Specific inhibition of mitochondrial oxidative stress suppresses inflammation and improves cardiac function in a rat pneumonia-related sepsis model

Using a mitochondria-targeted vitamin E (Mito-Vit-E) in a rat pneumonia-related sepsis model, we examined the role of mitochondrial reactive oxygen species in sepsis-mediated myocardial inflammation and subsequent cardiac contractile dysfunction. Sepsis was produced in adult male Sprague-Dawley rats via intratracheal injection of S. pneumonia (4 × 10(6) colony formation units per rat). A single dose of Mito-Vit-E, vitamin E, or control vehicle, at 21.5 μmol/kg, was administered 30 min postinoculation. Blood was collected, and heart tissue was harvested at various time points. Mito-Vit-E in vivo distribution was confirmed by mass spectrometry. In cardiac mitochondria, Mito-Vit-E improved total antioxidant capacity and suppressed H(2)O(2) generation, whereas vitamin E offered little effect. In cytosol, both antioxidants decreased H(2)O(2) levels, but only vitamin E strengthened antioxidant capacity. Mito-Vit-E protected mitochondrial structure and function in the heart during sepsis, demonstrated by reduction in lipid and protein oxidation, preservation of mitochondrial membrane integrity, and recovery of respiratory function. While both Mito-Vit-E and vitamin E suppressed sepsis-induced peripheral and myocardial production of proinflammatory cytokines (tumor necrosis factor-α, interleukin-1β, and interleukin-6), Mito-Vit-E exhibited significantly higher efficacy (P < 0.05). Stronger anti-inflammatory action of Mito-Vit-E was further shown by its near-complete inhibition of sepsis-induced myeloperoxidase accumulation in myocardium, suggesting its effect on neutrophil infiltration. Echocardiography analysis indicated that Mito-Vit-E ameliorated cardiac contractility of sepsis animals, shown by improved fractional shortening and ejection fraction. Together, our data suggest that targeted scavenging of mitochondrial reactive oxygen species protects mitochondrial function, attenuates tissue-level inflammation, and improves whole organ activities in the heart during sepsis.     

Effect of Roux-en-Y Gastric Bypass on the NLRP3 Inflammasome in Adipose Tissue from Obese Rats

Objective

Obesity is associated with low-grade chronic inflammation. We hypothesized that Roux-en-Y gastric bypass (RYGB) surgery would reduce activation of the NLRP3 inflammasome in metabolically active adipose tissue (AT) of obese rats, and this change would be related to decreases in body weight and improved glycemic control.

Methods

Omental, mesenteric and subcutaneous fat depots were collected from Sprague-Dawley rats: Sham control and RYGB; 90-days after surgery. NLRP3, caspase–1, apoptosis-associated speck-like protein (ASC), IL–1β, IL–18, IL–6 and MCP–1 gene and protein expression were quantified. Glucose metabolism was assessed by oral glucose tolerance test (OGTT).

Results

Compared to Sham surgery controls, RYGB surgery decreased IL–6, MCP–1, NLRP3, IL–18, caspase–1 and ASC in omental fat, and decreased IL–6, MCP1, IL–1β, IL–18, caspase–1 and ASC gene expression in mesenteric fat. We observed differential gene expression between visceral and subcutaneous fat for IL–6 and IL–1β, both being downregulated by RYGB in visceral, and upregulated in subcutaneous depots. These changes in gene expression were accompanied by a decrease in NLRP3, ASC, IL–18, caspase–1 and IL–1β protein expression in omental tissue. We found a positive correlation between caspase–1, ASC, MCP–1, IL–18 and IL–6 gene expression following surgery and glucose AUC response in omental fat, while the change in glucose AUC response correlated with caspase–1 gene expression in subcutaneous fat.

Conclusion

This study demonstrates that bariatric surgery reverses inflammation in visceral adipose tissue by suppressing NLRP3 inflammasome activation. These are the first data to implicate the NLRP3 inflammasome in diabetes remission after RYGB surgery.     

The Adaptor Protein Myd88 Is a Key Signaling Molecule in the Pathogenesis of Irinotecan-Induced Intestinal Mucositis

Intestinal mucositis is a common side effect of irinotecan-based anticancer regimens. Mucositis causes cell damage, bacterial/endotoxin translocation and production of cytokines including IL–1 and IL–18. These molecules and toll-like receptors (TLRs) activate a common signaling pathway that involves the Myeloid Differentiation adaptor protein, MyD88, whose role in intestinal mucositis is unknown. Then, we evaluated the involvement of TLRs and MyD88 in the pathogenesis of irinotecan-induced intestinal mucositis. MyD88-, TLR2- or TLR9-knockout mice and C57BL/6 (WT) mice were given either saline or irinotecan (75 mg/kg, i.p. for 4 days). On day 7, animal survival, diarrhea and bacteremia were assessed, and following euthanasia, samples of the ileum were obtained for morphometric analysis, myeloperoxidase (MPO) assay and measurement of pro-inflammatory markers. Irinotecan reduced the animal survival (50%) and induced a pronounced diarrhea, increased bacteremia, neutrophil accumulation in the intestinal tissue, intestinal damage and more than twofold increased expression of MyD88 (200%), TLR9 (400%), TRAF6 (236%), IL–1β (405%), IL–18 (365%), COX–2 (2,777%) and NF-κB (245%) in the WT animals when compared with saline-injected group (P<0.05). Genetic deletion of MyD88, TLR2 or TLR9 effectively controlled the signs of intestinal injury when compared with irinotecan-administered WT controls (P<0.05). In contrast to the MyD88^-/- and TLR2^-/- mice, the irinotecan-injected TLR9^-/- mice showed a reduced survival, a marked diarrhea and an enhanced expression of IL–18 versus irinotecan-injected WT controls. Additionally, the expression of MyD88 was reduced in the TLR2^-/- or TLR9^-/- mice. This study shows a critical role of the MyD88-mediated TLR2 and TLR9 signaling in the pathogenesis of irinotecan-induced intestinal mucositis.     

The complex roles of neutrophils in APAP‐induced liver injury

Acetaminophen (APAP) is a widely applied drug for the alleviation of pain and fever, which is also a dose‐depedent toxin. APAP‐induced acute liver injury has become one of the primary causes of liver failure which is an increasingly serious threat to human health. Neutrophils are the major immune cells in human serving as the first barrier against the invasion of pathogen. It has been reported that neutrophils patriciate in the occurrence and development of APAP‐induced liver injury. However, evolving evidences suggest that neutrophils also contribute to tissue repair and actively orchestrate resolution of inflammation. Here, we addressed the complex roles in APAP‐induced liver injury on the basis of brief introduction of neutrophil''s activation, recruitment and migration.

A lot of factors deeply influence the neutrophil''s activation, recruitment and migration in APAP‐induced liver injury. DAMPs like HMGB1 and ATP are released from damaged hepatocytes, recognized by RAGE, P2Y2 receptors, respectively, inducing the activation and recruitment of neutrophils. But the TLR9 expressed on neutrophils recognize mtDNA and then neutrophils could release miR‐223, which inhibits the neutrophil''s activation and recruitment. As for other immune cells, they are divided into cells that promote and inhibit the activation and recruitment of neutrophils. Firstly, macrophages express OPN after APAP treatment and then attract neutrophils. Moreover, HMGB1 could recognize TLR4 receptor expressed on macrophages, release IL‐23 to induce the release of IL‐17A in γδ T cells and finally activate and recruit neutrophils. However, Ly6C^hi monocytes inhibit the activation and recruitment of neutrophils via CCR2 and M‐CSF pathways. Macrophages could also generate MerTK to inhibit neutrophils. Furthermore, TNF‐α/LPS MDSCs could express iNOS to decrease the intrahepatic neutrophils infiltration and induce the apoptosis of activated neutrophil. Activated and recruited neutrophils then migrate to the site of injury to alleviate the inflammation and induce tissue repair. GRPR antagonist could downregulate the expression of CD11b/CD66b via activating MAPKs pathways, which significantly influence the migration of neutrophils to the injury site.     

MicroRNA-363-3p/sphingosine-1-phosphate receptor 1 axis inhibits sepsis-induced acute lung injury via the inactivation of nuclear factor kappa-B ligand signaling

Infection-associated inflammation and coagulation are critical pathologies in sepsis-induced acute lung injury (ALI). This study aimed to investigate the effects of microRNA-363-3p (miR-363-3p) on sepsis-induced ALI and explore the underlying mechanisms. A cecal ligation and puncture-induced septic mouse model was established. The results of this study suggested that miR-363-3p was highly expressed in lung tissues of septic mice. Knockdown of miR-363-3p attenuated sepsis-induced histopathological damage, the inflammation response and oxidative stress in lung tissues. Furthermore, knockdown of miR-363-3p reduced the formation of platelet-derived microparticles and thrombin generation in blood samples of septic mice. Downregulation of miR-363-3p suppressed sphingosine-1-phosphate receptor 1 (S1PR1) expression in lung tissues and subsequently inactivated the nuclear factor kappa-B ligand (NF-κB) signaling. A luciferase reporter assay confirmed that miR-363-3p directly targeted the 3’-untranslated region of the mouse S1pr1 mRNA. Collectively, our study suggests that inactivation of NF-κB signaling is involved in the miR-363-3p/S1PR1 axis-mediated protective effect on septic ALI.     

Toll-Like Receptor 9 Mediated Responses in Cardiac Fibroblasts

Altered cardiac Toll-like receptor 9 (TLR9) signaling is important in several experimental cardiovascular disorders. These studies have predominantly focused on cardiac myocytes or the heart as a whole. Cardiac fibroblasts have recently been attributed increasing significance in mediating inflammatory signaling. However, putative TLR9-signaling through cardiac fibroblasts remains non-investigated. Thus, our aim was to explore TLR9-signaling in cardiac fibroblasts and investigate the consequence of such receptor activity on classical cardiac fibroblast cellular functions. Cultivated murine cardiac fibroblasts were stimulated with different TLR9 agonists (CpG A, B and C) and assayed for the secretion of inflammatory cytokines (tumor necrosis factor α [TNFα], CXCL2 and interferon α/β). Expression of functional cardiac fibroblast TLR9 was proven as stimulation with CpG B and –C caused significant CXCL2 and TNFα-release. These responses were TLR9-specific as complete inhibition of receptor-stimulated responses was achieved by co-treatment with a TLR9-antagonist (ODN 2088) or chloroquine diphosphate. TLR9-stimulated responses were also found more potent in cardiac fibroblasts when compared with classical innate immune cells. Stimulation of cardiac fibroblasts TLR9 was also found to attenuate migration and proliferation, but did not influence myofibroblast differentiation in vitro. Finally, results from in vivo TLR9-stimulation with subsequent fractionation of specific cardiac cell-types (cardiac myocytes, CD45+ cells, CD31+ cells and cardiac fibroblast-enriched cell-fractions) corroborated our in vitro data and provided evidence of differentiated cell-specific cardiac responses. Thus, we conclude that cardiac fibroblast may constitute a significant TLR9 responder cell within the myocardium and, further, that such receptor activity may impact important cardiac fibroblast cellular functions.     

HIV-Derived ssRNA Binds to TLR8 to Induce Inflammation-Driven Macrophage Foam Cell Formation

Even though combined anti-retroviral therapy (cART) dramatically improves patient survival, they remain at a higher risk of being afflicted with non-infectious complications such as cardiovascular disease (CVD). This increased risk is linked to persistent inflammation and chronic immune activation. In this study, we assessed whether this complication is related to HIV-derived ssRNAs inducing in macrophages increases in TNFα release through TLR8 activation leading to foam cell formation. HIV ssRNAs induced foam cell formation in monocyte-derived macrophages (MDMs) in a dose-dependent manner. This response was reduced when either endocytosis or endosomal acidification was inhibited by dynasore or chloroquine, respectively. Using a flow cytometry FRET assay, we demonstrated that ssRNAs bind to TLR8 in HEK cells. In MDMs, ssRNAs triggered a TLR8-mediated inflammatory response that ultimately lead to foam cell formation. Targeted silencing of the TLR8 and MYD88 genes reduced foam cell formation. Furthermore, foam cell formation induced by these ssRNAs was blocked by an anti-TNFα neutralizing antibody. Taken together in MDMs, HIV ssRNAs are internalized; bind TLR8 in the endosome followed by endosomal acidification. TLR8 signaling then triggers TNFα release and ultimately leads to foam cell formation. As this response was inhibited by a blocking anti-TNFα antibody, drug targeting HIV ssRNA-driven TLR8 activation may serve as a potential therapeutic target to reduce chronic immune activation and inflammation leading to CVD in HIV+ patients.     

TLR2 and TLR7 mediate distinct immunopathological and antiviral plasmacytoid dendritic cell responses to SARS‐CoV‐2 infection

Understanding the molecular pathways driving the acute antiviral and inflammatory response to SARS‐CoV‐2 infection is critical for developing treatments for severe COVID‐19. Here, we find decreasing number of circulating plasmacytoid dendritic cells (pDCs) in COVID‐19 patients early after symptom onset, correlating with disease severity. pDC depletion is transient and coincides with decreased expression of antiviral type I IFNα and of systemic inflammatory cytokines CXCL10 and IL‐6. Using an in vitro stem cell‐based human pDC model, we further demonstrate that pDCs, while not supporting SARS‐CoV‐2 replication, directly sense the virus and in response produce multiple antiviral (interferons: IFNα and IFNλ1) and inflammatory (IL‐6, IL‐8, CXCL10) cytokines that protect epithelial cells from de novo SARS‐CoV‐2 infection. Via targeted deletion of virus‐recognition innate immune pathways, we identify TLR7‐MyD88 signaling as crucial for production of antiviral interferons (IFNs), whereas Toll‐like receptor (TLR)2 is responsible for the inflammatory IL‐6 response. We further show that SARS‐CoV‐2 engages the receptor neuropilin‐1 on pDCs to selectively mitigate the antiviral interferon response, but not the IL‐6 response, suggesting neuropilin‐1 as potential therapeutic target for stimulation of TLR7‐mediated antiviral protection.     

Cask methylation involved in the injury of insulin secretion function caused by interleukin1‐β

Islet inflammation severely impairs pancreatic β‐cell function, but the specific mechanisms are still unclear. Interleukin1‐β (IL‐1β), an essential inflammatory factor, exerts a vital role in multiple physio‐pathologic processes, including diabetes. Calcium/calmodulin‐dependent serine protein kinase (CASK) is an important regulator especially in insulin secretion process. This study aims to unveil the function of CASK in IL‐1β–induced insulin secretion dysfunction and the possible mechanism thereof. Islets of Sprague‐Dawley (SD) rats and INS‐1 cells stimulated with IL‐1β were utilized as models of chronic inflammation. Insulin secretion function associated with Cask and DNA methyltransferases (DNMT) expression were assessed. The possible mechanisms of IL‐1β‐induced pancreatic β‐cell dysfunction were also explored. In this study, CASK overexpression effectively improved IL‐1β‐induced islet β‐cells dysfunction, increased insulin secretion. DNA methyltransferases and the level of methylation in the promoter region of Cask were elevated after IL‐1β administration. Methyltransferase inhibitor 5‐Aza‐2’‐deoxycytidine (5‐Aza‐dC) and si‐DNMTs partially up‐regulated CASK expression and reversed potassium stimulated insulin secretion (KSIS) and glucose‐stimulated insulin secretion (GSIS) function under IL‐1β treatment in INS‐1 and rat islets. These results reveal a previously unknown effect of IL‐1β on insulin secretion dysfunction and demonstrate a novel pathway for Cask silencing based on activation of DNA methyltransferases via inducible nitric oxide synthase (iNOS) and modification of gene promoter methylation.     

Kaposi's Sarcoma-Associated Herpesvirus MicroRNAs Target IRAK1 and MYD88, Two Components of the Toll-Like Receptor/Interleukin-1R Signaling Cascade,To Reduce Inflammatory-Cytokine Expression

Kaposi''s sarcoma (KS)-associated herpesvirus (KSHV) is the causative agent of KS, an important AIDS-associated malignancy. KSHV expresses at least 18 different mature microRNAs (miRNAs). We identified interleukin-1 receptor (IL-1R)-associated kinase 1 (IRAK1) as a potential target of miR-K12-9 (miR-K9) in an array data set examining changes in cellular gene expression levels in the presence of KSHV miRNAs. Using 3′-untranslated region (3′UTR) luciferase reporter assays, we confirmed that miR-K9 and other miRNAs inhibit IRAK1 expression. In addition, IRAK1 expression is downregulated in cells transfected with miR-K9 and during de novo KSHV infection. IRAK1 is an important component of the Toll-like receptor (TLR)/IL-1R signaling cascade. The downregulation of IRAK1 by miR-K9 resulted in the decreased stimulation of NF-κB activity in endothelial cells treated with IL-1α and in B cells treated with a TLR7/8 agonist. Interestingly, miR-K9 had a greater effect on NF-κB activity than did a small interfering RNA (siRNA) targeting IRAK1 despite the more efficient downregulation of IRAK1 expression with the siRNA. We hypothesized that KSHV miRNAs may also be regulating a second component of the TLR/IL-1R signaling cascade, resulting in a stronger phenotype. Reanalysis of the array data set identified myeloid differentiation primary response protein 88 (MYD88) as an additional potential target. 3′UTR luciferase reporter assays and Western blot analysis confirmed the targeting of MYD88 by miR-K5. The presence of miR-K9 and miR-K5 inhibited the production of IL-6 and IL-8 upon the IL-1α stimulation of endothelial cells. These results demonstrate KSHV-encoded miRNAs regulating the TLR/IL-1R signaling cascade at two distinct points and suggest the importance of these pathways during viral infection.     

NLRP3 Gene Silencing Ameliorates Diabetic Cardiomyopathy in a Type 2 Diabetes Rat Model

Background

Nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome is associated with metabolic disorder and cell death, which are important triggers in diabetic cardiomyopathy (DCM). We aimed to explore whether NLRP3 inflammasome activation contributes to DCM and the mechanism involved.

Methods

Type 2 diabetic rat model was induced by high fat diet and low dose streptozotocin. The characteristics of type 2 DCM were evaluated by metabolic tests, echocardiography and histopathology. Gene silencing therapy was used to investigate the role of NLRP3 in the pathogenesis of DCM. High glucose treated H9c2 cardiomyocytes were used to determine the mechanism by which NLRP3 modulated the DCM. The cell death in vitro was detected by TUNEL and EthD-III staining. TXNIP-siRNA and pharmacological inhibitors of ROS and NF-kB were used to explore the mechanism of NLRP3 inflammasome activation.

Results

Diabetic rats showed severe metabolic disorder, cardiac inflammation, cell death, disorganized ultrastructure, fibrosis and excessive activation of NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), pro-caspase-1, activated caspase-1 and mature interleukin-1β (IL-1β). Evidence for pyroptosis was found in vivo, and the caspase-1 dependent pyroptosis was found in vitro. Silencing of NLRP3 in vivo did not attenuate systemic metabolic disturbances. However, NLRP3 gene silencing therapy ameliorated cardiac inflammation, pyroptosis, fibrosis and cardiac function. Silencing of NLRP3 in H9c2 cardiomyocytes suppressed pyroptosis under high glucose. ROS inhibition markedly decreased nuclear factor-kB (NF-kB) phosphorylation, thioredoxin interacting/inhibiting protein (TXNIP), NLRP3 inflammasome, and mature IL-1β in high glucose treated H9c2 cells. Inhibition of NF-kB reduced the activation of NLRP3 inflammasome. TXNIP-siRNA decreased the activation of caspase-1 and IL-1β.

Conclusion

NLRP3 inflammasome contributed to the development of DCM. NF-κB and TXNIP mediated the ROS-induced caspase-1 and IL-1β activation, which are the effectors of NLRP3 inflammasome. NLRP3 gene silencing may exert a protective effect on DCM.     

TNFalpha mediates sepsis-induced impairment of basal and leucine-stimulated signaling via S6K1 and eIF4E in cardiac muscle

Decreased translation initiation adversely impacts protein synthesis and contributes to the myocardial dysfunction produced by sepsis. Therefore, the purpose of the present study was to identify sepsis-induced changes in signal transduction pathways known to regulate translation initiation in cardiac muscle and to determine whether the stimulatory effects of leucine can reverse the observed defects. To address this aim, sepsis was produced by cecal ligation and puncture (CLP) in anesthetized rats and the animals studied in the fasted condition 24 h later. Separate groups of septic and time-matched control rats also received an oral gavage of leucine. To identify potential mechanisms responsible for regulating cap-dependent mRNA translation in cardiac muscle, several eukaryotic initiation factors (eIFs) were examined. Under basal conditions, hearts from septic rats demonstrated a redistribution of the rate-limiting factor eIF4E due to increased binding of the translational repressor 4E-BP1 with eIF4E. However, this change was independent of an alteration in the phosphorylation state of 4E-BP1. The phosphorylation of mTOR, S6K1, the ribosomal protein (rp) S6, and eIF4G was not altered in hearts from septic rats under basal conditions. In control rats, leucine failed to alter eIF4E distribution but increased the phosphorylation of S6K1 and S6. In contrast, in hearts from septic rats leucine acutely reversed the alterations in eIF4E distribution. However, the ability of leucine to increase S6K1 and rpS6 phosphorylation in septic hearts was blunted. Sepsis increased the content of tumor necrosis factor (TNF)-alpha in heart and pre-treatment of rats with a TNF antagonist prevented the above-mentioned sepsis-induced changes. These data indicate that oral administration of leucine acutely reverses sepsis-induced alterations eIF4E distribution observed under basal conditions but the anabolic actions of this amino acid on S6K1 and rpS6 phosphorylation remain blunted, providing evidence for a leucine resistance. Finally, TNFalpha, either directly or indirectly, appears to mediate the sepsis-induced defects in myocardial translation initiation.     

Conditional Knockout of Prolyl Hydroxylase Domain Protein 2 Attenuates High Fat-Diet-Induced Cardiac Dysfunction in Mice

Oxygen sensor prolyl hydroxylases (PHDs) play important roles in the regulation of HIF-α and cell metabolisms. This study was designed to investigate the direct role of PHD2 in high fat-diet (HFD)-induced cardiac dysfunction. In HFD fed mice, PHD2 expression was increased without significant changes in PHD1 and PHD3 levels in the heart. This was accompanied by a significant upregulation of myeloid differentiation factor 88 (MYD88) and NF-κB. To explore the role of PHD2 in HFD-induced cardiac dysfunction, PHD2 conditional knockout mice were fed a HFD for 16 weeks. Intriguingly, knockout of PHD2 significantly reduced MYD88 and NF-κb expression in HFD mouse hearts. Moreover, knockout of PHD2 inhibited TNFα and ICAM-1 expression, and reduced cell apoptosis and macrophage infiltration in HFD mice. This was accompanied by a significant improvement of cardiac function. Most importantly, conditional knockout of PHD2 at late stage in HFD mice significantly improved glucose tolerance and reversed cardiac dysfunction. Our studies demonstrate that PHD2 activity is a critical contributor to the HFD-induced cardiac dysfunction. Inhibition of PHD2 attenuates HFD-induced cardiac dysfunction by a mechanism involving suppression of MYD88/NF-κb pathway and inflammation.     

The stiffness‐controlled release of interleukin‐6 by cardiac fibroblasts is dependent on integrin α2β1

Cardiac fibroblasts are able to sense the rigidity of their environment. The present study examines whether the stiffness of the substrate in cardiac fibroblast culture can influence the release of interleukin‐6 (IL‐6), interleukin‐11 (IL‐11) and soluble receptor of IL‐6 (sIL‐6R). It also examines the roles of integrin α2β1 activation and intracellular signalling in these processes. Cardiac fibroblasts were cultured on polyacrylamide gels and grafted to collagen, with an elasticity of E = 2.23 ± 0.8 kPa (soft gel) and E = 8.28 ± 1.06 kPa (stiff gel, measured by Atomic Force Microscope). Flow cytometry and ELISA demonstrated that the fibroblasts cultured on the soft gel demonstrated higher expression of the α2 integrin subunit and increased α2β1 integrin count and released higher levels of IL‐6 and sIL‐6R than those on the stiff gel. Substrate elasticity did not modify fibroblast IL‐11 content. The silencing of the α2 integrin subunit decreased the release of IL‐6. Similar effects were induced by TC‐I 15 (an α2β1 integrin inhibitor). The IL‐6 levels in the serum and heart were markedly lower in α2 integrin‐deficient mice B6.Cg‐Itga2^{tm1.1Tkun/tm1.1Tkun} than wild type. Inhibition of Src kinase by AZM 475271 modifies the IL‐6 level. sIL‐6R secretion is not dependent on α2β1 integrin. Conclusion: The elastic properties of the substrate influence the release of IL‐6 by cardiac fibroblasts, and this effect is dependent on α2β1 integrin and kinase Src activation.     

The Epithelial αvβ3-Integrin Boosts the MYD88-Dependent TLR2 Signaling in Response to Viral and Bacterial Components

TLR2 is a cell surface receptor which elicits an immediate response to a wide repertoire of bacteria and viruses. Its response is usually thought to be proinflammatory rather than an antiviral. In monocytic cells TLR2 cooperates with coreceptors, e.g. CD14, CD36 and αMβ2-integrin. In an earlier work we showed that αvβ3-integrin acts in concert with TLR2 to elicit an innate response to HSV, and to lipopolysaccharide. This response is characterized by production of IFN-α and -β, a specific set of cytokines, and NF-κB activation. We investigated the basis of the cooperation between αvβ3-integrin and TLR2. We report that β3-integrin participates by signaling through Y residues located in the C-tail, known to be involved in signaling activity. αvβ3-integrin boosts the MYD88-dependent TLR2 signaling and IRAK4 phosphorylation in 293T and in epithelial, keratinocytic and neuronal cell lines. The replication of ICP0minus HSV is greatly enhanced by DN versions of MYD88, of Akt – a hub of this pathway, or by β3integrin-silencing. αvβ3-integrin enables the recruitment of TLR2, MAL, MYD88 at lipid rafts, the platforms from where the signaling starts. The PAMP of the HSV-induced innate response is the gH/gL virion glycoprotein, which interacts with αvβ3-integrin and TLR2 independently one of the other, and cross-links the two receptors. Given the preferential distribution of αvβ3-integrin to epithelial cells, we propose that αvβ3-integrin serves as coreceptor of TLR2 in these cells. The results open the possibility that TLR2 makes use of coreceptors in a variety of cells to broaden its spectrum of activity and tissue specificity.