Toll-like Receptor Signal Transduction Research Articles

Selenium Deficiency via the TLR4/TRIF/NF-κB Signaling Pathway Leading to Inflammatory Injury in Chicken Spleen.

The aim of the present study was to investigate the effect of selenium (Se) deficiency on the expression of the toll-like receptor (TLR) signal transduction pathway in the spleen of chickens and explore the relationship between the TLR4/TRIF/NF-κB signaling pathway and inflammatory spleen injury. A total of 200 one-day-old healthy broilers were allocated to two groups. The experimental group was fed a self-made low-Se diet (0.004mg/kg) while the control group was fed a complete formula feed (0.2mg/kg) for 15, 25, 35, 45, and 55days, respectively. We observed histopathological changes in the chicken spleens. The messenger RNA(mRNA) expression levels of 8 kinds of ChTLRs, myeloid differential protein-88 (MyD88), toll-interleukine-1 receptor domain-containing adapter-inducing interferon-β (TRIF), nuclear factor-κB (NF-κB), and cytokine (IL-6, TNF-α, IL-2, and IFN-γ) were detected via quantitative real-time PCR. Western blotting was used to detect the protein expression level of TLR4. Then principal component analysis (PCA) was used to analyze the correlation between the ChTLRs, MyD88, TRIF, and NF-κB. The results showed that the boundary between red pulp and white pulp was unclear, the number of lymphocytes decreased, and the nucleus was fragmented and dissolved in the experimental group at 25-55days. At 15-45days, the relative expression of TLR4 mRNA was higher than in the control group, and the difference was extremely significant on day 15 (P < 0.01).The relative expression of TRIF mRNA in the experimental group was higher than in the control group at 25-55days, and the relative expression of NF-κB mRNA in the experimental group was higher than in the control group at 15-45days. The relative expression of IL-6 mRNA in the experimental group was higher than in the control group at 15-45days. The protein expression level of TLR4 in the experimental group was higher than in the control group at 15-45days. The PCA results showed that there was a strong correlation between TLR4, TRIF, and component 1. The results suggest that TLR4 plays an important role in regulating the expression of inflammatory cytokines in the spleens of Se-deficient chickens, and Se deficiency may cause inflammatory injury through the TLR4/TRIF/NF-κB signaling pathway in chicken spleen.

The role of TLR4 and HO-1 in neuroinflammation after subarachnoid hemorrhage.

This review on the mechanisms of neuroinflammation following subarachnoid hemorrhage will focus mainly on toll‐like receptor 4 (TLR4), Heme Oxygenase‐1 (HO‐1), and the role of microglia and macrophages in this process. Vasospasm has long been the focus of research in SAH; however, clinical trials have shown that amelioration of vasospasm does not lead to an improved clinical outcome. This necessitates the need for novel avenues of research. Our work has demonstrated that microglial TLR4 and microglial HO‐1, not only affects cognitive dysfunction, but also circadian dysrhythmia in a mouse model of SAH. To attempt to translate these findings, we have also begun investigating macrophages in the cerebrospinal fluid of SAH patients. The goal of this review is to provide an update on the role of TLR4, HO‐1, and other signal transduction pathways in SAH‐induced neuroinflammation.

Targeting IRAK4 disrupts inflammatory pathways and delays tumor development in chronic lymphocytic leukemia

Interleukin-1 receptor-associated kinase 4 (IRAK4) plays a critical role in Toll-like receptor (TLR) signal transduction and innate immune responses. Recruitment and subsequent activation of IRAK4 upon TLR stimulation is mediated by the myeloid differentiation primary response 88 (MYD88) adaptor protein. Around 3% of chronic lymphocytic leukemia (CLL) patients have activating mutations of MYD88, a driver mutation in this disease. Here, we studied the effects of TLR activation and the pharmacological inhibition of IRAK4 with ND2158, an IRAK4 competitive inhibitor, as a therapeutic approach in CLL. Our in vitro studies demonstrated that ND2158 preferentially killed CLL cells in a dose-dependent manner. We further observed a decrease in NF-κB and STAT3 signaling, cytokine secretion, proliferation and migration of primary CLL cells from MYD88-mutated and -unmutated cases. In the Eµ-TCL1 adoptive transfer mouse model of CLL, ND2158 delayed tumor progression and modulated the activity of myeloid and T cells. Our findings show the importance of TLR signaling in CLL development and suggest IRAK4 as a therapeutic target for this disease.

The effect of fenofibrate, a PPARα activator on toll-like receptor-4 signal transduction in melanoma both in vitro and in vivo.

The anti-cancer effect of peroxisome proliferator-activated receptor (PPAR) α ligands on growth and metastatic potential of melanoma cells has been shown previously. However, themechanismunderlying these effects remains to be elucidated. Here, we investigated the effects of fenofibrate (PPAR ligand) on Toll-like receptor-4 (TLR-4) signaling in mice melanoma. Mice melanoma cells (B16F10) were treated with fenofibrate or LPS or LPS + fenofibrate or pre-treated with CLI-095 (a TLR4 inhibitor), followed by fenofibrate. In in vivo model, C57BL/6 mice were subcutaneously injected with B16F10 cells (with/without LPS pre-treatment), and fenofibrate was administrated after development of palpable tumors. Cell proliferation, the expression level of Tlr4, Myd88, Nf-κb1 genes, TLR-4 protein expression, TNF-α levels, and tumor volume were measured. Our results indicated that fenofibrate significantly inhibited the Tlr-4, Myd-88, and Nf-kb1 mRNA expression and TNF-α concentration in B16F10 LPS-stimulated cells. In addition, blocking TLR-4 signaling increased the anti-inflammatory potential of fenofibrate. Also fenofibrate can reduce LPS-induced tumor volume, Tlr-4, Myd-88, Nf-kb1 mRNA, and TLR-4 protein expression in tumor tissue and also TNF-α level in tumor tissue lysate. Our data indicate that fenofibrate may exert its anti-melanoma effects via interaction with TLR4-dependent signaling pathway (TLR-4/MyD-88/ NF-kB).

PS1145 TARGETED ACTIVATION OF THE PROTEIN PHOSPHATASES SHP-1 AND PP2A ABROGATES CONSTITUTIVE ACTIVATION OF SURVIVAL PATHWAYS IN LARGE GRANULAR LYMPHOCYTE LEUKEMIA (LGLL)

Background: Interleukin-1 receptor-associated kinase 4 (IRAK4) plays a critical role in Toll-like receptor (TLR) signal transduction and innate immune responses. Recruitment and subsequent activation of IRAK4 upon TLR stimulation is mediated by the myeloid differentiation primary response 88 (MYD88) adaptor protein. Around 3% of chronic lymphocytic leukemia (CLL) patients have activating mutations of MYD88, a driver mutation in this disease. Aims: Here, we studied the effects of TLR activation and 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: Our in vitro studies demonstrated that ND2158 preferentially killed CLL cells in a dose-dependent manner. We further observed a decrease in NF-κB and STAT3 signaling, cytokine secretion, proliferation and migration of primary CLL cells from MYD88-mutated and –unmutated cases. In the Eμ-TCL1 adoptive transfer mouse model of CLL, ND2158 delayed tumor progression and modulated the activity of myeloid and T cells. Summary/Conclusion: Our findings show the importance of TLR signaling in CLL development and suggest IRAK4 as a therapeutic target for this disease.

Elevated pre-activation basal level of nuclear NF-\u03baB in native macrophages accelerates LPS-induced translocation of cytosolic NF-\u03baB into the cell nucleus

Signaling via Toll-like receptor 4 (TLR4) in macrophages constitutes an essential part of the innate immune response to bacterial infections. Detailed and quantified descriptions of TLR4 signal transduction would help to understand and exploit the first-line response of innate immune defense. To date, most mathematical modelling studies were performed on transformed cell lines. However, properties of primary macrophages differ significantly. We therefore studied TLR4-dependent activation of NF-κB transcription factor in bone marrow-derived and peritoneal primary macrophages. We demonstrate that the kinetics of NF-κB phosphorylation and nuclear translocation induced by a wide range of bacterial lipopolysaccharide (LPS) concentrations in primary macrophages is much faster than previously reported for macrophage cell lines. We used a comprehensive combination of experiments and mathematical modeling to understand the mechanisms of this rapid response. We found that elevated basal NF-κB in the nuclei of primary macrophages is a mechanism increasing native macrophage sensitivity and response speed to the infection. Such pre-activated state of macrophages accelerates the NF-κB translocation kinetics in response to low agonist concentrations. These findings enabled us to refine and construct a new model combining both NF-κB phosphorylation and translocation processes and predict the existence of a negative feedback loop inactivating phosphorylated NF-κB.

Targeting IRAK4 Disrupts Inflammatory Pathways and Delays Tumor Development in Chronic Lymphocytic Leukemia

Interleukin-1 receptor-associated kinase 4 (IRAK4) plays a critical role in Toll-like receptor (TLR) signal transduction and the innate immune responses. Recruitment and subsequent activation of IRAK4 upon TLR stimulation is mediatedby the myeloid differentiation primary response 88 (MYD88) adaptor protein. Around 5% of chronic lymphocytic leukemia (CLL) patients have activating mutations of MYD88, a driver mutation in this disease. Gene expression profiling of 423 CLL cases, assessed by the computational GSEA analysis tool, 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. To assess the effects of this pathway in CLL, we stimulated these cells with a TLR agonists mix, containing Pam3CSK4, HKLM, FSL1 and ODN2006, which was able to induce cytokines secretion, increase in NF-KB activity and STAT3 signaling as well as cells proliferation and migration. Here, we study the pharmacological inhibition of IRAK4 with ND2158, an IRAK4 competitive inhibitor, as a therapeutic approach in CLL. ND2158 was able to efficiently decrease all TLR effects seen with the mix like STAT3 signaling and the secretion of CCL2, CCL3, CCL4, TNFα, IL1β, IL10 and IL6 cytokines (assessed by Luminex). 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 (checked by carboxyfluoresceinsuccinimidyl ester staining) and migration towards CXCL12 in MYD88-mutated and -unmutated CLL cases.ND2158 also induced a selective dose-dependent cytotoxic effect in CLL cells, without differences regarding their MYD88 mutational status. To further validate this results, we checked the effect of ND2158 in splenocytes from leukemic Eμ-TCL1 mice and similar antitumor effects were seen.In addition to the direct impact on tumor cells, we analyzed whether ND2158 also affects other immune cells that also signal via MYD88/IRAK4. Monocytes, broadly known for their importance in tumor supporting, were also modulated by TLR agonists, and a reduction in activation markers and cytokines secretion was achieved after ND2158 treatment.In vivo studies in Eμ-TCL1 leukemic mice showed a significant reduction in tumor load in CLL-affected organs (peripheral blood, spleen, lymph node and peritoneal cavity) associated with reduced PD-L1 expression levels 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. Corroborating the in vitro studies,ND2158 also modulated tumor-supporting monocytes. The effect of ND2158 was also assessed in CD8+ effector T cells, where a low proliferative (Ki-67+ percentage), exhausted phenotype (increase in TIGIT, CD244, CD160 and LAG3 markers) and less functional population (increase in the immune checkpoint protein PD-1) was found.Based on our in vitro data, it seems that ND2158 targets both MYD88 mutated and unmutated CLL cases to a similar degree. Since CLL progression in the TCL1 mouse model has not been reported to be driven by an activating MYD88 mutation, our in vivo results support the notion that IRAK4 inhibition might also be effective in CLL patients with an unmutated MYD88 status. To overcome the potential negative impact of IRAK4 inhibition on T cells, the combination treatment with drugs that improve T cell function and avoid their rapid exhaustion should be considered. As we observed increased expression of several inhibitory receptors on CD8+ T cells in ND2158-treated mice, including PD-1 and TIGIT, blocking these receptors with antibodies might be a successful strategy to overcome loss of T cell function and improve therapy outcome.In summary, our data show the importance of TLR signaling in CLL development and suggest IRAK4 as a novel treatment target for CLL that targets both the malignant cells and the tumor-supportive inflammatory milieu. DisclosuresNo relevant conflicts of interest to declare.

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

Abstract BACKGROUND &amp; 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.

Molecular cloning and functional analysis of tumor necrosis factor receptor-associated factor 6 (TRAF6) in thick shell mussel, Mytilus coruscus

Tumor necrosis factor receptor-associated factor 6 (TRAF6) is one of the key adapter molecules in Toll-like receptor signal transduction that triggers downstream cascades involved in innate immunity. Despite of the well study in vertebrates, there is few data ascribe to this TRAF member in invertebrates, especially in bivalves. In the present study, a novel TRAF6 homologue termed McTRAF6 was firstly characterized in Mytilus coruscus. Like its counterparts in mammals, McTRAF6 shared the domain topology containing one RING domain, two zinc finger domains, one coiled-coil region and a MATH domain. McTRAF6 transcripts predominantly expressed in gills, digestive glands and hemocytes in M. coruscus, and were significantly up-regulated in hemocytes after challenge with lipopolysaccharide (LPS) and polyinosine-polycytidylic acid (poly I:C). Further, the subcellular localization in cytoplasm and the activation of Nk-κB or ISRE luciferase reporter by overexpressed McTRAF6 were identified in HEK293T cells. These results collectively indicate that McTRAF6 is a member of TRAF6 subfamily and plays a potential role in immune defense system against pathogenic agents invasions in thick shell mussel. To our knowledge, this is the first report on component of TLR signaling pathway in thick shell mussel, providing further evidence for the existence of TLR pathway in M. coruscus and contribute to clarify the innate immune system of thick shell mussel.

Molecular characterization and expression analysis of TLR1 and TLR4 from the endangered fish Dabry's sturgeon (Acipenser dabryanus)

Toll-like receptors (TLRs) are important pattern recognition receptors (PRRs) of teleost innate immune system. However, information about TLRs is absent in Dabry's sturgeon (Acipenser dabryanus), one of the most primitive actinopterygii species. In the present study, the full lengths of adaTLR1 and adaTLR4 were cloned from Dabry's sturgeon using RT-PCR and RACE-PCR. The obtained adaTLR1 was 2957 bp in length, encoding a putative protein of 767 amino acids and adaTLR4 cDNA was 2902 bp in length, encoding a putative protein of 830 aa. Both adaTLR1 and adaTLR4 possessed several typical TLRs motifs, including signal peptides, leucine-rich repeat (LRR) motifs, a transmembrane domain and a TIR motifs. In addition, adaTLR4 contained three conserved boxes in its TIR motif, involving in TLRs signal transduction. A proline, important for LPS recognition of mammalian TLR4, was also found in adaTLR4. Physicochemical features of adaTLR1 and adaTRL4 were also analyzed. Quantitative realtime PCR showed that both transcripts were ubiquitously expressed in all 11 normal tissues selected, but they exhibited different expression patterns, with adaTLR1 highly expression in heart and adaTLR4 highly in skin. Further, adaTLR1 and adaTLR4 were up-regulated in the primary head-kidney leucocytes following LPS and polyI:C stimulation, indicating that both genes involved in the sturgeon immune response to LPS and polyI:C. To our best knowledge, this was the first report of these genes in sturgeon and these results provided the basis for further elucidating the ligand specificity and signaling pathway of fish TLRs.

An unexpected link between fatty acid synthase and cholesterol synthesis in proinflammatory macrophage activation

Different immune activation states require distinct metabolic features and activities in immune cells. For instance, inhibition of fatty acid synthase (FASN), which catalyzes the synthesis of long-chain fatty acids, prevents the proinflammatory response in macrophages; however, the precise role of this enzyme in this response remains poorly defined. Consistent with previous studies, we found here that FASN is essential for lipopolysaccharide-induced, Toll-like receptor (TLR)-mediated macrophage activation. Interestingly, only agents that block FASN upstream of acetoacetyl-CoA synthesis, including the well-characterized FASN inhibitor C75, inhibited TLR4 signaling, while those acting downstream had no effect. We found that acetoacetyl-CoA could overcome C75's inhibitory effect, whereas other FASN metabolites, including palmitate, did not prevent C75-mediated inhibition. This suggested an unexpected role for acetoacetyl-CoA in inflammation that is independent of its role in palmitate synthesis. Our evidence further suggested that acetoacetyl-CoA arising from FASN activity promotes cholesterol production, indicating a surprising link between fatty acid synthesis and cholesterol synthesis. We further demonstrate that this process is required for TLR4 to enter lipid rafts and facilitate TLR4 signaling. In conclusion, we have uncovered an unexpected link between FASN and cholesterol synthesis that appears to be required for TLR signal transduction and proinflammatory macrophage activation.

Measurement of TLR4 and CD14 Receptor Endocytosis Using Flow Cytometry.

After recognizing extracellular bacterial lipopolysaccharide (LPS), the toll-like receptor 4 (TLR4)-CD14 signaling complex initiates two distinct signaling pathways-one from the plasma membrane and the other from the signaling endosomes (Kagan et al., 2008). Understanding the early stages of TLR4 signal transduction therefore requires a robust and quantitative method to measure LPS-triggered TLR4 and CD14 receptor endocytosis, one of the earliest events of LPS detection. Here, we describe a flow cytometry-based method that we used recently to study the role of the ion channel TRPM7 in TLR4 endocytosis (Schappe et al., 2018). The assay relies on stimulating the cells with LPS and measuring the cell surface levels of TLR4 (or CD14) at various time points using flow cytometry. Although we detail the method specifically for TLR4 and CD14 from murine bone marrow-derived macrophages, it can be readily adapted to evaluate receptor endocytosis in a variety of other signaling contexts.

Regulation of Toll-like receptor signal transduction by Rho guanine nucleotide exchange factors

Regulation of Toll-like receptor signal transduction by Rho guanine nucleotide exchange factors

TLR4 regulates insulin-resistant proteins to increase apoptosis in the mouse retina.

Work in multiple organs has suggested that toll-like receptor 4 (TLR4) may play a role in insulin resistance. Additional studies have shown a negative role for TLR4 on retinal health. We have previously reported that β-adrenergic receptors can regulate both TLR4 signal transduction, as well as insulin signaling in the retina and in retinal endothelial cells. Thus, we hypothesized that TLR4 would regulate retinal insulin signaling. We used endothelial cell-specific TLR4 knockout mice, as well as TLR4-overexpressing mice for these studies. Western blotting and ELISA analyses were done for investigations of insulin receptor, insulin receptor substrate 1 (IRS-1) serine 307, and Akt phosphorylation, as well as cleaved caspase 3 levels in the mouse retina. We found that loss of TLR4 led to increased insulin receptor and Akt phosphorylation, as well as decreased IRS-1Ser307 levels. In support of these results, TLR4 overexpression decreased insulin signaling and the cleavage of caspase 3. Therefore, these results suggest that TLR4 plays a key role in insulin signaling in the retina. Reduction of TLR4 levels may be protective to the retina.

Role of TOLL-like receptor signal transduction pathway in inflammatory reaction induced by endoplasmic reticulum stress

Role of TOLL-like receptor signal transduction pathway in inflammatory reaction induced by endoplasmic reticulum stress

Cytokine secretory responses of MYD88 skeletal muscle specific knockout to pathogen exposure in vitro

The role of skeletal muscles in the immune response to pathogens is poorly understood. Though intact skeletal muscles contain toll like receptors (TLRs) and secrete cytokines in response to pathogen exposure, it is unknown whether the cytokines come from muscle fibers or other resident cells. The purpose of this study was to interrupt TLR transduction, only in muscle fibers, in order to isolate their contribution from other cell types. To accomplish this, we created a skeletal muscle specific Myd88 knockout mouse (Myd88skmKO). Myd88 is an adaptor protein, critical to TLR signal transduction in all known TLRs in muscle. To stimulate TLRs, we used lipopolysaccharide (LPS), a potent TLR2 and TLR4 ligand.Matched solei muscles from MYD88skmKO mice (n=18) and background strain‐matched wild type controls (n=8) were excised in oxygenated (95%O2/5%CO2) Krebs solution and placed under 1 g of tension in baths at 35°C. Following a 30‐min acclimation period (T0), the buffer was replaced and muscles were exposed to 1 μg/ml LPS. Bath samples at T0, 1 hr (T1) and 2 hr (T2) were collected, mixed with a protease inhibitor cocktail and stored at −80°C prior to Luminex multiplex analysis.Both Myd88skmKO and control mice secreted significant quantities of cytokines in response to LPS. The response was greater in control mice than MYD88skmKOs in IL‐6 (3.9 fold, p=0.0005), IL‐15 (0.7 fold, p&lt; 0.02) GM‐CSF (3.0 fold, p&lt; 0.02) IL‐12p40 (2.7 fold, p&lt; 0.02), IL12‐p70 (0.6 fold, p&lt; 0.02), and MCP‐1 (1.8 fold, p&lt; 0.05). At T2, control mice secreted greater IL‐6 (10.4 fold, p= 0.0002), GCS‐F (8.6 fold, p= 0.002), MCP‐1 (1.7 fold, p&lt; 0.01) IP‐10 (1.4 fold, p= 0.0145) and KC (4.3 fold, p&lt; 0.03). Interestingly, MYD88skmKOs mice secreted greater MIP‐1α at T1 (0.5 fold, p=0.02). Despite the much greater secretion in controls, LPS treated MYD88skmKOs solei still demonstrated significant secretion of some cytokines over T1 to T2 in IL‐6 (p= 0.0001), KC (p= 0.0001), MCP‐1 (P= 0.0001), MIP‐2 (p&lt;0.04) and GM‐CSF (p&lt; 0.04).In conclusion, solei from MYD88skmKO mice secreted significantly lower amounts of cytokines in response to LPS than controls, with the lone exception of MIP‐1α. This confirms that much of the cytokine secretory response to LPS in whole muscle comes from muscle fibers. However, muscles from Myd88skmKO mice still demonstrated the capacity to secrete some cytokines. This remaining cytokine release may be indicative of remaining MYD88‐independent TLR4 signaling, responses of non‐muscle resident cells or incomplete KO of the MYD88 protein in muscle fibers.Support or Funding InformationSupported by NIH RO1GM118895‐01 and the BK and Betty Stevens Endowment.

Progress on mechanisms for pathogensto evade NOD-like receptor and Toll-like receptor signaling pathways

The innate immune system provides a first line of defense against invading pathogens, in which the pattern recognition receptors (PRR) recognize pathogen-associated molecular patterns (PAMP) and initiate the downstream signaling pathways to eliminate the encountered pathogens. There are two main classes of such signaling pathways: NOD-like receptor (NLR) signaling pathway and Toll-like receptor (TLR) signaling pathway. The microbial pathogens under selective pressure have evolved numerous mechanisms to avoid and/or manipulate the NLR and TLR signal transduction for survival and replication. To evade the NLR signaling pathway, pathogens interfere and/or inhibit inflammasome activation in innate immune cells by producing virulence factors or reducing PAMPs expression. The mechanisms for pathogens to evade TLR signaling pathway include: inhibition of mitogen activated protein kinases (MAPKs) cascade reaction, inhibition of NF-КB activation, and interference of down-stream signal transduction by producing Toll/interleukin-1 receptor (TIR)-containing proteins which bind directly with TLRs or adaptor proteins in the signaling pathway.

Differential expression of microRNA in neonatal necrotizing enterocolitis based on high-throughput sequencing

Objective To analyze the differential expression of microRNA (miRNA) and its significance in patients with neonatal necrotizing enterocolitis (NEC). Methods Twenty-five patients diagnosed with NEC with Bell stage ≥Ⅱ, and 25 non-NEC patients as control group admitted to Guangzhou Women and Children's Medical Center between October 2014 and November 2015 were collected. White blood cells were extracted from the peripheral blood. Five samples were selected randomly each from NEC group and control group, and sequenced by second-generation Illumina high-throughput sequencing, screened for differentially expressed miRNA and analyzed for target genes prediction and biological function. The rest samples of the two groups were detected by real-time fluorescent quantitative polymerase chain reaction technology (RT-qPCR), the results were used to validate the results of high-throughput sequencing. Differentially expressed miRNA in the two groups of data was analyzed using DEGseq software. P<0.05 was considered statistically significant. P<0.01, q<0.001 and丨Log2 Ratio丨≥1 were taken as criteria for screening the differential expression. The differential expressions of miRNA in NEC group and control group were analyzed by cluster analysis using MeV4.6 software. Results A total of 482 miRNAs were differentially expressed in the two groups, with significant difference (P<0.05). Among them, 126 were known miRNAs with significantly differential expression in the two groups, with 58 being up-regulated and 68 being down-regulated. The results of up-regulated miRNAs (hsa-miR-223-5p, -183-3p, -222-5p) and down-regulated miRNAs (hsa-miR-23b-5p, -150-5p, -146a-3p, -1298-5p) were confirmed to be consistent with the results of sequencing. Bioinformatics analysis showed that the target genes with differential miRNA expression mainly involved Toll-like receptor signal transduction pathway, mitogen-activated protein kinase pathway, JAK-STAT and other signal transduction pathways. Conclusions There are significantly differential expressions of miRNAs in peripheral white blood cells of NEC neonates. These miRNs may be involved in the occurrence and development of NEC via adjusting different target genes to regulate the signal pathway. Key words: Enterocolitis, necrotizing; MicroRNAs; High-throughput nucleotide sequencing; Infant, newborn

TAK-242 Protects Against Apoptosis in Coronary Microembolization-Induced Myocardial Injury in Rats by Suppressing TLR4/NF-κB Signaling Pathway

Background/Aims: Myocardial apoptosis is heavily implicated in the myocardial injury caused by coronary microembolization (CME), and toll-like receptor 4 (TLR4) is considered to be involved in this apoptotic cascade. Therefore, the present study was designed to investigate the role of TLR4/NF-κB signaling pathway regulated by TAK-242, a selective TLR4 signal transduction inhibitor, in the myocardial apoptosis after CME in rats. Methods: Forty-five rats were randomized (random number) into three groups: sham, CME and CME + TAK-242 (n = 15 per group).CME was induced by injecting polyethylene microspheres (42µm) into the left ventricular except the sham group. CME + TAK-242 group was treated with TAK-242 (2mg/kg) via the tail vein 30 minutes before CME modeling. Cardiac function was evaluated 6 hours after operation. Tissue biopsy was stained with HBFP to measure the size of micro-infarction area. TUNEL staining was used to detect myocardial apoptosis. Western blot and qPCR were used to evaluate the expression of TLR4, MyD88, NF-κB p65, p-IκBα and Cleaved caspase-3. Results: Cardiac function in the CME group and CME + TAK-242 group were significantly decreased compared with the sham group (P < 0.05) and the micro-infarction area, the apoptotic index, the expression of TLR4, NF-κB p65, p-IκBα and Cleaved caspase-3 were increased significantly (P < 0.05). Cardiac function in the CME + TAK-242 group was significantly improved compared with the CME group (P < 0.05) and the micro-infarction area, the apoptotic index, the expression of TLR4, MyD88, NF-κB p65, p-IκBα and Cleaved caspase-3 were decreased significantly (P < 0.05). Conclusions: TAK-242 can effectively improve CME-induced cardiac dysfunction by regulating TLR4/NF-κB signaling pathway and then reducing the myocardial apoptosis.

Effects of porcine MyD88 knockdown on the expression of TLR4 pathway-related genes and proinflammatory cytokines.

As a critical adapter protein in Toll-like receptor (TLR)/Interleukin (IL)-1R signalling pathway, myeloid differentiation protein 88 (MyD88) plays an important role in immune responses and host defence against pathogens. The present study was designed to provide a foundation and an important reagent for the mechanistic study of MyD88 and its role TLR/IL-1R signalling pathways in porcine immunity. Lentivirus-mediated RNAi was used to generate a porcine PK15 cell line with a silenced MyD88 gene and quantitative real-time PCR (qPCR) and Western blotting were used to detect changes in the expression of critical genes in the Toll-like receptor 4 (TLR4) signalling pathway. ELISA was used to measure the levels of seven proinflammatory cytokines–interleukin-1β (IL-1β), tumour necrosis factor-α (TNF-α), IL-6, IL-8, IL-12, macrophage inflammatory protein (MIP)-1α and MIP-1β–in cell culture supernatants after MyD88 silencing. We successfully obtained a PK15 cell line with 61% MyD88 mRNA transcript down-regulated. In PK15 cells with MyD88 silencing, the transcript levels of TLR4 and IL-1β were significantly reduced, whereas there were no significant changes in the expression levels of cluster of differentiation antigen 14 (CD14), interferon-α (IFN-α) or TNF-α. The ELISA results showed that the levels of most cytokines were not significantly changed apart from IL-8 without stimulation, which was significantly up-regulated. When cells were induced by lipopolysaccharide (LPS) (0.1 μg/ml) for 6 h, the global level of seven proinflammatory cytokines up-regulated and the level of IL-1β, TNF-α, IL-6, IL-8 and IL-12 of Blank and negative control (NC) group up-regulated more significantly than RNAi group (P<0.05), which revealed that the MyD88 silencing could reduce the TLR4 signal transduction which inhibited the release of proinflammatory cytokines and finally leaded to immunosuppression.