Toll-like Receptor 4-mediated Inflammation Research Articles

Arctigenin mitigates insulin resistance by modulating the IRS2/GLUT4 pathway via TLR4 in type 2 diabetes mellitus mice.

Arctigenin (AR), extracted from Arctium lappa L. (Burdock), is a folk herbal medicine used to treat diabetes. However, its mechanism of action has remained elusive. In this study, type 2 diabetes mellitus (T2DM) mice received AR orally for 10weeks to evaluate its therapeutic effect based on changes in glucose and lipid metabolism, histological examination of target tissues, and liver immunohistochemistry. Furthermore, HepG2 insulin-resistant cells were established to verify the mechanism of AR against diabetes. The results showed that AR treatment reduced blood glucose and lipid levels, reversing liver as well as pancreas tissue damage in T2DM mice. AR reduced the levels of pro-inflammatory cytokines in the serum of T2DM mice, as well as those in insulin-resistant HepG2 cell supernatants, while increasing interleukin-10 (IL-10) levels. The levels of p-p65, phospho-c-Jun N-terminal kinase (p-JNK), induced nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) were reduced in the liver tissue of T2DM mice, accompanied by an upregulation of glucose transporter 4 (GLUT4) and insulin receptor substrate 2 (IRS-2). In vitro studies further showed that AR downregulated toll-like receptor 4-mediated inflammation, while upregulating insulin pathway-related proteins and ultimately improving glucose uptake in insulin-resistant HepG2 cells. In conclusion, AR protected mice from insulin resistance, and its therapeutic effect was likely associated with inhibition of toll-like receptor 4 inflammatory signaling to reactivate IRS-2/GLUT4.

Forkhead box protein A2 alleviates toll-like receptor 4-mediated inflammation, endoplasmic reticulum stress, autophagy, and apoptosis induced by lipopolysaccharide in bovine hepatocytes

Lipopolysaccharide (LPS) is an important stimulus of inflammation via binding to toll-like receptor 4 (TLR4), but the role of TLR4 in LPS-induced cellular homeostasis disruption indicated by the increased level of endoplasmic reticulum (ER) stress, autophagy, and apoptosis is unknown in the liver of dairy cows. Previous studies show that forkhead box protein A2 (FOXA2) is an important transcriptional factor to maintain cellular metabolic homeostasis, but the mechanisms by which FOXA2 mediates cellular homeostasis disruption in response to LPS remains unclear. To achieve the aims, hepatocytes separated from dairy cows at ∼160 d in milk were pretreated with a specific TLR4 inhibitor TAK-242 for 12 h, followed by LPS treatment for another 12 h to investigate the role of TLR4 in LPS-induced disruption of cellular homeostasis. The results indicated that LPS-induced nuclear factor-κB (NF-κB)-mediated inflammatory cascades, ER stress, autophagy, and apoptosis via activating TLR4 and downregulating FOXA2 expression in bovine hepatocytes. The application of TLR4 inhibitor alleviated LPS-induced inflammation through inactivating NF-κB proinflammatory pathway, restored cell homeostasis by decreasing the level of ER stress, autophagy, and apoptosis, and upregulated FOXA2 expression. Furthermore, we also elevated FOXA2 expression with an overexpression plasmid to clarify its molecular role in response to LPS challenge. FOXA2 overexpression reduced LPS-caused inflammation by inhibiting NF-κB signaling pathway. Also, FOXA2 could alleviate ER stress to block unfolded protein response and suppress autophagic flux. In addition, FOXA2 enhanced mitochondrial membrane potential via reducing pro-apoptotic protein BAX, CASPASE3, and Cleaved CASPASE3 expression and elevating anti-apoptotic protein BCL-2 expression to mitigate LPS-induced apoptosis. Taken together, these findings suggested that FOXA2 is a mediator to alleviate TLR4-controlled inflammation, ER stress, autophagy, and apoptosis in LPS-treated bovine hepatocytes, it could serve as a potential target to intervene cell homeostasis disruption caused by LPS in the liver of dairy cows.

Toll-like receptor 4-mediated inflammation triggered by extracellular IFI16 is enhanced by lipopolysaccharide binding.

Damage-associated molecular patterns (DAMPs) are endogenous molecules activating the immune system upon release from injured cells. Here we show that the IFI16 protein, once freely released in the extracellular milieu of chronically inflamed tissues, can function as a DAMP either alone or upon binding to lipopolysaccharide (LPS). Specifically, using pull-down and saturation binding experiments, we show that IFI16 binds with high affinity to the lipid A moiety of LPS. Remarkably, IFI16 DAMP activity is potentiated upon binding to subtoxic concentrations of strong TLR4-activating LPS variants, as judged by TLR4-MD2/TIRAP/MyD88-dependent IL-6, IL-8 and TNF-α transcriptional activation and release in stimulated monocytes and renal cells. Consistently, using co-immunoprecipitation (co-IP) and surface plasmon resonance (SPR) approaches, we show that IFI16 is a specific TLR4-ligand and that IFI16/LPS complexes display a faster stimulation turnover on TLR4 than LPS alone. Altogether, our findings point to a novel pathomechanism of inflammation involving the formation of multiple complexes between extracellular IFI16 and subtoxic doses of LPS variants, which then signal through TLR4.

Hyperglycaemia and Ischaemia Impair Wound Healing via Toll-like Receptor 4 Pathway Activation in vitro and in an Experimental Murine Model

Diabetes mellitus has reached epidemic proportions. Foot ulceration is a multifactorial complication of diabetes associated with marked morbidity and mortality. Innate immune Toll-like receptor 4 (TLR4) mediated inflammation has been implicated in the systemic pathogenesis of diabetes and may contribute to impairment of wound healing. This study investigates the effect of high glucose and hypoxic conditions on TLR4 activation and signalling in vitro and in vivo. Fibroblasts cultured at physiological glucose concentration (5.5 mM) were exposed to glucose concentrations from 0 mM to 25 mM, with duplicates placed in a hypoxic chamber. TLR4 inhibition was assessed in the 25 mM glucose groups. Diabetes was induced in wild type (WT) and TLR4 knockout (KO) C57BL/6 mice by intraperitoneal injection of low dose streptozocin (STZ). Hindlimb ischaemia was induced by femoral artery ligation four weeks post streptozocin, and a full thickness 4 mm skin wound inflicted below the knee. Wound healing was assessed via digital planimetry on days 3, 7, and 14 post surgery. Hypoxic and high glucose (25 mM) conditions led to an increase in TLR4 protein expression, apoptosis, and interleukin (IL)-6 release. Inhibition with a TLR4 neutralising antibody and specific TLR4 antagonist ameliorated the effects of high glucose and ischaemia (p < .05). In vivo, wound healing was significantly impaired in the diabetic ischaemic group at day 14 (p < .05). Diabetic ischaemic wounds in TLR4 KO mice exhibited significantly improved healing rates compared with those in WT mice at all time points. Hypoxia stimulates upregulation of TLR4 protein expression and this effect is exaggerated by hyperglycaemia. In TLR4 KO mice, there is a significant improvement in the healing of diabetic ischaemic wounds compared with WT. It is suggested that a synergistic effect between hypoxia and hyperglycaemia impairing wound healing exists, through TLR4 mediated inflammation.

Hyperglycaemia and Ischaemia Impair Wound Healing via Toll-like Receptor 4 Pathway Activation in vitro and in an Experimental Murine Model

Diabetes mellitus has reached epidemic proportions. Foot ulceration is a multifactorial complication of diabetes associated with marked morbidity and mortality. Innate immune Toll-like receptor 4 (TLR4) mediated inflammation has been implicated in the systemic pathogenesis of diabetes and may contribute to impairment of wound healing. This study investigates the effect of high glucose and hypoxic conditions on TLR4 activation and signalling invitro and invivo. Fibroblasts cultured at physiological glucose concentration (5.5mM) were exposed to glucose concentrations from 0mM to 25mM, with duplicates placed in a hypoxic chamber. TLR4 inhibition was assessed in the 25mM glucose groups. Diabetes was induced in wild type (WT) and TLR4 knockout (KO) C57BL/6 mice by intraperitoneal injection of low dose streptozocin (STZ). Hindlimb ischaemia was induced by femoral artery ligation four weeks post streptozocin, and a full thickness 4mm skin wound inflicted below the knee. Wound healing was assessed via digital planimetry on days 3, 7, and 14 post surgery. Hypoxic and high glucose (25mM) conditions led to an increase in TLR4 protein expression, apoptosis, and interleukin (IL)-6 release. Inhibition with a TLR4 neutralising antibody and specific TLR4 antagonist ameliorated the effects of high glucose and ischaemia (p<.05). Invivo, wound healing was significantly impaired in the diabetic ischaemic group at day 14 (p<.05). Diabetic ischaemic wounds in TLR4 KO mice exhibited significantly improved healing rates compared with those in WT mice at all time points. Hypoxia stimulates upregulation of TLR4 protein expression and this effect is exaggerated by hyperglycaemia. In TLR4 KO mice, there is a significant improvement in the healing of diabetic ischaemic wounds compared with WT. It is suggested that a synergistic effect between hypoxia and hyperglycaemia impairing wound healing exists, through TLR4 mediated inflammation.

Globo-series glycosphingolipids enhance Toll-like receptor 4-mediated inflammation and play a pathophysiological role in diabetic nephropathy.

Alteration of glycosphingolipid (GSL) expression plays key roles in the pathogenesis and pathophysiology of many important human diseases, including cancer, diabetes and glycosphingolipidosis. Inflammatory processes are involved in development and progression of diabetic nephropathy, a major complication of type 2 diabetes mellitus. GSLs are known to play roles in inflammatory responses in various diseases, and levels of renal GSLs are elevated in mouse models of diabetic nephropathy; however, little is known regarding the pathophysiological role of these GSLs in this disease process. We studied proinflammatory activity of GSLs in diabetic nephropathy using spontaneously diabetic mouse strain KK. Mice were fed a high-fat diet (HFD) (60% kcal from fat) or normal diet (ND) (4.6% kcal from fat) for a period of 8 wk. HFD-feeding resulted in quantitative and qualitative changes of renal globo-series GSLs (particularly Gb3Cer), upregulation of TNF-α, and induction of renal inflammation. Gb3Cer/Gb4Cer treatment enhanced inflammatory responses via TLR4 in TLR4/MD-2 complex expressing cells, including HEK293T, mouse bone marrow-derived macrophages (BMDMs) and human monocytes. Our findings suggest that HFD-induced increase of Gb3Cer/Gb4Cer positively modulate TLR4-mediated inflammatory response, and that such GSLs play an important pathophysiological role in diabetic nephropathy.

Tie2 signalling through Erk1/2 regulates TLR4 driven inflammation

The inflammatory response is essential for eradication of lipopolysaccharide (LPS) presenting microbial invaders but requires exquisite regulation to prevent detrimental vascular inflammation. Endothelial cells play active roles in both the initiation of inflammation, through the detection of LPS by Toll-like Receptor 4 (TLR4), and the resolution of inflammation, through the actions of the receptor tyrosine kinase, Tie2. The process by which Tie2 attenuates LPS-TLR4 driven inflammation is poorly understood. To investigate the effects of Tie2 on TLR4 signalling, Nf-κB activation was monitored in cells expressing Tie2 mutants harboring tyrosine (Y) to phenylalanine (F) substitutions in the cytoplasmic domain. Tie2 attenuated LPS induced Nf-κB activation in a manner requiring Tie2 kinase activation, the carboxy-terminal tyrosine residue Y1100 and downstream Erk1/2 signalling. Tyrosine 1100 was also required for the Tie2 dependent decrease in expression of the TLR4 signalling proteins, TRAF6 and IRAK1 and stabilization of the Nf-κB inhibitor, IκBα. In contrast, upregulation of known TLR4 antagonist miRNA-146b-5p required all three tyrosine phosphorylation sites in Tie2. Finally, we confirmed in an in vivo model that activation of Tie2 signalling reduces LPS mediated inflammation. Our results show that Y1100 initiated Erk1/2 signalling is essential for the anti-inflammatory effect of Tie2 on TLR4 mediated inflammation.

Mincle suppresses Toll-like receptor 4 activation.

Regulation of Toll-like receptor responses is critical for limiting tissue injury and autoimmunity in both sepsis and sterile inflammation. We found that Mincle, a C-type lectin receptor, regulates proinflammatory Toll-like receptor 4 signaling. Specifically, Mincle ligation diminishes Toll-like receptor 4-mediated inflammation, whereas Mincle deletion or knockdown results in marked hyperresponsiveness to lipopolysaccharide in vitro, as well as overwhelming lipopolysaccharide-mediated inflammation in vivo. Mechanistically, Mincle deletion does not up-regulate Toll-like receptor 4 expression or reduce interleukin 10 production after Toll-like receptor 4 ligation; however, Mincle deletion decreases production of the p38 mitogen-activated protein kinase-dependent inhibitory intermediate suppressor of cytokine signaling 1, A20, and ABIN3 and increases expression of the Toll-like receptor 4 coreceptor CD14. Blockade of CD14 mitigates the increased sensitivity of Mincle(-/-) leukocytes to Toll-like receptor 4 ligation. Collectively, we describe a major role for Mincle in suppressing Toll-like receptor 4 responses and implicate its importance in nonmycobacterial models of inflammation.

Toll-like receptor-4 mediated inflammation is involved in the cardiometabolic alterations induced by intermittent hypoxia.

Objective. Intermittent hypoxia (IH) is a major component of sleep apnea syndrome as its cardiometabolic complications have been mainly attributed to IH. The pathophysiology is still poorly understood but there are some similarities with the obesity-associated cardiometabolic complications. As the latter results from inflammation involving toll-like receptor-4 (TLR4) signaling, we assessed this pathway in the cardiometabolic consequences of IH. Methods. Lean adult male TLR4-deficient (TLR4−/−) mice and their controls (C57BL/6 mice) were exposed to either IH (FiO2 21-5%, 1 min cycle, 8 h/day) or air (normoxic mice) for 4 weeks. Animals were assessed at 1-week exposure for insulin tolerance test and after 4-week exposure for morphological and inflammatory changes of the epididymal fat and thoracic aorta. Results. IH induced insulin resistance, morphological and inflammatory changes of the epididymal fat (smaller pads and adipocytes, higher release of TNF-α and IL-6) and aorta (larger intima-media thickness and higher NFκB-p50 activity). All these alterations were prevented by TLR4 deletion. Conclusion. IH induces metabolic and vascular alterations that involve TLR4 mediated inflammation. These results confirm the important role of inflammation in the cardiometabolic consequences of IH and suggest that targeting TLR4/NFκB pathway could represent a further therapeutic option for sleep apnea patients.

Semaphorin 3A inactivation suppresses ischemia-reperfusion-induced inflammation and acute kidney injury.

Recent studies show that guidance molecules that are known to regulate cell migration during development may also play an important role in adult pathophysiologic states. One such molecule, semaphorin3A (sema3A), is highly expressed after acute kidney injury (AKI) in mice and humans, but its pathophysiological role is unknown. Genetic inactivation of sema3A protected mice from ischemia-reperfusion-induced AKI, improved tissue histology, reduced neutrophil infiltration, prevented epithelial cell apoptosis, and increased cytokine and chemokine excretion in urine. Pharmacological-based inhibition of sema3A receptor binding likewise protected against ischemia-reperfusion-induced AKI. In vitro, sema3A enhanced toll-like receptor 4-mediated inflammation in epithelial cells, macrophages, and dendritic cells. Moreover, administration of sema3A-treated, bone marrow-derived dendritic cells exacerbated kidney injury. Finally, sema3A augmented cisplatin-induced apoptosis in kidney epithelial cells in vitro via expression of DFFA-like effector a (cidea). Our data suggest that the guidance molecule sema3A exacerbates AKI via promoting inflammation and epithelial cell apoptosis.