corneal epitheliopathy in DED.

Control of cytokine mRNA degradation by the histone deacetylase inhibitor ITF2357 in rheumatoid arthritis fibroblast-like synoviocytes: beyond transcriptional regulation

BackgroundHistone deacetylase inhibitors (HDACi) suppress cytokine production in immune and stromal cells of patients with rheumatoid arthritis (RA). Here, we investigated the effects of the HDACi givinostat (ITF2357) on the transcriptional and post-transcriptional regulation of inflammatory markers in RA fibroblast-like synoviocytes (FLS).MethodsThe effects of ITF2357 on the expression and messenger RNA (mRNA) stability of IL-1β-inducible genes in FLS were analyzed using array-based qPCR and Luminex. The expression of primary and mature cytokine transcripts, the mRNA levels of tristetraprolin (TTP, or ZFP36) and other AU-rich element binding proteins (ARE-BP) and the cytokine profile of fibroblasts derived from ZFP36+/+ and ZFP36−/− mice was measured by qPCR. ARE-BP silencing was performed by small interfering RNA (siRNA)-mediated knockdown, and TTP post-translational modifications were analyzed by immunoblotting.ResultsITF2357 reduced the expression of 85% of the analyzed IL-1β-inducible transcripts, including cytokines (IL6, IL8), chemokines (CXCL2, CXCL5, CXCL6, CXCL10), matrix-degrading enzymes (MMP1, ADAMTS1) and other inflammatory mediators. Analyses of mRNA stability demonstrated that ITF2357 accelerates IL6, IL8, PTGS2 and CXCL2 mRNA degradation, a phenomenon associated with the enhanced transcription of TTP, but not other ARE-BP, and the altered post-translational status of TTP protein. TTP knockdown potentiated cytokine production in RA FLS and murine fibroblasts, which in the latter case was insensitive to inhibition by ITF2357 treatment.ConclusionsOur study identifies that regulation of cytokine mRNA stability is a predominant mechanism underlying ITF2357 anti-inflammatory properties, occurring via regulation of TTP. These results highlight the therapeutic potential of ITF2357 in the treatment of RA.

Human Bone Marrow-Derived Myeloid Dendritic Cells Show an Immature Transcriptional and Functional Profile Compared to Their Peripheral Blood Counterparts and Separate from Slan+ Non-Classical Monocytes.

The human bone marrow (BM) gives rise to all distinct blood cell lineages, including CD1c+ (cDC2) and CD141+ (cDC1) myeloid dendritic cells (DC) and monocytes. These cell subsets are also present in peripheral blood (PB) and lymphoid tissues. However, the difference between the BM and PB compartment in terms of differentiation state and immunological role of DC is not yet known. The BM may represent both a site for development as well as a possible effector site and so far, little is known in this light with respect to different DC subsets. Using genome-wide transcriptional profiling we found clear differences between the BM and PB compartment and a location-dependent clustering for cDC2 and cDC1 was demonstrated. DC subsets from BM clustered together and separate from the corresponding subsets from PB, which similarly formed a cluster. In BM, a common proliferating and immature differentiating state was observed for the two DC subsets, whereas DC from the PB showed a more immune-activated mature profile. In contrast, BM-derived slan+ non-classical monocytes were closely related to their PB counterparts and not to DC subsets, implying a homogenous prolife irrespective of anatomical localization. Additional functional tests confirmed these transcriptional findings. DC-like functions were prominently exhibited by PB DC. They surpassed BM DC in maturation capacity, cytokine production, and induction of CD4+ and CD8+ T cell proliferation. This first study on myeloid DC in healthy human BM offers new information on steady state DC biology and could potentially serve as a starting point for further research on these immune cells in healthy conditions as well as in diseases.

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.

An update on the NLRP3 inflammasome and influenza: the road to redemption or perdition?

Inflammation is an integral aspect of influenza A virus (IAV) infection. It is critical to induce an antiviral environment to reduce viral replication and dissemination, while also being essential to the development and maturation of adaptive immunity, which ultimately resolves infection. Conversely, excessive pulmonary inflammation and cellular influx are characteristic of lethal IAV infections. It has become increasingly apparent that the innate immune inflammasome complex is a crucial moderator in IAV disease pathogenesis. It is responsible for the maturation and secretion of prototypic inflammatory cytokines, Interleukin (IL)-1β and IL-18, and the induction of pyroptotic cell death. This short review will examine recent work on the regulation and targeting of the NLRP3 inflammasome as a means of intrinsic and extrinsic modulation of inflammasome-mediated inflammation. It is encouraging that recent studies suggest 'starving' the inflammasome of substrate, or directly inhibiting activity, may be the means to reducing host inflammatory responses to IAV infection and to directing a positive disease outcome.

Chronic Low Grade Inflammation in Type 2 Diabetes—Activation of the Inflammasomes by Circulating Metabolites

Type 2 diabetes (T2D) is associated with chronic low-grade inflammation. We studied its mechanisms by addressing the role of inflammasomes in monocytes and their activation by circulating metabolites to produce inflammatory cytokines IL1β and IL18. Blood samples were collected from patients with T2D. Serum was filtered at 3 kDa to remove macromolecules. Inflammasome activation was analyzed by gene expression of various Nod Like Receptors by RT-quantitative PCR as well as IL1β and IL18 secretion from these monocytes in culture (ELISA). Twenty-one patients (38% women) were included in 2016. They were 57±10 years old, BMI were 33.1±6.3 kg/m2. They had T2D for 14±9 years and HbA1c at 9.5±1.2%. Their C Reactive Protein levels was 8.1±8.0 mg/L. Each subject was sampled twice at 4 days of interval: in hyperglycemia (2.56±0.86g/L) then in normoglycemia (1.23±0.41g/L). Two inflammasomes were transcripted: NLRP1 and NLRC4, with significant high level whether in normoglycemia (fold-change 1.47, p=0.01 and 1.33, p=0.02) and hyperglycemia (fold-change 1.41, p=0.02 and 1.31, p=0.04). This transcriptional priming seems to have a functional consequence with IL1β production by monocytes of some T2D patients. Healthy monocytes were collected from voluntary blood donors (Glycaemia: 0.97±0.17g/L). Incubation of them with filtered serum of T2D patients recapitulated the priming signal. The same two inflammasomes were transcripted: NLRP1 and NLRC4, whether in normoglycemia (fold-change 1.35, p=0.002 and 1.23, p=0.02) and hyperglycemia (fold-change 1.31, p=0.01 and 1.30, p=0.052). Although it did not induce activation of mature cytokines. We have shown the activation of NLRP1 and NLRC4 inflammasomes and their secretion of inflammatory cytokine in T2D patients. Some soluble serum metabolites seem directly involved in this inflammatory cascade. Our next objective will be to identify these molecules, which are potential biomarkers of predictive of chronic immune activation in T2D. Disclosure M. Monlun: None. V. Rigalleau: None. L. Blanco: None. K. Mohammedi: Speaker's Bureau; Self; Novo Nordisk Inc.. Other Relationship; Self; Novo Nordisk Inc.. Speaker's Bureau; Self; Sanofi. Other Relationship; Self; Sanofi, Takeda Development Centre Europe Ltd., Boehringer Ingelheim Pharmaceuticals, Inc.. P. Blanco: None.

Mitochondrial SLC25 proteins interact with NLRP3 to regulate inflammasome function

Abstract The NLRP3 protein is a key initiator of inflammation in humans. NLRP3 becomes activated by a multitude of danger signals, including pathogens, markers of metabolic dysfunction, and internalized particulates. Upon activation, NLRP3 nucleates formation of a multiprotein complex called the inflammasome, in which caspase-1 activity mediates processing of the pro-inflammatory cytokines IL-1β and IL-18 and induces pyroptosis, a pro-inflammatory form of cell death that serves to release the mature cytokines along with other inflammatory mediators, thus initiating the inflammatory response. While multiple regulators of the NLRP3 inflammasome have been described, specific ligand(s) of NLRP3 and its mechanism of activation remain largely unknown. We performed a proteomics screen using co-immunoprecipitation (IP) and mass spectrometry to identify cellular proteins that bind NLRP3. Using this screen, we identified multiple NLRP3 interactors, including a family of solute carrier (SLC) proteins that localize to the inner mitochondrial membrane. Co-IP experiments verified that four of these proteins, namely SLC25A1, a citrate transporter, SLC25A6, an ADP/ATP translocase, SLC25A11, a 2-oxoglutarate/malate carrier, and SLC25A13, a calcium-dependent glutamate transporter, specifically interact with NLRP3. Because of its linkage with known metabolic disease, we further assessed the role of SCL25A13 in inflammasome function. When ectopically expressed, SLC25A13 enhanced IL-1β release from cells, while CRISPR/Cas9 knockout of SLC25A13 from THP-1 macrophages partially abrogated NLRP3-dependent pyroptosis. Our studies reveal a novel role for mitochondrial SLC25 proteins in regulation of the NLRP3 inflammasome.

HIV replication is associated to inflammasomes activation, IL-1β, IL-18 and caspase-1 expression in GALT and peripheral blood.

BackgroundHuman immunodeficiency virus (HIV) promotes an inflammatory process, leading to the progressive loss of the functional capacity of the immune system. The HIV infection induces alterations in several tissues, but mainly in the gut-associated lymphoid tissue (GALT). However, the degree of GALT deterioration varies among infected individuals. In fact, it has been shown that HIV-controllers, who spontaneously control viral replication, exhibit a lower inflammatory response, and a relative normal frequency and function of most of the immune cells. Inflammasomes are molecular complexes involved in the inflammatory response, being NLRP1, NLRP3, NLRC4, AIM2 and Pyrin inflammasomes, the best characterized so far. These complexes regulate the maturation of cytokines of the IL-1 family, including IL-1β and IL-18. These cytokines have been associated with immune activation and expansion of HIV target cells, promoting viral replication. Interesting, some reports indicate that HIV induces the activation of the NLRP3 inflammasome, but the role of this, and other inflammasomes during HIV infection, especially in GALT, remains unclear.ObjectiveTo compare the relative expression of inflammasome components and the proinflammatory response related to their activity, between HIV-progressors and HIV-controllers.MethodsGALT biopsies and peripheral blood mononuclear cells (PBMCs) from 15 HIV-controllers and 15 HIV-progressors were obtained. The relative expression of the following inflammasome components were evaluated by RT-PCR: NLRP3, NLRC4, NLRP1, AIM2, ASC, Caspase-1, IL-1β and IL-18. In addition, plasma concentration of IL-18 was evaluated as an indicator of baseline proinflammatory status. Finally, in supernatants of PBMCs in vitro stimulated with inflammasome agonists, the concentrations of IL-1β and IL-18 were quantified by ELISA.ResultsHIV-progressors exhibited higher expression of IL-1β, IL-18 and caspase-1 genes in GALT and PBMCs compared with HIV-controllers. In addition, HIV-progressors had also increased expression of ASC in PBMCs. When plasma levels were evaluated, IL-18 was increased in HIV-progressors. Interesting, these patients also showed an increased production of IL-1β in supernatants of PBMCs stimulated in vitro with the agonists of AIM2, NLRP1 and NLRC4 inflammasomes. Finally, the expression of caspase-1, NLRP1, IL-1β and IL-18 in GALT or peripheral blood was correlated with CD4+ T-cell count and viral load.ConclusionOur results suggest that during HIV-infection, the required signals to induce the expression of different components of the inflammasomes are produced, both in GALT and in periphery. The activation of these molecular complexes could increase the number of target cells, favoring HIV replication and cell death, promoting the disease progression.

Stimulation of P2Y11 receptor modulates cardiac fibroblasts secretome towards immunomodulatory and protective roles after simulated Ischemia/Reperfusion injury

Ischemia/Reperfusion (I/R) injuries are involved in many myocardial pathologic processes. Cardiac fibroblasts (CF) respond to this stress by excessive proliferation and secretion of soluble factors, e.g. ATP, leading to purinergic receptors activation. We previously showed that P2Y11 receptor (P2Y11R) played an immunomodulatory role in human dendritic cells (DC). We aimed to investigate the role of human CF (hCF) P2Y11R stressed by simulated I/R (Hypoxia/Reoxygenation or H/R) on immune response modulation and cardiac protection. Adult ventricular hCF were subjected to H/R (5 h at 1% O 2 and 24 h at 21% O 2 ), and P2Y11R specific agonist (NF546) and/or antagonist (NF340) were added at the onset of reoxygenation. Resulting hCF secretome (hCFS) was analyzed by bioluminescence and Mutiplex assays. DCs maturation markers and cytokines secretion were analyzed by flow cytometry and ELISA after 48 h incubation with hCFS and LPS stimulation. Human cardiomyocytes (CM) were subjected to 5 h/1 h H/R and hCFS was added at the onset of reoxygenation. CM viability/mortality was assessed by MTT assay and intracellular ATP quantification. hCF P2Y11R stimulation with NF546 significantly decreased H/R-induced proliferation and pro-inflammatory factors secretion. hCFS alone increased CD83, CD25 and CD86 expression in DC, suggesting a switch of DC from immature to mature phenotype. Under LPS stimulation, DCs seemed to be pro-inflammatory (high IL-12/IL-10 ratio) but hCFS induced a switch to tolerogenic profile (significant decrease of this ratio). hCFS prevented H/R-induced CM death via Akt phosphorylation. P2Y11R inhibition in hCF induced a complete loss of hCFS-induced protection in CM. Our data demonstrate a paracrine link between CF, CM and DC after I/R suggesting a key role of CF in the cellular responses to reperfusion. Therefore, P2Y11R modulates myocardial I/R injuries by acting on CF secretome.

Identification of interactions among host and bacterial proteins and evaluation of their role early during Shigella flexneri infection.

Shigella species cause diarrhoea by invading and spreading through the epithelial layer of the human colon. The infection triggers innate immune responses in the host that the bacterium combats by translocating into the host cell cytosol via a type 3 secretion system bacterial effector proteins that interfere with host processes. We previously demonstrated that interaction of the Shigella type 3 secreted effector protein IcsB with the host protein Toca-1 inhibits the innate immune response microtubule-associated protein light-chain 3 (LC3)-associated phagocytosis, and that IcsB interaction with Toca-1 is required for inhibition of this host response. Here, we show that Toca-1 in vitro precipitated not only IcsB, but also the type 3 secreted proteins OspC3, IpgD and IpaB. OspC3 and IpgD precipitation with Toca-1 was dependent on IcsB. Early during infection, most of these proteins localized near intracellular Shigella. We examined whether interactions among these proteins restrict innate host cell responses other than LC3-associated phagocytosis. In infected cells, OspC3 blocks production and secretion of the mature pro-inflammatory cytokine IL-18; however, we found that interaction of OspC3 with IcsB, either directly or indirectly via Toca-1, was not required for OspC3-mediated restriction of IL-18 production. These results indicate that interactions of the host protein Toca-1 with a subset of type 3 effector proteins contribute to the established function of some, but not all involved, effector proteins.