Role Of Toll-like Receptors Research Articles

Toll-Like Receptor Agonists Modulate Wound Regeneration in Airway Epithelial Cells.

Background: Impaired regeneration of airway epithelium may lead to persistence of inflammation and remodelling. Regeneration of injured epithelium is a complex phenomenon and the role of toll-like receptors (TLRs) in the stimulation of respiratory virus products in this process has not been established. Objective: This study was undertaken to test the hypothesis that the wound repair process in airway epithelium is modulated by microbial products via toll-like receptors. Methods: Injured and not-injured bronchial epithelial cells (ECs) (BEAS-2B line) were incubated with the TLR agonists poly(I:C), lipopolisacharide (LPS), allergen Der p1, and supernatants from virus-infected epithelial cells, either alone or in combination with TLR inhibitors. Regeneration and immune response in injured and not-injured cells were studied. Results: Addition of either poly(I:C) or LPS to ECs induced a marked inhibition of wound repair. Supernatants from RV1b-infected cells also decreased regeneration. Preincubation of injured and not-injured ECs with TLR inhibitors decreased LPS and poly(I:C)-induced repair inhibition. TGF-β and RANTES mRNA expression was higher in injured ECs and IFN-α, IFN-β, IL-8, and VEGF mRNA expression was lower in damaged epithelium as compared to not-injured. Stimulation with poly(I:C) increased IFN-α and IFN-β mRNA expression in injured cells, and LPS stimulation decreased interferons mRNA expression both in not-injured and injured ECs. Conclusion: Regeneration of the airway epithelium is modulated by microbial products via toll-like receptors.

The Role of Toll-Like Receptor 2 and 4 Innate Immunity Pathways in Intracortical Microelectrode-Induced Neuroinflammation.

We have recently demonstrated that partial inhibition of the cluster of differentiation 14 (CD14) innate immunity co-receptor pathway improves the long-term performance of intracortical microelectrodes better than complete inhibition. We hypothesized that partial activation of the CD14 pathway was critical to a neuroprotective response to the injury associated with initial and sustained device implantation. Therefore, here we investigated the role of two innate immunity receptors that closely interact with CD14 in inflammatory activation. We implanted silicon planar non-recording neural probes into knockout mice lacking Toll-like receptor 2 (Tlr2−/−), knockout mice lacking Toll-like receptor 4 (Tlr4−/−), and wildtype (WT) control mice, and evaluated endpoint histology at 2 and 16 weeks after implantation. Tlr4−/− mice exhibited significantly lower BBB permeability at acute and chronic time points, but also demonstrated significantly lower neuronal survival at the chronic time point. Inhibition of the Toll-like receptor 2 (TLR2) pathway had no significant effect compared to control animals. Additionally, when investigating the maturation of the neuroinflammatory response from 2 to 16 weeks, transgenic knockout mice exhibited similar histological trends to WT controls, except that knockout mice did not exhibit changes in microglia and macrophage activation over time. Together, our results indicate that complete genetic removal of Toll-like receptor 4 (TLR4) was detrimental to the integration of intracortical neural probes, while inhibition of TLR2 had no impact within the tests performed in this study. Therefore, approaches focusing on incomplete or acute inhibition of TLR4 may still improve intracortical microelectrode integration and long term recording performance.

Toll-like receptor 9 negatively regulates pancreatic islet beta cell growth and function in a mouse model of type 1 diabetes

Aims/hypothesisInnate immune effectors interact with the environment to contribute to the pathogenesis of the autoimmune disease, type 1 diabetes. Although recent studies have suggested that innate immune Toll-like receptors (TLRs) are involved in tissue development, little is known about the role of TLRs in tissue development, compared with autoimmunity. We aimed to fill the knowledge gap by investigating the role of TLR9 in the development and function of islet beta cells in type 1 diabetes, using NOD mice.MethodsWe generated Tlr9−/− NOD mice and examined them for type 1 diabetes development and beta cell function, including insulin secretion and glucose tolerance. We assessed islet and beta cell number and characterised CD140a expression on beta cells by flow cytometry. We also tested beta cell function in Tlr9−/− C57BL/6 mice. Finally, we used TLR9 antagonists to block TLR9 signalling in wild-type NOD mice to verify the role of TLR9 in beta cell development and function.ResultsTLR9 deficiency promoted pancreatic islet development and beta cell differentiation, leading to enhanced glucose tolerance, improved insulin sensitivity and enhanced first-phase insulin secretory response. This was, in part, mediated by upregulation of CD140a (also known as platelet-derived growth factor receptor-α [PDGFRα]). In the absence of TLR9, induced by either genetic targeting or treatment with TLR9 antagonists, which had similar effects on ontogenesis and function of beta cells, NOD mice were protected from diabetes.Conclusions/interpretationOur study links TLR9 and the CD140a pathway in regulating islet beta cell development and function and indicates a potential therapeutic target for diabetes prevention and/or treatment.

The Role of Toll-Like Receptor in Inflammation and Tumor Immunity.

Toll-like receptors (TLRs) activation enables host to recognize a large number of pathogen-associated molecule patterns (PAMPs), ignite immune cells to discriminate between self and non-self, and then promote the following innate and adaptive immune responses. Accumulated clinical/preclinical evidences have proven TLRs to be critical role in the autoimmune diseases, including inflammatory and tumor-associated diseases. Activation of TLRs is becoming or has been a target for cancer treatment. It is shown that TLRs can induce preferable anti-tumor effect by eliciting inflammatory cytokines expression and cytotoxic T lymphocytes (CTLs) response. As adjuvant, TLRs agonists can launch a strong immune response to assist cancer radiotherapy and bio-chemotherapy. On the other hand, tumor-associated antigens acting as PAMPs, can also activate TLRs and induce tumor gene-related programmed cell death, including apoptosis, autophagy and programmed necrosis. While there are also arguments that the excessive TLRs expression will promote tumor deterioration in various organisms, as the TLR-induced inflammation will accelerate the cancer cells boost in the tumor microenvironment (TME). However, the effect of TLRs acting on cancers is still not quite clear today. In this review, we will summarize the recent researches of TLRs in cancer treatment and their role in TME, giving a brief overview on future expectation.

Role of Toll-like receptor 1, 2 and 4 on thromboxane B2 production by different microbial products

Role of Toll-like receptor 1, 2 and 4 on thromboxane B2 production by different microbial products

Role of Toll-like receptor 2 in mediating the production of cytokines and human beta-defensins in oral mucosal epithelial cell response to Leptospiral infection.

Pathogenic Leptospira spp. is the causative agent of leptospirosis. Oral mucosal cavity is one of portal entry for this bacterium. Oral mucosal epithelium provides a physical barrier and secretes cytokines, chemokines and antimicrobial peptides (AMPs) in response to microbial infection. Human β-defensins (hBDs); hBD1, hBD2, and hBD3 are predominantly AMPs expressed in the oral cavity. Toll-like receptors (TLRs) have been reported in hBD regulation. TLR2 recognizes leptospiral lipopolysaccharide, and plays a key role in the early control of leptospirosis. The aim of this study is to investigate the role of TLR2 in mediating the production of cytokines and hBDs in oral mucosal epithelial cell response to leptospiral infection. Cultivated oral mucosal epithelial cells were prepared, characterized, and compared with oral mucosal tissues. The TLR1-10 and hBD mRNA expressions were examined. Pro-inflammatory cytokine and hBD1-3 expressions in response to leptospires were determined by quantitative (q) RT-PCR. The cultivated oral epithelium expressed TLR2 and hBD1-3. The induction of IL-βIL-8, TNF-α, and hBD2 were increased in response to Leptospira via TLR2 recognition. The characteristics of primary epithelial cells and tissue were similar in terms of TLR expression. All primary epithelial cells expressed TLR2 and hBD1-3. We used primary epithelial cells to study response to L. interrogans. Our results yielded the first evidence that human TLR2 regulates hBD2 expression in oral mucosa epithelial responded to L. interrogans. Expression of hBD2 may act to neutralize the virulence or prevent the invasion of L. interrogans at the portal of entry.

MiR-182-5p Attenuates High-Fat -Diet-Induced Nonalcoholic Steatohepatitis in Mice

Introduction and Aim: Patients with NASH have increased risk for sepsis or cardiovascular disease after Liver transplantation. An important role of Toll-like receptor (TLR) 4 in the pathogenesis of nonalcoholic steatohepatitis (NASH) was demonstrated. Here, we study the role of miR-182-5p in TLR4 expression and high-fat-diet (HFD)-induced NASH in vitro and in vivo. Methods: Following transfection with a miR-182-5p mimic, the effect of miR-182-5p on TLR4 in RAW264.7 and HepG2 cells was investigated. Following administration of the miR-182-5p mimic into the livers of HFD-induced NASH mice, we determined the in vivo expression of TLR4, TNFα, and IL-6 and assessed the histologic features of the livers. Results: Following lipopolysaccharide (LPS) treatment of RAW264.7 cells, real-time RT-PCR and western blot results indicated decreases levels of TLR4 mRNA and protein in the miR-182-5p group as compared with levels observed in controls, with similar trends were observed in TNFα and IL-6 protein levels. Following oleic acid (OA) treatment of HepG2 cells, TLR4, TNFα, and IL-6 levels were significantly decreased in the miR-182-5p group as compared with levels observed in controls. Following miR-182-5p administration, TLR4 mRNA and protein levels decreased along with those of TNFα and IL-6 proteins, and the liver weight/body weight ratio of treated mice was less than that observed in controls. Furthermore, hematoxylin and eosin staining showed that the miR-182-5p-treated group exhibited low adipose-cell cross-sectional areas, and Oil Red O staining showed decreases in the size of lipid droplets in the miR-182-5p-treated group. Conclusions: miR-182-5p ameliorated HFD-induced NASH by suppressing TLR4.

Conjugation of chitosan oligosaccharides enhances immune response to porcine circovirus vaccine by activating macrophages

Porcine circovirus type 2 (PCV2)-associated diseases have led to great economic losses to the pig industry. Our lab previously found that conjugation of chitosan oligosaccharides (COS) or via a carrier protein enhanced the immunogenicity of PCV2 vaccine against infectious pathogens. However, precise mechanisms and signal transduction pathways underlying the efficacy of COS conjugation remains poorly defined. In this study, to better understand the effects and mechanism of COS conjugates maintain the adjuvant potential in vivo, we investigated its augmentation of macrophage function, including cell activation, NO production, cytokine production and phagocytosis. Additionally, the role of Toll-like receptors (TLR) proteins in this process was also assessed. The results indicate that, as compared to the PCV and PCV/COS, conjugation of COS effectively enhanced the NO production, cytokines generation and phagocytosis activity of macrophages. Noticeably, the generation of NO and proinflammatory cytokines was closely related to the TLR2/4 signaling pathways, strongly suggesting that conjugation of COS regulates innate and adaptive immunity by activation of macrophages, resulting in immune enhancement. In summary, the present study provides a potential mechanism of COS conjugation as a novel adjuvant to improve immune responses against various diseases.

Synergistic induction of interferon-γ by interleukin-2, interleukin-12 and poly(I:C) in a human natural killer cell line

Natural killer (NK) cells express various Toll-like receptors (TLRs). Little is known about the role of TLRs in direct NK cell activation. To clarify possible synergistic roles of cytokine and TLR signaling, human NK cell line KHYG-1 was stimulated with agonists for TLR1-9. IFN-γ production was not significantly induced following stimulation with single TLR agonists. Of the nine TLR agonists tested, only poly(I:C) strongly upregulated IFN-γ production by synergistic interleukin-2 (IL-2) and IL-12 stimulation. The role of TLR3 signaling was also examined. An inhibitor of Toll-IL-1 receptor domain-containing adaptor inducing IFN-β (TRIF) blocked this synergistic action. However, TLR3 expression was unchanged in the presence of IL-2 and IL-12. Our findings suggest a possible role for type 1 cytokines in NK cell IFN-γ production against viral infections.

Abstract LB-083: Targeting IRAK4 disrupts inflammatory pathways and tumor microenvironment in chronic lymphocytic leukemia regardless MYD88 mutational status

Abstract BACKGROUND & AIM. Interleukin-1 receptor-associated kinase 4 (IRAK4) plays a critical role in Toll-like receptor (TLR) signal transduction and the innate immune response. Recruitment of IRAK4 to these receptors and its subsequent activation is facilitated by the MYD88 adaptor protein. Approximately 2-5% of chronic lymphocytic leukemia (CLL) patients have activating mutations of MYD88, a driver mutation in this disease. Here we study the pharmacological inhibition of IRAK4 with ND2158, an IRAK4 competitive inhibitor, as a therapeutic approach in CLL. METHODS. TLR stimulation was performed with an agonist mix of Pam3CSK4, HKLM, FSL1 and ODN2006. Cytokine secretion and CLL proliferation were assessed by Luminex system and carboxyfluorescein succinimidyl ester staining, respectively. For in vivo studies, the Eμ-TCL1 adoptive transfer mouse model was used. RESULTS. TLR repertoire analysis in CLL cells revealed high expression levels of TLR1, TLR7 and TLR10. Gene expression profiling identified enrichment of gene sets related to cytokines and inflammation in the subgroup carrying mutations in MYD88. Accordingly, CLL cells from individuals with MYD88 mutations showed higher p65 NF-κB activity and secreted higher basal levels of CCL2, CCL3 and CCL4 cytokines compared with unmutated cases. ND2158 induced a selective dose-dependent cytotoxic effect in CLL cells, without differences regarding their MYD88 mutational status. In the context of TLR stimulation, ND2158 was able to efficiently decrease the secretion of CCL2, CCL3, CCL4, TNFα, IL1β and IL6 cytokines. NF-κB signaling was also modulated by the drug, as observed by the downregulation of IκBα phosphorylation protein level, p65 translocation to the nucleus and decreased p65 DNA binding activity. ND2158 also impaired CLL proliferation and migration towards CXCL12. In vitro experiments with splenocytes from leukemic Eμ-TCL1 mice corroborated the ND2158 antitumor effects. In vivo studies showed a significant reduction in tumor load in peripheral blood, lymph node, spleen and peritoneal cavity in mice treated with 100 mg/kg/day of the IRAK4 inhibitor. A decrease in spleen weight and size was also detected after the treatment. As monocytes have been shown to support CLL survival, we investigated ND2158 impact on this cell type. A significant reduction in monocyte absolute counts in spleen and skewing to inflammatory Ly6C+ monocytes in peritoneal cavity was detected after ND2158 treatment, thereby disrupting the support for tumor cells. CONCLUSION. Our findings support pharmacological inhibition of IRAK4 as a potential therapeutic strategy in CLL cells regardless of their MYD88 mutational status, indicating that TLR signaling plays an important role in CLL cells development and CLL tumor microenvironment. Citation Format: Laia Rosich, Neus Gimenez, Ralph Schulz, Morihiro Higashi, Marta Aymerich, Monica Lopez, Manel Juan, Martina Seiffert, Elias Campo, Dolors Colomer. Targeting IRAK4 disrupts inflammatory pathways and tumor microenvironment in chronic lymphocytic leukemia regardless MYD88 mutational status [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-083.

Role of Toll-like Receptor 4 Expressed by Fibroblasts in Allograft Fibrosis in Mouse Orthotopic Tracheal Transplantation

Development of chronic lung allograft dysfunction involves various alloimmune-independent insults including those mediated by Toll-like receptor (TLR) signaling, which is known to activate alloimmune responses. We hypothesized that TLR signaling may also contribute to the activation of fibroblasts and promoting allograft airway fibrosis. Mouse orthotopic tracheal transplants were conducted between major histocompatibility complex (MHC)-mismatched Balb/c donor and wild-type C3H or C3H-derived TLR4 mutant recipients (nonfunctional TLR4). Immunohistochemistry on day 21 showed significantly smaller alpha-smooth muscle actin (α-SMA)-positive areas in TLR4 mutant recipients than wild-type recipients (P = .01). No difference was found for CD3+ T-cell infiltration. Proliferation of alloreactive T cells derived from the recipient spleen showed no difference between TLR4 mutant and wild-type recipients in a mixed lymphocyte reaction. The effect of TLR4 signaling was examined in primary pulmonary fibroblast cultures both with lipopolysaccharide (LPS) and transforming growth factor (TGF)-β1. Stimulation with LPS significantly increased expression of α-SMA mRNA in wild-type fibroblasts cultured with TGF-β1 compared with the control without LPS (P = .001). Taken together, these findings suggest disruption of TLR signaling leads to reduced activation of fibroblasts without affecting T-cell infiltration and proliferation in this model. TLR4-mediated activation of fibroblasts may be a potentially important mechanism of allograft remodeling.

Role of Toll-like receptor 9 signaling on activation of nasal polyp-derived fibroblasts and its association with nasal polypogenesis.

Nasal polyposis is characterized by persistent inflammation and remodeling in sinonasal mucosa. Toll-like receptor 9 (TLR9) is a DNA receptor of the innate immune system that plays a pivotal role in fibrosis and inflammatory responses. The aim of this study is to explore the expression, activity, and potential pathogenic role of TLR9 signaling in tissue remodeling in nasal polyp-derived fibroblasts (NPDFs). Fibrotic and inflammatory responses elicited by type A CpG oligonucleotides were examined in the NPDFs by a combination of real-time quantitative polymerase chain reaction, Western blot analysis, enzyme-linked immunosorbent assay, and immunofluorescence staining. For these experiments, the NPDFs were stimulated with different TLR9 agonists (CpG A and B) and blocked with inhibitors (MyD88 inhibitor and chloroquine). TLR9 expression was significantly higher in nasal polyposis (NP) tissues compared to control or chronic rhinosinusitis (CRS) mucosa. In the NPDFs, TLR9 showed intracellular localization and expression of TLR9 was increased after treatment with CpG A. CpG A increased production of α-smooth muscle actin (α-SMA), fibronectin, and matrix metalloproteinases (MMPs) (MMP1, MMP2, and MMP9) in the NPDFs, while MyD88 inhibitor and chloroquine, which are known to block the TLR9 signaling pathway, inhibited their production. CpG A also produced type I interferons (IFN-α and IFN-β), which were inhibited by MyD88 inhibitor. Our data indicates that CpG A-induced fibroblast activation and cytokine production were mediated via TLR9 stimulation in NPDFs. Disrupting this process with an inhibitor targeting TLR9 or its downstream signaling pathways could represent a novel approach to CRS with NP (CRSwNP) therapy.

Role of toll-like receptor 4 (TLR4)-mediated interleukin-6 (IL-6) production in chemotherapy-induced mucositis.

Despite significant advances in our ability to treat cancer, cytotoxic chemotherapy continues to be the mainstay treatment for many solid tumours. Chemotherapy is commonly associated with a raft of largely manageable adverse events; however, gastrointestinal (GI) toxicity (also termed mucositis) remains a significant challenge with little in the way of preventative and therapeutic options. The inability to manage GI complications likely reflects our incomplete understanding of its aetiology and the idiosyncrasies of each chemotherapeutic agent. This review highlights aims to provide a narrative for the involvement of Toll-like receptor (TLR4) in the development of chemotherapy-induced GI mucositis, an already emerging theme within this field. Particular focus will be placed upon the signalling interaction between TLR4 and interleukin (IL)-6. This parallels recent preclinical findings showing that TLR4 knockout mice, which are protected from developing severe GI mucositis, completely lack an IL-6 response. As such, we suggest that this signalling pathway presents as a novel mechanism with potential for therapeutic intervention.

Toll-like receptor 2 signaling induces interferon regulatory factor 8 expression in spinal cord microglia after peripheral nerve injury

Although it is well known that spinal cord microglia activation plays a causal role in the development of neuropathic pain after peripheral nerve injury (PNI), the mechanisms of this activation have not been completely elucidated. We have previously shown that toll-like receptor 2 (TLR2) is involved in PNI-induced spinal cord microglia activation. A more recent study showed that PNI induces interferon regulatory factor 8 (IRF8), which is a key transcription factor responsible for microglia activation. In this study, we investigated the putative role of TLR2 in IRF8 expression in spinal cord microglia after PNI. In TLR2 knockout mice, nerve injury-induced spinal cord IRF8 expression was severely compromised. In addition, an intrathecal injection of lipoteichoic acid (LTA), a TLR2 agonist, induced IRF8 mRNA expression in the spinal cord. Lastly, TLR2 stimulation by LTA induced IRF8 mRNA expression in primary spinal cord glial cells. TLR2-induced IRF8 expression was dependent on p38 MAPK and NF-κB activation. Taken together, these data show that TLR2 activation induces IRF8 expression in spinal cord microglia after PNI.

Role of toll-like receptor 4 Asp299Gly polymorphism in the development of cardiovascular diseases in HIV-infected patients

Cardiovascular diseases (CVDs) are one of the main causes of morbimortality in HIV-infected patients on suppressive antiretroviral therapy. The objective of this work was to evaluate the role of single nucleotide polymorphisms (SNPs) in lipopolysaccharide (LPS) Toll-like receptor 4 (TLR4) and CVDs occurrence in HIV-infected patients. Additionally, the functional consequences of carrying these SNPs were analyzed. The association of TLR4 SNPs, Asp299Gly/Thr399Ile with CVDs occurrence was analyzed using multivariate logistic regression models. Clinical, immunological, and traditional cardiovascular risk factors were used as covariates. The monocyte phenotype and response were assessed by multiparametric flow cytometry comparing carriers with noncarriers of this SNP. Asp299Gly SNP, assayed in 253 HIV-infected patients, was independently associated with the occurrence of CVDs after adjusting for CD4+ T-cell nadir, HCV-coinfection, bacterial pneumonia, diabetes mellitus, and traditional cardiovascular risk factors [odds ratio (confidence interval 95%) = 3.672 (1.061-12.712), P = 0.04). Carriers of Asp299Gly SNP showed higher percentage of patrolling and intermediate monocytes producing a proinflammatory combination of cytokines compared with noncarriers (P = 0.037 and P = 0.046, respectively). Intermediate monocyte subset levels correlated with soluble interleukin-6 levels only in carriers (r = 0.89; P = 0.01). TLR4 Asp299Gly polymorphism is independently associated with the occurrence of CVDs in HIV-infected patients. The proinflammatory profile associated to this variant could be involved in the development of atherosclerotic pathologies.

Role of Toll-Like Receptor 4 on Osteoblast Metabolism and Function.

Inflammation is a process whose main function is to fight against invading pathogens or foreign agents. Nonetheless, it is widely accepted that inflammation takes part in multiple processes in a physiological or pathophysiological context. Among these processes the inflammation has been closely related to bone metabolism. It is well-known that in systemic inflammatory diseases such as rheumatoid arthritis the inflammatory environment contributes to the reduction of the bone mineral density. This has been further evidenced in different animals models of osteoporosis where the deletion of key inflammatory molecules dramatically reduced the bone loss. On the contrary, it is also well-known that certain degree of inflammation is required to allow bone fractures healing. In fact, excessive use of anti-inflammatory drugs inhibits bone fracture consolidation. The innate immune responses (IIRs) contribute to the development and maintenance of the inflammation. These responses have been observed in cells of the musculoskeletal system. Chondrocytes and osteoblasts are equipped with the molecular repertoire necessary to setting up these IIR, including the expression of several toll-like receptors. Specifically, toll-like receptor 4 (TLR4) activation in mesenchymal stem cells, osteoblasts, and osteocytes has been involved in catabolic and anabolic process. Accordingly, in this review we have summarized the current knowledge about the physiology of TLR4, including its signaling, and its endogenous agonists. In addition we have focused on its role on osteoblast metabolism and function.

Role of Toll-like receptor 4 in cardiomyocyte apoptosis induced by hypoxia-reoxygenation in newborn rats

Objective To evaluate the role of Toll-like receptor 4 (TLR4) in cardiomyocyte apoptosis induced by hypoxia-reoxygenation (H/R) in newborn rats. Methods Cardiomyocytes obtained from newborn Sprague-Dawley rats, aged 1-3 days, were primarily cultured in DMEM culture medium supplemented with 20% fetal bovine serum and divided into 4 groups (n=10 each) using a random number table: control group (group C), H/R group, negative control group (con-siRNA group) and TLR4-siRNA group.Cardiomyocytes were routinely cultured for 24 h in group C. Cardiomyocytes were subjected to hypoxia for 2 h followed by 4 h of reoxygenation in group H/R.Cardiomyocytes were transfected with siRNA and TLR4-siRNA at the final concentration of 80 nmol/L in con-siRNA group and TLR4-siRNA group, respectively.After successful establishment of H/R injury model, the cell survival rate was measured by MTT assay, flow cytometry was used to detect apoptosis in cardiomyocytes, the expression of TLR4 mRNA was determined by real-time polymerase chain reaction, and the expression of TLR4, Bcl-2, Bax and caspase-3 was detected by Western blot.The apoptosis rate was calculated. Results Compared with group C, the cell survival rate was significantly decreased, the expression of TLR4 protein and mRNA, caspase-3 and Bax was up-regulated, the apoptosis rate was increased and the expression of Bcl-2 was down-regulated in the other three groups (P<0.01). Compared with H/R and con-siRNA groups, the cell survival rate was significantly increased, the expression of TLR4 protein and mRNA, caspase-3 and Bax was down-regulated, the apoptosis rate was decreased and the expression of Bcl-2 was up-regulated in group TLR4-siRNA (P<0.01). Conclusion TLR4 is involved in cardiomyocyte apoptosis induced by H/R in newborn rats. Key words: Toll-like receptor 4; RNA, small interfering; Myocytes, cardiac; Anoxia; Apoptosis

Evidence that TLR4 Is Not a Receptor for Saturated Fatty Acids but Mediates Lipid-Induced Inflammation by Reprogramming Macrophage Metabolism

Chronic inflammation is a hallmark of obesity and is linked to the development of numerous diseases. The activation of toll-like receptor 4 (TLR4) by long-chain saturated fatty acids (lcSFAs) is an important process in understanding how obesity initiates inflammation. While experimental evidence supports an important role for TLR4 in obesity-induced inflammation invivo, via a mechanism thought to involve direct binding to and activation of TLR4 by lcSFAs, several lines of evidence argue against lcSFAs being direct TLR4 agonists. Using multiple orthogonal approaches, we herein provide evidence that while loss-of-function models confirm that TLR4 does, indeed, regulate lcSFA-induced inflammation, TLR4 is not a receptor for lcSFAs. Rather, we show that TLR4-dependent priming alters cellular metabolism, gene expression, lipid metabolic pathways, and membrane lipid composition, changes that are necessary for lcSFA-induced inflammation. These results reconcile previous discordant observations and challenge the prevailing view of TLR4's role in initiating obesity-induced inflammation.

Association of Tolllike receptor2 and 9 gene variants with pulmonary tuberculosis: exploration in a northern Indian population.

Tuberculosis (TB) is a disease of global importance. There is an increasing recognition of the role of Toll like receptors, important pattern recognition receptors of host immune system, in determining the susceptibility or resistance to TB in various populations. In an attempt to examine the importance of Toll like receptors in immune response to Mycobacterium tuberculosis infection, we explored two variants each of TLR2 and TLR9 in a population residing in Uttar Pradesh, India. Genotyping was performed to detect -196 to -174 del polymorphism and G2258A SNP (Arg753Gln, rs5743708) in TLR2 gene and -T1237C (rs5743836) and G2848A (rs352140) SNP in TLR9 gene in patients with pulmonary TB and healthy controls. The A allele of G2848A SNP in TLR9 gene was found with a marginally higher frequency among TB patients as compared to healthy controls, suggesting that A allele at position 2848 of TLR9 gene may be associated with susceptibility to TB in North Indian population [p = 0.05, Mantel-Haenszel OR = 1.34, 95% CI (1.0-1.82)].

Protective effect of Lactobacillus reuteri DSM 17938 against experimental necrotizing enterocolitis is mediated by Toll-like receptor 2.

Lactobacillus reuteri DSM 17938 (LR 17938) has been shown to reduce the incidence and severity of necrotizing enterocolitis (NEC). It is unclear if preventing NEC by LR 17938 is mediated by Toll-like receptor 2 (TLR2), which is known to mediate proinflammatory responses to bacterial cell wall components. NEC was induced in newborn TLR2-/- or wild-type (WT) mice by the combination of gavage-feeding cow milk-based formula and exposure to hypoxia and cold stress. Treatment groups were administered formula supplemented with LR 17938 or placebo (deMan-Rogosa-Sharpe media). We observed that LR 17938 significantly reduced the incidence of NEC and reduced the percentage of activated effector CD4+T cells, while increasing Foxp3+ regulatory T cells in the intestinal mucosa of WT mice with NEC, but not in TLR2-/- mice. Dendritic cell (DC) activation by LR 17938 was mediated by TLR2. The percentage of tolerogenic DC in the intestine of WT mice was increased by LR 17938 treatment during NEC, a finding not observed in TLR2-/- mice. Furthermore, gut levels of proinflammatory cytokines IL-1β and IFN-γ were decreased after treatment with LR 17938 in WT mice but not in TLR2-/- mice. In conclusion, the combined in vivo and in vitro findings suggest that TLR2 receptors are involved in DC recognition and DC-priming of T cells to protect against NEC after oral administration of LR 17938. Our studies further clarify a major mechanism of probiotic LR 17938 action in preventing NEC by showing that neonatal immune modulation of LR 17938 is mediated by a mechanism requiring TLR2. NEW & NOTEWORTHY Lactobacillus reuteri DSM 17938 (LR 17938) has been shown to protect against necrotizing enterocolitis (NEC) in neonates and in neonatal animal models. The role of Toll-like receptor 2 (TLR2) as a sensor for gram-positive probiotics, activating downstream anti-inflammatory responses is unclear. Our current studies examined TLR2 -/- mice subjected to experimental NEC and demonstrated that the anti-inflammatory effects of LR 17938 are mediated via a mechanism requiring TLR2.