Inhibition Of Toll-like Receptor Research Articles

Tinopanoids K-T, clerodane diterpenoids with anti-inflammatory activity from Tinospora crispa

A total of 17 structurally diverse clerodane diterpenoids, including ten undescribed clerodane diterpenoids (tinopanoids K-T, 1–10) and seven known compounds (11–17), were isolated from the vines and leaves of Tinospora crispa. Compound 3 has not only bear the dominant substituents of γ-hydroxy-α, β-unsaturated-γ-lactone with anti-inflammatory activity, but also a ternary epoxy structure at C-3/C-4. The planar structures and relative configurations of the clerodane diterpenoids were elucidated by spectroscopic data interpretation. The absolute configurations of compounds 1, 4, 8 and 13 were determined by single-crystal X-ray crystallographic, while that of compound 3 was determined using computed ECD data and single crystal X-ray diffraction of related p-bromobenzoate ester (3a). Subsequently, all compounds were evaluated for their inhibitory effect on nitric oxide (NO) production of LPS-activated BV-2 cells, and compounds 3 and 8 exhibited better NO inhibitory potency, with IC50 values of 5.6 and 13.8 μM than the positive control minocycline (Mino, IC50 = 22.9 μM). The corresponding results of western blot analysis and qRT-PCR revealed that compound 3 can significantly inhibit the inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) protein expressions, mRNA levels of pro-inflammatory cytokins of tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6) and interleukin 1β (IL-1β). The underlying mechanism by which compound 3 exerted anti-neuroinflammatory effects was investigated by western blot and immunofluorescence assay, which suggested compound 3 inhibited LPS induced neuroinflammation via the suppression of toll-like receptor 4 (TLR4) dependent Signal Transducer and Activator of Transcription 3 (Stat3) and mitogen-activated protein kinase (MAPK) signaling pathways, and the activation of Heme Oxygenase-1 (HO-1) mediated signals.

Inhibition of toll-like receptor 4 activation by apigenin and chrysin via competition for sites and conformational changes

The activation of toll-like receptor 4 (TLR4) signaling is crucial for initiating and coordinating the immune response against infections, and is proved as a vital target for inflammatory diseases. Herein, TLR4 with sufficient amount and functional activity was generated by heterologous expression and used to investigate the mechanism of apigenin (Api)/chrysin (Chr) inhibition of TLR4 activation. The results demonstrated that Api/Chr exhibited a strong fluorescence quenching effect through a static quenching and a high binding affinity (Ka > 105 L·mol−1) with TLR4, indicating the potential of Api/Chr as a TLR4 inhibitor. Additionally, the binding of Api/Chr induced a loose and unstable conformation of TLR4 with evidence like the decreased hydrophobicity of the tryptophan microenvironment, decreased α-helix content and increased free sulfhydryl content, resulting in reduced stability of the TLR4. The computer simulations revealed that Api/Chr occupied the myeloid differentiation factor 2 (MD-2) binding region, preventing MD-2 from binding to TLR4. Furthermore, the accuracy of the binding site between Api/Chr and TLR4 was confirmed through genetic mutations. Overall, the mechanism by which Api/Chr inhibited TLR4 activation was elucidated at the macroscopic and molecular levels, providing the worthful information concerning the future therapeutic application of Api/Chr as a natural TLR4 inhibitor.

Inhibition of Toll-like receptor 4 and Interleukin-1 receptor prevent SARS-CoV-2 mediated kidney injury

Acute kidney injury (AKI) is a common and severe complication of the coronavirus disease 2019 (COVID-19). Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) directly affects the glomerular and tubular epithelial cells to induce AKI; however, its pathophysiology remains unclear. Here, we explored the underlying mechanisms and therapeutic targets of renal involvement in COVID-19. We developed an in vitro human kidney cellular model, including immortalized tubular epithelial and endothelial cell lines, demonstrating that SARS-CoV-2 directly triggers cell death. To identify the molecular targets in the process of SARS-CoV-2-mediated cell injury, we performed transcriptional analysis using RNA sequencing. Tubular epithelial cells were more prone to dying by SARS-CoV-2 than endothelial cells; however, SARS-CoV-2 did not replicate in renal cells, distinct from VeroE6/transmembrane protease serine 2 cells. Transcriptomic analysis revealed increased inflammatory and immune-related gene expression levels in renal cells incubated with SARS-CoV-2. Toll-like receptor (TLR) 3 in renal cells recognized viral RNA and underwent cell death. Furthermore, analysis of upstream regulators identified several key transcriptional regulators. Among them, inhibition of the interleukin-1 receptor (IL-1R) and TLR4 pathways protects tubular epithelial and endothelial cells from injury via regulation of the signal transducer and activator of transcription protein-3/nuclear factor-kB pathway. Our results reveal that SARS-CoV-2 directly injures renal cells via the proinflammatory response without viral replication, and that IL-1R and TLR4 may be used as therapeutic targets for SARS-CoV-2 mediated kidney injury.

Abstract P1053: Circulating Mitochondrial DNA Level Is A Biomarker Of Acute Myocardial Infarction And Mediates Cardiac Ischemia-reperfusion Injury

Introduction: Ischemia/reperfusion (I/R) injury occurs after reperfusion, causing significant myocardial damage. Circulating mitochondrial DNA (mtDNA) levels are elevated in acute myocardial infarction patients, and act as Damage Associated Molecular Patterns (mtDNA DAMP) that induce inflammation via immune processes, including the toll-like receptor 9 (TLR9) pathway. However, mtDNA DAMP levels have not been measured in ST-elevation myocardial infarction (STEMI) patients before and after percutaneous coronary intervention (PCI) and the role of mtDNA DAMPs in I/R injury remains unknown. Hypothesis: Pre- and post-PCI levels of mtDNA DAMPs serve as markers of STEMI related cardiac injury. Limiting mtDNA DAMP levels by increasing autophagy or blocking their downstream effects on TLR9 will decrease cardiac I/R injury. Methods: Troponin and mtDNA DAMP levels were measured pre- and 24 hours post- PCI in 55 STEMI patients (90%-100% blockage) and compared to 37 healthy controls. MtDNA DAMP levels in cell-free plasma were measured by real-time qPCR. Using a C57BL6J mouse model of I/R injury, Tat-Beclin (TB, an autophagy inducer), ODN2088 (a TLR9 inhibitor) or negative control were given at reperfusion and infarct size, cardiac function, and mtDNA DAMP levels were determined. Results: Pre- PCI mtDNA DAMP levels were increased ~200 folds (p<0.0001) in STEMI patients compared to healthy controls; post- PCI mtDNA DAMP levels were significantly decreased in the majority of patients. In contrast, troponin T levels increased, suggesting that mtDNA DAMP levels are early markers of STEMI. Interestingly, a greater decrease in mtDNA DAMP levels was associated with shorter symptom-to-PCI time. In mice, both TB induction of autophagy and TLR9 blockage by ODN2088 decreased infarct size by ~50% and preserved systolic function. As expected, TB induction of autophagy decreased mtDNA DAMP levels, while ODN2088 had no impact on mtDNA DAMP levels. Conclusions: Circulating mtDNA DAMP level is an early marker of STEMI and may predict the success of early PCI. Moreover, limiting the downstream effects of mtDNA DAMPs via induction of autophagy or blocking their action on TLR9 ameliorates cardiac I/R injury, and thus may provide a viable therapeutic avenue for STEMI patients.

Ozone attenuates chemotherapy-induced peripheral neuropathy via upregulating the AMPK-SOCS3 axis.

Chemotherapy-induced peripheral neuropathy (CIPN) is a severe adverse reaction to chemotherapeutics, which seriously affects the outcome of chemotherapy and patients' quality of life. Although it is commonly seen, it lacks effective treatment. Our previous study found that ozone could alleviate neuropathic pain. Damage-associated molecular patterns (DAMPs) or Toll-like receptor 4 (TLR4) or tissue factor (TF)-mediated neuroinflammation and microcirculation disturbance is the main reason for CIPN. Suppressors of cytokine signaling (SOCS) 3 is an endogenous negative feedback regulator of inflammation via TLR4 inhibition. Oxaliplatin (L-OHP) was used to establish mice's CIPN model. Nociceptive responses were assessed by observing the ICR mice's incidence of foot regression in mechanical indentation response experiments. Cell signaling assays were performed by Western blotting and immunohistochemistry. The mouse leukemia cells of monocyte-macrophage line RAW 264.7 were cultured to investigate the effects of ozone administration on macrophage. Ozone decreased the expression of TF in the blood and sciatic nerve. It upregulated the adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK)-SOCS3 axis to relieve CIPN and inhibit TF expression in vivo. SOCS3 expression was induced by ozone to inhibit the p38/TF signaling in RAW 246.7 cells. Ozone also prevented L-OHP-induced sciatic nerve demyelination. Microglia activation was inhibited, and c-Fos and calcitonin gene-related peptide (CGRP) expression was decreased in the spinal dorsal horn via ozone. In this study, we demonstrated that ozone could alleviate CIPN by upregulating the AMPK-SOCS3 axis to inhibit TF expression, which is a potential treatment for CIPN.

Activation of cell-free mtDNA-TLR9 signaling mediates chronic stress-induced social behavior deficits

Inflammation and social behavior deficits are associated with a number of neuropsychiatric disorders. Chronic stress, a major risk factor for depression and other mental health conditions is known to increase inflammatory responses and social behavior impairments. Disturbances in mitochondria function have been found in chronic stress conditions, however the mechanisms that link mitochondrial dysfunction to stress-induced social behavior deficits are not well understood. In this study, we found that chronic restraint stress (RS) induces significant increases in serum cell-free mitochondrial DNA (cf-mtDNA) levels in mice, and systemic Deoxyribonuclease I (DNase I) treatment attenuated RS-induced social behavioral deficits. Our findings revealed potential roles of mitophagy and Mitochondrial antiviral-signaling protein (MAVS) in mediating chronic stress-induced changes in cf-mtDNA levels and social behavior. Furthermore, we showed that inhibition of Toll-like receptor 9 (TLR9) attenuates mtDNA-induced social behavior deficits. Together, these findings show that cf-mtDNA-TLR9 signaling is critical in mediating stress-induced social behavior deficits.

DNMT1-mediated DNA methylation in toll-like receptor 4 (TLR4) inactivates NF-κB signal pathway-triggered pyroptotic cell death and cellular inflammation to ameliorate lipopolysaccharides (LPS)-induced osteomyelitis

Toll-like receptor 4 (TLR4) plays a critical role in various human diseases, and was associated with pyroptotic cell death and inflammatory responses. DNA methylation, which has stable and reversible properties, has been reported to alter the expression of target genes, including TLR4. However, the role of methylated TLR4 in osteomyelitis (OM) and the underlying molecular mechanisms remain unclear. RNA sequencing was used to identify differentially expressed genes and associated signaling pathways. RT-qPCR, Western blot, emzyme-linked immunosorbent assay (ELISA), cell counting kit-8 (CCK-8) and LDH assay kit were used to detect mRNA and protein expression of relevant genes, cell viability and the LDH activity, respectively. TLR4 methylation was detected by methylation-specific PCR (MSP) and verified by Chromatin immunoprecipitation (ChIP). Here, we found that DNA methyltransferase-1 (DNMT1)-mediated TLR4 demethylation significantly suppressed lipopolysaccharides (LPS)-induced pyroptosis and inflammatory response by inhibiting the TLR4/nuclear transcription factor-kappa B (NF-κB) axis. First, we confirmed TLR4 as the study target by mRNA transcriptome sequencing analysis, and TLR4 was observably high-expressed in both OM patients and LPS-treated osteoblastic MC3T3-E1. Then, we found that downregulation of DNMT1 blocked TLR4 promoter methylation modification, resulting in upregulation of TLR4. Simultaneously, functional experiments indicated that suppression of TLR4 or overexpression of DNMT1 promoted cell proliferation and inhibited cell pyroptosis and inflammation in LPS-induced MC3T3-E1, while upregulation of TLR4 restored the effects of DNMT1 silencing on OM progression. In addition, TLR4 elevated phosphorylation of IκB-α and NF-κB p65 in the NF-κB signal pathway, and inhibition of TLR4 or the NF-κB inhibitor PDTC reversed the influence of inhibition of DNMT1. In conclusion, our study demonstrated that DNMT1-mediated TLR4 DNA methylation alleviated LPS-induced OM by inhibiting the NF-κB signaling pathway.

TAK-242, a toll-like receptor 4 antagonist, against brain injury by alleviates autophagy and inflammation in rats.

Inhibition of Toll-like receptor 4 (TLR4)-mediated inflammatory pathways exerts a critical effect on neuronal death; therefore, it is a possible new therapeutic approach for traumatic brain injury (TBI). Resatorvid (TAK-242) is a novel small-molecule compound widely used to inhibit TLR4-mediated pathways, but the protective mechanism of TAK-242 in TBI remains unclear. Herein, we analyzed the neuroprotective effects of TAK-242 in rats after TBI. The rat model of brain injury was established using a modified Free-fall device, and the rats were injected with TAK-242 (0.5 mg/kg) through the caudal vein before TBI. The rats were allocated into four groups: a sham group, a TBI group, a TBI + vehicle group, and a TBI + TAK-242 group. The brain tissue was extracted for histology and determination of the expression of autophagy-related proteins and inflammatory mediators. TAK-242 pretreatment significantly reduced the damage to hippocampal neurons. Neuronal autophagy increased after brain injury, whereas TAK-242 significantly reduced autophagy marker protein LC3-II in the hippocampus. In addition, TAK-242 pretreatment significantly downregulated NF-κB p65, TNF-α, and IL-1β in the hippocampus. In conclusion, TAK-242 significantly reduced hippocampal neuronal damage by inhibiting autophagy and neuroinflammatory activity, possibly via the NF-κB signaling pathway.

Disulfiram blocks inflammatory TLR4 signaling by targeting MD-2

Toll-like receptor 4 (TLR4) sensing of lipopolysaccharide (LPS), the most potent pathogen-associated molecular pattern of gram-negative bacteria, activates NF-κB and Irf3, which induces inflammatory cytokines and interferons that trigger an intense inflammatory response, which is critical for host defense but can also cause serious inflammatory pathology, including sepsis. Although TLR4 inhibition is an attractive therapeutic approach for suppressing overexuberant inflammatory signaling, previously identified TLR4 antagonists have not shown any clinical benefit. Here, we identify disulfiram (DSF), an FDA-approved drug for alcoholism, as a specific inhibitor of TLR4-mediated inflammatory signaling. TLR4 cell surface expression, LPS sensing, dimerization and signaling depend on TLR4 binding to MD-2. DSF and other cysteine-reactive drugs, previously shown to block LPS-triggered inflammatory cell death (pyroptosis), inhibit TLR4 signaling by covalently modifying Cys133 of MD-2, a key conserved residue that mediates TLR4 sensing and signaling. DSF blocks LPS-triggered inflammatory cytokine, chemokine, and interferon production by macrophages in vitro. In the aggressive N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model of Parkinson's disease (PD) in which TLR4 plays an important role, DSF markedly suppresses neuroinflammation and dopaminergic neuron loss, and restores motor function. Our findings identify a role for DSF in curbing TLR4-mediated inflammation and suggest that DSF and other drugs that target MD-2 might be useful for treating PD and other diseases in which inflammation contributes importantly to pathogenesis.

Yang-Xin-Shu-Mai granule alleviates atherosclerosis by regulating macrophage polarization via the TLR9/MyD88/NF-κB signaling pathway

Ethnopharmacological relevancePrevious studies have found that Yang-Xin-Shu-Mai granule (YXSMG) has certain advantages in the treatment of stable coronary heart disease. However, YXSMG can inhibit the progression of atherosclerotic plaque and stabilize vulnerable plaque needs to be further explored and studied. This research, mass spectrometry analysis, network pharmacology, in vivo and in vitro experimental studies were conducted to explore the mechanism of YXSMG on atherosclerosis. Aim of the studyTo decipher the mechanism of atherosclerotic plaque, stabilization for YXSMG by analysis of its active ingredients and biological network and activity in whole animal and at cellular and molecular levels. MethodsThe active components of YXSMG were determined using high performance liquid chromatography-mass spectrometry/mass spectrometry (HPLC-MS/MS) analysis. The ‘Disease-Compound-Target-Pathway’ network diagram was constructed using network pharmacology, and the stability of binding between core targets and core compounds was analyzed with molecular docking. After intervention with YXSMG, the pathology of aortic plaque, inflammation in the surrounding tissue, expression of TLR9/MyD88/NF-κB pathway protein in plaque and M1/M2 polarization of plaque macrophages were evaluated in vivo in apolipoprotein E-deficient (ApoE−/−) mice fed with high-fat diet. To verify whether it suppressed inflammation by inhibiting Toll-like receptor 9 (TLR9) reprogramming of macrophage polarization, we used RAW264.7 macrophages treated with specific TLR9 agonist (ODN1826) and inhibitor (ODN2088). ResultsFive active compounds were identified in YXSMG: catechin, formononetin, tanshinone IIA, cryptotanshinone and glycitein. Network pharmacology studies revealed TLR9 as one of the core targets of YXSMG intervention in atherosclerosis. Computer simulation of molecular docking showed that TLR9 could interact with the core compound to form a stable complex. In vivo experiments confirmed that YXSMG could significantly inhibit atherosclerotic plaque, reduce levels of blood lipids and inflammatory factors, downregulate TLR9/MyD88/NF-κB pathway protein and inhibit aortic sinus macrophages polarization to M1, but promote their polarization to M2 to inhibit inflammation. In vitro experiments revealed that YXSMG could downregulate expression of TLR9 gene and protein in ODN1826-activated RAW264.7 macrophages. ODN2088 had a synergistic effect with YXSMG on the TLR9/MyD88/NF-κB signaling pathway, and reprogrammed macrophages polarization from M1 to M2 by inhibiting TLR9, thus reducing immuno-inflammatory response. ConclusionYXSMG can reduce the level of blood lipid and improve the size of atherosclerotic plaque and inflammatory infiltration in ApoE−/− mice fed with high fat. It is concluded that YXSMG can improve the mechanism of atherosclerotic plaque by inhibiting TLR9/MyD88/NF-κB pathway reprogramming macrophage M1/M2 polarization and reducing arterial inflammation.

S14G-Humanin ameliorates ovalbumin-induced airway inflammation in asthma mediated by inhibition of toll-like receptor 4 (TLR4) expression and the nuclear factor κ-B (NF-κB)/early growth response protein-1 (Egr-1) pathway.

Asthma is a chronic inflammatory disease with a high morbidity rate in children and significantly impacts their healthy growth. It is reported that Th2 cell-mediated airway inflammation and activated oxidative stress are involved in the pathogenesis of asthma. S14G-humanin (HNG) is a derivative of Humanin with higher activity. The present study proposes to explore the potential treating property of HNG on asthma. An asthma model was constructed in mice using ovalbumin (OVA), the mice were treated with 2.5 mg/kg and 5 mg/kg HNG for 16 days. Dramatically increased lung weight index, elevated number of monocytes, eosinophils, and neutrophils, promoted production of Th2 cytokines including interleukin-4 (IL-4), interleukin-5 (IL-5), and interleukin-13 (IL-13), and severe histological pathology were observed in OVA-challenged mice, all of which were extremely alleviated by 2.5 mg/kg and 5 mg/kg HNG. Furthermore, the increased malondialdehyde (MDA) level and declined superoxide dismutase (SOD) activity in OVA-challenged mice were abolished by 2.5 mg/kg and 5 mg/kg HNG. Lastly, the upregulated TLR4, p-NF-κB p65, and early growth response 1 (Egr-1) in lung tissues of OVA-challenged mice were pronouncedly downregulated by 2.5 mg/kg and 5 mg/kg HNG. Collectively, our data suggested that HNG ameliorated airway inflammation in asthma partially due to NF-κB and Egr-1-mediated responses.

Downregulation of XBP1s aggravates lipopolysaccharide-induced inflammation by promoting NF-κB and NLRP3 pathways’ activation in goat endometrial epithelial cells

After delivery, bacterial contamination and uterine tissue degeneration in animals can lead to the development of uterine diseases, such as endometritis, accompanied by endoplasmic reticulum stress (ERS). Increasing evidence suggests that spliced X-box binding protein 1 (XBP1s), a critical component of ERS, is involved in several pathological processes in various organisms. However, the specific molecular mechanisms by which XBP1s mediates the inflammatory response in goat endometrial epithelial cells (gEECs) remain largely unknown. In the present study, XBP1s protein was induced into the nucleus in the lipopolysaccharide (LPS, 5 μg/mL)-induced inflammatory response of gEECs. Lipopolysaccharide-induced expression and nucleation of XBP1s were reduced by the inhibition of Toll-like receptor 4 (TLR4) using TAK-242 (1 μM; a TLR4 inhibitor). Expression and nucleation of XBP1s were similarly reduced when the activity of inositol-requiring enzyme 1α (IRE1α) was inhibited using 4μ8C (10 μM; an IRE1α inhibitor). In addition, inhibition of IRE1a increased IL-1β, TNF-α, and IL-8 levels and secretion of IL-6 induced by LPS. Notably, phosphorylation of nuclear factor kappa-B (NF-κB) P65 protein and expression of NOD-like receptor thermal protein domain associated protein 3 (NLRP3) were similarly increased. Furthermore, knockdown of XBP1s in gEECs consistently promoted NF-κB P65 protein phosphorylation, NLRP3 protein expression, and inflammatory cytokine secretion. In summary, the current results suggest that in the LPS-induced inflammatory response in gEECs, LPS generates intracellular signaling cascades in gEECs via TLR4, which may promote XBP1s protein expression and nucleation by activating IRE1a. However, downregulation of XBP1s expression exacerbates inflammation by promoting activation of the NF-κB and NLRP3 inflammatory vesicle pathways. These results will potentially contribute to the treatment and prevention of endometritis in ruminants.

Correction: Anti-Inflammatory Activity of Odina wodier Roxb, an Indian Folk Remedy, through Inhibition of Toll-Like Receptor 4 Signaling Pathway

Correction: Anti-Inflammatory Activity of Odina wodier Roxb, an Indian Folk Remedy, through Inhibition of Toll-Like Receptor 4 Signaling Pathway

Fecal Microbiota Transplantation Alleviated Paclitaxel-Induced Peripheral Neuropathy by Interfering with Astrocytes and TLR4/p38MAPK Pathway in Rats.

Paclitaxel-induced peripheral neuropathy (PIPN) constitutes a refractory and progressive adverse consequence of paclitaxel treatment, causing pain and sensory anomalies in cancer survivors. Although the gut-brain axis is involved in multiple disorders including cancer, its impact on peripheral pain conditions remains elusive. Thus, we assessed the importance of gut microbiota and related mechanisms in PIPN. By implementing fecal microbiota transplantation (FMT) in a rat PIPN model (ie, rats treated with paclitaxel; hereafter as PIPN rats), we explored the effect of gut microbiota on PIPN rats using multiple methods, including different behavioral tests, 16S ribosomal DNA (rDNA) sequencing, and biochemical techniques. Sequencing of 16S rDNA revealed that the abundance of genera Bacteroides and UCG-005 increased, while that of genera Turicibacter, Clostridium sensu stricto 1 and Corynebacterium decreased in the PIPN rats. However, when treated with FMT using fecal from normal rats, the mechanical allodynia and thermal hyperalgesia in PIPN rats were significantly alleviated. In addition, FMT treatment reduced the expression of toll-like receptor 4 (TLR4), phospho-p38 mitogen-activated protein kinase (p-p38MAPK), and the astrocytic marker glial fibrillary acidic protein in the colon and spinal dorsal horn. TAK242 (a TLR4 inhibitor) significantly alleviated the behavioral hypersensitivity of PIPN rats and inhibited the TLR4/p38MAPK pathway in astrocytes in these rats. The gut microbiota played a critical role in PIPN. Future therapies treating PIPN should consider microbe-based treatment as an option.

1496-P: Resatorvid, an Inhibitor of Toll-Like Receptor-4, Protects Allogenic Islets from Inflammatory and Immune Response

Purpose: Loss of islet mass by instant blood mediated inflammatory reaction (IBMIR) and immunological rejection are major hurdles currently facing the field of islet transplantation. Toll-like receptor 4 (TLR4) plays a central role in inflammation and shaping adaptive immune response. We investigated the protective effects of resatorvid (TAK-242) using in vitro models of islet transplantation. Methods: Soluble TAK-242 or TAK-242 conjugated to the surface of islets utilizing copper-free click chemistry with a cleavable chemical linker were used. Islets were mixed with allogenic blood for a period of 3 hours and plasma levels of miRNA-375 and miRNA-200c were analyzed by RT-qPCR. Allogenic peripheral blood mononuclear cells were cultured with islets. T cell and monocyte activation were monitored by flow cytometry. Cytokine and miRNA levels in culture supernatant were measured by Luminex assay and RT-qPCR respectively. Results: The presence of TAK-242 in soluble form as well as released by cleavable linker from islet surface significantly reduced the release of miRNA-375 (p<0.01) and miRNA-200c (p<0.01) indicating less islet damage. In allogenic co-culture of islets and PBMCs, TAK-242 inhibited the expression of CD69 in T cells as well as decreased (p<0.05) the expression of CD86 and CD80 in monocytes. The production of cytokines IL-1β (p<0.001), IL-6 (p=0.01), IL-8 (p<0.05), IP10 (p<0.05), MCP-1 (p<0.001) and TNFα (p=0.005) in culture supernatant was decreased in the presence of soluble TAK-242 as well as TAK-242 released from islet surfaces. Conclusions: Inhibition of TLR4 using small molecule, TAK-242, protected islets from innate damage due to IBMIR as well as adaptive responses of activation of T cells and monocytes. These results validate the future study of inhibition of TLR4 using in vivo transplant model(s) to further investigate the efficacy of TLR4 inhibition to improve islet transplant survival and function. Disclosure J.D.Mattke: None. B.Naziruddin: None. B.Kane: None.

480-P: Synergistic Effects of Hyperglycemia and Dyslipidemia on Diabetic Neuropathy—High Glucose Aggravates Oxidized LDL-Induced Schwann Cell Death via Hyperactivation of TLR4

Diabetic neuropathy is a progressive complication of diabetes mellitus, with persistent pain and loss of sensory function. Oxidized LDL (oxLDL), an oxidized form of LDL, has been shown to play a significant role in the development of neuronal dysfunction. Circulating oxLDL is elevated in type 2 diabetes, leading to higher cardiovascular risk. However, the combined effects of hyperglycemia and oxLDL on the progression of diabetic neuropathy remain unknown. Here, we investigated the synergistic effects of high glucose on oxLDL-induced schwann cell death and its detailed mechanism. Immortalized adult mouse Schwann (IMS32) cells were treated with oxLDL (0, 150, 300 μg/mL) under normal glucose (NG, 5.5 mM) or high glucose (HG, 25 mM) conditions for 24 hours. Following treatment, we evaluated cell viability, gene, and protein expression of Toll-like receptor 4 (TLR4), a receptor for oxLDL. Under NG conditions, 300 μg/mL oxLDL, but not 150 μg/mL reduced the cell viability. Moreover, under HG conditions, both 150 μg/mL and 300 μg/mL oxLDL decreased the cell viability. Likewise, the gene and protein expression of TLR4 was synergistically upregulated by the combination of oxLDL and HG. We further evaluated the effects of TLR4 inhibition on the cell viability and apoptotic caspase-3 activity. The cells were pretreated with TAK-242, a TLR4 inhibitor, for 2 hours prior to 150 μg/mL of oxLDL treatment under NG or HG conditions. TAK-242 pretreatment attenuated the oxLDL-induced cell death in HG conditions. The caspase-3 activity was increased by oxLDL in HG conditions, but not in NG. Furthermore, the increased caspase-3 activity by oxLDL and HG was abolished by TAK-242. These results indicate that high glucose and oxLDL induce activation of TLR4, leading to exacerbated schwann cell apoptosis. The dysregulation of TLR4 signal in diabetes complicated with dyslipidemia may contribute to the pathogenesis of diabetic neuropathy. Disclosure W.Nihei: None. A.Kato: None. T.Himeno: None. M.Kondo: None. J.Nakamura: None. H.Kamiya: Research Support; Kissei Pharmaceutical Co., Ltd., Ono Pharmaceutical Co., Ltd., Eli Lilly Japan K.K., Merck & Co., Inc., Sumitomo Dainippon Pharma Co., Ltd., Takeda Pharmaceutical Co., Ltd., Mitsubishi Tanabe Pharma Corporation, Japan Tobacco Inc., Novo Nordisk, Taisho Pharmaceutical Holdings Co., Ltd., Speaker's Bureau; Sanofi, Novo Nordisk, Sumitomo Dainippon Pharma Co., Ltd., Boehringer Ingelheim Japan, Inc., Eli Lilly Japan K.K., Daiichi Sankyo, Ono Pharmaceutical Co., Ltd., Kissei Pharmaceutical Co., Ltd., Mitsubishi Tanabe Pharma Corporation, Kowa Company, Ltd., Novartis Pharma K.K., Merck & Co., Inc., Sanwa Kagaku Kenkyusho, Otsuka Pharmaceutical Co., Ltd., Taisho Pharmaceutical Holdings Co., Ltd., Astellas Pharma Inc., Terumo Corporation, Fukuda Denshi, LifeScan Diabetes Institute, EA Pharma Co., Ltd, KAKEN PHARMACEUTICAL CO., LTD, AstraZeneca. K.Sango: None. K.Kato: None.

Toll-like receptor 4-mediated microglial inflammation exacerbates early white matter injury following experimental subarachnoid hemorrhage.

Neuroinflammation has been reported to be associated with white matter injury (WMI) after subarachnoid hemorrhage (SAH). As the main resident immune cells of the brain, microglia can be activated into proinflammatory and anti-inflammatory phenotypes. Toll-like receptor 4 (TLR4), expressed on the surface of the microglia, plays a key role in microglial inflammation. However, the relationship between TLR4, microglial polarization, and WMI following SAH remains unclear. In this study, a total of 121 male adult C57BL/6 wild-type (WT) mice, 20 WT mice at postnatal day 1 (P1), and 41 male adult TLR4 gene knockout (TLR4-/-) mice were used to investigate the potential role of TLR4-induced microglial polarization in early WMI after SAH by radiological, histological, microstructural, transcriptional, and cytological evidence. The results indicated that microglial inflammation was associated with myelin loss and axon damage, shown as a decrease in myelin basic protein (MBP), as well as increase in degraded myelin basic protein (dMBP) and amyloid precursor protein (APP). Gene knockout of TLR4 revised microglial polarization toward the anti-inflammatory phenotype and protected the white matter at an early phase after SAH (24 h), as shown through reduction of toxic metabolites, preservation of myelin, reductions in APP accumulation, reductions in white matter T2 hyperintensity, and increases in FA values. Cocultures of microglia and oligodendrocytes, the cells responsible for myelin production and maintenance, were established to further elucidate the relationship between microglial polarization and WMI. In vitro, TLR4 inhibition decreased the expression of microglial MyD88 and phosphorylated NF-κB, thereby inhibiting M1 polarization and mitigating inflammation. Decrease in TLR4 in the microglia increased preservation of neighboring oligodendrocytes. In conclusion, microglial inflammation has dual effects on early WMI after experimental SAH. Future explorations on more clinically relevant methods for modulating neuroinflammation are warranted to combat stroke with both WMI and gray matter destruction.

Bidirectional crosstalk of the cAMP/ROS-dependent signaling pathways in inflammatory macrophage: An activation of formononetin

Bacterial lipopolysaccharide (LPS) is a toxic stimulant to macrophage inflammation. Inflammation intersects cell metabolism and often directs host immunopathogenesis stress. We aim here at pharmacological discovering of formononetin (FMN) action, to which anti-inflammatory signaling spans across immune membrane receptors and second messenger metabolites. In ANA-1 macrophage stimulated by LPS, and simultaneous treatment with FMN, results show the Toll-like receptor 4 (TLR4) and estrogen receptor (ER) signals, in concert with reactive oxygen species (ROS) and cyclic adenosine monophosphate (cAMP), respectively. LPS stimulates inactivation of the ROS-dependent nuclear factor erythroid 2-related factor 2 (Nrf2) by upregulating TLR4, but it does not affect cAMP. However, FMN treatment not only activates Nrf2 signaling by TLR4 inhibition, but also it activates cAMP-dependent protein kinase activities by upregulating ER. The cAMP activity gives rise to phosphorylation (p-) of protein kinase A, liver kinase B1 and 5’-AMP activated protein kinase (AMPK). Moreover, bidirectional signal crosstalk is amplified between p-AMPK and ROS, as FMN combinational validation with AMPK activator/inhibitor/target small-interfering RNA or ROS scavenger. The signal crosstalk is well positioned serving as the ˈplug-inˈ knot for rather long signaling axis, and the immune-to-metabolic circuit via ER/TLR4 signal transduction. Collectively, convergence of the FMN-activated signals drives significant reduction of cyclooxygenase-2, interleukin-6 and NLR family pyrin domain-containing protein 3, in LPS-stimulated cell. Although anti-inflammatory signaling is specifically related to the immune-type macrophage, the p-AMPK antagonizing effect arises from FMN combination with ROS scavenger H-bond donors. Information of our work assists in predictive traits against macrophage inflammatory challenges, using phytoestrogen discoveries.

Escherichia coli enhances Th17/Treg imbalance via TLR4/NF-κB signaling pathway in oral lichen planus

Oral lichen planus (OLP) is a T-cell-mediated immunoinflammatory disease. Several studies have proposed that Escherichia coli (E. coli) may participate in the progress of OLP. In this study, we examined the functional role of E. coli and its supernatant via toll-like receptor 4 (TLR4)/nuclear factor-kappab (NF-κB) signaling pathway in regulating T helper (Th) 17/ regulatory T (Treg) balance and related cytokines and chemokines profile in OLP immune microenvironment. We discovered that E. coli and supernatant could activate the TLR4/NF-κB signaling pathway in human oral keratinocytes (HOKs) and OLP-derived T cells and increase the expression of interleukin (IL)-6, IL-17, C-C motif chemokine ligand (CCL) 17 and CCL20, thereby increasing the expression of retinoic acid-related orphan receptor (RoRγt) and the proportion of Th17 cells. Furthermore, the co-culture experiment revealed that HOKs treated with E. coli and supernatant increased T cell proliferation and migration, which promoted HOKs apoptosis. TLR4 inhibitor (TAK-242) successfully reversed the effect of E. coli and its supernatant. Consequently, E. coli and supernatant activated the TLR4/NF-κB signaling pathway in HOKs and OLP-derived T cells, leading to increased cytokines and chemokines expression and Th17/Treg imbalance in OLP.

Differential effects of dexmedetomidine on Gram-positive and Gram-negative bacterial killing and phagocytosis

Dexmedetomidine is a commonly used sedative in perioperative and intensive care settings with purported immunomodulatory properties. Since its effects on immune functions against infections have not been extensively studied, we tested the effects of dexmedetomidine on Gram-positive [Staphylococcus aureus and Enterococcus faecalis] and Gram-negative bacteria [Escherichia coli], and on effector functions of human monocytes THP-1 cells against them. We evaluated phagocytosis, reactive oxygen species (ROS) formation, and CD11b activation, and performed RNA sequencing analyses. Our study revealed that dexmedetomidine improved Gram-positive but mitigated Gram-negative bacterial phagocytosis and killing in THP-1 cells. The attenuation of Toll-like receptor 4 (TLR4) signaling by dexmedetomidine was previously reported. Thus, we tested TLR4 inhibitor TAK242. Similar to dexmedetomidine, TAK242 reduced E. coli phagocytosis but enhanced CD11b activation. The reduced TLR4 response potentially increases CD11b activation and ROS generation and subsequently enhances Gram-positive bacterial killing. Conversely, dexmedetomidine may inhibit the TLR4-signaling pathway and mitigate the alternative phagocytosis pathway induced by TLR4 activation through LPS-mediated Gram-negative bacteria, resulting in worsened bacterial loads. We also examined another α2 adrenergic agonist, xylazine. Because xylazine did not affect bacterial clearance, we proposed that dexmedetomidine may have an off-target effect on bacterial killing process, potentially involving crosstalk between CD11b and TLR4. Despite its potential to attenuate inflammation, we provide a novel insight into potential risks of dexmedetomidine use during Gram-negative infections, highlighting the differential effect of dexmedetomidine on Gram-positive and Gram-negative bacteria.