Promotes Inflammasome Activation Research Articles

Characterization of AB598, a CD39 Enzymatic Inhibitory Antibody for the Treatment of Solid Tumors.

AB598 is a CD39 inhibitory antibody being pursued for the treatment of solid tumors in combination with chemotherapy and immunotherapy. CD39 metabolizes extracellular ATP (eATP), an alarmin capable of promoting anti-tumor immune responses, into adenosine, an immuno-inhibitory metabolite. By inhibiting CD39, the consumption of eATP is reduced, resulting in a pro-inflammatory milieu in which eATP can activate myeloid cells to promote anti-tumor immunity. The preclinical characterization of AB598 provides a mechanistic rationale for combining AB598 with chemotherapy in the clinic. Chemotherapy can induce ATP release from tumor cells and, when preserved by AB598, both chemotherapy-induced eATP and exogenously added ATP promote the function of monocyte-derived dendritic cells via P2Y11 signaling. Inhibition of CD39 in the presence of ATP can promote inflammasome activation in in vitro-derived macrophages, an effect mediated by P2X7. In a MOLP8 murine xenograft model, AB598 results in full inhibition of intratumoral enzymatic activity, an increase in intratumoral ATP, a decrease of extracellular CD39 on tumor cells, and ultimately, control of tumor growth. In cynomolgus monkeys, systemically dosed AB598 results in effective enzymatic inhibition in tissues, full peripheral and tissue target engagement, and a reduction in cell surface CD39 both in tissues and in the periphery. Taken together, these data support a promising therapeutic strategy of harnessing the eATP generated by standard-of-care chemotherapies to prime the tumor microenvironment for a productive anti-tumor immune response.

NLRP3 Cys126 palmitoylation by ZDHHC7 promotes inflammasome activation

Nucleotide oligomerization domain (NOD)-like receptor protein 3 (NLRP3) inflammasome hyperactivation contributes to many human chronic inflammatory diseases, and understanding how NLRP3 inflammasome is regulated can provide strategies to treat inflammatory diseases. Here, we demonstrate that NLRP3 Cys126 is palmitoylated by zinc finger DHHC-type palmitoyl transferase 7 (ZDHHC7), which is critical for NLRP3-mediated inflammasome activation. Perturbing NLRP3 Cys126 palmitoylation by ZDHHC7 knockout, pharmacological inhibition, or modification site mutation diminishes NLRP3 activation in macrophages. Furthermore, Cys126 palmitoylation is vital for inflammasome activation invivo. Mechanistically, ZDHHC7-mediated NLRP3 Cys126 palmitoylation promotes resting NLRP3 localizing on the trans-Golgi network (TGN) and activated NLRP3 on the dispersed TGN, which is indispensable for recruitment and oligomerization of the adaptor ASC (apoptosis-associated speck-like protein containing a CARD). The activation of NLRP3 by ZDHHC7 is different from the termination effect mediated by ZDHHC12, highlighting versatile regulatory roles of S-palmitoylation. Our study identifies an important regulatory mechanism of NLRP3 activation that suggests targeting ZDHHC7 or the NLRP3 Cys126 residue as a potential therapeutic strategy to treat NLRP3-related human disorders.

LRRK2 G2019S Promotes Colon Cancer Potentially via LRRK2-GSDMD Axis-Mediated Gut Inflammation.

Leucine-rich repeat kinase 2 (LRRK2) is a serine-threonine protein kinase belonging to the ROCO protein family. Within the kinase domain of LRRK2, a point mutation known as LRRK2 G2019S has emerged as the most prevalent variant associated with Parkinson's disease. Recent clinical studies have indicated that G2019S carriers have an elevated risk of cancers, including colon cancer. Despite this observation, the underlying mechanisms linking LRRK2 G2019S to colon cancer remain elusive. In this study, employing a colitis-associated cancer (CAC) model and LRRK2 G2019S knock-in (KI) mouse model, we demonstrate that LRRK2 G2019S promotes the pathogenesis of colon cancer, characterized by increased tumor number and size in KI mice. Furthermore, LRRK2 G2019S enhances intestinal epithelial cell proliferation and inflammation within the tumor microenvironment. Mechanistically, KI mice exhibit heightened susceptibility to DSS-induced colitis, with inhibition of LRRK2 kinase activity ameliorating colitis severity and CAC progression. Our investigation also reveals that LRRK2 G2019S promotes inflammasome activation and exacerbates gut epithelium necrosis in the colitis model. Notably, GSDMD inhibitors attenuate colitis in LRRK2 G2019S KI mice. Taken together, our findings offer experimental evidence indicating that the gain-of-kinase activity in LRRK2 promotes colorectal tumorigenesis, suggesting LRRK2 as a potential therapeutic target in colon cancer patients exhibiting hyper LRRK2 kinase activity.

BRCC3-Mediated NLRP3 Deubiquitylation Promotes Inflammasome Activation and Atherosclerosis in Tet2 Clonal Hematopoiesis.

Clonal hematopoiesis (CH) has emerged as an independent risk factor for atherosclerotic cardiovascular disease, with activation of macrophage inflammasomes as a potential underlying mechanism. The NLRP3 (NLR family pyrin domain containing 3) inflammasome has a key role in promoting atherosclerosis in mouse models of Tet2 CH, whereas inhibition of the inflammasome product interleukin-1β appeared to particularly benefit patients with TET2 CH in CANTOS (Cardiovascular Risk Reduction Study [Reduction in Recurrent Major CV Disease Events]). TET2 is an epigenetic modifier that decreases promoter methylation. However, the mechanisms underlying macrophage NLRP3 inflammasome activation in TET2 (Tet methylcytosine dioxygenase 2) 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 in murine macrophages acted synergistically with cholesterol loading in cell culture and with hypercholesterolemia in vivo to increase JNK1 (c-Jun N-terminal kinase 1) phosphorylation and NLRP3 inflammasome activation. The mechanism of JNK (c-Jun N-terminal kinase) 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 (BRCA1/BRCA2-containing complex subunit 3). Accelerated atherosclerosis and neutrophil extracellular trap formation (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. Human TET2-/- macrophages displayed increased JNK1 and NLRP3 inflammasome activation, especially after cholesterol loading, with reversal by holomycin treatment, indicating human relevance. 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 cardiovascular disease in TET2 CH.

Inflammasomes and Atherosclerosis: a Mixed Picture.

The CANTOS (Canakinumab Anti-inflammatory Thrombosis Outcome Study) and colchicine trials suggest an important role of inflammasomes and their major product IL-1β (interleukin 1β) in human atherosclerotic cardiovascular disease. Moreover, studies in mouse models indicate a causal role of inflammasomes and IL-1β in atherosclerosis. However, recent studies have led to a more granular view of the role of inflammasomes in atherosclerosis. Studies in hyperlipidemic mouse models suggest that prominent activation of the NLRP3 inflammasome requires a second hit such as defective cholesterol efflux, defective DNA repair, clonal hematopoiesis or diabetes. Similarly in humans some mutations promoting clonal hematopoiesis increase coronary artery disease risk in part by promoting inflammasome activation. Recent studies in mice and humans point to a wider role of the AIM2 (absent in melanoma 2) inflammasome in promoting cardiovascular disease including in some forms of clonal hematopoiesis and diabetes. These developments suggest a precision medicine approach in which treatments targeting inflammasomes or IL-1β might be best employed in clinical settings involving increased inflammasome activation.

Gasdermin-D activation promotes NLRP3 activation and host resistance to Leishmania infection

Intracellular parasites from the Leishmania genus cause Leishmaniasis, a disease affecting millions of people worldwide. NLRP3 inflammasome is key for disease outcome, but the molecular mechanisms upstream of the inflammasome activation are still unclear. Here, we demonstrate that despite the absence of pyroptosis, Gasdermin-D (GSDMD) is active at the early stages of Leishmania infection in macrophages, allowing transient cell permeabilization, potassium efflux, and NLRP3 inflammasome activation. Further, GSDMD is processed into a non-canonical 25 kDa fragment. Gsdmd–/– macrophages and mice exhibit less NLRP3 inflammasome activation and are highly susceptible to infection by several Leishmania species, confirming the role of GSDMD for inflammasome-mediated host resistance. Active NLRP3 inflammasome and GSDMD are present in skin biopsies of patients, demonstrating activation of this pathway in human leishmaniasis. Altogether, our findings reveal that Leishmania subverts the normal functions of GSDMD, an important molecule to promote inflammasome activation and immunity in Leishmaniasis.

Cytosolic galectin-4 enchains bacteria, restricts their motility, and promotes inflammasome activation in intestinal epithelial cells

Galectin-4, a member of the galectin family of animal glycan-binding proteins (GBPs), is specifically expressed in gastrointestinal epithelial cells and is known to be able to bind microbes. However, its function in host-gut microbe interactions remains unknown. Here, we show that intracellular galectin-4 in intestinal epithelial cells (IECs) coats cytosolic Salmonella enterica serovar Worthington and induces the formation of bacterial chains and aggregates. Galectin-4 enchains bacteria during their growth by binding to the O-antigen of lipopolysaccharides. Furthermore, the binding of galectin-4 to bacterial surfaces restricts intracellular bacterial motility. Galectin-4 enhances caspase-1 activation and mature IL-18 production in infected IECs especially when autophagy is inhibited. Finally, orally administered S. enterica serovar Worthington, which is recognized by human galectin-4 but not mouse galectin-4, translocated from the intestines to mesenteric lymph nodes less effectively in human galectin-4-transgenic mice than in littermate controls. Our results suggest that galectin-4 plays an important role in host-gut microbe interactions and prevents the dissemination of pathogens. The results of the study revealed a novel mechanism of host-microbe interactions that involves the direct binding of cytosolic lectins to glycans on intracellular microbes.

TRIM50 promotes NLRP3 inflammasome activation by directly inducing NLRP3 oligomerization.

Tripartite motif protein (TRIM) 50 is a new member of the tripartite motif family, and its biological function and the molecular mechanism it is involved in remain largely unknown. The NOD-like receptor family protein (NLRP)3 inflammasome is actively involved in a wide array of biological processes while mechanisms of its regulation remain to be fully clarified. Here, we demonstrate the role of TRIM50 in NLRP3 inflammasome activation. In contrast to the conventional E3 ligase functions of TRIM proteins, TRIM50 mediates direct oligomerization of NLRP3, thereby suppressing its ubiquitination and promoting inflammasome activation. Mechanistically, TRIM50 directly interacts with NLRP3 through its RING domain and induces NLRP3 oligomerization via its coiled-coil domain. Finally, we show that TRIM50 promotes NLRP3 inflammasome-mediated diseases in mice. We thus reveal a novel regulatory mechanism of NLRP3 via TRIM50 and suggest that modulating TRIM50 might represent a therapeutic strategy for NLRP3-dependent pathologies.

USP3 deubiquitinates and stabilizes the adapter protein ASC to regulate inflammasome activation.

Inflammasomes are essential components of the innate immune system and its defense against infections, whereas the dysregulation of inflammasome activation has a detrimental effect on human health. The activation of inflammasomes is subjected to tight regulation to maintain immune homeostasis, yet the underlying mechanism remains elusive. Here, we identify USP3 as a direct deubiquitinating enzyme (DUB) for ASC, the central adapter mediating the assembly and activation of most inflammasomes. USP3 removes the K48-linked ubiquitination on ASC and strengthens its stability by blocking proteasomal degradation. Additionally, USP3 promotes inflammasome activation, and this function was confirmed in mouse models of aluminum (Alum)-induced peritonitis, F. novicida infection and flagellin-induced pneumonia in vivo. Our work unveils that USP3 functions as a key regulator of ASC ubiquitination and maintains the physiological role of ASC in mediating inflammasome activation, and we propose a new mechanism by which the ubiquitination of ASC regulates inflammasome activation.

Toxoplasma gondii Rhoptry Protein 7 (ROP7) Interacts with NLRP3 and Promotes Inflammasome Hyperactivation in THP-1-Derived Macrophages.

Toxoplasma gondii is a common opportunistic protozoan pathogen that can parasitize the karyocytes of humans and virtually all other warm-blooded animals. In the host’s innate immune response to T. gondii infection, inflammasomes can mediate the maturation of pro-IL-1β and pro-IL-18, which further enhances the immune response. However, how intercellular parasites specifically provoke inflammasome activation remains unclear. In this study, we found that the T. gondii secretory protein, rhoptry protein 7 (ROP7), could interact with the NACHT domain of NLRP3 through liquid chromatography-mass spectrometry analysis and co-immunoprecipitation assays. When expressing ROP7 in differentiated THP-1 cells, there was significant up-regulation in NF-κB and continuous release of IL-1β. This process is pyroptosis-independent and leads to inflammasome hyperactivation through the IL-1β/NF-κB/NLRP3 feedback loop. The loss of ROP7 in tachyzoites did not affect parasite proliferation in host cells but did attenuate parasite-induced inflammatory activity. In conclusion, these findings unveil that a T. gondii-derived protein is able to promote inflammasome activation, and further study of ROP7 will deepen our understanding of host innate immunity to parasites.

Trikafta Rescues CFTR and Lowers Monocyte P2X7R-induced Inflammasome Activation in Cystic Fibrosis.

Rationale: Cystic fibrosis (CF) is caused by mutations in the CFTR (CF transmembrane conductance regulator) gene and is characterized by sustained inflammation. ATP triggers IL-1β secretion via P2X7R (P2X7 receptor) and activation of the NLRP3 (NOD-, LRR-, and pyrin domain-containing protein 3) inflammasome. Objectives: To explore the effect of the CFTR modulator elexacaftor/tezacaftor/ivacaftor (Trikafta) on CFTR expression and the ATP/P2X7R signaling axis in monocytes and on circulating proinflammatory markers. Methods: Inflammatory mediators were detected in blood from 42 patients with CF before and after 3 months of Trikafta therapy. Markers of inflammasome activation and IL-1β secretion were measured in monocytes before and after stimulation with ATP and LPS, in the presence or absence of the P2X7R inhibitor A438079. Measurements and Main Results: P2X7R is overexpressed in CF monocytes, and receptor inhibition decreased NLRP3 expression, caspase-1 activation, and IL-1β secretion. In vitro and invivo, P2X7R expression is regulated by CFTR function and intracellular chloride (Cl-) levels. Trikafta therapy restored CFTR expression yet decreased P2X7R in CF monocytes, resulting in normalized Cl- and potassium efflux, and reduced intracellular calcium levels. CFTR modulator therapy decreased circulating levels of ATP and LPS and reduced inflammasome activation and IL-1β secretion. Conclusions: P2X7R expression is regulated by intracellular Cl- levels and in CF monocytes promotes inflammasome activation. Trikafta therapy significantly increased CFTR protein expression and reduced ATP/P2X7R-induced inflammasome activation. P2X7R may therefore be a promising target for reducing inflammation in patients with CF who are noneligible for Trikafta or other CFTR modulator therapy.

Sensing low intracellular potassium by NLRP3 results in a stable open structure that promotes inflammasome activation.

The NLRP3 inflammasome is activated by a wide range of stimuli and drives diverse inflammatory diseases. The decrease of intracellular K+ concentration is a minimal upstream signal to most of the NLRP3 activation models. Here, we found that cellular K+ efflux induces a stable structural change in the inactive NLRP3, promoting an open conformation as a step preceding activation. This conformational change is facilitated by the specific NLRP3 FISNA domain and a unique flexible linker sequence between the PYD and FISNA domains. This linker also facilitates the ensemble of NLRP3PYD into a seed structure for ASC oligomerization. The introduction of the NLRP3 PYD-linker-FISNA sequence into NLRP6 resulted in a chimeric receptor able to be activated by K+ efflux–specific NLRP3 activators and promoted an in vivo inflammatory response to uric acid crystals. Our results establish that the amino-terminal sequence between PYD and NACHT domain of NLRP3 is key for inflammasome activation.

FUNDC1 Regulates Autophagy by Inhibiting ROS-NLRP3 Signaling to Avoid Apoptosis in the Lung in a Lipopolysaccharide-Induced Mouse Model.

The incidence and mortality of acute respiratory distress syndrome (ARDS) are high, but the relevant mechanism for this disorder remains unclear. Autophagy plays an important role in the development of ARDS. The mitochondrial outer membrane protein FUNDC1 is involved in hypoxia-mediated mitochondrial autophagy, which may contribute to ARDS development. This study explored whether FUNDC1 regulates autophagy by inhibiting ROS-NLRP3 signaling to avoid apoptosis in the lung in a lipopolysaccharide-induced mouse model. In this study, FUNDC1 knockout mice were constructed, and a lipopolysaccharide-induced mouse model was generated. HE staining of pathological sections from the lung, wet/dry lung measurements, myeloperoxidase concentration/neutrophil counts in BALF and survival time of mice were examined to determine the effect of modeling. The release of cytokines (TNF-α, IL-1β, IL-6, and IL-10) in response to LPS in the BALF and plasma was assessed using ELISA. The effects of oxidative stress (malondialdehyde, superoxide dismutase, catalase, glutathione peroxidase) in lung tissue in response to LPS were detected by biochemical analysis. Oxidative stress damage was validated by iNOS staining, and apoptosis was assessed by TUNEL staining after LPS. Finally, the expression of autophagy-associated proteins and inflammasome-associated proteins in lung tissue after LPS intervention was analyzed by western blot. We found that wild-type control, FUNDC1 knockout control, lipopolysaccharide-induced wild-type, and FUNDC1 knockout mouse models were used to investigate whether FUNDC1-mediated autophagy is involved in lung injury and its possible molecular mechanisms. Compared with the normal control group, lung tissue FUNDC1 and LC3 II increased and p62/SQSTM1 decreased after LPS intervention, and increased ROS levels led to a decrease in corresponding antioxidant enzymes along with an increased inflammatory response and apoptosis. Levels of autophagy in lipopolysaccharide-induced mice deficient in FUNDC1 were significantly decreased, but the expression of ROS and inflammatory factors in lung tissue was more severe than in lipopolysaccharide-induced wild-type mice, and the survival rate was significantly decreased. Western blot analysis showed that autophagy was significantly inhibited in the FUNDC1 KO+LPS group, and there was a significant increase in NLRP3, caspase-1, IL-1β, and ASC compared with the lipopolysaccharide-induced wild-type group. In summary, lipopolysaccharide-induced wild-type mice exhibit ROS-dependent activation of autophagy, and knocking out FUNDC1 promotes inflammasome activation and exacerbates lung injury.

FXR/TGR5 mediates inflammasome activation and host resistance to bacterial infection

Bacterial infections are a major cause of chronic infections and mortality. Innate immune control is crucial for protection against bacterial pathogens. Bile acids facilitate intestinal absorption of lipid-soluble nutrients and modulate various metabolic pathways through the farnesoid X receptor (FXR) and Takeda G-protein-coupled receptor 5 (TGR5). Here, we identified a new role of FXR and TGR5 in promoting inflammasome activation during bacterial infection. Caspase-1/11 activation and release of cleaved interleukin (IL)-1β in FXR- and TGR5-deficient mouse bone marrow-derived macrophages upon Listeria monocytogenes or Escherichia coli infection was significantly reduced. In contrast, FXR- or TGR5-deficiency did not affect the transcription of caspase-1/11 and IL-1β. Inflammasome activation is critical for host immune defense against bacterial infections. Consistent with this, the deletion of FXR or TGR5 impaired effective clearance of L. monocytogenes or E. coli in vitro and in vivo, which was associated with greater mortality and bacterial burden than that of wild-type mice. Pretreatment with an FXR agonist decreased bacterial burden in vitro and increased survival in vivo. Thus, FXR and TGR5 promote inflammasome-mediated antimicrobial responses and may represent novel antibacterial therapeutic targets.

Exosome-inflammasome crosstalk and their roles in inflammatory responses.

Inflammasome is a complex of multiple proteins found in cytoplasm of the cells activated by infectious and/or non-infectious stimuli. This complex involves caspase-1 activation, leading to unconventional secretion of interleukin-1β (IL-1β) and IL-18 and inflammatory cascade. Exosome is the nanoscale membrane-bound extracellular vesicle that plays significant roles in intercellular communications by carrying bioactive molecules, e.g., proteins, RNAs, microRNAs (miRNAs), DNAs, from one cell to the others. In this review, we provide the update information on the crosstalk between exosome and inflammasome and their roles in inflammatory responses. The effects of inflammasome activation on exosomal secretion are summarized. On the other hand, the (dual) effects of exosomes on inhibiting and promoting inflammasome activation are discussed. Finally, perspectives on therapeutic roles of exosomes in human diseases and future direction of the research on exosome-inflammasome crosstalk are provided.

SIRT3-mediated deacetylation of NLRC4 promotes inflammasome activation.

Salmonella typhimurium (S. typhimurium) infection of macrophage induces NLRC4 inflammasome-mediated production of the pro-inflammatory cytokines IL-1β. Post-translational modifications on NLRC4 are critical for its activation. Sirtuin3 (SIRT3) is the most thoroughly studied mitochondrial nicotinamide adenine dinucleotide (NAD+) -dependent deacetylase. We wondered whether SIRT3 mediated-deacetylation could take part in NLRC4 inflammasome activation.Methods: We initially tested IL-1β production and pyroptosis after cytosolic transfection of flagellin or S. typhimurium infection in wild type and SIRT3-deficient primary peritoneal macrophages via immunoblotting and ELISA assay. These results were confirmed in SIRT3-deficient immortalized bone marrow derived macrophages (iBMDMs) which were generated by CRISPR-Cas9 technology. In addition, in vivo experiments were conducted to confirm the role of SIRT3 in S. typhimurium-induced cytokines production. Then NLRC4 assembly was analyzed by immune-fluorescence assay and ASC oligomerization assay. Immunoblotting, ELISA and flow cytometry were performed to clarify the role of SIRT3 in NLRP3 and AIM2 inflammasomes activation. To further investigate the mechanism of SIRT3 in NLRC4 activation, co-immunoprecipitation (Co-IP), we did immunoblot, cellular fractionation and in-vitro deacetylation assay. Finally, to clarify the acetylation sites of NLRC4, we performed liquid chromatography-mass spectrometry (LC-MS) and immunoblotting analysis.Results: SIRT3 deficiency led to significantly impaired NLRC4 inflammasome activation and pyroptosis both in vitro and in vivo. Furthermore, SIRT3 promotes NLRC4 inflammasome assembly by inducing more ASC speck formation and ASC oligomerization. However, SIRT3 is dispensable for NLRP3 and AIM2 inflammasome activation. Moreover, SIRT3 interacts with and deacetylates NLRC4 to promote its activation. Finally, we proved that deacetylation of NLRC4 at Lys71 or Lys272 could promote its activation.Conclusions: Our study reveals that SIRT3 mediated-deacetylation of NLRC4 is pivotal for NLRC4 activation and the acetylation switch of NLRC4 may aid the clearance of S. typhimurium infection.

Short-chain fatty acids bind to apoptosis-associated speck-like protein to activate inflammasome complex to prevent Salmonella infection.

Short-chain fatty acids (SCFAs) produced by gastrointestinal microbiota regulate immune responses, but host molecular mechanisms remain unknown. Unbiased screening using SCFA-conjugated affinity nanobeads identified apoptosis-associated speck-like protein (ASC), an adaptor protein of inflammasome complex, as a noncanonical SCFA receptor besides GPRs. SCFAs promoted inflammasome activation in macrophages by binding to its ASC PYRIN domain. Activated inflammasome suppressed survival of Salmonella enterica serovar Typhimurium (S. Typhimurium) in macrophages by pyroptosis and facilitated neutrophil recruitment to promote bacterial elimination and thus inhibit systemic dissemination in the host. Administration of SCFAs or dietary fibers, which are fermented to SCFAs by gut bacteria, significantly prolonged the survival of S. Typhimurium-infected mice through ASC-mediated inflammasome activation. SCFAs penetrated into the inflammatory region of the infected gut mucosa to protect against infection. This study provided evidence that SCFAs suppress Salmonella infection via inflammasome activation, shedding new light on the therapeutic activity of dietary fiber.

Cardiolipin dynamics regulate proinflammatory cytokine production in methicillin-resistant Staphylococcus aureus--infected macrophages

Abstract During the immune response, mitochondria serve as a hub for innate immune signaling molecules, such as mitochondrial antiviral signaling protein (MAVS) and Nod Like Receptor Family Pyrin Domain Containing 3 (NLRP3). Cardiolipin, a class of phospholipids found primarily in the inner mitochondrial membrane (IMM), regulates mitochondrial function and cellular stress. In lipopolysaccharide (LPS)-stimulated macrophages, CL translocates from the IMM to the outer mitochondrial membrane (OMM) and recruits NLRP3 to promote inflammasome activation. However, the role of CL in macrophage antibacterial responses remains ill defined. Here, we have made CL-deficient RAW264.7 (RAW) cells by stably knocking down Cardiolipin Synthase (Crls1). Upon infection with methicillin-resistant Staphylococcus aureus (MRSA), Crls1 KD RAW cells showed markedly decreased expression and production of cytokines IL-6, IL-1β, and IFN-β compared to control cells, while TNF-α levels remained similar. For IL-6 and IFN-β, transcript and protein levels in Crls1 KD RAW cells were comparable to unstimulated controls. Surprisingly, these cells showed normal MRSA killing and NF-kB p65 nuclear translocation. These findings suggest a novel role for CL in transcript-level regulation of cytokines during infection. To further investigate CL-dependent innate immune functions, we will use RAW cells expressing a GFP-tagged CL-binding protein, Stomatin-like Protein-2-GFP, to track CL dynamics at membrane-level resolution using 3D structured illumination microscopy. This approach supports earlier studies that describe increased CL OMM localization in response to LPS. Our data suggest an underappreciated role for CL in macrophage responses to MRSA infection.

Brucella abortus Infection Elicited Hepatic Stellate Cell-Mediated Fibrosis Through Inflammasome-Dependent IL-1β Production.

In human brucellosis, the liver is frequently affected. Brucella abortus triggers a profibrotic response on hepatic stellate cells (HSCs) characterized by inhibition of MMP-9 with concomitant collagen deposition and TGF-β1 secretion through type 4 secretion system (T4SS). Taking into account that it has been reported that the inflammasome is necessary to induce a fibrotic phenotype in HSC, we hypothesized that Brucella infection might create a microenvironment that would promote inflammasome activation with concomitant profibrogenic phenotype in HSCs. B. abortus infection induces IL-1β secretion in HSCs in a T4SS-dependent manner. The expression of caspase-1 (Casp-1), absent in melanoma 2 (AIM2), Nod-like receptor (NLR) containing a pyrin domain 3 (NLRP3), and apoptosis-associated speck-like protein containing a CARD (ASC) was increased in B. abortus-infected HSC. When infection experiments were performed in the presence of glyburide, a compound that inhibits NLRP3 inflammasome, or A151, a specific AIM2 inhibitor, the secretion of IL-1β was significantly inhibited with respect to uninfected controls. The role of inflammasome activation in the induction of a fibrogenic phenotype in HSCs was determined by performing B. abortus infection experiments in the presence of the inhibitors Ac-YVAD-cmk and glyburide. Both inhibitors were able to reverse the effect of B. abortus infection on the fibrotic phenotype in HSCs. Finally, the role of inflammasome in fibrosis was corroborated in vivo by the reduction of fibrotic patches in liver from B. abortus-infected ASC, NLRP, AIM2, and cCasp-1/11 knock-out (KO) mice with respect to infected wild-type mice.

ROS-induced NLRP3 inflammasome priming and activation mediate PCB 118- induced pyroptosis in endothelial cells.

Growing epidemiological evidence has shown that exposure to polychlorinated biphenyls (PCBs) is harmful to the cardiovascular system. However, how PCB 118-induced oxidative stress mediates endothelial dysfunction is not fully understood. Here, we explored whether and how PCB 118 exposure-induced oxidative stress leads to NLRP3 inflammasome-dependent pyroptosis in endothelial cells. As expected, PCB 118 was cytotoxic to HUVECs and induced caspase-1 activation and cell membrane disruption, which are characteristics of pyroptosis. Moreover, PCB 118-induced pyroptosis may have been due to the activation of the NLRP3 infammasomes. PCB 118 also induced excessive reactive oxygen species (ROS) in HUVECs. The ROS scavenger (±)-α-tocopherol and the NFκB inhibitor BAY11-7082 reversed the upregulation of NLRP3 expression and the increase in NLRP3 inflammasome activation induced by PCB 118 exposure in HUVECs. Additionally, PCB 118-induced oxidative stress and pyroptosis were dependent on Aryl hydrocarbon receptor (AhR) activation and subsequent cytochrome P450 1A1 upregulation, which we confirmed by using the AhR selective antagonist CH 223191. These data suggest that PCB 118 exposure induces NLRP3 inflammasome activation and subsequently leads to pyroptosis in endothelial cells in vitro and in vivo. AhR-mediated ROS production play a central role in PCB 118-induced pyroptosis by priming NFκB-dependent NLRP3 expression and promoting inflammasome activation.