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Leptospirosis is a worldwide re-emerging zoonosis caused by pathogenic Leptospira spp. All vertebrate species can be infected; humans are sensitive hosts whereas other species, such as rodents, may become long-term renal carrier reservoirs. Upon infection, innate immune responses are initiated by recognition of Microbial Associated Molecular Patterns (MAMPs) by Pattern Recognition Receptors (PRRs). Among MAMPs, the lipopolysaccha-ride (LPS) is recognized by the Toll-Like-Receptor 4 (TLR4) and activates both the MyD88-dependent pathway at the plasma membrane and the TRIF-dependent pathway after TLR4 internalization. We previously showed that leptospiral LPS is not recognized by the human-TLR4, whereas it signals through mouse-TLR4 (mTLR4), which mediates mouse resistance to acute leptospirosis. However, although resistant, mice are known to be chronically infected by leptospires. Interestingly, the leptospiral LPS has low endotoxicity in mouse cells and is an agonist of TLR2, the sensor for bacterial lipoproteins. Here, we investigated the signaling properties of the leptospiral LPS in mouse macrophages. Using confocal micros-copy and flow cytometry, we showed that the LPS of L. interrogans did not induce internali-zation of mTLR4, unlike the LPS of Escherichia coli. Consequently, the LPS failed to induce the production of the TRIF-dependent nitric oxide and RANTES, both important antimicro-bial responses. Using shorter LPS and LPS devoid of TLR2 activity, we further found this mTLR4-TRIF escape to be dependent on both the co-purifying lipoproteins and the full-length O antigen. Furthermore, our data suggest that the O antigen could alter the binding of the leptospiral LPS to the co-receptor CD14 that is essential for TLR4-TRIF activation. Overall , we describe here a novel leptospiral immune escape mechanism from mouse macro-phages and hypothesize that the LPS altered signaling could contribute to the stealthiness and chronicity of the leptospires in mice.

Recent genome-wide association studies (GWAS) have identified a strong association signal of microRNA137 host gene ( MIR137 ) with schizophrenia. MIR137 dysfunction results in downregulation of presynaptic target gene complexin 1 ( CPLX1 ) and impairs synaptic plasticity in the hippocampus. In this study, we aimed to investigate whether the variants of MIR137 and CPLX1 confer susceptibility to schizophrenia in Han Chinese. This study employed 736 patients with schizophrenia patients and 751 well-matched healthy subjects for genetic analysis, and genotyped 12 SNPs within MIR137 and CPLX1 . SZDB database was used to performed brain eQTL analysis. There were no significant differences of CPLX1 expression in hippocampus, prefrontal cortex or stratum between the schizophrenia patients and control subjects. No significant differences were observed in allele and genotype frequencies in studied SNPs between the case and control groups. Gene interaction analysis showed that MIR137 SNP rs1625579 did not affect schizophrenia susceptibility in interaction with the CPLX1 polymorphic variants. Our findings do not support MIR137 and CPLX1 conferring susceptibility to schizophrenia in Han Chinese.

Toll-like receptor (TLR) 4 belongs to the TLR family of receptors inducing pro-inflammatory responses to invading pathogens. TLR4 is activated by lipopolysaccharide (LPS, endotoxin) of Gram-negative bacteria and sequentially triggers two signaling cascades: the first one involving TIRAP and MyD88 adaptor proteins is induced in the plasma membrane, whereas the second engaging adaptor proteins TRAM and TRIF begins in early endosomes after endocytosis of the receptor. The LPS-induced internalization of TLR4 and hence also the activation of the TRIF-dependent pathway is governed by a GPI-anchored protein, CD14. The endocytosis of TLR4 terminates the MyD88-dependent signaling, while the following endosome maturation and lysosomal degradation of TLR4 determine the duration and magnitude of the TRIF-dependent one. Alternatively, TLR4 may return to the plasma membrane, which process is still poorly understood. Therefore, the course of the LPS-induced pro-inflammatory responses depends strictly on the rates of TLR4 endocytosis and trafficking through the endo-lysosomal compartment. Notably, prolonged activation of TLR4 is linked with several hereditary human diseases, neurodegeneration and also with autoimmune diseases and cancer. Recent studies have provided ample data on the role of diverse proteins regulating the functions of early, late, and recycling endosomes in the TLR4-induced inflammation caused by LPS or phagocytosis of E. coli. In this review, we focus on the mechanisms of the internalization and intracellular trafficking of TLR4 and CD14, and also of LPS, in immune cells and discuss how dysregulation of the endo-lysosomal compartment contributes to the development of diverse human diseases.

Traditionally viewed as an autodigestive pathway, autophagy also facilitates cellular secretion; however, the mechanisms underlying these processes remain unclear. Here, we demonstrate that components of the autophagy machinery specify secretion within extracellular vesicles (EVs). Using a proximity-dependent biotinylation proteomics strategy, we identify 200 putative targets of LC3-dependent secretion. This secretome consists of a highly interconnected network enriched in RNA-binding proteins (RBPs) and EV cargoes. Proteomic and RNA profiling of EVs identifies diverse RBPs and small non-coding RNAs requiring the LC3-conjugation machinery for packaging and secretion. Focusing on two RBPs, heterogeneous nuclear ribonucleoprotein K (HNRNPK) and scaffold-attachment factor B (SAFB), we demonstrate that these proteins interact with LC3 and are secreted within EVs enriched with lipidated LC3. Furthermore, their secretion requires the LC3-conjugation machinery, neutral sphingomyelinase 2 (nSMase2) and LC3-dependent recruitment of factor associated with nSMase2 activity (FAN). Hence, the LC3-conjugation pathway controls EV cargo loading and secretion.Leidal et al. show that the LC3-conjugation pathway, which is part of the autophagy machinery, controls extracellular vesicle cargo loading and secretion of RNA-binding proteins.

Type I IFNs are key cytokines mediating innate antiviral immunity. cGMP-AMP synthase, ritinoic acid-inducible protein 1 (RIG-I)–like receptors, and Toll-like receptors recognize microbial double-stranded (ds)DNA, dsRNA, and LPS to induce the expression of type I IFNs. These signaling pathways converge at the recruitment and activation of the transcription factor IRF-3 (IFN regulatory factor 3). The adaptor proteins STING (stimulator of IFN genes), MAVS (mitochondrial antiviral signaling), and TRIF (TIR domain-containing adaptor inducing IFN-β) mediate the recruitment of IRF-3 through a conserved pLxIS motif. Here we show that the pLxIS motif of phosphorylated STING, MAVS, and TRIF binds to IRF-3 in a similar manner, whereas residues upstream of the motif confer specificity. The structure of the IRF-3 phosphomimetic mutant S386/396E bound to the cAMP response element binding protein (CREB)-binding protein reveals that the pLxIS motif also mediates IRF-3 dimerization and activation. Moreover, rotavirus NSP1 (nonstructural protein 1) employs a pLxIS motif to target IRF-3 for degradation, but phosphorylation of NSP1 is not required for its activity. These results suggest a concerted mechanism for the recruitment and activation of IRF-3 that can be subverted by viral proteins to evade innate immune responses.

Non-apoptotic forms of cell death can trigger sterile inflammation through the release of danger-associated molecular patterns, which are recognized by innate immune receptors. However, despite years of investigation the mechanisms which initiate inflammatory responses after heart transplantation remain elusive. Here, we demonstrate that ferrostatin-1 (Fer-1), a specific inhibitor of ferroptosis, decreases the level of pro-ferroptotic hydroperoxy-arachidonoyl-phosphatidylethanolamine, reduces cardiomyocyte cell death and blocks neutrophil recruitment following heart transplantation. Inhibition of necroptosis had no effect on neutrophil trafficking in cardiac grafts. We extend these observations to a model of coronary artery ligation-induced myocardial ischemia reperfusion injury where inhibition of ferroptosis resulted in reduced infarct size, improved left ventricular systolic function, and reduced left ventricular remodeling. Using intravital imaging of cardiac transplants, we uncover that ferroptosis orchestrates neutrophil recruitment to injured myocardium by promoting adhesion of neutrophils to coronary vascular endothelial cells through a TLR4/TRIF/type I IFN signaling pathway. Thus, we have discovered that inflammatory responses after cardiac transplantation are initiated through ferroptotic cell death and TLR4/Trif-dependent signaling in graft endothelial cells. These findings provide a platform for the development of therapeutic strategies for heart transplant recipients and patients, who are vulnerable to ischemia reperfusion injury following restoration of coronary blood flow.

Autophagy is a highly conserved process from yeast to mammals in which intracellular materials are engulfed by a double-membrane organelle called autophagosome and degrading materials by fusing with the lysosome. The process of autophagy is regulated by sequential recruitment and function of autophagy-related (Atg) proteins. Genetic hierarchical analyses show that the ULK1 complex comprised of ULK1-FIP200-ATG13-ATG101 translocating from the cytosol to autophagosome formation sites as a most upstream ATG factor; this translocation is critical in autophagy initiation. However, how this translocation occurs remains unclear. Here, we show that ULK1 is palmitoylated by palmitoyltransferase ZDHHC13 and translocated to the autophagosome formation site upon autophagy induction. We find that the ULK1 palmitoylation is required for autophagy initiation. Moreover, the ULK1 palmitoylated enhances the phosphorylation of ATG14L, which is required for activating PI3-Kinase and producing phosphatidylinositol 3-phosphate, one of the autophagosome membrane’s lipids. Our results reveal how the most upstream ULK1 complex translocates to the autophagosome formation sites during autophagy. It was unknown how the most upstream Atg protein transits from the cytosol to autophagosome formation sites. Here, the authors show that ULK1 palmitoylation by ZDHHC13 recruits the complex to the formation site and enhances ATG14L phosphorylation.

Recent genome-wide association studies (GWASs) have identified a locus on chromosome 1p13 strongly associated with both plasma low-density lipoprotein cholesterol (LDL-C) and myocardial infarction (MI) in humans. Here we show through a series of studies in human cohorts and human-derived hepatocytes that a common noncoding polymorphism at the 1p13 locus, rs12740374, creates a C/EBP (CCAAT/enhancer binding protein) transcription factor binding site and alters the hepatic expression of the SORT1 gene. With small interfering RNA (siRNA) knockdown and viral overexpression in mouse liver, we demonstrate that Sort1 alters plasma LDL-C and very low-density lipoprotein (VLDL) particle levels by modulating hepatic VLDL secretion. Thus, we provide functional evidence for a novel regulatory pathway for lipoprotein metabolism and suggest that modulation of this pathway may alter risk for MI in humans. We also demonstrate that common noncoding DNA variants identified by GWASs can directly contribute to clinical phenotypes. Blood lipids and the heart Lipid concentration in the blood is a major risk factor for coronary artery disease, and one that can be targeted for therapeutic intervention. A genome-wide association study (GWAS) in more than 100,000 individuals of European ancestry has been used to identify 95 genetic variants linked to plasma lipids. Among associated loci are those involved in cholesterol metabolism, known targets of cholesterol-lowering drugs and novel loci that contribute to normal variation in lipid traits and to extreme lipid phenotypes. One locus identified in the study as being associated with both plasma low-density lipoprotein cholesterol and coronary artery disease forms the focus of a second paper in this issue. The locus, on chromosome 1p13, is shown to create a binding site for C/EBP transcription factors and to alter SORT1 gene expression in the liver. Modulating Sort1 levels in the mouse liver alters plasma lipoprotein levels, potentially explaining why variation at this locus is associated with heart disease. This finding identifies the sortilin pathway as a possible target for therapeutic intervention and illustrates how GWAS results can be used as a production line for drug targets. A non-coding polymorphism at a locus associated with myocardial infarction in humans creates a CCAAT/enhancer binding protein transcription factor binding site and alters the hepatic expression of the SORT1 gene. These authors show that modulating Sort1 levels in mouse liver alters levels of plasma low-density lipoprotein cholesterol and very low-density lipoprotein, potentially explaining why polymorphisms at this locus are associated with heart disease.

Membrane fission, which facilitates compartmentalization of biological processes into discrete, membrane-bound volumes, is essential for cellular life. Proteins with specific structural features including constricting rings, helical scaffolds, and hydrophobic membrane insertions are thought to be the primary drivers of fission. In contrast, here we report a mechanism of fission that is independent of protein structure—steric pressure among membranebound proteins. In particular, random collisions among crowded proteins generate substantial pressure, which if unbalanced on the opposite membrane surface can dramatically increase membrane curvature, leading to fission. Using the endocytic protein epsin1 N-terminal homology domain (ENTH), previously thought to drive fission by hydrophobic insertion, our results show that membrane coverage correlates equally with fission regardless of the hydrophobicity of insertions. Specifically, combining FRET-based measurements of membrane coverage with multiple, independent measurements of membrane vesiculation revealed that fission became spontaneous as steric pressure increased. Further, fission efficiency remained equally potent when helices were replaced by synthetic membrane-binding motifs. These data challenge the view that hydrophobic insertions drive membrane fission, suggesting instead that the role of insertions is to anchor proteins strongly to membrane surfaces, amplifying steric pressure. In line with these conclusions, even green fluorescent protein (GFP) was able to drive fission efficiently when bound to the membrane at high coverage. Our conclusions are further strengthened by the finding that intrinsically disordered proteins, which have large hydrodynamic radii yet lack a defined structure, drove fission with substantially greater potency than smaller, structured proteins.

TLR2 promotes NLRP3 inflammasome activation via an early MyD88-IRAK1-dependent pathway that provides a priming signal (signal 1) necessary for activation of the inflammasome by a second potassium-depleting signal (signal 2). Here we show that TLR3 binding to dsRNA promotes post-translational inflammasome activation through intermediate and late TRIF/RIPK1/FADD-dependent pathways. Both pathways require the scaffolding but not the catalytic function of caspase-8 or RIPK1. Only the late pathway requires kinase competent RIPK3 and MLKL function. Mechanistically, FADD/caspase-8 scaffolding function provides a post-translational signal 1 in the intermediate pathway, whereas in the late pathway it helps the oligomerization of RIPK3, which together with MLKL provides both signal 1 and 2 for inflammasome assembly. Cytoplasmic dsRNA activates NLRP3 independent of TRIF, RIPK1, RIPK3 or mitochondrial DRP1, but requires FADD/caspase-8 in wildtype macrophages to remove RIPK3 inhibition. Our study provides a comprehensive analysis of pathways that lead to NLRP3 inflammasome activation in response to dsRNA. Inflammasome activation requires a complex and incompletely understood network of signalling events. Here the authors characterize step-by-step contributions of TLR3, caspase-8, RIPK3 and MLKL to the activation of NLRP3 inflammasome in response to double-stranded RNA.