Sirtuin 3-induced macrophage autophagy in regulating NLRP3 inflammasome activation

Defective autophagy of monocytes or macrophages might result in NLRP3 inflammasome activation and cause vascular metabolic inflammation. However, the mechanism underlying the initiation of the autophagy response to hyperlipidaemia remains unclear. Sirtuin 3 (SIRT3), an NAD-dependent deacetylase, is sensitive to the metabolic status and mediates adaptation responses. In this study, we investigated the role of SIRT3-mediated autophagy in regulating NLRP3 inflammasome activation. We determined that the inhibition of autophagy and the activation of the NLRP3 inflammasome were concomitant with reduced SIRT3 levels both in peripheral blood monocytes from obese humans and in palmitate-treated THP-1 cells. Furthermore, we demonstrated that SIRT3 could form a molecular complex with ATG5, while SIRT3 overexpression altered the acetylation of endogenous ATG5. ATG5 acetylation inhibited autophagosome maturation and induced NLRP3 inflammasome activation. In parallel, SIRT3 overexpression in THP-1 cells decreased the palmitate-induced generation of mitochondrial reactive oxygen species, restored autophagy, and attenuated NLRP3 inflammasome activation. The incubation of human aortic endothelial cells (HAECs) with macrophage-conditioned medium (MCM) induced HAEC expression of vascular cell adhesion molecule-1, intercellular adhesion molecule 1, α-smooth muscle actin, and collagen-1. The effect of MCM could be reversed by the addition of neutralizing anti-IL-1β antibody or the overexpression of SIRT3. Consistent with this, en face analyses displayed a marked increase in α-SMC-positive endothelial cells in SIRT3^?/? mice with acute hyperlipidaemia. Taken together, these findings revealed that SIRT3-deficient macrophages displayed impaired autophagy and accelerated NLRP3 inflammasome activation and endothelial dysfunction.     

Ligand-dependent kinase activity of MERTK drives efferocytosis in human iPSC-derived macrophages

Removal of apoptotic cells by phagocytes (also called efferocytosis) is a crucial process for tissue homeostasis. Professional phagocytes express a plethora of surface receptors enabling them to sense and engulf apoptotic cells, thus avoiding persistence of dead cells and cellular debris and their consequent effects. Dysregulation of efferocytosis is thought to lead to secondary necrosis and associated inflammation and immune activation. Efferocytosis in primarily murine macrophages and dendritic cells has been shown to require TAM RTKs, with MERTK and AXL being critical for clearance of apoptotic cells. The functional role of human orthologs, especially the exact contribution of each individual receptor is less well studied. Here we show that human macrophages differentiated in vitro from iPSC-derived precursor cells express both AXL and MERTK and engulf apoptotic cells. TAM RTK agonism by the natural ligand growth-arrest specific 6 (GAS6) significantly enhanced such efferocytosis. Using a newly-developed mouse model of kinase-dead MERTK, we demonstrate that MERTK kinase activity is essential for efferocytosis in peritoneal macrophages in vivo. Moreover, human iPSC-derived macrophages treated in vitro with blocking antibodies or small molecule inhibitors recapitulated this observation. Hence, our results highlight a conserved MERTK function between mice and humans, and the critical role of its kinase activity in homeostatic efferocytosis.Subject terms: Immune cell death, Peritoneal macrophages     

Small molecule-driven mitophagy-mediated NLRP3 inflammasome inhibition is responsible for the prevention of colitis-associated cancer

Nonresolving inflammation in the intestine predisposes individuals to the development of colitis-associated cancer (CAC). Inflammasomes are thought to mediate intestinal homeostasis, and their dysregulation contributes to inflammatory bowel diseases and CAC. However, few agents have been reported to reduce CAC by targeting inflammasomes. Here we show that the small molecule andrographolide (Andro) protects mice against azoxymethane/dextran sulfate sodium-induced colon carcinogenesis through inhibiting the NLRP3 inflammasome. Administration of Andro significantly attenuated colitis progression and tumor burden. Andro also inhibited NLRP3 inflammasome activation in macrophages both in vivo and in vitro, as indicated by reduced expression of cleaved CASP1, disruption of NLRP3-PYCARD-CASP1 complex assembly, and lower IL1B secretion. Importantly, Andro was found to trigger mitophagy in macrophages, leading to a reversed mitochondrial membrane potential collapse, which in turn inactivated the NLRP3 inflammasome. Moreover, downregulation of the PIK3CA-AKT1-MTOR-RPS6KB1 pathway accounted for Andro-induced autophagy. Finally, Andro-driven inhibition of the NLRP3 inflammasome and amelioration of murine models for colitis and CAC were significantly blocked by BECN1 knockdown, or by various autophagy inhibitors. Taken together, our findings demonstrate that mitophagy-mediated NLRP3 inflammasome inhibition by Andro is responsible for the prevention of CAC. Our data may help guide decisions regarding the use of Andro in patients with inflammatory bowel diseases, which ultimately reduces the risk of CAC.     

Macrophage Specific Caspase-1/11 Deficiency Protects against Cholesterol Crystallization and Hepatic Inflammation in Hyperlipidemic Mice

Background & Aims

While non-alcoholic steatohepatitis (NASH) is characterized by hepatic steatosis combined with inflammation, the mechanisms triggering hepatic inflammation are unknown. In Ldlr^-/- mice, we have previously shown that lysosomal cholesterol accumulation in Kupffer cells (KCs) correlates with hepatic inflammation and cholesterol crystallization. Previously, cholesterol crystals have been shown to induce the activation of inflammasomes. Inflammasomes are protein complexes that induce the processing and release of pro-inflammatory cytokines IL-1b and IL-18 via caspase-1 activation. Whereas caspase-1 activation is independent of caspase-11 in the canonical pathway of inflammasome activation, caspase-11 was found to trigger caspase-1-dependent IL-1b and IL-18 in response to non-canonical inflammasome activators. So far, it has not been investigated whether inflammasome activation stimulates the formation of cholesterol crystals. We hypothesized that inflammasome activation in KCs stimulates cholesterol crystallization, thereby leading to hepatic inflammation.

Methods

Ldlr ^-/- mice were transplanted (tp) with wild-type (Wt) or caspase-1/11^-/- (dKO) bone marrow and fed either regular chow or a high-fat, high-cholesterol (HFC) diet for 12 weeks. In vitro, bone marrow derived macrophages (BMDM) from wt or caspase-1/11^-/- mice were incubated with oxLDL for 24h and autophagy was assessed.

Results

In line with our hypothesis, caspase-1/11^-/--tp mice had less severe hepatic inflammation than Wt-tp animals, as evident from liver histology and gene expression analysis in isolated KCs. Mechanistically, KCs from caspase-1/11^-/--tp mice showed less cholesterol crystals, enhanced cholesterol efflux and increased autophagy. In wt BMDM, oxLDL incubation led to disturbed autophagy activity whereas BMDM from caspase-1/11^-/- mice had normal autophagy activity.

Conclusion

Altogether, these data suggest a vicious cycle whereby disturbed autophagy and decreased cholesterol efflux leads to newly formed cholesterol crystals and thereby maintain hepatic inflammation during NASH by further activating the inflammasome.     

Ezetimibe ameliorates steatohepatitis via AMP activated protein kinase-TFEB-mediated activation of autophagy and NLRP3 inflammasome inhibition

Impairment in macroautophagy/autophagy flux and inflammasome activation are common characteristics of nonalcoholic steatohepatitis (NASH). Considering the lack of approved agents for treating NASH, drugs that can enhance autophagy and modulate inflammasome pathways may be beneficial. Here, we investigated the novel mechanism of ezetimibe, a widely prescribed drug for hypercholesterolemia, as a therapeutic option for ameliorating NASH. Human liver samples with steatosis and NASH were analyzed. For in vitro studies of autophagy and inflammasomes, primary mouse hepatocytes, human hepatoma cells, mouse embryonic fibroblasts with Ampk or Tsc2 knockout, and human or primary mouse macrophages were treated with ezetimibe and palmitate. Steatohepatitis and fibrosis were induced by feeding Atg7 wild-type, haploinsufficient, and knockout mice a methionine- and choline-deficient diet with ezetimibe (10 mg/kg) for 4 wk. Human livers with steatosis or NASH presented impaired autophagy with decreased nuclear TFEB and increased SQSTM1, MAP1LC3-II, and NLRP3 expression. Ezetimibe increased autophagy flux and concomitantly ameliorated lipid accumulation and apoptosis in palmitate-exposed hepatocytes. Ezetimibe induced AMPK phosphorylation and subsequent TFEB nuclear translocation, related to MAPK/ERK. In macrophages, ezetimibe blocked the NLRP3 inflammasome-IL1B pathway in an autophagy-dependent manner and modulated hepatocyte-macrophage interaction via extracellular vesicles. Ezetimibe attenuated lipid accumulation, inflammation, and fibrosis in liver-specific Atg7 wild-type and haploinsufficient mice, but not in knockout mice. Ezetimibe ameliorates steatohepatitis by autophagy induction through AMPK activation and TFEB nuclear translocation, related to an independent MTOR ameliorative effect and the MAPK/ERK pathway. Ezetimibe dampens NLRP3 inflammasome activation in macrophages by modulating autophagy and a hepatocyte-driven exosome pathway.     

Mitochondrial damage contributes to Pseudomonas aeruginosa activation of the inflammasome and is downregulated by autophagy

The nucleotide-binding domain, leucine-rich repeat containing family caspase recruitment domain containing 4 (NLRC4) inflammasome can be activated by pathogenic bacteria via products translocated through the microbial type III secretion apparatus (T3SS). Recent work has shown that activation of the NLRP3 inflammasome is downregulated by autophagy, but the influence of autophagy on NLRC4 activation is unclear. We set out to determine how autophagy might influence this process, using the bacterium Pseudomonas aeruginosa, which activates the NLRC4 inflammasome via its T3SS. Infection resulted in T3SS-dependent mitochondrial damage with increased production of reactive oxygen intermediates and release of mitochondrial DNA. Inhibiting mitochondrial reactive oxygen release or degrading intracellular mitochondrial DNA abrogated NLRC4 inflammasome activation. Moreover, macrophages lacking mitochondria failed to activate NLRC4 following infection. Removal of damaged mitochondria by autophagy significantly attenuated NLRC4 inflammasome activation. Mitochondrial DNA bound specifically to NLRC4 immunoprecipitates and transfection of mitochondrial DNA directly activated the NLRC4 inflammasome; oxidation of the DNA enhanced this effect. Manipulation of autophagy altered the degree of inflammasome activation and inflammation in an in vivo model of P. aeruginosa infection. Our results reveal a novel mechanism contributing to NLRC4 activation by P. aeruginosa via mitochondrial damage and release of mitochondrial DNA triggered by the bacterial T3SS that is downregulated by autophagy.     

Small molecule-driven mitophagy-mediated NLRP3 inflammasome inhibition is responsible for the prevention of colitis-associated cancer

Nonresolving inflammation in the intestine predisposes individuals to the development of colitis-associated cancer (CAC). Inflammasomes are thought to mediate intestinal homeostasis, and their dysregulation contributes to inflammatory bowel diseases and CAC. However, few agents have been reported to reduce CAC by targeting inflammasomes. Here we show that the small molecule andrographolide (Andro) protects mice against azoxymethane/dextran sulfate sodium-induced colon carcinogenesis through inhibiting the NLRP3 inflammasome. Administration of Andro significantly attenuated colitis progression and tumor burden. Andro also inhibited NLRP3 inflammasome activation in macrophages both in vivo and in vitro, as indicated by reduced expression of cleaved CASP1, disruption of NLRP3-PYCARD-CASP1 complex assembly, and lower IL1B secretion. Importantly, Andro was found to trigger mitophagy in macrophages, leading to a reversed mitochondrial membrane potential collapse, which in turn inactivated the NLRP3 inflammasome. Moreover, downregulation of the PIK3CA-AKT1-MTOR-RPS6KB1 pathway accounted for Andro-induced autophagy. Finally, Andro-driven inhibition of the NLRP3 inflammasome and amelioration of murine models for colitis and CAC were significantly blocked by BECN1 knockdown, or by various autophagy inhibitors. Taken together, our findings demonstrate that mitophagy-mediated NLRP3 inflammasome inhibition by Andro is responsible for the prevention of CAC. Our data may help guide decisions regarding the use of Andro in patients with inflammatory bowel diseases, which ultimately reduces the risk of CAC.     

Targeting the NLRP3 Inflammasome to Reduce Diet-Induced Metabolic Abnormalities in Mice

Although the molecular links underlying the causative relationship between chronic low-grade inflammation and insulin resistance are not completely understood, compelling evidence suggests a pivotal role of the nucleotide-binding oligomerization domain (NOD)-like receptor pyrin domain containing 3 (NLRP3) inflammasome. Here we tested the hypothesis that either a selective pharmacological inhibition or a genetic downregulation of the NLRP3 inflammasome results in reduction of the diet-induced metabolic alterations. Male C57/BL6 wild-type mice and NLRP3^−/− littermates were fed control diet or high-fat, high-fructose diet (HD). A subgroup of HD-fed wild-type mice was treated with the NLRP3 inflammasome inhibitor BAY 11-7082 (3 mg/kg intraperitoneally [IP]). HD feeding increased plasma and hepatic lipids and impaired glucose homeostasis and renal function. Renal and hepatic injury was associated with robust increases in profibrogenic markers, while only minimal fibrosis was recorded. None of these metabolic abnormalities were detected in HD-fed NLRP3^−/− mice, and they were dramatically reduced in HD-mice treated with the NLRP3 inflammasome inhibitor. BAY 11-7082 also attenuated the diet-induced increase in NLRP3 inflammasome expression, resulting in inhibition of caspase-1 activation and interleukin (IL)-1β and IL-18 production (in liver and kidney). Interestingly, BAY 11-7082, but not gene silencing, inhibited nuclear factor (NF)-κB nuclear translocation. Overall, these results demonstrate that the selective pharmacological modulation of the NLRP3 inflammasome attenuates the metabolic abnormalities and the related organ injury/dysfunction caused by chronic exposure to HD, with effects similar to those obtained by NLRP3 gene silencing.     

Glucose regulates hypoxia-induced NLRP3 inflammasome activation in macrophages

Although the intimate linkage between hypoxia and inflammation is well known, the mechanism underlying this linkage has not been fully understood. Nucleotide-binding oligomerization domain-like receptor (NLR) family pyrin domain containing 3 (NLRP3) inflammasome is an intracellular multiprotein complex that regulates interleukin-1β (IL-1β) secretion and pyroptosis, and is implicated in the pathogenesis of sterile inflammatory diseases. Here, we investigated the regulatory mechanism of NLRP3 inflammasome activation in response to hypoxia in macrophages. Severe hypoxia (0.1% O₂) induced the processing of pro-IL-1β, pro-caspase-1, and gasdermin D, as well as the release of IL-1β and lactate dehydrogenase in lipopolysaccharide (LPS)-primed murine macrophages, indicating that hypoxia induces NLRP3 inflammasome-driven inflammation and pyroptosis. NLRP3 deficiency and a specific caspase-1 blockade inhibited hypoxia-induced IL-1β release. Hypoxia-induced IL-1β release and cell death were augmented under glucose deprivation, and an addition of glucose in the media negatively regulated hypoxia-induced IL-1β release. Under hypoxia and glucose deprivation, hypoxia-induced glycolysis was not driven and subsequently, the intracellular adenosine triphosphates (ATPs) were depleted. Atomic absorption spectrometry analysis showed a reduction of intracellular K⁺ concentrations, indicating the K⁺ efflux occurring under hypoxia and glucose deprivation. Furthermore, hypoxia and glucose deprivation-induced IL-1β release was significantly prevented by inhibition of K⁺ efflux and K_ATP channel blockers. In vivo experiments further revealed that IL-1β production was increased in LPS-primed mice exposed to hypoxia (9.5% O₂), which was prevented by a deficiency of NLRP3, an apoptosis-associated speck-like protein containing a caspase recruitment domain, and caspase-1. Our results demonstrate that NLRP3 inflammasome can sense intracellular energy crisis as a danger signal induced by hypoxia and glucose deprivation, and provide new insights into the mechanism underlying hypoxia-induced inflammation.     

NLRP3 inflammasome suppression improves longevity and prevents cardiac aging in male mice

While NLRP3‐inflammasome has been implicated in cardiovascular diseases, its role in physiological cardiac aging is largely unknown. During aging, many alterations occur in the organism, which are associated with progressive impairment of metabolic pathways related to insulin resistance, autophagy dysfunction, and inflammation. Here, we investigated the molecular mechanisms through which NLRP3 inhibition may attenuate cardiac aging. Ablation of NLRP3‐inflammasome protected mice from age‐related increased insulin sensitivity, reduced IGF‐1 and leptin/adiponectin ratio levels, and reduced cardiac damage with protection of the prolongation of the age‐dependent PR interval, which is associated with atrial fibrillation by cardiovascular aging and reduced telomere shortening. Furthermore, old NLRP3 KO mice showed an inhibition of the PI3K/AKT/mTOR pathway and autophagy improvement, compared with old wild mice and preserved Nampt‐mediated NAD⁺ levels with increased SIRT1 protein expression. These findings suggest that suppression of NLRP3 prevented many age‐associated changes in the heart, preserved cardiac function of aged mice and increased lifespan.     

Encephalomyocarditis Virus Viroporin 2B Activates NLRP3 Inflammasome

Nod-like receptors (NLRs) comprise a large family of intracellular pattern- recognition receptors. Members of the NLR family assemble into large multiprotein complexes, termed the inflammasomes. The NLR family, pyrin domain-containing 3 (NLRP3) is triggered by a diverse set of molecules and signals, and forms the NLRP3 inflammasome. Recent studies have indicated that both DNA and RNA viruses stimulate the NLRP3 inflammasome, leading to the secretion of interleukin 1 beta (IL-1β) and IL-18 following the activation of caspase-1. We previously demonstrated that the proton-selective ion channel M2 protein of influenza virus activates the NLRP3 inflammasome. However, the precise mechanism by which NLRP3 recognizes viral infections remains to be defined. Here, we demonstrate that encephalomyocarditis virus (EMCV), a positive strand RNA virus of the family Picornaviridae, activates the NLRP3 inflammasome in mouse dendritic cells and macrophages. Although transfection with RNA from EMCV virions or EMCV-infected cells induced robust expression of type I interferons in macrophages, it failed to stimulate secretion of IL-1β. Instead, the EMCV viroporin 2B was sufficient to cause inflammasome activation in lipopolysaccharide-primed macrophages. While cells untransfected or transfected with the gene encoding the EMCV non-structural protein 2A or 2C expressed NLRP3 uniformly throughout the cytoplasm, NLRP3 was redistributed to the perinuclear space in cells transfected with the gene encoding the EMCV 2B or influenza virus M2 protein. 2B proteins of other picornaviruses, poliovirus and enterovirus 71, also caused the NLRP3 redistribution. Elevation of the intracellular Ca²⁺ level, but not mitochondrial reactive oxygen species and lysosomal cathepsin B, was important in EMCV-induced NLRP3 inflammasome activation. Chelation of extracellular Ca²⁺ did not reduce virus-induced IL-1β secretion. These results indicate that EMCV activates the NLRP3 inflammasome by stimulating Ca²⁺ flux from intracellular storages to the cytosol, and highlight the importance of viroporins, transmembrane pore-forming viral proteins, in virus-induced NLRP3 inflammasome activation.     

NLRP3 inflammasome negatively regulates podocyte autophagy in diabetic nephropathy

Inflammasome mechanisms are recognized as a key pathophysiology of diabetic nephropathy (DN). The nucleotide-oligomerization domain-like receptor 3 (NLRP3) inflammasome has attracted the most attention. Autophagy as a conserved intracellular catabolic pathway plays essential roles in the maintenance of podocytes. Although autophagy was involved in preventing excessive inflammatory responses in kidney diseases, a clear understanding of the regulation of NLRP3 inflammasome on autophagy in glomerular damage in DN is still lacking. In this study, we focused on the effect of the activation of NLRP3 inflammasome on the suppression of podocyte autophagy and aimed to investigate the role of autophagy in podocyte injury in DN. Podocyte autophagy has been confirmed to be inhibited in high-fat diet/streptozotocin (HFD/STZ)-induced DN mice, and NLRP3 has been found to be upregulated in both mice and human DN biopsies and in vitro. Activation of NLRP3 inflammasome exacerbated podocyte autophagy and reduced podocyte nephrin expression, while silencing of NLRP3 efficiently restored podocyte autophagy and ameliorated podocyte injury induced by high glucose. The results showed that NLRP3 was a negative regulator of autophagy and suggested that restoration of podocyte autophagy by inactivation of NLRP3 under high glucose could reduce podocyte injury. Proper modification of autophagy and inflammasome has the potential to benefit the kidney in DN.     

Tris DBA ameliorates IgA nephropathy by blunting the activating signal of NLRP3 inflammasome through SIRT1‐ and SIRT3‐mediated autophagy induction

Tris (dibenzylideneacetone) dipalladium (Tris DBA), a small‐molecule palladium complex, can inhibit cell growth and proliferation in pancreatic cancer, lymphocytic leukaemia and multiple myeloma. Given that this compound is particularly active against B‐cell malignancies, we have been suggested that it can alleviate immune complexes (ICs)–mediated conditions, especially IgA nephropathy (IgAN). The therapeutic effects of Tris DBA on glomerular cell proliferation and renal inflammation and mechanism of action were examined in a mouse model of IgAN. Treatment of IgAN mice with Tris DBA resulted in markedly improved renal function, albuminuria and renal pathology, including glomerular cell proliferation, neutrophil infiltration, sclerosis and periglomerular inflammation in the renal interstitium, together with (Clin J Am Soc Nephrol. 2011, 6, 1301‐1307) reduced mitochondrial ROS generation; (Am J Physiol‐Renal Physiol. 2011. 301, F1218‐F1230) differentially regulated autophagy and NLRP3 inflammasome; (Clin J Am Soc Nephrol. 2012, 7, 427‐436) inhibited phosphorylation of JNK, ERK and p38 MAPK signalling pathways, and priming signal of the NLRP3 inflammasome; and (Free Radic Biol Med. 2013, 61, 285‐297) blunted NLRP3 inflammasome activation through SIRT1‐ and SIRT3‐mediated autophagy induction, in renal tissues or cultured macrophages. In conclusion, Tris DBA effectively ameliorated the mouse IgAN model and targeted signalling pathways downstream of ICs‐mediated interaction, which is a novel immunomodulatory strategy. Further development of Tris DBA as a therapeutic candidate for IgAN is warranted.     

Mitochondrial damage contributes to Pseudomonas aeruginosa activation of the inflammasome and is downregulated by autophagy

The nucleotide-binding domain, leucine-rich repeat containing family caspase recruitment domain containing 4 (NLRC4) inflammasome can be activated by pathogenic bacteria via products translocated through the microbial type III secretion apparatus (T3SS). Recent work has shown that activation of the NLRP3 inflammasome is downregulated by autophagy, but the influence of autophagy on NLRC4 activation is unclear. We set out to determine how autophagy might influence this process, using the bacterium Pseudomonas aeruginosa, which activates the NLRC4 inflammasome via its T3SS. Infection resulted in T3SS-dependent mitochondrial damage with increased production of reactive oxygen intermediates and release of mitochondrial DNA. Inhibiting mitochondrial reactive oxygen release or degrading intracellular mitochondrial DNA abrogated NLRC4 inflammasome activation. Moreover, macrophages lacking mitochondria failed to activate NLRC4 following infection. Removal of damaged mitochondria by autophagy significantly attenuated NLRC4 inflammasome activation. Mitochondrial DNA bound specifically to NLRC4 immunoprecipitates and transfection of mitochondrial DNA directly activated the NLRC4 inflammasome; oxidation of the DNA enhanced this effect. Manipulation of autophagy altered the degree of inflammasome activation and inflammation in an in vivo model of P. aeruginosa infection. Our results reveal a novel mechanism contributing to NLRC4 activation by P. aeruginosa via mitochondrial damage and release of mitochondrial DNA triggered by the bacterial T3SS that is downregulated by autophagy.     

The NLRP3 Inflammasome and IL-1β Accelerate Immunologically Mediated Pathology in Experimental Viral Fulminant Hepatitis

Viral fulminant hepatitis (FH) is a severe disease with high mortality resulting from excessive inflammation in the infected liver. Clinical interventions have been inefficient due to the lack of knowledge for inflammatory pathogenesis in the virus-infected liver. We show that wild-type mice infected with murine hepatitis virus strain-3 (MHV-3), a model for viral FH, manifest with severe disease and high mortality in association with a significant elevation in IL-1β expression in the serum and liver. Whereas, the viral infection in IL-1β receptor-I deficient (IL-1R1^-/-) or IL-1R antagonist (IL-1Ra) treated mice, show reductions in virus replication, disease progress and mortality. IL-1R1 deficiency appears to debilitate the virus-induced fibrinogen-like protein-2 (FGL2) production in macrophages and CD45⁺Gr-1^high neutrophil infiltration in the liver. The quick release of reactive oxygen species (ROS) by the infected macrophages suggests a plausible viral initiation of NLRP3 inflammasome activation. Further experiments show that mice deficient of p47^phox, a nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunit that controls acute ROS production, present with reductions in NLRP3 inflammasome activation and subsequent IL-1β secretion during viral infection, which appears to be responsible for acquiring resilience to viral FH. Moreover, viral infected animals in deficiencies of NLRP3 and Caspase-1, two essential components of the inflammasome complex, also have reduced IL-1β induction along with ameliorated hepatitis. Our results demonstrate that the ROS/NLRP3/IL-1β axis institutes an essential signaling pathway, which is over activated and directly causes the severe liver disease during viral infection, which sheds light on development of efficient treatments for human viral FH and other severe inflammatory diseases.     

Autophagy deficiency in myeloid cells increases susceptibility to obesity-induced diabetes and experimental colitis

Autophagy, which is critical for the proper turnover of organelles such as endoplasmic reticulum and mitochondria, affects diverse aspects of metabolism, and its dysregulation has been incriminated in various metabolic disorders. However, the role of autophagy of myeloid cells in adipose tissue inflammation and type 2 diabetes has not been addressed. We produced mice with myeloid cell-specific deletion of Atg7 (autophagy-related 7), an essential autophagy gene (Atg7 conditional knockout [cKO] mice). While Atg7 cKO mice were metabolically indistinguishable from control mice, they developed diabetes when bred to ob/w mice (Atg7 cKO-ob/ob mice), accompanied by increases in the crown-like structure, inflammatory cytokine expression and inflammasome activation in adipose tissue. Mφs (macrophages) from Atg7 cKO mice showed significantly higher interleukin 1 β release and inflammasome activation in response to a palmitic acid plus lipopolysaccharide combination. Moreover, a decrease in the NAD⁺:NADH ratio and increase in intracellular ROS content after treatment with palmitic acid in combination with lipopolysaccharide were more pronounced in Mφs from Atg7 cKO mice, suggesting that mitochondrial dysfunction in autophagy-deficient Mφs leads to an increase in lipid-induced inflammasome and metabolic deterioration in Atg7 cKO-ob/ob mice. Atg7 cKO mice were more susceptible to experimental colitis, accompanied by increased colonic cytokine expression, T helper 1 skewing and systemic bacterial invasion. These results suggest that autophagy of Mφs is important for the control of inflammasome activation in response to metabolic or extrinsic stress, and autophagy deficiency in Mφs may contribute to the progression of metabolic syndrome associated with lipid injury and colitis.     

Autophagy in microglia degrades extracellular β-amyloid fibrils and regulates the NLRP3 inflammasome

Accumulation of β-amyloid (Aβ) and resultant inflammation are critical pathological features of Alzheimer disease (AD). Microglia, a primary immune cell in brain, ingests and degrades extracellular Aβ fibrils via the lysosomal system. Autophagy is a catabolic process that degrades native cellular components, however, the role of autophagy in Aβ degradation by microglia and its effects on AD are unknown. Here we demonstrate a novel role for autophagy in the clearance of extracellular Aβ fibrils by microglia and in the regulation of the Aβ-induced NLRP3 (NLR family, pyrin domain containing 3) inflammasome using microglia specific atg7 knockout mice and cell cultures. We found in microglial cultures that Aβ interacts with MAP1LC3B-II via OPTN/optineurin and is degraded by an autophagic process mediated by the PRKAA1 pathway. We anticipate that enhancing microglial autophagy may be a promising new therapeutic strategy for AD.     

TRPV1 channel mediates NLRP3 inflammasome-dependent neuroinflammation in microglia

Multiple sclerosis (MS) is a chronic inflammatory autoimmune disease in the central nervous system (CNS). The NLRP3 inflammasome is considered an important regulator of immunity and inflammation, both of which play a critical role in MS. However, the underlying mechanism of NLRP3 inflammasome activation is not fully understood. Here we identified that the TRPV1 (transient receptor potential vanilloid type 1) channel in microglia, as a Ca²⁺ influx-regulating channel, played an important role in NLRP3 inflammasome activation. Deletion or pharmacological blockade of TRPV1 inhibited NLRP3 inflammasome activation in microglia in vitro. Further research revealed that TRPV1 channel regulated ATP-induced NLRP3 inflammasome activation through mediating Ca²⁺ influx and phosphorylation of phosphatase PP2A in microglia. In addition, TRPV1 deletion could alleviate mice experimental autoimmune encephalomyelitis (EAE) and reduce neuroinflammation by inhibiting NLRP3 inflammasome activation. These data suggested that the TRPV1 channel in microglia can regulate NLRP3 inflammasome activation and consequently mediate neuroinflammation. Meanwhile, our study indicated that TRPV1–Ca²⁺–PP2A pathway may be a novel regulator of NLRP3 inflammasome activation, pointing to TRPV1 as a potential target for CNS inflammatory diseases.Subject terms: Neuroimmunology, Neuroimmunology