Mincle-GSDMD-mediated release of IL-1ß small extracellular vesicles from hepatic macrophages in ethanol-induced liver injury.

BACKGROUND: Macrophage-inducible C-type lectin (Mincle) is expressed on hepatic macrophages and senses ethanol (EtOH)-induced danger signals released from dying hepatocytes and promotes IL-1ß production. However, it remains unclear what and how EtOH-induced Mincle ligands activate downstream signaling events to mediate IL-1ß release and contribute to alcohol-associated liver disease (ALD). In this study, we investigated the association of circulating ß-glucosylceramide (ß-GluCer), an endogenous Mincle ligand, with severity of ALD and examined the mechanism by which ß-GluCer engages Mincle on hepatic macrophages to release IL-1ß in the absence of cell death and exacerbates ALD. METHOD AND RESULTS: Concentrations of ß-GluCer were increased in serum of patients with severe AH and correlated with disease severity. Challenge of hepatic macrophages with lipopolysaccharide and ß-GluCer induced formation of a Mincle and Gsdmd-dependent secretory complex containing chaperoned full-length gasdermin D (Hsp90-CDC37-NEDD4) with polyubiquitinated pro-IL-1ß and components of the Caspase 8-NLRP3 inflammasome loaded as cargo in small extracellular vesicles (sEVs). Gao-binge EtOH exposure to wild-type, but not Mincle-/- and Gsdmd-/-, mice increased release of IL-1ß-containing sEVs from liver explant cultures. Myeloid-specific deletion of Gsdmd similarly decreased the formation of sEVs by liver explant cultures and protected mice from EtOH-induced liver injury. sEVs collected from EtOH-fed wild-type, but not Gsdmd-/-, mice promoted injury of cultured hepatocytes and, when injected into wild-type mice, aggravated Gao-binge EtOH-induced liver injury. CONCLUSION: ß-GluCer functions as a danger-associated molecular pattern activating Mincle-dependent gasdermin D-mediated formation and release of IL-1ß-containing sEVs, which in turn exacerbate hepatocyte cell death and contribute to the pathogenesis of ALD.

TH17 cells promote CNS inflammation by sensing danger signals via Mincle.

The C-type lectin receptor Mincle is known for its important role in innate immune cells in recognizing pathogen and damage associated molecular patterns. Here we report a T cell-intrinsic role for Mincle in the pathogenesis of experimental autoimmune encephalomyelitis (EAE). Genomic deletion of Mincle in T cells impairs TH17, but not TH1 cell-mediated EAE, in alignment with significantly higher expression of Mincle in TH17 cells than in TH1 cells. Mechanistically, dying cells release ß-glucosylceramide during inflammation, which serves as natural ligand for Mincle. Ligand engagement induces activation of the ASC-NLRP3 inflammasome, which leads to Caspase8-dependent IL-1ß production and consequentially TH17 cell proliferation via an autocrine regulatory loop. Chemical inhibition of ß-glucosylceramide synthesis greatly reduces inflammatory CD4+ T cells in the central nervous system and inhibits EAE progression in mice. Taken together, this study indicates that sensing of danger signals by Mincle on TH17 cells plays a critical role in promoting CNS inflammation.

IL-1R-IRAKM-Slc25a1 signaling axis reprograms lipogenesis in adipocytes to promote diet-induced obesity in mice.

Toll-like receptors/Interleukin-1 receptor signaling plays an important role in high-fat diet-induced adipose tissue dysfunction contributing to obesity-associated metabolic syndromes. Here, we show an unconventional IL-1R-IRAKM-Slc25a1 signaling axis in adipocytes that reprograms lipogenesis to promote diet-induced obesity. Adipocyte-specific deficiency of IRAKM reduces high-fat diet-induced body weight gain, increases whole body energy expenditure and improves insulin resistance, associated with decreased lipid accumulation and adipocyte cell sizes. IL-1ß stimulation induces the translocation of IRAKM Myddosome to mitochondria to promote de novo lipogenesis in adipocytes. Mechanistically, IRAKM interacts with and phosphorylates mitochondrial citrate carrier Slc25a1 to promote IL-1ß-induced mitochondrial citrate transport to cytosol and de novo lipogenesis. Moreover, IRAKM-Slc25a1 axis mediates IL-1ß induced Pgc1a acetylation to regulate thermogenic gene expression in adipocytes. IRAKM kinase-inactivation also attenuates high-fat diet-induced obesity. Taken together, our study suggests that the IL-1R-IRAKM-Slc25a1 signaling axis tightly links inflammation and adipocyte metabolism, indicating a potential therapeutic target for obesity.

Inhibition of IRAK4 kinase activity improves ethanol-induced liver injury in mice.

BACKGROUNDS & AIMS: Alcohol-related liver disease (ALD) is a major cause of chronic liver disease worldwide with limited therapeutic options. Interleukin-1 receptor associated kinase 4 (IRAK4), the master kinase of Toll-like receptor (TLR)/IL-1R-mediated signalling activation, is considered a novel therapeutic target in inflammatory diseases, but has not been investigated in the context of ALD. METHODS: IRAK4 phosphorylation and IRAK1 protein were analysed in liver from alcohol-related hepatitis patients and healthy controls. IRAK4 kinase activity-inactive knock-in (Irak4 KI) mice and bone marrow chimeric mice were exposed to chronic ethanol-induced liver injury. IL-1ß-induced IRAK4-mediated signalling and acute phase response were investigated in cultured hepatocytes. IRAK1/4 inhibitor was used to test the therapeutic potential for ethanol-induced liver injury in mice. RESULTS: Increased IRAK4 phosphorylation and reduced IRAK1 protein were found in livers of patients with alcoholic hepatitis. In the chronic ethanol-induced liver injury mouse model, hepatic inflammation and hepatocellular damage were attenuated in Irak4 KI mice. IRAK4 kinase activity promotes expression of acute phase proteins in response to ethanol exposure, including C-reactive protein and serum amyloid A1 (SAA1). SAA1 and IL-1ß synergistically exacerbate ethanol-induced cell death ex vivo. Pharmacological blockage of IRAK4 kinase abrogated ethanol-induced liver injury, inflammation, steatosis, as well as acute phase gene expression and protein production in mice. CONCLUSIONS: Our data elucidate the critical role of IRAK4 kinase activity in the pathogenesis of ethanol-induced liver injury in mice and provide preclinical validation for use of an IRAK1/4 inhibitor as a new potential therapeutic strategy for the treatment of ALD. LAY SUMMARY: Herein, we have identified the role of IRAK4 kinase activity in the development of alcohol-induced liver injury in mice. Hepatocyte-specific IRAK4 is associated with an acute phase response and release of proinflammatory cytokines/chemokines, which synergistically exacerbate alcohol-induced hepatocyte cell death ex vivo. Pharmacological inhibition of IRAK4 kinase activity effectively attenuates alcohol-induced liver injury in mice and could have therapeutic implications.

TLR-stimulated IRAKM activates caspase-8 inflammasome in microglia and promotes neuroinflammation.

NLRP3 inflammasome plays a critical spatiotemporal role in the pathogenesis of experimental autoimmune encephalomyelitis (EAE). This study reports a mechanistic insight into noncanonical NLRP3 inflammasome activation in microglia for the effector stage of EAE. Microglia-specific deficiency of ASC (apoptosis-associated speck-like protein containing a C-terminal caspase-activation and recruitment [CARD] domain) attenuated T cell expansion and neutrophil recruitment during EAE pathogenesis. Mechanistically, TLR stimulation led to IRAKM-caspase-8-ASC complex formation, resulting in the activation of caspase-8 and IL-1ß release in microglia. Noncanonical inflammasome-derived IL-1ß produced by microglia in the CNS helped to expand the microglia population in an autocrine manner and amplified the production of inflammatory cytokines/chemokines. Furthermore, active caspase-8 was markedly increased in the microglia in the brain tissue from patients with multiple sclerosis. Taken together, our study suggests that microglia-derived IL-1ß via noncanonical caspase-8-dependent inflammasome is necessary for microglia to exert their pathogenic role during CNS inflammation.

Integrin CD11b negatively regulates Mincle-induced signaling via the Lyn-SIRPα-SHP1 complex.

During mycobacteria infection, anti-inflammatory responses allow the host to avoid tissue damage caused by overactivation of the immune system; however, little is known about the negative modulators that specifically control mycobacteria-induced immune responses. Here we demonstrate that integrin CD11b is a critical negative regulator of mycobacteria cord factor-induced macrophage-inducible C-type lectin (Mincle) signaling. CD11b deficiency resulted in hyperinflammation following mycobacterial infection. Activation of Mincle by mycobacterial components turns on not only the Syk signaling pathway but also CD11b signaling and induces formation of a Mincle-CD11b signaling complex. The activated CD11b recruits Lyn, SIRPα and SHP1, which dephosphorylate Syk to inhibit Mincle-mediated inflammation. Furthermore, the Lyn activator MLR1023 effectively suppressed Mincle signaling, indicating the possibility of Lyn-mediated control of inflammatory responses. These results describe a new role for CD11b in fine-tuning the immune response against mycobacterium infection.

Mincle activation enhances neutrophil migration and resistance to polymicrobial septic peritonitis.

Sepsis is a systemic inflammatory response to bacterial infection. The therapeutic options for treating sepsis are limited. Impaired neutrophil recruitment into the infection site is directly associated with severe sepsis, but the precise mechanism is unclear. Here, we show that Mincle plays a key role in neutrophil migration and resistance during polymicrobial sepsis. Mincle-deficient mice exhibited lower survival rates in experimental sepsis from cecal ligation and puncture and Escherichia coli-induced peritonitis. Mincle deficiency led to higher serum inflammatory cytokine levels and reduced bacterial clearance and neutrophil recruitment. Transcriptome analyses revealed that trehalose dimycolate, a Mincle ligand, reduced the expression of G protein-coupled receptor kinase 2 (GRK2) in neutrophils. Indeed, GRK2 expression was upregulated, but surface expression of the chemokine receptor CXCR2 was downregulated in blood neutrophils from Mincle-deficient mice with septic injury. Moreover, CXCL2-mediated adhesion, chemotactic responses, and F-actin polymerization were reduced in Mincle-deficient neutrophils. Finally, we found that fewer Mincle-deficient neutrophils infiltrated from the blood circulation into the peritoneal fluid in bacterial septic peritonitis compared with wild-type cells. Thus, our results indicate that Mincle plays an important role in neutrophil infiltration and suggest that Mincle signaling may provide a therapeutic target for treating sepsis.

Mincle-mediated translational regulation is required for strong nitric oxide production and inflammation resolution.

In response to persistent mycobacteria infection, the host induces a granuloma, which often fails to eradicate bacteria and results in tissue damage. Diverse host receptors are required to control the formation and resolution of granuloma, but little is known concerning their regulatory interactions. Here we show that Mincle, the inducible receptor for mycobacterial cord factor, is the key switch for the transition of macrophages from cytokine expression to high nitric oxide production. In addition to its stimulatory role on TLR-mediated transcription, Mincle enhanced the translation of key genes required for nitric oxide synthesis through p38 and eIF5A hypusination, leading to granuloma resolution. Thus, Mincle has dual functions in the promotion and subsequent resolution of inflammation during anti-mycobacterial defence using both transcriptional and translational controls.

Tripartite motif-containing protein 30 modulates TCR-activated proliferation and effector functions in CD4+ T cells.

To avoid excessive activation, immune signals are tightly controlled by diverse inhibitory proteins. TRIM30, a tripartite motif (TRIM)-containing protein is one of such inhibitors known to function in macrophages. To define the roles of TRIM30, we generated Trim30 knockout (Trim30-/-) mice. Trim30 deletion caused no major developmental defects in any organs, nor showed any discernable defect in the activation of macrophages. But, Trim30-/- mice showed increased CD4/CD8 ratio when aged and Trim30-/- CD4+ T cells exhibited an abnormal response upon TCR activation, in particular in the absence of a costimulatory signal. Adoptive transfer of wild-type and Trim30-/- CD4+ T cells together into lymphopenic hosts confirmed higher proliferation of the Trim30-/- CD4+ T cells in vivo. Despite the enhanced proliferation, Trim30-/- T cells showed decreased levels of NF-κB activation and IL-2 production compared to wild-type cells. These results indicate a distinct requirement for TRIM30 in modulation of NF-κB activation and cell proliferation induced by TCR stimulation.