IL-1β Production In Response Research Articles

Impact of IL-1β on lung pathology caused by Mycobacterium abscessus infection and its association with IL-17 production

Mycobacterium abscessus (MAB), a non-tuberculous mycobacterium (NTM), causes chronic pulmonary inflammation in humans. The NLRP3 inflammasome is a multi-protein complex that triggers IL-1β maturation and pyroptosis through the cleavage of caspase-1. In this study, we investigated the roles of NLRP3 and IL-1β in the host’s defense against MAB. The IL-1β production by MAB was completely abolished in NLRP3, but not NLRC4, deficient macrophages. The NLRP3 inflammasome components, which are ASC and caspase-1 were also found to be essential for IL-1β production in response to MAB. NLRP3 and IL-1β deficiency did not affect the intracellular growth of MAB in macrophages, and the bacterial burden in lungs of NLRP3- and IL-1β-deficient mice was also comparable to the burden observed in WT mice. In contrast, IL-1β deficiency ameliorated lung pathology in MAB-infected mice. Notably, the lung homogenates of IL-1β-deficient mice had reduced levels of IL-17, but not IFN-γ and IL-4 when compared with WT counterparts. Furthermore, in vitro co-culture analysis showed that IL-1β signaling was essential for IL-17 production in response to MAB. Finally, we observed that the anti-IL-17 antibody administration moderately mitigated MAB-induced lung pathology. These findings indicated that IL-1β production contribute to MAB-induced lung pathology via the elevation of IL-17 production.

TLR8-activating miR-146a-3p is an intermediate signal contributing to fetal membrane inflammation in response to bacterial LPS.

Preterm birth is the largest contributor to neonatal morbidity and is often associated with chorioamnionitis, defined as inflammation/infection of the fetal membranes (FMs). Chorioamnionitis is characterised by neutrophil infiltration of the FMs and is associated with elevated levels of the neutrophil chemoattractant, interleukin (IL)-8 and the proinflammatory cytokine, IL-1β. While FMs can respond to infections through innate immune sensors, such as toll-like receptors (TLRs), the downstream mechanisms by which chorioamnionitis arises are not fully understood. A novel group of non-classical microRNAs (miR-21a, miR-29a, miR-146a-3p, Let-7b) function as endogenous danger signals by activating the ssRNA viral sensors TLR7 and TLR8. In this study, the pro-inflammatory roles of TLR7/TLR8-activating miRs were examined as mediators of FM inflammation in response to bacterial lipopolysaccharide (LPS) using an in vitro human FM explant system, an in vivo mouse model of pregnancy, and human clinical samples. Following LPS exposure, miR-146a-3p was significantly increased in both human FM explants and wild-type mouse FMs. Expression of miR-146a-3p was also significantly elevated in FMs from women with preterm birth and chorioamnionitis. FM IL-8 and inflammasome-mediated IL-1β production in response to LPS was dependent on miR-146a-3p and TLR8 downstream of TLR4 activation. In wild-type mice, LPS exposure increased FM IL-8 and IL-1β production and induced preterm birth. In TLR7-/-/TLR8-/- mice, LPS exposure was able to initiate but not sustain preterm birth, and FM inflammation was reduced. Together, we demonstrate a novel signalling mechanism at the maternal-fetal interface in which TLR8-activating miR-146a-3p acts as an intermediate danger signal to drive FM inflammasome-dependent and -independent mechanisms of inflammation and, thus, may play a role in chorioamnionitis and subsequent preterm birth.

Acetyl-CoA synthetase 2 induces pyroptosis and inflammation of renal epithelial tubular cells in sepsis-induced acute kidney injury by upregulating the KLF5/NF-κB pathway

BackgroundPyroptosis of the renal tubular epithelial cells (RTECs) and interstitial inflammation are central pathological characteristics of acute kidney injury (AKI). Pyroptosis acts as a pro-inflammatory form of programmed cell death and is mainly dependent on activation of the NLRP3 inflammasome. Previous studies revealed that acetyl-CoA synthetase 2 (ACSS2) promotes inflammation during metabolic stress suggesting that ACSS2 might regulate pyroptosis and inflammatory responses of RTECs in AKI.Methods and resultsThe expression of ACSS2 was found to be significantly increased in the renal epithelial cells of mice with lipopolysaccharide (LPS)-induced AKI. Pharmacological and genetic strategies demonstrated that ACSS2 regulated NLRP3-mediated caspase-1 activation and pyroptosis through the stimulation of the KLF5/NF-κB pathway in RTECs. The deletion of ACSS2 attenuated renal tubular pathological injury and inflammatory cell infiltration in an LPS-induced mouse model, and ACSS2-deficient mice displayed impaired NLRP3 activation-mediated pyroptosis and decreased IL-1β production in response to the LPS challenge. In HK-2 cells, ACSS2 deficiency suppressed NLRP3-mediated caspase-1 activation and pyroptosis through the downregulation of the KLF5/NF-κB pathway. The KLF5 inhibitor ML264 suppressed NF-κB activity and NLRP3-mediated caspase-1 activation, thus protecting HK-2 cells from LPS-induced pyroptosis.ConclusionOur results suggested that ACSS2 regulates activation of the NLRP3 inflammasome and pyroptosis by inducing the KLF5/NF-κB pathway in RTECs. These results identified ACSS2 as a potential therapeutic target in AKI.Graphical We found that the expression of ACSS2 was significantly increased in RTECs in septic AKI. ACSS2-deficient mice displayed resistance to renal damage in this model. ACSS2 regulated NLRP3-mediated caspase-1 activation and pyroptosis through the KLF5/NF-κB pathway in RTECs. Pharmacological inhibition of KLF5 suppressed NLRP3 activation and caspase-1 cleavage by downregulating of NF-κB. Our results suggest that ACSS2 is a pro-pathogenic mediator of cell pyroptosis of RTECs and renal inflammation in sepsis-induced AKI.

P109 Organic Cation Transporter (OCTN)-1 variants shape innate immunity and predict individual response to vedolizumab In ulcerative colitis patients

Abstract Background Limited evidence is available about predictors of response to specific medications for ulcerative colitis (UC). OCTN1, an organic cation transporter, with its variants, could modulate the inflammatory response and potentially modify drug response to immune-modulatory therapy (anti-TNFα and vedolizumab), perhaps predicting individual response. Methods We first confirmed the ability of OCTN1 to differentially modulate IL1β secretion under different stimuli in a human leukemia monocytic cell line (THP-1) and in human peripheral blood mononuclear cells. We therefore prospectively enrolled a cohort of UC patients starting either anti-TNFα or vedolizumab, determined their OCTN1 genotype and evaluated their clinical features at baseline and clinical response to therapy or disease remission (according to clinical Mayo score) after six weeks (T1) and six months (T2). We finally enrolled a cohort of UC patients in clinical and endoscopic remission (according to full Mayo score) under immune-modulatory therapy (anti-TNFα or vedolizumab) for at least two years. Results OCTN1-deficient THP1 cells showed reduced IL-1β secretion in response to peptidoglycan (PGN) and Fusobacterium nucleatum (Fn), whilst primary monocytes bearing OCTN1 503F variant displayed an increased production of IL-1β in response to PGN and live bacteria. We therefore enrolled 90 patients, 50 starting anti-TNFα and 40 vedolizumab. Patients with 503F genotype showed higher rates of pancolitis as compared to 503L genotype. Among patients with 503L genotype, vedolizumab results highly associated with long-term response (OR 8.92, 95%CI 0.50-11.0; p=0.019), whilst conversely co-presence of 503F genotype and vedolizumab therapy disclosed an opposite role on the long-term response rate (OR 0.02, 95%CI 0.00-0.81; p=0.038).The population of UC patients in clinical and endoscopic remission was composed by 92 patients, 63 under anti-TNFα and 29 under vedolizumab. Of note, similar trends of disease extension were observed across genotypes: higher rates of pancolitis were observed in the 503F population as compared to 503L (62.5% vs 44%). Furthermore, among patients in remission with 503F genotype, the percentage of subjects receiving vedolizumab was sensibly lower than among patients with 503L genotype (87.5% anti-TNFα vs12.5% vedolizumab among 503F genotype; 56% anti-TNFα vs 44% vedolizumab among 503L genotype). Conclusion Mutated OCTN1 genotype (503F) favors an increased IL1β secretion from human stimulated leukocytes, thus suggesting a worse disease course with higher rate of pancolitis and lower response to vedolizumab as compared to 503L.

A1 is induced by pathogen ligands to limit myeloid cell death and NLRP3 inflammasome activation.

Programmed cell death pathways play an important role in innate immune responses to infection. Activation of intrinsic apoptosis promotes infected cell clearance; however, comparatively little is known about how this mode of cell death is regulated during infections and whether it can induce inflammation. Here, we identify that the pro-survival BCL-2 family member, A1, controls activation of the essential intrinsic apoptotic effectors BAX/BAK in macrophages and monocytes following bacterial lipopolysaccharide (LPS) sensing. We show that, due to its tight transcriptional and post-translational regulation, A1 acts as a molecular rheostat to regulate BAX/BAK-dependent apoptosis and the subsequent NLRP3 inflammasome-dependent and inflammasome-independent maturation of the inflammatory cytokine IL-1β. Furthermore, induction of A1 expression in inflammatory monocytes limits cell death modalities and IL-1β activation triggered by Neisseria gonorrhoeae-derived outer membrane vesicles (NOMVs). Consequently, A1-deficient mice exhibit heightened IL-1β production in response to NOMV injection. These findings reveal that bacteria can induce A1 expression to delay myeloid cell death and inflammatory responses, which has implications for the development of host-directed antimicrobial therapeutics.

#3581 ACSS2-INDUCED FATTY ACID SYNTHESIS PROMOTES NLRP3 INFLAMMASOME AND PYROPTOSIS OF RENAL TUBULAR CELLS IN SEPSIS-INDUCED ACUTE KIDNEY INJURY

Abstract Background and Aims Cellular fatty acid metabolism was supposed to be tightly associated with immune responses. Pyroptosis is a form of programmed cell death dependent on the NLRP3 inflammasome activation. Previous studies have revealed that acyl-CoA synthetase 2 (ACSS2) promoted fatty acid synthesis under metabolic stress. However, whether ACSS2-mediated fatty acid metabolism can regulate pyroptosis and inflammatory responses of renal tubular cells in AKI remain unclear. Method lipopolysaccharide (LPS) was intraperitoneally injected to establish the sepsis mouse model, and in vitro HK-2 cell culture model was achieved by LPS stimulation. HE and PAS staining were carried out to evaluate pathological injury of kidneys in mice. The renal tissue immunostaining was conducted to detect IL-1β expression and macrophage distribution in kidney tissues of mice. Gene expression of inflammatory factors was evaluated by real-time PCR. Western Blot was conducted to the activation of the NLRP3 pathway and pyroptosis in vivo and in vitro. Results Here, we demonstrated that the expression of ACSS2 was significantly increased in the renal epithelial cells of mice with Lipopolysaccharide (LPS)-induced AKI when compared to wild-type mice. ACSS2 regulates NLRP3-mediated caspase-1 activation and IL-1β production through the stimulation of fatty acid synthase (FASN) in renal epithelial cells. The deletion of ACSS2 attenuated renal tubular pathological injury and inflammatory cell infiltration in an LPS-induced mouse model. Consistently, ACSS2-deficient mice displayed impaired FASN-mediated lipid synthesis and decreased IL-1β production in response to the LPS challenge. In HK-2 cells, ACSS2 deficiency suppressed NLRP3-mediated caspase-1 activation and decreased fatty acid synthesis through the downregulation of FASN. The treatment with the chemical inhibitor C75 suppressed NLRP3-mediated caspase-1 activation and pyroptosis of HK-2 cells under LPS treatment in renal tubular cells. Conclusion Our results suggested that ACSS2 regulated the NLRP3 inflammasome activation and pyroptosis by inducing the FASN-mediated fatty acid synthesis pathway in renal epithelial cells. These results identified ACSS2 as a potential therapeutic target in AKI.

BPOZ-2 is a negative regulator of the NLPR3 inflammasome contributing to SARS-CoV-2-induced hyperinflammation.

Inflammation play important roles in the initiation and progression of acute lung injury (ALI), acute respiratory distress syndrome (ARDS), septic shock, clotting dysfunction, or even death associated with SARS-CoV-2 infection. However, the pathogenic mechanisms underlying SARS-CoV-2-induced hyperinflammation are still largely unknown. The animal model of septic shock and ALI was established after LPS intraperitoneal injection or intratracheal instillation. Bone marrow-derived macrophages (BMDMs) from WT and BPOZ-2 KO mouse strains were harvested from the femurs and tibias of mice. Immunohistology staining, ELISA assay, coimmunoprecipitation, and immunoblot analysis were used to detect the histopathological changes of lung tissues and the expression of inflammatory factors and protein interaction. We show a distinct mechanism by which the SARS-CoV-2 N (SARS-2-N) protein targets Bood POZ-containing gene type 2 (BPOZ-2), a scaffold protein for the E3 ubiquitin ligase Cullin 3 that we identified as a negative regulator of inflammatory responses, to promote NLRP3 inflammasome activation. We first demonstrated that BPOZ-2 knockout (BPOZ-2 KO) mice were more susceptible to lipopolysaccharide (LPS)-induced septic shock and ALI and showed increased serum IL-1β levels. In addition, BMDMs isolated from BPOZ-2 KO mice showed increased IL-1β production in response to NLRP3 stimuli. Mechanistically, BPOZ-2 interacted with NLRP3 and mediated its degradation by recruiting Cullin 3. In particular, the expression of BPOZ-2 was significantly reduced in lung tissues from mice infected with SARS-CoV-2 and in cells overexpressing SARS-2-N. Importantly, proinflammatory responses triggered by the SARS-2-N were significantly blocked by BPOZ-2 reintroduction. Thus, we concluded that BPOZ-2 is a negative regulator of the NLPR3 inflammasome that likely contributes to SARS-CoV-2-induced hyperinflammation.

Involvement of the p38 MAPK-NLRC4-Caspase-1 Pathway in Ionizing Radiation-Enhanced Macrophage IL-1β Production.

Macrophages are abundant immune cells in the tumor microenvironment and are crucial in regulating tumor malignancy. We previously reported that ionizing radiation (IR) increases the production of interleukin (IL)-1β in lipopolysaccharide (LPS)-treated macrophages, contributing to the malignancy of colorectal cancer cells; however, the mechanism remained unclear. Here, we show that IR increases the activity of cysteine-aspartate-specific protease 1 (caspase-1), which is regulated by the inflammasome, and cleaves premature IL-1β to mature IL-1β in RAW264.7 macrophages. Irradiated RAW264.7 cells showed increased expression of NLRC4 inflammasome, which controls the activity of caspase-1 and IL-1β production. Silencing of NLRC4 using RNA interference inhibited the IR-induced increase in IL-1β production. Activation of the inflammasome can be regulated by mitogen-activated protein kinase (MAPK)s in macrophages. In RAW264.7 cells, IR increased the phosphorylation of p38 MAPK but not extracellular signal-regulated kinase and c-Jun N-terminal kinase. Moreover, a selective inhibitor of p38 MAPK inhibited LPS-induced IL-1β production and NLRC4 inflammasome expression in irradiated RAW264.7 macrophages. Our results indicate that IR-induced activation of the p38 MAPK-NLRC4-caspase-1 activation pathway in macrophages increases IL-1β production in response to LPS.

Innate Memory Following Intravenous BCG in Neonatal Pigs

Abstract In adults and rodents, Bacillus Calmette-Guerin (BCG) inoculation alters innate immune cells via epigenetic and metabolic modifications resulting in heightened responses to subsequent microbial exposure (innate memory). Neonatal BCG vaccination can correlate to improved disease resistance in some human populations, but studies with human infants are extremely limited. Pigs are a relevant biomedical model for human disease and offer a path to directly asses neonatal innate memory to improve disease resilience. BCG administration in a weaned (3 week old) pig model elicited monocyte memory only with intravenous (IV) but not subcutaneous administration. Therefore, to evaluate induction of innate memory in neonates, 3–5 day old piglets received intravenous (IV) BCG or mock and ex vivo monocyte memory was assessed. At 3 and 6 weeks post-BCG, monocytes from the IV-BCG group had elevated TNF but not IL-1β production in response to LPS stimulation ex vivo, indicating neonatal BCG exposure induced innate memory. Interestingly, broncho-alveolar lavage cells from IV-BCG group had minimal differences compared to mock group in response to LPS, suggesting IV-BCG alters responses in peripheral monocytes differently than tissue resident myeloid-lineage cells. Overall, IV-BCG administration induced monocyte memory in neonatal pigs, highlighting piglets as a valuable model for continued study on the utility of BCG induced innate memory to improve disease resilience. This work was funded by USDA-NIFA AFRI grant # 2019-07511 as part of the joint USDA-NIFA-NIH Dual Purpose with Dual Benefit: Research in Biomedicine and Agriculture Using Agriculturally Important Domestic Animal Species Program and USDA-ARS CRIS project 5030-32000-225-000D

Senecavirus a 3D Interacts with NLRP3 to Induce IL-1β Production by Activating NF-κB and Ion Channel Signals.

ABSTRACTSenecavirus A (SVA) infection induces inflammation in animals, such as fever, diarrhea, vesicles and erosions, and even death. The inflammatory cytokine interleukin-1β (IL-1β) plays a pivotal role in inflammatory responses to combat microbes. Although SVA infection can produce inflammatory clinical symptoms, the modulation of IL-1β production by SVA infection remains unknown at present. Here, both in vitro and in vivo, SVA robustly induced IL-1β production in macrophages and pigs. Infection performed in NOD-, LRR-, and pyrin domain-containing three (NLRP3) knockdown cells indicated that NLRP3 is essential for SVA-induced IL-1β secretion. Importantly, we identified that the 1 to 154 amino acid (aa) portion of SVA 3D binds to the NLRP3 NACHT domain to activate NLRP3 inflammasome assembly and IL-1β secretion. In addition, the SVA 3D protein interacts with IKKα and IKKβ to induce NF-κB activation, which facilitates pro-IL-1β transcription. Meanwhile, 3D induces p65 nucleus entry. Moreover, SVA 3D induces calcium influx and potassium efflux, which triggers IL-1β secretion. Ion channels might be related to 3D binding with NLRP3, resulting in NLRP3-ASC complex assembly. We found that 3D protein expression induced tissue hemorrhage and swelling in the mice model. Consistently, expression of 3D in mice caused IL-1β maturation and secretion. In the natural host of pigs, we confirmed that 3D also induced IL-1β production. Our data reveal a novel mechanism underlying the activation of the NLRP3 inflammasome after SVA 3D expression, which provides clues for controlling pig’s inflammation during the SVA infection.IMPORTANCE Inflammation refers to the response of the immune system to viral, bacterial, and fungal infections or other foreign particles in the body, which can involve the production of a wide array of soluble inflammatory mediators. The NLRP3 inflammasome is one of the best-characterized inflammasome leading to IL-1β production and maturation. Senecavirus A (SVA) is an oncolytic virus that can cause fever, vesicles and erosions, severe fatal diarrhea, and even the sudden death of piglets. In this study, we demonstrated that 1 to 154 aa of SVA polymerase protein 3D interacts with the NACHT domain of NLRP3 to induce IL-1β production via the NF-κB signaling pathway and ion channel signal. Our study unveils the mechanism underlying the regulation of inflammasome assembly and production of IL-1β in response to SVA infection that will help better understand the modulation of host inflammation in pathogens invasion and development of the vaccine.

Human fetal membrane IL-1β production in response to bacterial components is mediated by uric-acid induced NLRP3 inflammasome activation

Inflammatory interleukin-1β (IL-1β) is an important mediator of preterm birth. IL-1β secretion is mediated by the inflammasome that processes pro-IL-1β into its active form. However the mechanisms involved at the level of the fetal membrane (FM) are not fully understood. This study sought to determine the FM compartment involved in IL-1β production in response to bacterial components and to evaluate the mechanism of inflammasome activation. Since IL-18 is also mediated by the inflammasome and IL-8 is a chemoattractant that contributes to neutrophil recruitment in chorioamnionitis, we also evaluated the production of these factors. A human explant system was used to evaluate the response of the chorion, amnion, and intact FMs to the bacterial components lipopolysaccharide (LPS), peptidoglycan (PGN), or muramyl dipeptide (MDP). The chorion was the major source of IL-1β and IL-8 production in response to LPS, PGN, and MDP. LPS, PGN, and MDP induced FM IL-1β and IL-18 secretion in a non-pyroptotic manner through activation of the NLRP3 inflammasome with contributions from ATP release through Pannexin-1, and ROS signaling. Since LPS, PGN, and MDP are not known to activate NLRP3 directly, the role of uric acid as a potential mediator was assessed. FMs produced elevated uric acid in response to LPS, PGN and MDP. FM IL-1β secretion was inhibited by allopurinol, which blocks uric acid production, for LPS and PGN, and to a lesser degree, MDP. These findings shed light on the mechanisms by which fetal membrane inflammation and subsequent preterm birth may arise.

CLEC-1 Acts as a Negative Regulator of Dectin-1 Induced Host Inflammatory Response Signature in Aspergillus fumigatus Keratitis.

PurposeC-type lectin-like receptor-1 (CLEC-1) is a member of the Dectin-1 cluster of pattern recognition receptors (PRRs). It is involved in host immunity, has immunoregulatory function, and supports allograft tolerance. Our study aimed to describe the role of CLEC-1 in response to fungal keratitis, in situ, in vivo, and in vitro.MethodsQuantitative polymerase chain reaction (qRT-PCR) and immunofluorescence were used to detect the expression of CLEC-1 in corneas of patients with Aspergillus fumigatus (A. fumigatus) keratitis. In vitro and in vivo experiments were designed in THP-1 macrophages and C57BL/6 mouse models, respectively. The expression of CLEC-1 in corneas of mice model was determined by qRT-PCR, Western blot, and immunofluorescence. CLEC-1 overexpression in mouse corneas was achieved by intrastromal injection of adeno-associated virus (AAV) vectors. Disease response was evaluated by slit-lamp photography, clinical score, and colony forming unit (CFU). Bioluminescence imaging system image acquisition, myeloperoxidase (MPO) assays, immunofluorescence staining, qRT-PCR, and Western blot were used to investigate the role of CLEC-1. To further define the role of CLEC-1, we used lentivirus vectors to overexpress CLEC-1 or/and Dectin-1 in THP-1 macrophages.ResultsThe expression of CLEC-1 was increased in corneas of patients with A. fumigatus keratitis. In corneas of mice from the A. fumigatus keratitis model, the expression of CLEC-1 was decreased in the acute inflammatory stage and increased during convalescence. Following Natamycin treatment, CLEC-1 was upregulated in A. fumigatus keratitis mice. Compared with normal C57BL/6 mice, overexpression of CLEC-1 converted the characteristic susceptible response to resistance, as demonstrated by slit-lamp photography and clinical score. In vivo studies revealed decreased MPO levels and neutrophils recruitment and higher fungal load after the upregulation of CLEC-1. Compared with control corneas, CLEC-1 overexpression impaired corneal pro-inflammatory cytokine IL-1β production.ConclusionsThese findings demonstrate that CLEC-1 may act as a negative regulator of Dectin-1 induced host inflammatory response via suppressing neutrophils recruitment and production of pro-inflammatory cytokine IL-1β production in response to A. fumigatus keratitis.

273 Bacterial components induce fetal membrane non-pyroptotic IL-1β production through activation of the NLRP3 inflammasome

Bacterial infections increase the risk of chorioamnionitis and preterm birth; however the mechanisms involved are not fully understood. Inflammatory interleukin-1β (IL-1β) is an important mediator of preterm birth, and its secretion is mediated by the inflammasome that processes pro-IL-1β into its active form. This study sought to determine the fetal membrane (FM) compartment involved in IL-1β production in response to bacterial components and to evaluate the mechanism of inflammasome activation. An ex vivo human FM explant system was used to evaluate the response of the amnion, chorion, and intact FMs to media or the bacterial components lipopolysaccharide (LPS), peptidoglycan (PGN), flagellin, iE-DAP, or MDP (n=8). After 24 hours, IL-1β secretion was quantified by ELISA, the pyroptotic marker Gasdermin-D (GSDMD) evaluated by Western blot, and viability measured using a LDH assay. Inflammasome function was assessed using inhibitors to NLRP3 (MCC950), caspase-1 (Z-WEHD-FMK), Pannexin-1 (carbenoxolone), and ROS (DPI) (n=8-12). LPS, PGN, and MDP significantly increased IL-1β secretion from intact FM explants by 57.0±22.4-; 38.3±16.4-; and 28.4±11.9-fold respectively (p<0.05). Flagellin and iE-DAP had no significant effect. The chorion, not the amnion, was the source of this IL-1β response. Inhibitors to NLRP3, Pannexin-1, and ROS significantly inhibited LPS, PGN, and MDP-induced IL-1β secretion from intact FM explants (p<0.05), while the caspase-1 inhibitor significantly reduced LPS and MDP, but not PGN-induced, IL-1β (p<0.05). There was no significant difference in GSDMD activity or LDH secretion by intact FMs under all treatment conditions. The chorion is the major source of IL-1β production following FM exposure to bacterial components. Bacterial LPS, PGN, and MDP induced human FM IL-1β secretion through activation of the NLRP3 inflammasome with contributions from ATP release through Pannexin-1 and ROS signaling. Further investigation is needed to determine the role of alternate caspases and non-pyroptotic mechanisms of FM IL-1β production and release.

Binding of Macrophage Receptor MARCO, LDL, and LDLR to Disease-Associated Crystalline Structures.

Endogenous and exogenous crystalline structures are involved in various pathologies and diseases in humans by inducing sterile inflammation, mechanical stress, or obstruction of excretory organs. The best studied of these diseases is gout, in which crystallization of uric acid in the form of monosodium urate (MSU) mainly in synovial fluid of the joints leads to sterile inflammation. Though some of these diseases have been described for centuries, little is known about if and how the immune system recognizes the associated crystals. Thus, in this study we aimed at identifying possible recognition molecules of MSU using liquid chromatography-mass spectrometry (LC-MS) analysis of MSU-binding serum proteins. Among the strongest binding proteins, we unexpectedly found two transmembrane receptors, namely macrophage receptor with collagenous structure (MARCO) and low-density lipoprotein (LDL) receptor (LDLR). We show that recombinant versions of both human and mouse MARCO directly bind to unopsonized MSU and several other disease-associated crystals. Recombinant LDLR binds many types of crystals mainly when opsonized with serum proteins. We show that this interaction is predominantly mediated by LDL, which we found to bind to all crystalline structures tested except for cholesterol crystals. However, murine macrophages lacking LDLR expression do neither show altered phagocytosis nor interleukin-1β (IL-1β) production in response to opsonized crystals. Binding of LDL to MSU has previously been shown to inhibit the production of reactive oxygen species (ROS) by human neutrophils. We extend these findings and show that LDL inhibits neutrophil ROS production in response to most crystals tested, even cholesterol crystals. The inhibition of neutrophil ROS production only partly correlated with the inhibition of IL-1β production by peripheral blood mononuclear cells (PBMCs): LDL inhibited IL-1β production in response to large MSU crystals, but not small MSU or silica crystals. This may suggest distinct upstream signals for IL-1β production depending on the size or the shape of the crystals. Together, we identify MARCO and LDLR as potential crystal recognition receptors, and show that LDL binding to diverse disease-associated crystalline structures has variable effects on crystal-induced innate immune cell activation.

Unveiling the Crucial Role of Type IV Secretion System and Motility of Helicobacter pylori in IL-1β Production via NLRP3 Inflammasome Activation in Neutrophils.

Helicobacter pylori is a gram-negative, microaerophilic, and spiral-shaped bacterium and causes gastrointestinal diseases in human. IL-1β is a representative cytokine produced in innate immune cells and is considered to be a key factor in the development of gastrointestinal malignancies. However, the mechanism of IL-1β production by neutrophils during H. pylori infection is still unknown. We designed this study to identify host and bacterial factors involved in regulation of H. pylori-induced IL-1β production in neutrophils. We found that H. pylori-induced IL-1β production is abolished in NLRP3-, ASC-, and caspase-1/11-deficient neutrophils, suggesting essential role for NLRP3 inflammasome in IL-1β response against H. pylori. Host TLR2, but not TLR4 and Nod2, was also required for transcription of NLRP3 and IL-1β as well as secretion of IL-1β. H. pylori lacking cagL, a key component of the type IV secretion system (T4SS), induced less IL-1β production in neutrophils than did its isogenic WT strain, whereas vacA and ureA were dispensable. Moreover, T4SS was involved in caspase-1 activation and IL-1β maturation in H. pylori-infected neutrophils. We also found that FlaA is essential for H. pylori-mediated IL-1β production in neutrophils, but not dendritic cells. TLR5 and NLRC4 were not required for H. pylori-induced IL-1β production in neutrophils. Instead, bacterial motility is essential for the production of IL-1β in response to H. pylori. In conclusion, our study shows that host TLR2 and NLRP3 inflammasome and bacterial T4SS and motility are essential factors for IL-1β production by neutrophils in response to H. pylori.

Role reversal: adaptive immunity instructs inflammasome activation for anti-viral defence.

Signalling by innate immune cells is critical to shaping the adaptive immune response to microbial infection. In this issue of The EMBO Journal, Labzin etal reveal that the adaptive immune system can instruct the innate response to adenovirus infection. In human macrophages, antibody-coated adenovirus triggers a novel TRIM21-dependent pathway that activates the NLRP3 inflammasome and the secretion of IL-1β.

85 Linking Diabetes and Dementia Risk: A Role for Aβ-42 Induced Activation of the NLRP3 Inflammasome? Results from the ENBIND Study

Abstract Background Midlife Type 2 Diabetes Mellitus (T2DM) is associated with a greater risk of dementia in later life. The innate immune NLRP3 inflammasome, an innate immune receptor, has been implicated in both T2DM and Alzheimer’s Dementia (AD) and can become activated by Amyloid-β 42 (Aβ-42). We analysed inflammasome-dependent cytokine responses in midlife T2DM and correlated these with cognitive and gait performance Methods Peripheral Blood Mononuclear Cells (PBMCs) of otherwise healthy patients with T2DM (n=30; 52 ± 8 yrs) and matched controls (n=15; 52.5 ± 7.86 yrs) were incubated for 18h with the following NLRP3 ligands: (i) Lipopolysaccharide (LPS), (ii) Aβ42 or (ii) LPS &amp; Aβ42. Cell supernatants were analysed for production of the inflammasome-dependent cytokine IL-1β. Cognitive function was assessed using the MoCA and gait speed assessed under self-selected, maximal and cognitive dual-task conditions. Mann-Whitney, Wilcoxon tests and linear regression of log-transformed data were used to analyse results Results Incubation of PBMCs from all participants with NLRP3 agonists was associated with significant production of IL-1β (all p&lt;0.001). There were no statistically significant between-group differences in IL-1β production. MoCA score did not predict IL-1β production by PBMCs. However, poorer performance on the cognitive dual-task gait paradigm was associated with significantly greater IL-1β production in response to LPS &amp; Aβ42 in those with T2DM (p=0.015), which persisted after robust control for demographic, cognitive and cardiovascular covariates (p=0.003). The same association was not seen for healthy controls (p=0.58). Conclusion Both LPS &amp; Aβ42 induce significant IL-1β production in PBMCs which did not differ between those with midlife T2DM and healthy controls. Poorer performance on the cognitive dual task gait paradigm was associated with significantly greater IL-1β production in response to LPS &amp; Aβ42. Given the value of dual task gait in predicting cognitive decline, longitudinal follow up of this cohort may provide insight into the underlying pathophysiological mechanisms and potential biomarkers of dementia risk in T2DM.

Toll-like Receptor 2/IL-1β signaling is required for sensing and responding to a commensal bacterium at the ocular surface

Abstract Conjunctiva-associated lymphoid tissue (CALT) resembles a conventional mucosal immune tissue, and harbors a variety of immune cells. CALT is exposed to diverse microorganisms that come in contact with the ocular surface. Previously, we demonstrated that colonization of the ocular mucosa with the commensal bacterium, Corynebacterium mastitidis (C. mast), results in increased resistance of the ocular surface to infectious fungal and bacterial pathogens. Further, we showed that this effect is due to IL-17 produced by Vγ4+γδ T cells that respond to C. mast in an IL-1-dependent fashion. In this study, we show that the toll-like receptor 2 (TLR2) and IL-1β are important for IL-17 production, activation and proliferation of Vg4+ γδ T cells. Using in vivo and in vitro approaches, we show that cells of TLR2 KO mice have reduced production of IL-1β in response to C. mast. These mice also fail to recruit Vg4+γδ T cells and neutrophils to the conjunctiva. We interpret these data to mean that TLR2 is required for sensing this commensal on the conjunctiva and its draining lymph nodes to activate the inflammasome and consequent IL-1 production, which in turn drives the IL-17 response. Our data highlight the importance and the role of the innate receptor TLR2 in the generation of the ocular mucosal immune response and in maintenance of the homeostatic γδ T cell response at the ocular surface.

Gamma Herpesvirus manipulates HVEM regulated IL-1β pathway to evade immune responses

Abstract The herpes virus entry mediator (HVEM) a member of the TNFR superfamily, is a key immunoregulator with both proinflammatory and inhibitory signaling functions. Both α and β-herpes viruses exploit HVEM to establish persistent infections, however little is known about whether the oncogenic γ-herpesviruses modulate HVEM. Here, we investigate the role of HVEM in a mouse model of γ-herpesvirus (MHV68) infection. Control C57BL/6 and HVEM-deficient (Tnfrsf14−/−) mice were intranasally infected with MHV68. We found that virus replication in the lungs of Tnfrsf14−/− mice increased significantly indicating HVEM expression is required to control MHV68 infection. We found that alveolar macrophages were drastically reduced in Tnfrsf14−/− mice compared to C57/BL6 controls suggesting their survival requires HVEM signaling. Importantly, we showed that IL-1β levels in the lungs of MHV68 infected Tnfrsf14−/−mice were significantly higher than C57BL/6 controls. Bone marrow-derived macrophage from Tnfrsf14−/− showed that HVEM is necessary to prevent IL-1β production in response to inflammasome activator. Antibodies that block IL-1R or IL-1β drastically increased the viral load in the lungs suggested that IL-1β is necessary to control viral replication. Taken together, our results highlight the role for HVEM in innate immune response evasion of MHV68 that could involve the inflammasome.

MEK2 Negatively Regulates Lipopolysaccharide-Mediated IL-1β Production through HIF-1α Expression.

LPS-activated macrophages require metabolic reprogramming and glucose uptake mediated by hypoxia-inducible factor (HIF)-1 α and glucose transporter 1 (Glut1) expression for proinflammatory cytokine production, especially IL-1β. This process is tightly regulated through activation of MAPK kinases, including the MEK/ERK pathway as well as several transcription factors including HIF-1α. Although MAPK kinase (MEK) 2 deficiency had no significant effect on NO, TNF-α, or IL-12 production in response to LPS challenge, MEK2-deficient murine bone marrow-derived macrophages (BMDMs) exhibited lower IL-10 production. Importantly, MEK2-deficient BMDMs exhibited a preserved ERK1/2 phosphorylation, higher HIF-1α and Glut1 levels, and substantially increased IL-1β as well as IL-6 production in response to LPS stimulation. Knockdown of HIF-1α expression via short interference RNA decreased the level of HIF-1α expression in MEK2-deficient BMDMs and decreased IL-1β production in response to LPS treatment. Furthermore, we performed gain of function experiments by overexpressing MEK2 protein in RAW264.7 cells. LPS stimulation of MEK2 overexpressed in RAW264.7 cells led to a marked decreased IL-1β production. Finally, we investigated the role of Mek1 and Mek2 double and triple mutation on ERK phosphorylation, HIF-1α expression, and IL-1β production. We found that MEK2 is the major kinase, which inversely proportionally regulates HIF-1α and IL-1β expression independent of ERK activation. Our findings demonstrate a novel regulatory function for MEK2 in response to TLR4 activation in IL-1β production through modulating HIF-1α expression.