NLRP3 Inflammasome In Macrophages Research Articles

Pericancerous cross-presentation to cytotoxic T lymphocytes impairs immunotherapeutic efficacy in hepatocellular carcinoma.

Hyperprogressive disease can occur in cancer patients receiving immune checkpoint blockade (ICB) therapy, but whether and how reactive cytotoxic T lymphocytes (CTLs) exert protumorigenic effects in this context remain elusive. Herein, our study reveals that pericancerous macrophages cross-present antigens to CD103+ CTLs in hepatocellular carcinoma (HCC) via the endoplasmic reticulum (ER)-associated degradation machinery-mediated cytosolic pathway. This process leads to the retention of CD103+ CTLs in the pericancerous area, whereby they activate NLRP3 inflammasome in macrophages, promoting hepatoma progression and resistance to immunotherapy. Our single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics analysis of HCC patients shows that despite their tissue-resident effector phenotype, the aggregation of CD103+ CTLs predicts unfavorable clinical outcomes for HCC patients receiving multiple types of treatment. Correspondingly, therapeutic strategies that redistribute CD103+ CTLs can disrupt this pathogenic interplay with macrophages, enhancing the efficacy of ICB treatment against HCC.

Macrophage NLRP3 inflammasome mediates the effects of sympathetic nerve on cardiac remodeling in obese rats

Macrophage NLRP3 inflammasome mediates the effects of sympathetic nerve on cardiac remodeling in obese rats

Targeting S100A9-TLR2 axis controls macrophage NLRP3 inflammasome activation in fatty liver ischemia reperfusion injury

Abstract Liver ischemia reperfusion (IR) injury significantly impacts clinical outcomes by increasing the risk of hepatic dysfunction after liver surgery. Fatty livers are more susceptible to IR stress. Recent studies have demonstrated that S100A9 plays a crucial role in both IR injury and the progression of liver steatosis. Nevertheless, the precise mechanisms underlying these effects remain unclear. In our study, transcriptome analysis of fatty livers subjected to IR insult in mice identified S100A9 as an important mediator. Employing loss-of-function approaches, we investigated the immune regulatory function of S100A9 and its downstream signaling in fatty liver IR injury. As expected, S100A9 emerged as one of the most significantly upregulated genes during the reperfusion stage in fatty livers. Genetic knockdown of S100A9 markedly ameliorated liver pathological damage, evidenced by reduced macrophage/neutrophil infiltration as well as the decreased expression of proinflammatory factors. Transcriptome/functional studies revealed that S100A9 triggered liver inflammatory response via regulating Toll-like receptor 2 (TLR2)/Activating transcription factor 4 (ATF4) signaling. Additionally, TLR2 expression was notably increased in macrophages from ischemic fatty livers. In vitro, recombinant S100A9-stimulated macrophages exhibited the elevated production of proinflammatory factors and TLR2/ATF4 pathway activation. Intriguingly, S100A9 facilitated ATF4 nuclear translocation and enhanced NEK7/NLRP3 inflammasome activation in macrophages. In conclusion, our study identified S100A9 as a key regulator responsible for macrophage NLRP3 inflammasome activation and subsequent inflammatory injury in fatty liver IR process. Targeting TLR2/ATF4 signaling may offer a novel therapeutic strategy for mitigating S100A9-mediated liver injury.

GDF3 promotes adipose tissue macrophage-mediated inflammation via altered chromatin accessibility during aging.

Age-related susceptibility to sepsis and endotoxemia is poorly defined, although hyperactivation of the immune system and the expansion of the visceral adipose as an immunological reservoir are underlying features. Macrophages from older organisms exhibit substantial changes, including chronic NLRP3 inflammasome activation, genomic remodeling and a dysfunctional, amplified inflammatory response upon new exposure to pathogen. However, the mechanisms by which old macrophages maintain their inflammatory phenotype during endotoxemia remains elusive. We previously identified Gdf3 , a TGFβ superfamily cytokine, as a top-regulated gene by age and the NLRP3 inflammasome in adipose tissue macrophages (ATMs). Here, we demonstrate that endotoxemia increases inflammatory (CD11c + ) ATMs in a Gdf3- dependent manner in old mice. Lifelong systemic or myeloid-specific deletion of Gdf3 leads to reduced endotoxemia- induced inflammation, with decreased CD11c + ATMs and inflammatory cytokines, and protection from hypothermia. Moreover, acute blockade of Gdf3 using JQ1, a BRD4 inhibitor, phenocopies old mice with lifelong Gdf3- deficiency. We show that GDF3 promotes the inflammatory phenotype in ATMs by phosphorylating SMAD2/3. Mechanistically, the differential chromatin landscape of ATMs from old mice with or without myeloid-driven Gdf3 indicates that GDF3- SMAD2/3 signaling axis shifts the chromatin accessibility of ATMs towards an inflammatory state during aging. Furthermore, pharmaceutical inhibition of SMAD3 with a specific inhibitor of SMAD3 (SIS3) mimics Gdf3 deletion. SIS3 reduces endotoxemia-mediated inflammation with fewer CD11c + ATMs and less severe hypothermia in old, but not young mice, as well as reduced mortality. In human adipose tissue, age positively correlates with GDF3 level, while inflammation correlates with pSMAD2/3 level. Overall, these results highlight the importance of GDF3-SMAD2/3 axis in driving inflammation in older organisms and identify this signaling axis as a promising therapeutic target for mitigating endotoxemia-related inflammation in the aged.

Human NLRP3 inflammasome activation leads to formation of condensate at the microtubule organizing center.

The NLRP3 inflammasome is a multi-protein molecular machine that mediates inflammatory responses in innate immunity. Its dysregulation has been linked to a large number of human diseases. Using cryogenic fluorescence-guided focused-ion-beam (cryo-FIB) milling and electron cryo-tomography (cryo-ET), we obtained 3-D images of the NLRP3 inflammasome in situ at various stages of its activation at macromolecular resolution. The cryo-tomograms unexpectedly reveal dense condensates of the human macrophage NLRP3 inflammasome that form within and around the microtubule organizing center (MTOC). We also find that following activation, the trans-Golgi network disperses and 50-nm NLRP3-associated vesicles appear which likely ferry NLRP3 to the MTOC. At later time points after activation, the electron-dense condensates progressively solidify and the cells undergo pyroptosis with widespread damaged mitochondria and autophagasomal structures.

Targeting the NLRP3 inflammasome in cochlear macrophages protects against hearing loss in chronic suppurative otitis media

The activation of the NLRP3 inflammasome has been linked to several inflammatory and autoinflammatory diseases. Despite cases of potential hearing improvement in immune-mediated diseases, direct evidence of the efficacy of targeting this mechanism in the inner ear is still lacking. Previously, we discovered that macrophages are associated with Sensorineural Hearing loss (SNHL) in Chronic Suppurative Otitis Media (CSOM), the leading cause of this permanent hearing loss in the developing world and incurring costs of $4 to $11 billion dollars in the United States. However, the underlying mechanism remained unknown. Here, we investigate how macrophages drive permanent hearing loss in CSOM. We first confirmed the occurrence of NLRP3 inflammasome activation in cochlear macrophages in CSOM. We then revealed that Outer Hair Cells (OHCs) were protected in CSOM by macrophage depletion and subsequently confirmed the same protection in the NLRP3 knockout condition. Furthermore, we showed that therapeutic inhibition of NLRP3 inflammasome activation and downstream inhibition of IL-1β protects OHCs in CSOM. Collectively, our data demonstrates that the main driver for hearing loss in CSOM is NLRP3 inflammasome activation in cochlear macrophages and this is therapeutically targetable, leading the way for the development of interventions to prevent the leading cause of permanent hearing loss and a costly disease in the developed world.

Gasdermin D and Gasdermin E Are Dispensable for Silica-Mediated IL-1β Secretion from Mouse Macrophages.

Silica crystals activate the NLRP3 inflammasome in macrophages, resulting in the caspase-1-dependent secretion of the proinflammatory cytokine IL-1β. Caspase-1-mediated cleavage of gasdermin D (GSDMD) triggers the formation of GSDMD pores, which drive pyroptotic cell death and facilitate the rapid release of IL-1β. However, the role of GSDMD in silica-induced lung injury is unclear. In this study, we show that although silica-induced lung injury is dependent on the inflammasome adaptor ASC and IL-1R1 signaling, GSDMD is dispensable for acute lung injury. Although the early rapid secretion of IL-1β in response to ATP and nigericin was GSDMD dependent, GSDMD was not required for IL-1β release at later time points. Similarly, secretion of IL-1β from macrophages in response to silica and alum proceeded in a GSDMD-independent manner. We further found that gasdermin E did not contribute to macrophage IL-1β secretion in the absence of GSDMD invitro and was also not necessary for silica-induced acute lung injury invivo. These findings demonstrate that GSDMD and gasdermin E are dispensable for IL-1β secretion in response to silica invitro and in silica-induced acute lung injury invivo.

Butyrate and propionate are microbial danger signals that activate the NLRP3 inflammasome in human macrophages upon TLR stimulation

Short-chain fatty acids (SCFAs) are immunomodulatory compounds produced by the microbiome through dietary fiber fermentation. Although generally considered beneficial for gut health, patients suffering from inflammatory bowel disease (IBD) display poor tolerance to fiber-rich diets, suggesting that SCFAs may have contrary effects under inflammatory conditions. To investigate this, we examined the effect of SCFAs on human macrophages in the presence of Toll-like receptor (TLR) agonists. In contrast to anti-inflammatory effects under steady-state conditions, we found that butyrate and propionate activated the NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) inflammasome in the presence of TLR agonists. Mechanistically, these SCFAs prevented transcription of FLICE-like inhibitory protein (cFLIP) and interleukin-10 (IL-10) through histone deacetylase (HDAC) inhibition, triggering caspase-8-dependent NLRP3 inflammasome activation. SCFA-driven NLRP3 activation was potassium efflux independent and did not result in cell death but rather triggered hyperactivation and IL-1β release. Our findings demonstrate that butyrate and propionate are bacterially derived danger signals that regulate NLRP3 inflammasome activation through epigenetic modulation of the inflammatory response.

Discovery of natural product derivative triptolidiol as a direct NLRP3 inhibitor by reducing K63-specific ubiquitination.

NLRP3 is up-regulated in inflammatory and autoimmune diseases. The development of NLRP3 inhibitors is challenged by the identification of compounds with distinct mechanisms of action avoiding side effects and toxicity. Triptolide is a natural product with multiple anti-inflammatory activities, but a narrow therapeutic window. Natural product triptolide derivatives were screened for NLRP3 inhibitors in human THP-1 and mouse bone marrow-derived macrophages. The efficacy of potent NLRP3 inhibitors was evaluated in LPS-induced acute lung injury and septic shock models. Triptolidiol was identified as a selective inhibitor of NLRP3 with high potency. Triptolidiol inactivated the NLRP3 inflammasome in human THP-1 and mouse primary macrophages primed with LPS. Triptolidiol specifically inhibited pro-caspase 1 cleavage downstream of NLRP3, but not AIM2 or NLRC4 inflammasomes. Based on the structure-activity relationship study, the C8-β-OH group was critical for its binding to NLRP3. Triptolidiol exhibited a submicromolar KD for NLRP3, binding to residue C280. This binding prevented the interaction of NLRP3 with NEK7, the key regulator of NLRP3 inflammasome oligomerization and assembly, but not with the inflammasome adaptor protein ASC. Triptolidiol decreased the K63-specific ubiquitination of NLRP3, leading NLRP3 to a "closed" inactive conformation. Intraperitoneal administration of triptolidiol significantly attenuated LPS-induced acute lung injury and lethal septic shock. Triptolidiol is a novel NLRP3 inhibitor that regulates inflammasome assembly and activation by decreasing K63-linked ubiquitination. Triptolidiol has novel structural features that make it distinct from reported NLRP3 inhibitors and represents a viable therapeutic lead for inflammatory diseases.

Mycobacterium tuberculosis Utilizes Serine/Threonine Kinase PknF to Evade NLRP3 Inflammasome-driven Caspase-1 and RIPK3/Caspase-8 Activation in Murine Dendritic Cells.

Dendritic cells (DCs) are crucial for initiating the acquired immune response to infectious diseases such as tuberculosis. Mycobacterium tuberculosis has evolved strategies to inhibit activation of the NLRP3 inflammasome in macrophages via its serine/threonine protein kinase, protein kinase F (PknF). It is not known whether this pathway is conserved in DCs. In this study, we show that the pknF deletion mutant of M. tuberculosis (MtbΔpknF) compared with wild-type M. tuberculosis-infected cells induces increased production of IL-1β and increased pyroptosis in murine bone marrow-derived DCs (BMDCs). As shown for murine macrophages, the enhanced production of IL-1β postinfection of BMDCs with MtbΔpknF is dependent on NLRP3, ASC, and caspase-1/11. In contrast to macrophages, we show that MtbΔpknF mediates RIPK3/caspase-8-dependent IL-1β production in BMDCs. Consistently, infection with MtbΔpknF results in increased activation of caspase-1 and caspase-8 in BMDCs. When compared with M. tuberculosis-infected cells, the IL-6 production by MtbΔpknF-infected cells was unchanged, indicating that the mutant does not affect the priming phase of inflammasome activation. In contrast, the activation phase was impacted because the MtbΔpknF-induced inflammasome activation in BMDCs depended on potassium efflux, chloride efflux, reactive oxygen species generation, and calcium influx. In conclusion, PknF is important for M. tuberculosis to evade NLRP3 inflammasome-mediated activation of caspase-1 and RIPK3/caspase-8 pathways in BMDCs.

UK5099 Inhibits the NLRP3 Inflammasome Independently of its Long-Established Target Mitochondrial Pyruvate Carrier.

Targeting NLRP3 inflammasome has been recognized as a promising therapeutic strategy for the treatment of numerous common diseases. UK5099, a long-established inhibitor of mitochondrial pyruvate carrier (MPC), is previously found to inhibit macrophage inflammatory responses independent of MPC expression. However, the mechanisms by which UK5099 inhibit inflammatory responses remain unclear. Here, it is shown that UK5099 is a potent inhibitor of the NLRP3 inflammasome in both mouse and human primary macrophages. UK5099 selectively suppresses the activation of the NLRP3 but not the NLRC4 or AIM2 inflammasomes. Of note, UK5099 retains activities on NLRP3 in macrophages devoid of MPC expression, indicating this inhibitory effect is MPC-independent. Mechanistically, UK5099 abrogates mitochondria-NLRP3 interaction and in turn inhibits the assembly of the NLRP3 inflammasome. Further, a single dose of UK5099 persistently reduces IL-1β production in an endotoxemia mouse model. Importantly, structure modification reveals that the inhibitory activities of UK5099 on NLRP3 are unrelated to the existence of the activated double bond within the UK5099 molecule. Thus, this study uncovers a previously unknown molecular target for UK5099, which not only offers a new candidate for the treatment of NLRP3-driven diseases but also confounds its use as an MPC inhibitor in immunometabolism studies.

A Dual-Function CD47-Targeting Nano-Drug Delivery System Used to Regulate Immune and Anti-Inflammatory Activities in the Treatment of Atherosclerosis.

Atherosclerosis is a primary contributor to cardiovascular disease. Current studies have highlighted the association between the immune system, particularly immune cells, and atherosclerosis, although treatment options and clinical trials remain scarce. Immunotherapy for cardiovascular disease is still in its infancy. Bruton's tyrosine kinase (BTK), widely expressed in various immune cells, represents a promising therapeutic target for atherosclerosis by modulating the anti-inflammatory function of immune cells. This study introduces a polydopamine-based nanocarrier system to deliver the BTK inhibitor, ibrutinib, to atherosclerotic plaques with an active targeting property via an anti-CD47 antibody. Leveraging polydopamine's pH-sensitive reversible disassembly, the system offers responsive, controlled release within the pathologic microenvironment. This allows precise and efficient ibrutinib delivery, concurrently inhibiting the activation of the NF-κB pathway in B cells and the NLRP3 inflammasome in macrophages within the plaques. This treatment also modulates both the immune cell microenvironment and inflammatory conditions in atherosclerotic lesions, thereby conveying promising therapeutic effects for atherosclerosis in vivo. This strategy also provides a novel option for atherosclerosis treatment.

Dual Regulation of Nicotine on NLRP3 Inflammasome in Macrophages with the Involvement of Lysosomal Destabilization, ROS and α7nAChR.

Nicotine, the primary alkaloid in tobacco products, has been shown to have immunoregulatory function in at least 20 diseases. The biological mechanism of action of nicotine immunoregulation is complex, resulting in an improvement of some disease states and exacerbation of others. Given the central role of the NLRP3 inflammasome in macrophages among multiple inflammatory diseases, this study examined how nicotine alters NLRP3 inflammasome activation in macrophages. NLRP3 inflammasome activation was examined mechanistically in the context of different nicotine dosages. We show NLRP3 inflammasome activation, apoptosis-associated speck-like protein (ASC) expression, caspase-1 activity and subsequent IL-1β secretion were positively correlated with nicotine in a dose-dependent relationship, and destabilization of lysosomes and ROS production were also involved. At high concentrations of nicotine surpassing 0.25mM, NLRP3 inflammasome activity declined, along with increased expression of the anti-inflammatory Alpha7 nicotinic acetylcholine receptor (α7nAChR) and the inhibition of TLR4/NF-κB signaling. Consequently, high doses of nicotine also reduced ASC expression, caspase-1 activity and IL-1β secretion in macrophages. Collectively, these results suggest a dual regulatory function of nicotine on NLRP3 inflammasome activation in macrophages, that is involved with the pro-inflammatory effects of lysosomal destabilization and ROS production. We also show nicotine mediates anti-inflammatory effects by activating α7nAChR at high doses.

Abstract 108: Inflammasome Activation Via BRCC3-mediated NLRP3 Deubiquitylation Promotes Atherosclerosis In Tet2 Clonal Hematopoiesis

Clonal hematopoiesis (CH) has emerged as an independent risk factor for atherosclerotic cardiovascular disease (CVD) with activation of macrophage inflammasomes as a potential underlying mechanism. The NLRP3 inflammasome has a key role in promoting atherosclerosis in mouse models of Tet2 CH, while inhibition of the inflammasome product IL-1β appeared to particularly benefit patients with TET2 CH in CANTOS. TET2 is an epigenetic modifier that decreases promoter methylation. However, the mechanisms underlying macrophage NLRP3 inflammasome activation in TET2 deficiency and potential links with epigenetic modifications are poorly understood. We used cholesterol-loaded TET2 deficient murine and embryonic stem cell derived isogenic human macrophages to evaluate mechanisms of NLRP3 inflammasome activation in vitro and hypercholesterolemic Ldlr -/- mice modeling TET2 CH to assess the role of NLRP3 inflammasome activation in atherosclerosis. Tet2 deficiency acted synergistically with cholesterol loading in cell culture and with hypercholesterolemia in vivo to increase JNK1 phosphorylation and NLRP3 inflammasome activation. The mechanism of JNK activation in TET2 deficiency was increased promoter methylation and decreased expression of the JNK-inactivating dual specificity phosphatase, Dusp10 . Active Tet1-deadCas9 targeted editing of Dusp10 promoter methylation abolished cholesterol-induced inflammasome activation in Tet2 -deficient macrophages. Increased JNK1 signaling led to NLRP3 deubiquitylation and activation by the deubiquitinase BRCC3. Accelerated atherosclerosis and NETosis in Tet2 CH mice were reversed by holomycin, a BRCC3 deubiquitinase inhibitor, and also by hematopoietic deficiency of Abro1 , an essential scaffolding protein in the BRCC3-containing cytosolic complex. Hypercholesterolemia and TET2 deficiency converge on a common pathway of NLRP3 inflammasome activation mediated by JNK1 activation and BRCC3-mediated NLRP3 deubiquitylation with potential therapeutic implications for the prevention of CVD in TET2 CH.

The IRG1–itaconate axis protects from cholesterol-induced inflammation and atherosclerosis

Atherosclerosis is fueled by a failure to resolve lipid-driven inflammation within the vasculature that drives plaque formation. Therapeutic approaches to reverse atherosclerotic inflammation are needed to address the rising global burden of cardiovascular disease (CVD). Recently, metabolites have gained attention for their immunomodulatory properties, including itaconate, which is generated from the tricarboxylic acid-intermediate cis-aconitate by the enzyme Immune Responsive Gene 1 (IRG1/ACOD1). Here, we tested the therapeutic potential of the IRG1-itaconate axis for human atherosclerosis. Using single-cell RNA sequencing (scRNA-seq), we found that IRG1 is up-regulated in human coronary atherosclerotic lesions compared to patient-matched healthy vasculature, and in mouse models of atherosclerosis, where it is primarily expressed by plaque monocytes, macrophages, and neutrophils. Global or hematopoietic Irg1-deficiency in mice increases atherosclerosis burden, plaque macrophage and lipid content, and expression of the proatherosclerotic cytokine interleukin (IL)-1β. Mechanistically, absence of Irg1 increased macrophage lipid accumulation, and accelerated inflammation via increased neutrophil extracellular trap (NET) formation and NET-priming of the NLRP3-inflammasome in macrophages, resulting in increased IL-1β release. Conversely, supplementation of the Irg1-itaconate axis using 4-octyl itaconate (4-OI) beneficially remodeled advanced plaques and reduced lesional IL-1β levels in mice. To investigate the effects of 4-OI in humans, we leveraged an ex vivo systems-immunology approach for CVD drug discovery. Using CyTOF and scRNA-seq of peripheral blood mononuclear cells treated with plasma from CVD patients, we showed that 4-OI attenuates proinflammatory phospho-signaling and mediates anti-inflammatory rewiring of macrophage populations. Our data highlight the relevance of pursuing IRG1-itaconate axis supplementation as a therapeutic approach for atherosclerosis in humans.

VCP Inhibition Augments NLRP3 Inflammasome Activation.

Lysosomal membrane permeabilization caused either via phagocytosis of particulates or the uptake of protein aggregates can trigger the activation of NLRP3 inflammasome- an intense inflammatory response that drives the release of the pro-inflammatory cytokine IL-1β by regulating the activity of CASPASE 1. The maintenance of lysosomal homeostasis and lysosomal membrane integrity is facilitated by the AAA+ ATPase, VCP/p97 (VCP). However, the relationship between VCP and NLRP3 inflammasome activity remains unexplored. Here, we demonstrate that the VCP inhibitors, DBeQ and ML240 elicit the activation of NLRP3 inflammasome in bone marrow-derived macrophages (BMDMs) when used as activation stimuli. Moreover, genetic inhibition of VCP or VCP chemical inhibition enhances lysosomal membrane damage and augments LLoME-associated NLRP3 inflammasome activation in BMDMs. Similarly, VCP inactivation also augments NLRP3 inflammasome activation mediated by aggregated alpha-synuclein fibrils and lysosomal damage. These data suggest that VCP is a participant in the complex regulation of NLRP3 inflammasome activation.

Stigmasterol alleviates neuropathic pain by reducing Schwann cell-macrophage cascade in DRG by modulating IL-34/CSF1R.

This study aimed to investigate the potential therapeutic applications of stigmasterol for treating neuropathic pain. Related mechanisms were investigated by DRG single-cell sequencing analysis and the use of specific inhibitors in cellular experiments. In animal experiments, 32 male Sprague-Dawley rats were randomly divided into the sham operation group, CCI group, ibuprofen group, and stigmasterol group. We performed behavioral tests, ELISA, H&E staining and immunohistochemistry, and western blotting. Cell communication analysis by single-cell sequencing reveals that after peripheral nerve injury, Schwann cells secrete IL-34 to act on CSF1R in macrophages. After peripheral nerve injury, the mRNA expression levels of CSF1R pathway and NLRP3 inflammasome in macrophages were increased in DRG. Invitro studies demonstrated that stigmasterol can reduce the secretion of IL-34 in LPS-induced RSC96 Schwann cells; stigmasterol treatment of LPS-induced Schwann cell-conditioned medium (L-S-CM) does not induce the proliferation and migration of RAW264.7 macrophages; L-S-CM reduces CSF1R signaling pathway (CSF1R, P38MAPK, and NFκB) activation, NLRP3 inflammasome activation, and ROS production. Invivo experiments have verified that stigmasterol can reduce thermal and cold hyperalgesia in rat chronic compressive nerve injury (CCI) model; stigmasterol can reduce IL-1β, IL-6, TNF-α, CCL2, SP, and PGE2 in serum of CCI rats; immunohistochemistry and western blot confirmed that stigmasterol can reduce the levels of IL-34/CSF1R signaling pathway and NLRP3 inflammasome in DRG of CCI rats. Stigmasterol alleviates neuropathic pain by reducing Schwann cell-macrophage cascade in DRG by modulating IL-34/CSF1R axis.

A stress sensor, IRE1α, is required for bacterial-exotoxin-induced interleukin-1β production in tissue-resident macrophages

Cholera toxin (CT), a bacterial exotoxin composed of one A subunit (CTA) and five B subunits (CTB), functions as an immune adjuvant. CTB can induce production of interleukin-1β (IL-1β), a proinflammatory cytokine, in synergy with a lipopolysaccharide (LPS), from resident peritoneal macrophages (RPMs) through the pyrin and NLRP3 inflammasomes. However, how CTB or CT activates these inflammasomes in the macrophages has been unclear. Here, we clarify the roles of inositol-requiring enzyme 1 alpha (IRE1α), an endoplasmic reticulum (ER) stress sensor, in CT-induced IL-1β production in RPMs. In RPMs, CTB is incorporated into the ER and induces ER stress responses, depending on GM1, a cell membrane ganglioside. IRE1α-deficient RPMs show a significant impairment of CT- or CTB-induced IL-1β production, indicating that IRE1α is required for CT- or CTB-induced IL-1β production in RPMs. This study demonstrates the critical roles of IRE1α in activation of both NLRP3 and pyrin inflammasomes in tissue-resident macrophages.

MS4A6D Promotes carrageenan-induced footpad swelling in mice through enhancing macrophages-derived inflammation

Our previous work has demonstrated that the tetraspan MS4A6D interacts with MHC-II to be a complex that promotes macrophage activation (Mol Immunol. 2023; 160: 121–132), however, the exact role of MS4A6D in controlling macrophage-derived inflammation is still poorly understood. Here, we showed that Ms4a6d-deficient (Ms4a6d-/-) mice manifested a lower level of footpad swelling induced by subcutaneous injection of 100 μL of 1% Carrageenan (CGN, w/v) plus CaCl2 (50 mM), a phenomenon that is similar to Nlrp3-/-, Casp-1-/-, and Ilr1-/- mice. Mechanistically, F4/80+ macrophages infiltrated in the footpad tissues of the Ms4A6d-/- mice was significantly lower than that of the WT littermates, leading to dramatically lower levels of proIL-1β in vivo. Moreover, macrophages from Ms4a6d-/- mice also showed a dramatical reduction of Il-1β secretion following NLRP3 inflammsome activation in vitro. Interestingly, both Ms4a6dC237G mutant (Interruption of MS4A6D homodimerization) and Ms4a6dY241G mutant (deletion of heITAM motif) mice also significantly inhibited CGN-induced footpad swelling due to lower levels of Il-1β secretion in vivo. Collectively, MS4A6D aggravates CGN-induced footpad swelling in mice by enhancing NLRP3 inflammasome in macrophages and inducing the release of IL-1β, indicating that MS4A6D promotes the progression of acute inflammation.

Widespread S-persulfidation in activated macrophages as a protective mechanism against oxidative-inflammatory stress

Acute inflammatory responses often involve the production of reactive oxygen and nitrogen species by innate immune cells, particularly macrophages. How activated macrophages protect themselves in the face of oxidative-inflammatory stress remains a long-standing question. Recent evidence implicates reactive sulfur species (RSS) in inflammatory responses; however, how endogenous RSS affect macrophage function and response to oxidative and inflammatory insults remains poorly understood. In this study, we investigated the endogenous pathways of RSS biogenesis and clearance in macrophages, with a particular focus on exploring how hydrogen sulfide (H2S)-mediated S-persulfidation influences macrophage responses to oxidative-inflammatory stress. We show that classical activation of mouse or human macrophages using lipopolysaccharide and interferon-γ (LPS/IFN-γ) triggers substantial production of H2S/RSS, leading to widespread protein persulfidation. Biochemical and proteomic analyses revealed that this surge in cellular S-persulfidation engaged ∼2% of total thiols and modified over 800 functionally diverse proteins. S-persulfidation was found to be largely dependent on the cystine importer xCT and the H2S-generating enzyme cystathionine γ-lyase and was independent of changes in the global proteome. We further investigated the role of the sulfide-oxidizing enzyme sulfide quinone oxidoreductase (SQOR), and found that it acts as a negative regulator of S-persulfidation. Elevated S-persulfidation following LPS/IFN-γ stimulation or SQOR inhibition was associated with increased resistance to oxidative stress. Upregulation of persulfides also inhibited the activation of the macrophage NLRP3 inflammasome and provided protection against inflammatory cell death. Collectively, our findings shed light on the metabolism and effects of RSS in macrophages and highlight the crucial role of persulfides in enabling macrophages to withstand and alleviate oxidative-inflammatory stress.