Toll‐like receptors hit calcium

Mitochondrial Ca²⁺ uptake is a multifarious signal that controls both the activity of matrix dehydrogenases and the sensitivity to apoptotic and necrotic challenges. Recent evidence indicates that mitochondria also play a role in triggering inflammation, as mitochondrial DNA, when released by the cell, is an important damage‐associated molecular pattern (DAMP). Now, Toll‐like receptors (TLRs) are shown to close the loop, by affecting in turn mitochondrial activity. Two studies by Shintani and colleagues, one in this issue of EMBO reports 1 2 , identify a new TLR transduction mechanism that impinges directly on mitochondrial function. Upon binding of CpG oligodeoxynucleotides, TLR9—which in non‐immune cells is retained in the ER—inhibits SERCA2, thus reducing Ca²⁺ transfer to the mitochondria and aerobic metabolism.     

Immune complex negatively regulates Toll‐like receptor 9‐mediated immune responses in B cells through the inhibitory Fc‐gamma receptor IIb

Because inappropriate activation of Toll‐like receptor 9 (TLR9) may induce pathological damage, negative regulation of the TLR9‐triggered immune response has attracted considerable attention. Nonpathogenic immune complex (IC) has been demonstrated to have beneficial therapeutic effects in some kinds of autoimmune diseases. However, the role of IC in the regulation of TLR9‐triggered immune responses and the underlying mechanisms remain unclear. In this study, it was demonstrated that IC stimulation of B cells not only suppresses CpG‐oligodeoxynucleotide (CpG‐ODN)‐induced pro‐inflammatory IL‐6 and IgM κ production, but also attenuates CD40 and CD80 expression. Furthermore, our results suggest that the receptor for the Fc portion of IgG (FcγR) IIb is involved in the suppressive effect of IC on TLR9‐mediated CD40, CD80 and IL‐6 expression. Finally, it was found that IC down‐regulates TLR9 expression in CpG‐ODN activated B cells. Our results provide an outline of a new pathway for the negative regulation of TLR9‐triggered immune responses in B cells via FcγRIIb. A new mechanistic explanation of the therapeutic effect of nonpathogenic IC on inflammatory and autoimmune diseases is also provided.     

LPS or ethanol triggers clathrin‐ and rafts/caveolae‐dependent endocytosis of TLR4 in cortical astrocytes

Toll‐like receptor 4 (TLR4) activation and signalling in glial cells play critical roles in neurological disorders and in alcohol‐induced brain damage. TLR4 endocytosis upon lipopolysaccharide (LPS) stimulation regulates which signalling pathway is activated, the MyD88‐dependent or the TIR‐domain‐containing adapter‐inducing interferon‐β (TRIF)‐dependent pathway. However, it remains elusive whether ethanol‐induced TLR4 signalling is associated with receptor internalization and trafficking, and which endocytic pathway(s) are used in cortical astrocytes. Using the adenoviral over‐expression of TLR4^GFP, confocal microscopy and the imagestream technique, we show that upon ethanol or LPS stimulation, TLR4 co‐localizes with markers of the clathrin and caveolin endocytic pathways, and that this endocytosis is dependent on dynamin. Using chlorpromazin and filipin as inhibitors of the clathrin and rafts/caveolae endocytic pathways, respectively, we demostrate that TRIF‐dependent signalling relies on an intact clathrin pathway, whereas disruption of rafts/caveolae inhibits the MyD88‐ and TRIF‐dependent signalling pathways. Immunofluorescence studies also suggest that lipid rafts and clathrin cooperate for appropriate TLR4 internalization. We also show that ethanol can trigger similar endocytic pathways as LPS does, although ethanol delays clathrin internalization and alters TLR4 vesicular trafficking. Our results provide new insights into the effects of ethanol or LPS on TLR4 signalling in cortical astrocytes, events that may underlie neuroinflammation and brain damage.

     

MicroRNA‐129‐1‐3p protects cardiomyocytes from pirarubicin‐induced apoptosis by down‐regulating the GRIN2D‐mediated Ca2+ signalling pathway

Pirarubicin (THP), an anthracycline anticancer drug, is a first‐line therapy for various solid tumours and haematologic malignancies. However, THP can cause dose‐dependent cumulative cardiac damage, which limits its therapeutic window. The mechanisms underlying THP cardiotoxicity are not fully understood. We previously showed that MiR‐129‐1‐3p, a potential biomarker of cardiovascular disease, was down‐regulated in a rat model of THP‐induced cardiac injury. In this study, we used Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genome (KEGG) pathway enrichment analyses to determine the pathways affected by miR‐129‐1‐3p expression. The results linked miR‐129‐1‐3p to the Ca²⁺ signalling pathway. TargetScan database screening identified a tentative miR‐129‐1‐3p‐binding site at the 3′‐UTR of GRIN2D, a subunit of the N‐methyl‐D‐aspartate receptor calcium channel. A luciferase reporter assay confirmed that miR‐129‐1‐3p directly regulates GRIN2D. In H9C2 (rat) and HL‐1 (mouse) cardiomyocytes, THP caused oxidative stress, calcium overload and apoptotic cell death. These THP‐induced changes were ameliorated by miR‐129‐1‐3p overexpression, but exacerbated by miR‐129‐1‐3p knock‐down. In addition, miR‐129‐1‐3p overexpression in cardiomyocytes prevented THP‐induced changes in the expression of proteins that are either key components of Ca²⁺ signalling or important regulators of intracellular calcium trafficking/balance in cardiomyocytes including GRIN2D, CALM1, CaMKⅡδ, RyR2‐pS2814, SERCA2a and NCX1. Together, these bioinformatics and cell‐based experiments indicate that miR‐129‐1‐3p protects against THP‐induced cardiomyocyte apoptosis by down‐regulating the GRIN2D‐mediated Ca²⁺ pathway. Our results reveal a novel mechanism underlying the pathogenesis of THP‐induced cardiotoxicity. The miR‐129‐1‐3p/Ca²⁺ signalling pathway could serve as a target for the development of new cardioprotective agents to control THP‐induced cardiotoxicity.     

Toll‐like receptor 4: A promising therapeutic target for pneumonia caused by Gram‐negative bacteria

Gram‐negative bacteria (GNB) emerge as important pathogens causing pulmonary infection, which can develop into sepsis due to bacterial resistance to antibiotics. GNB pneumonia poses a huge social and economic burden all over the world. During GNB infection in the lung, Toll‐like receptor 4 (TLR4) can form a complex with MD2 and CD14 after recognizing lipopolysaccharide of GNB, initiate the MyD88‐ and TRIF‐dependent signalling pathways and stimulate host non‐specific immune response. In this review, we summarize recent progress in our understanding of the role of TLR4 in GNB pneumonia. The latest experimental results, especially in TLR4 knockout animals, suggest a promising potential of targeting TLR4 signalling pathway for the treatment of GNB pneumonia. Furthermore, we highlight the benefits of Traditional Chinese Medicine as novel candidates for the therapy of GNB pneumonia due to the modulation of TLR4 signalling pathway. Finally, we discuss the promise and challenge in the development of TLR4‐based drugs for GNB pneumonia.     

The TLR4‐IRE1α pathway activation contributes to palmitate‐elicited lipotoxicity in hepatocytes

Lipotoxicity induced by saturated fatty acids (SFAs) plays a pathological role in the development of non‐alcoholic fatty liver disease (NAFLD); however, the exact mechanism(s) remain to be clearly elucidated. Toll‐like receptor (TLR) 4 plays a fundamental role in activating the innate immune system. Intriguingly, hepatocytes express TLR4 and machinery for TLR4 signalling pathway. That liver‐specific TLR4 knockout mice are protective against diet‐induced NAFLD suggests that hepatocyte TLR4 signalling pathway plays an important role in NAFLD pathogenesis. Herein, using cultured hepatocytes, we sought to directly examine the role of TLR4 signalling pathway in palmitate‐elicited hepatotoxicity and to elucidate underlying mechanism(s). Our data reveal that palmitate exposure up‐regulates TLR4 expression at both mRNA and protein levels in hepatocytes, which are associated with NF‐κB activation. The inhibition of TLR4 signalling pathway through both pharmacological and genetic approaches abolished palmitate‐induced cell death, suggesting that TLR4 signalling pathway activation contributes to palmitate‐induced hepatotoxicity. Mechanistic investigations demonstrate that inositol‐requiring enzyme 1α (IRE1α), one of three major signal transduction pathways activated during endoplasmic reticulum (ER) stress, is the downstream target of palmitate‐elicited TLR4 activation and mechanistically implicated in TLR4 activation‐triggered cell death in response to palmitate exposure. Collectively, our data identify that the TLR4‐IRE1α pathway activation contributes to palmitate‐elicited lipotoxicity in hepatocytes. Our findings suggest that targeting TLR4‐IRE1α pathway can be a potential therapeutic choice for the treatment of NAFLD as well as other metabolic disorders, with lipotoxicity being the principal pathomechanism.     

iTRAQ proteomic analysis of N‐acetylmuramic acid mediated anti‐inflammatory capacity in LPS‐induced RAW 264.7 cells

Lactobacillus acidophilus probiotic bacteria have lasting beneficial health effects in the gastrointestinal tract, including protecting against pathogens, improving immunomodulation, and producing beneficial bacteria‐derived molecules. In lipopolysaccharide (LPS) induced RAW 264.7 cells treated with peptidoglycan or N‐acetylmuramic acid (NAM) from L. acidophilus, 390 differentially expressed proteins (8.76%) were identified by iTRAQ analysis, 257 (5.77%) of which were upregulated and 133 (2.99%) were downregulated under LPS‐induced conditions. Most of these proteins were grouped into the following inflammation‐related cellular signaling: lysosome pathway, calcium signaling pathway, and Toll‐like receptor (TLR) signaling pathway. Among them, clathrin, SERCA, and interleukin 1 receptor antagonist were differentially expressed to a significant degree in peptidoglycan or NAM pretreated RAW 264.7 cells. Bioinformatics analysis indicated that NAM may mediate an anti‐inflammatory process via a Ca²⁺‐dependent NF‐κB pathway. These observations reveal new insights into the molecular mechanisms involved in the suppression of LPS‐induced macrophage inflammation by L. acidophilus.     

Relation between TLR4/NF‐κB signaling pathway activation by 27‐hydroxycholesterol and 4‐hydroxynonenal,and atherosclerotic plaque instability

It is now thought that atherosclerosis, although due to increased plasma lipids, is mainly the consequence of a complicated inflammatory process, with immune responses at the different stages of plaque development. Increasing evidence points to a significant role of Toll‐like receptor 4 (TLR4), a key player in innate immunity, in the pathogenesis of atherosclerosis. This study aimed to determine the effects on TLR4 activation of two reactive oxidized lipids carried by oxidized low‐density lipoproteins, the oxysterol 27‐hydroxycholesterol (27‐OH) and the aldehyde 4‐hydroxynonenal (HNE), both of which accumulate in atherosclerotic plaques and play a key role in the pathogenesis of atherosclerosis. Secondarily, it examined their potential involvement in mediating inflammation and extracellular matrix degradation, the hallmarks of high‐risk atherosclerotic unstable plaques. In human promonocytic U937 cells, both 27‐OH and HNE were found to enhance cell release of IL‐8, IL‐1β, and TNF‐α and to upregulate matrix metalloproteinase‐9 (MMP‐9) via TLR4/NF‐κB‐dependent pathway; these actions may sustain the inflammatory response and matrix degradation that lead to atherosclerotic plaque instability and to their rupture. Using specific antibodies, it was also demonstrated that these inflammatory cytokines increase MMP‐9 upregulation, thus enhancing the release of this matrix‐degrading enzyme by macrophage cells and contributing to plaque instability. These innovative results suggest that, by accumulating in atherosclerotic plaques, the two oxidized lipids may contribute to plaque instability and rupture. They appear to do so by sustaining the release of inflammatory molecules and MMP‐9 by inflammatory and immune cells, for example, macrophages, through activation of TLR4 and its NF‐κB downstream signaling.     

miR‐181a and miR‐150 regulate dendritic cell immune inflammatory responses and cardiomyocyte apoptosis via targeting JAK1–STAT1/c‐Fos pathway

The immune inflammatory response plays a crucial role in many cardiac pathophysiological processes, including ischaemic cardiac injury and the post‐infarction repair process. MicroRNAs (miRNAs) regulate the development and function of dendritic cells (DCs), which are key players in the initiation and regulation of immune responses; however, the underlying regulatory mechanisms remain unclear. Here, we used the supernatants of necrotic primary cardiomyocytes (Necrotic‐S) to mimic the myocardial infarction (MI) microenvironment to investigate the role of miRNAs in the regulation of DC‐mediated inflammatory responses. Our results showed that Necrotic‐S up‐regulated the DC maturation markers CD40, CD83 and CD86 and increased the production of inflammatory cytokines, concomitant with the up‐regulation of miR‐181a and down‐regulation of miR‐150. Necrotic‐S stimulation activated the JAK/STAT pathway and promoted the nuclear translocation of c‐Fos and NF‐κB p65, and silencing of STAT1 or c‐Fos suppressed Necrotic‐S‐induced DC maturation and inflammatory cytokine production. The effects of Necrotic‐S on DC maturation and inflammatory responses, its activation of the JAK/STAT pathway and the induction of cardiomyocyte apoptosis under conditions of hypoxia were suppressed by miR‐181a or miR‐150 overexpression. Taken together, these data indicate that miR‐181a and miR‐150 attenuate DC immune inflammatory responses via JAK1–STAT1/c‐Fos signalling and protect cardiomyocytes from cell death under conditions of hypoxia.     

Listeria monocytogenes induces IFNβ expression through an IFI16‐, cGAS‐ and STING‐dependent pathway

Listeria monocytogenes is a gram‐positive facultative intracellular bacterium, which replicates in the cytoplasm of myeloid cells. Interferon β (IFNβ) has been reported to play an important role in the mechanisms underlying Listeria disease. Although studies in murine cells have proposed the bacteria‐derived cyclic‐di‐AMP to be the key bacterial immunostimulatory molecule, the mechanism for IFNβ expression during L. monocytogenes infection in human myeloid cells remains unknown. Here we report that in human macrophages, Listeria DNA rather than cyclic‐di‐AMP is stimulating the IFN response via a pathway dependent on the DNA sensors IFI16 and cGAS as well as the signalling adaptor molecule STING. Thus, Listeria DNA is a major trigger of IFNβ expression in human myeloid cells and is sensed to activate a pathway dependent on IFI16, cGAS and STING.     

Identification of key genes and pathways associated with different immune statuses of hepatitis B virus infection

We aimed to identify key genes and pathways associated with different immune statuses of hepatitis B virus (HBV) infection. The gene expression and DNA methylation profiles were analysed in different immune statuses of HBV infection. Differentially expressed genes (DEGs) and differentially methylated genes (DMGs) were identified, followed by their functional and integrative analyses. The differential expression of IgG Fc receptors (FcγRs) in chronic HBV‐infected patients and immune cells during different stages of HBV infection was investigated. Toll‐like receptor (TLR) signalling pathway (including TLR6) and leucocyte transendothelial migration pathway (including integrin subunit beta 1) were enriched during acute infection. Key DEGs, such as FcγR Ib and FcγR Ia, and interferon‐alpha inducible protein 27 showed correlation with alanine aminotransferase levels, and they were differentially expressed between acute and immune‐tolerant phases and between immune‐tolerant and immune‐clearance phases. The integrative analysis of DNA methylation profile showed that lowly methylated and highly expressed genes, including cytotoxic T lymphocyte‐associated protein 4 and mitogen‐activated protein kinase 3 were enriched in T cell receptor signalling pathway during acute infection. Highly methylated and lowly expressed genes, such as Ras association domain family member 1 and cyclin‐dependent kinase inhibitor 2A were identified in chronic infection. Furthermore, differentially expressed FcγR Ia, FcγR IIa and FcγR IIb, CD3^?CD56⁺CD16⁺ natural killer cells and CD14^highCD16⁺ monocytes were identified between immune‐tolerant and immune‐clearance phases by experimental validation. The above genes and pathways may be used to distinguish different immune statuses of HBV infection.     

Rictor regulates the vasculogenic mimicry of melanoma via the AKT‐MMP‐2/9 pathway

Vasculogenic mimicry (VM)‐positive melanomas are usually associated with poor prognosis. Rictor, the key component of the rapamycin‐insensitive complex of mTOR (mTORC2), is up‐regulated in several cancers, especially in melanomas with poor prognosis. The aim of this study was to investigate the role of Rictor in the regulation of VM and the mechanism underlying this possible regulation. VM channels were found in 35 of 81 tested melanoma samples and high Rictor expression correlated with VM structures. Moreover, Kaplan–Meier survival curves indicated that VM structures and high Rictor expression correlated with shorter survival in patients with melanoma. In vitro, Rictor knockdown by short hairpin RNA (shRNA) significantly inhibited the ability of A375 and MUM‐2B melanoma cells to form VM structures, as evidenced by most tubes remaining open. Cell cycle analysis revealed that Rictor knockdown blocked cell growth and resulted in the accumulation of cells in G2/M phase, and cell migration and invasion were greatly affected after Rictor down‐regulation. Western blotting assays indicated that down‐regulating Rictor significantly inhibited the phosphorylation of AKT at Ser⁴⁷³ and Thr³⁰⁸, which subsequently inhibited the expression and activity of downstream MMP‐2/9, as confirmed by real‐time PCR and gelatin Zymography. MK‐2206, a small‐molecule inhibitor of AKT, similarly inhibited the activity of AKT and secretion of MMP‐2/9, further supporting that Rictor down‐regulation inhibits the phosphorylation of AKT and activity of downstream MMP‐2/9 to affect VM formation. In conclusion, Rictor plays an important role in melanoma VM via the Rictor—AKT—MMP‐2/9 signalling pathway.     

Toll-like receptor 2 signalling: Significance in megakaryocyte development through wnt signalling cross-talk and cytokine induction

TLR2 is a toll-like receptor protein which is involved in innate immune responses. TLR2 recognize several virus, fungal and bacterial pathogens, upon their uptake cause internalization and cellular activation. During this process several cytokines participate including interleukins, IL6 and IL12. Interestingly, TLR2 is expressed on megakaryocytes (MKs) and platelets, which is crucial for immune mediated platelet activation. The role of TLR2 on MKs is not completely understood. We observed TLR2 induction leads to MK maturation and is involved in production of ROS which is essential for MK development. In Dami cells, TLR2 up-regulation causes increase in the cytokine production, particularly IL-6, which has been shown to stimulate CFU formation and CD41 expression. Additionally, TLR2 ligand induces wnt β-catenin signalling pathway components suggesting a cross talk between wnt and TLR pathway leading to maturation of MKs. This study shows TLR2 signalling induce cytokine production and regulate wnt signalling thereby cause maturation of MKs.     

Distinct roles of ATR and DNA‐PKcs in triggering DNA damage responses in ATM‐deficient cells

The cellular response to DNA double‐strand breaks involves direct activation of ataxia telangiectasia mutated (ATM) and indirect activation of ataxia telangiectasia and Rad3 related (ATR) in an ATM/Mre11/cell‐cycle‐dependent manner. Here, we report that the crucial checkpoint signalling proteins—p53, structural maintainance of chromosomes 1 (SMC1), p53 binding protein 1 (53BP1), checkpoint kinase (Chk)1 and Chk2—are phosphorylated rapidly by ATR in an ATM/Mre11/cell‐cycle‐independent manner, albeit at low levels. We observed the sequential recruitment of replication protein A (RPA) and ATR to the sites of DNA damage in ATM‐deficient cells, which provides a mechanistic basis for the observed phosphorylations. The recruitment of ATR and consequent phosphorylations do not require Mre11 but are dependent on Exo1. We show that these low levels of phosphorylation are biologically important, as ATM‐deficient cells enforce an early G2/M checkpoint that is ATR‐dependent. ATR is also essential for the late G2 accumulation that is peculiar to irradiated ATM‐deficient cells. Interestingly, phosphorylation of KRAB associated protein 1 (KAP‐1), a protein involved in chromatin remodelling, is mediated by DNA‐dependent protein kinase catalytic subunit (DNA‐PKcs) in a spatio‐temporal manner in addition to ATM. We posit that ATM substrates involved in cell‐cycle checkpoint signalling can be minimally phosphorylated independently by ATR, while a small subset of proteins involved in chromatin remodelling are phosphorylated by DNA‐PKcs in addition to ATM.     

TLR3/TRIF signalling pathway regulates IL‐32 and IFN‐β secretion through activation of RIP‐1 and TRAF in the human cornea

Toll‐like receptor‐3 (TLR3) and RNA helicase retinoic‐acid‐inducible protein‐1 (RIG‐I) serve as cytoplasmic sensors for viral RNA components. In this study, we investigated how the TLR3 and RIG‐I signalling pathway was stimulated by viral infection to produce interleukin (IL)‐32‐mediated pro‐inflammatory cytokines and type I interferon in the corneal epithelium using Epstein–Barr virus (EBV)‐infected human cornea epithelial cells (HCECs/EBV) as a model of viral keratitis. Increased TLR3 and RIG‐I that are responded to EBV‐encoded RNA 1 and 2 (EBER1 and EBER2) induced the secretion of IL‐32‐mediated pro‐inflammatory cytokines and IFN‐β through up‐regulation of TRIF/TRAF family proteins or RIP‐1. TRIF silencing or TLR3 inhibitors more efficiently inhibited sequential phosphorylation of TAK1, TBK1, NF‐κB and IRFs to produce pro‐inflammatory cytokines and IFN‐β than RIG‐I‐siRNA transfection in HCECs/EBV. Blockade of RIP‐1, which connects the TLR3 and RIG‐I pathways, significantly blocked the TLR3/TRIF‐mediated and RIG‐I‐mediated pro‐inflammatory cytokines and IFN‐β production in HCECs/EBV. These findings demonstrate that TLR3/TRIF‐dependent signalling pathway against viral RNA might be a main target to control inflammation and anti‐viral responses in the ocular surface.     

Involvement of the Toll-like receptor 9 signaling pathway in the induction of innate immunity by baculovirus

We have previously shown that mice inoculated intranasally with a wild-type baculovirus (Autographa californica nuclear polyhedrosis virus [AcNPV]) are protected from a lethal challenge by influenza virus. However, the precise mechanism of induction of this protective immune response by the AcNPV treatment remained unclear. Here we show that AcNPV activates immune cells via the Toll-like receptor 9 (TLR9)/MyD88-dependent signaling pathway. The production of inflammatory cytokines was severely reduced in peritoneal macrophages (PECs) and splenic CD11c(+) dendritic cells (DCs) derived from mice deficient in MyD88 or TLR9 after cultivation with AcNPV. In contrast, a significant amount of alpha interferon (IFN-alpha) was still detectable in the PECs and DCs of these mice after stimulation with AcNPV, suggesting that a TLR9/MyD88-independent signaling pathway might also participate in the production of IFN-alpha by AcNPV. Since previous work showed that TLR9 ligands include bacterial DNA and certain oligonucleotides containing unmethylated CpG dinucleotides, we also examined the effect of baculoviral DNA on the induction of innate immunity. Transfection of the murine macrophage cell line RAW264.7 with baculoviral DNA resulted in the production of the inflammatory cytokine, while the removal of envelope glycoproteins from viral particles, UV irradiation of the virus, and pretreatment with purified baculovirus envelope proteins or endosomal maturation inhibitors diminished the induction of the immune response by AcNPV. Together, these results indicate that the internalization of viral DNA via membrane fusion mediated by the viral envelope glycoprotein, as well as endosomal maturation, which releases the viral genome into TLR9-expressing cellular compartments, is necessary for the induction of the innate immune response by AcNPV.