Glucosylation endows nanoparticles with TLR4 agonist capability to trigger macrophage polarization and augment antitumor immunity.

Carbohydrates have emerged as promising candidates for immunomodulation, however, how to present them to immune cells and achieve potent immunostimulatory efficacy remains challenging. Here, we proposed and established an effective way of designing unique glyconanoparticles that can amplify macrophage-mediated immune responses through structural mimicry and multiple stimulation. We demonstrate that surface modification with glucose can greatly augment the immunostimulatory efficacy of nanoparticles, comparing to mannose and galactose. In vitro studies show that glucosylation improved the pro-inflammatory efficacy of iron oxide nanoparticles (IONPs) by up to 300-fold, with the immunostimulatory activity of glucosylated IONPs even surpassing that of LPS under certain conditions. In vivo investigation show that glucosylated IONPs elicited increased antitumor immunity and achieved favorable therapeutic outcomes in multiple murine tumor models. Mechanistically, we proposed that glucosylation potentiated the immunostimulatory effect of IONPs by amplifying toll-like receptors 4 (TLR4) activation. Specifically, glucosylated IONPs directly interacted with the TLR4-MD2 complex, resulting in M1 macrophage polarization and enhanced antitumor immunity via activation of NF-κB, MAPK, and STAT1 signaling pathways. Our work provides a simple modification strategy to endow nanoparticles with potent TLR4 agonist effects, which may shed new light on the development of artificial immune modulators for cancer immunotherapy.

Glycogen synthase kinase-3ß mediates toll-like receptors 4/nuclear factor kappa-B-activated cerebral ischemia-reperfusion injury through regulation of fat mass and obesity-associated protein.

BACKGROUND: Glycogen synthase kinase-3ß (GSK3ß), fat mass and obesity-associated protein (FTO), and toll-like receptors 4 (TLR4) take on critical significance in different biological processes, whereas their interactions remain unclear. The objective was the investigation of the interaction effect in cerebral ischemia-reperfusion (I/R) injury. METHODS: The function of the cerebral cortex in the mouse middle cerebral artery occlusion (MCAO) model (each group n = 6) and P12 cells oxygen-glucose deprivation/reoxygenation (OGD/R) model was analyzed using short hairpin GSK3ß lentivirus and overexpression of FTO lentivirus (in vitro), TLR4 inhibitor (TAK242), and LiCl to regulate GSK3ß, FTO, TLR4 expression, and GSK3ß activity, respectively. RESULTS: After GSK3ß knockdown in the OGD/R model of PC12 cells, the levels of TLR4 and p-p65 were lower than in the control, and the level of FTO was higher than in the control. Knockdown GSK3ß reversed the OGD/R-induced nuclear factor kappa-B transfer to the intranuclear nuclei. As indicated by the result, TLR4 expression was down-regulated by overexpressed FTO, and TLR4 expression was up-regulated notably after inhibition of FTO with the use of R-2HG. After the inhibition of the activity of GSK3ß in vivo, the reduction of FTO in mice suffering from MCAO was reversed. CONCLUSIONS: Our research shows that GSK3ß/FTO/TLR4 pathway contributes to cerebral I/R injury.

Assessing cell chimerism in an acute graft-versus-host disease model established in TLR4 knockout mice.

BACKGROUND: Graft-versus-host disease (GVHD) is a major complication after allogeneic hematopoietic stem cell transplantation. OBJECTIVE: To elucidate the role of Toll-like receptor 4 (TLR4), the major receptor for bacterial lipopolysaccharide, in the development of GVHD, we constructed a GVHD model in TLR4 knockout (TLR4-/-) mice and monitored the cell chimerism. METHODS: In this study, we used polymerase chain reaction to identify whether TLR4 knockout (TLR4-/-) mice were established. Before transplantation, we pretreated mice with irradiation so as to obtain an appropriate irradiation dose. Flow cytometry was applied to measure the chimerism status, the distributions of antigen-presenting cells (APCs), and T-cells in TLR4+/+ and TLR4-/- recipient mice. RESULTS: The general condition of TLR4-/- recipients was better than that of TLR4+/+ recipients, and the TLR4-/- recipient mice showed less severe GVHD manifestations than the TLR4+/+ recipient mice. Most of the APCs and T-cells in the host mouse spleen were derived from donor cells, and CD4+ T-cells, including memory T-cells, were in the majority in host mice. CONCLUSION: In general, our data show that TLR4 deletion attenuated GVHD development, which suggests that TLR4 could be used as a novel target and therapeutic paradigm in GVHD therapies.

ANTECEDENTES: La enfermedad de injerto contra huésped (EICH) es una complicación importante después del trasplante alogénico de células madre hematopoyéticas. OBJETIVOS: Para dilucidar el papel de TLR4, el principal receptor de LPS bacteriano, en el desarrollo de GVHD, construimos un modelo de GVHD en ratones knockout para TLR4 (TLR4-/-) y monitoreamos el quimerismo celular. MÉTODOS: En este estudio, usamos PCR para identificar si se establecieron ratones knockout para TLR4 (TLR4-/-). Antes del trasplante, pretratamos a los ratones con irradiación para obtener la dosis de irradiación adecuada. Se aplicó citometría de flujo para medir el estado de quimerismo, las distribuciones de APC y células T en ratones receptores TLR4+/+ y TLR4-/-. RESULTADOS: El estado general de los receptores de TLR4-/- fue mejor que el de los receptores de TLR4+/+, y los ratones receptores de TLR4-/- mostraron manifestaciones de GVHD menos graves que los ratones receptores de TLR4+/+. La mayoría de las APC y las células T en el bazo del ratón huésped se derivaron de las células del donante, y las células T CD4+, incluidas las células T de memoria, se encontraban en su mayoría en los ratones huéspedes. CONCLUSIÓN: En general, nuestros datos muestran que la eliminación de TLR4 atenuó el desarrollo de GVHD, lo que sugiere que TLR4 podría usarse como un nuevo objetivo y paradigma terapéutico en las terapias de GVHD.

Associations Between Serum Soluble Toll-like Receptors 4 and 9 and Breast Cancer in Egyptian Patients.

BACKGROUND: Toll-like receptors (TLRs) play an important role in regulation of immune cells and are vital in tumorigenesis due to its crucial role in inflammatory microenvironment regulation, as they promote the synthesis and release of inflammatory cytokines and chemokines. Toll-like receptors 4 and TLRs 9 were found to be highly expressed in breast cancer. The aim of this study is to investigate the soluble toll-like receptors 4 and 9 (sTLR4 and sTLR9) as potential biomarkers for diagnosis and prognosis of breast cancer and their association with the clinicopathological parameters of breast cancer. PATIENTS AND METHOD: In this retrospective case-control study, 186 female subjects were recruited and divided into three groups, Group I: 62 healthy control, Group II: 62 subjects diagnosed with non-metastatic breast cancer, and Group III: 62 subjects diagnosed with metastatic breast cancer. Enzyme-linked immunosorbent assay (ELISA) technique was used to quantify the levels of sTLR4 and sTLR9 in serum. RESULTS: Both non-metastatic and metastatic groups showed significant higher levels of both serum sTLR4 and sTLR9 expression compared to healthy controls. Only sTLR9 was significantly increased among metastatic patients compared to non-metastatic group. Serum levels of sTLR9 and sTLR4 were still significantly associated with breast cancer in a multiple logistic regression model (P = <.001). ROC curves showed that both sTLR4 and sTLR9 can be a significant parameter to discriminate between normal females and breast cancer patients. CONCLUSION: Soluble toll-like receptors 4 and sTLR9 are over-expressed in patients with metastatic and non-metastatic BC than in benign cases. The expression levels of sTLR4 and TLR9 have clinical interest as indicators of tumor aggressiveness suggested to be prognostic biomarkers. Toll-like receptors may represent therapeutic targets in breast cancer.

Cathelicidin (LL-37) causes expression of inflammatory factors in coronary artery endothelial cells of Kawasaki disease by activating TLR4-NF-κB-NLRP3 signaling.

BACKGROUND: Kawasaki disease (KD) is a type of vasculitis with an unidentified etiology. Cathelicidin (LL-37) may be involved in the development of the KD process; therefore, further research to investigate the molecular mechanism of LL-37 involvement in KD is warranted. METHODS: Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß, NLRP3, and LL-37 in the sera of healthy subjects, children with KD, and children with pneumonia. Subsequently, human recombinant LL-37 or/and toll-like receptors 4 (TLR4)-specific inhibitor TAK-242 stimulated human coronary artery endothelial cells (HCAECs), CCK-8 was used to detect cell proliferation, flow cytometry to detect apoptosis, transmission electron microscopy to observe cytoskeletal changes, Transwell to measure cell migration ability, ELISA to detect inflammatory factor levels, Western blot analysis to analyze protein levels of toll-like receptors 4 (TLR4) and NF-κB p-65, and quantitative real-time polymerase chain reaction (qRT-PCR) to determine LL-37, NLRP3 mRNA levels. RESULTS: In this study, we found that the level of LL-37 was highly expressed in the serum of children with KD, and after LL-37 stimulation, apoptosis was significantly increased in HCAECs, and the expression levels of TLR4, NLRP3 and inflammatory factors in cells were significantly enhanced. Intervention with the TLR4-specific inhibitor TAK-242 significantly alleviated the LL-37 effects on cellular inflammation, TLR4, NLRP3 promotion effect. CONCLUSIONS: Our data suggest that LL-37 induces an inflammatory response in KD coronary endothelial cells via TLR4-NF-κB-NLRP3, providing a potential target for the treatment of KD.

Regulation of Nur77-TLR4/MyD88 signaling pathway is required for Ginsenoside Rc ameliorates hepatic fibrosis regression by deactivating hepatic stellate cells.

HSCs (hepatic stellate cells) contribute to the excessive extracellular matrix (ECM) deposition plays a key role in the progression of hepatic fibrosis. The present study focused on the hepatoprotective effect of Ginsenoside Rc (Rc), one of the protopanaxadiol type ginsenoside, which has contributed to reverse activated HSCs to improve hepatic fibrosis via regulating Nur77-TLR4/MyD88 signaling pathway. We established the hepatic fibrosis model by intraperitoneal injection of carbon tetrachloride (CCl4). And HSCs were stimulated with TGF-ß, followed by silencing of Nur77, and then incubated in Rc. Rc significantly alleviated histopathological changes, reduced serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels. Rc could upregulate the Nur77 and downregulate fibrosis markers in the liver of mice, including decreasing the expressions of α-SMA, Collagen-I, the ratio of TIMP-1/MMP-13. Rc significantly increased the expression of Nur77 and suppressed the production of ECM in HSCs. Rc inhibited TLR4 signaling pathway, consequently reversing the inflammatory response, including the production of MyD88, IRAK1, IRAK4 and IL-23. When Nur77 was knocked in TGF-ß-stimulated HSCs, TLR4 and α-SMA production were increased. Rc suppressed these activatory effects in Nur77 knockdown HSCs. Rc reduced inflammatory reaction by regulating the Nur77-TLR4 signaling pathway while suppressing the fibrogenesis suggesting, underscoring a promising approach of Rc for the treatment in hepatic fibrosis. Targeting Nur77-TLR4 signaling in HSCs would be the potential strategy for Rc against hepatic fibrosis.

[Mechanism of electroacupuncture penetration needling for relieving synovial inflammation of knee osteoarthritis through TLR4/MyD88/NF-κB signal pathway].

OBJECTIVE: To observe the effects of electroacupuncture (EA) penetration needling on Toll-like receptors 4/myeloid differentiation factor 88/nuclear factor-kappa B (TLR4/MyD88/NF-κB) signaling pathway in rat synovium and the serum-related inflammatory factors, so as to explore the mechanism of EA penetration needling on synovial inflammation in rats with knee osteoarthritis (KOA). METHODS: SD male rats were randomly divided into sham-operation group, model group, EA+penetration needling group, and conventional EA group, with 16 rats in each group. The rats model was prepared by anterior cruciate ligment transection and these rats were forced to exercise for 8 weeks after operation. After successful modeling, in the EA+penetration needling group, the needles were inserted at "Dubi" (ST35) "Neixiyan" (EX-LE4), and at "Xuehai"(SP10) "Liangqiu"(ST34) on the right hind limb, towards each other, 5-8 mm in depth, respectively. In the conventional EA group, the needles were inserted at ST35 and EX-LE4 on the right hind limb, obliquely, at 30° angle to the skin, 3-5 mm in depth; and were inserted at SP10 and ST34 on the right hind limb perpendicularly, 3-5 mm in depth. In these two groups, electric stimulation was operated with dense-disperse wave, 2 Hz/10 Hz in frequency and 0.5-1.5 mA in intensity, retained for 20 min in each treatment. The treatment was given once daily, 10 days as 1 course of treatment, and 2 courses were required at the interval of 2 days. After the intervention, the knee joint effusion was observed by musculoskeletal ultrasound; the contents of IL-1ß, IL-6 and TNF-α in serum were determined by ELISA; the morphological changes in the synovium were observed after H.E. staining; the positive expression of NF-κB p65 in the synovial membrane was detected by immunohistochemical method; the expression levels of TLR4, MyD88, TRAF-6 and NF-κB p65 proteins in the synovial membrane were determined by Western blot. RESULTS: Compared with the sham-operation group, in the model group, the knee joint effusion was obviously increased, the synovial lining cells were distributed irregularly, the cells were disarranged, the pannus was formed largely, and a great number of the inflammatory cells were infiltrated; the contents of serum IL-1ß, IL-6 and TNF-α, the positive expression of NF-κB p65, the protein expression levels of TLR4, MyD88, TRAF-6 and NF-κB p65 in the synovial tissue were increased (P<0.05). Compared with the model group, the knee joint effusion was reduced, the synovial lining cells were proliferated, a small number of the inflammatory cells were infiltrated, and the pannus was formed lightly; the contents of serum IL-1ß, IL-6 and TNF-α, the positive expression of NF-κB p65, the protein expression levels of TLR4, MyD88, TRAF-6 and NF-κB p65 in the synovial tissue were lower (P<0.05) in the EA+penetration needling group and the conventional EA group. In the conventional EA group, the knee joint effusion was increased, the synovial lining cells were proliferated, the inflammatory cells were infiltrated largely, and the pannus was formed increasingly; the contents of serum IL-1ß, IL-6 and TNF-α, and the protein expression levels of TLR4, MyD88 and NF-κB p65 in the synovial tissue were increased when compared with the EA+penetration needling group (P<0.05). CONCLUSION: The EA+penetration needling can significantly relieve the synovial inflammatory reaction and the knee joint effusion in KOA rats. The mechanism is probably related to down-regulating the downstream inflammatory cascade through inhibiting the transduction of TLR4/MyD88/NF-κB signaling pathway.

Remifentanil modulates the TLR4­mediated MMP­9/TIMP1 balance and NF­κB/STAT3 signaling in LPS­induced A549 cells.

Remifentanil is a widely used in general anesthetic that has been found to suppress the inflammatory response in aortic endothelial cells. Therefore, it was hypothesized that remifentanil can inhibit inflammatory dysfunction in lung epithelial cells to alleviate acute lung injury (ALI). The present study aimed to examine the effects of remifentanil on inflammatory injury, MMP-9/tissue inhibitor of metalloproteinase 1 (TIMP1) balance and the potential associated regulatory pathways in A549 cells. Lipopolysaccharide (LPS) was used to treat A549 cells to establish ALI models. The possible roles of different concentrations of remifentanil in cell viability was then determined by CCK-8 and Lactate dehydrogenase release assay. Apoptosis was assessed by flow cytometry analysis and western blotting. Inflammation and oxidative stress were measured by ELISA and corresponding kits respectively. Subsequently, the effects of remifentanil on Toll-like receptor 4 (TLR4) expression and the MMP-9/TIMP1 balance were assessed by western blotting and ELISA. In addition, the effects of remifentanil on NF-κB/STAT3 signaling were evaluated by measuring the protein expression levels of associated pathway components and the degree of NF-κB nuclear translocation using western blotting and immunofluorescence respectively. Remifentanil was found to increase cell viability whilst reducing apoptosis, inflammation and oxidative stress in the LPS-treated cells. In addition, TLR4 inhibitor CLI-095 suppressed MMP-9 expression and secretion while potentiating TIMP1 expression and secretion in LPS-challenged cells. Remifentanil treatment was able to modulate TLR4 to mediate LPS-induced MMP-9/TIMP1 imbalance and suppress the phosphorylation of NF-κB/STAT3 signaling components, in addition to inhibiting NF-κB nuclear translocation. Taken together, remifentanil downregulated TLR4 to reduce MMP-9/TIMP1 imbalance to inhibit inflammatory dysfunction in LPS-treated A549 cells, by regulating NF-κB/STAT3 signaling.

Regulation of SIRT1-TLR2/TLR4 pathway in cell communication from macrophages to hepatic stellate cells contribute to alleviates hepatic fibrosis by Luteoloside.

Regulating macrophage-hepatic stellate cells (HSCs) crosstalk through SIRT1-TLR2/TLR4 has contributed to the essence of new pharmacologic strategies to improve hepatic fibrosis. We investigated how Luteoloside (LUT), one of the flavonoid monomers isolated from Eclipta prostrata (L.) L., modulates macrophage-HSCs crosstalk during hepatic fibrosis. HSC-T6 or rat peritoneal macrophages were activated by TGF-ß or LPS/ATP, and then treated with LUT or Sirtinol (SIRT1 inhibitor) for 6 h. Further, HSCs were cultured with the conditioned medium from the LPS/ATP activated peritoneal macrophages. In HSC-T6 or peritoneal macrophages, LUT could decrease the expressions of α-SMA, Collagen-I, the ratio of TIMP-1/MMP-13. LUT also significantly increased the expressions of SIRT1 and ERRα. And LUT significantly suppressed the releases of pro-inflammatory cytokines, including NLRP3, ASC, caspase-1, IL-1ß, and regulated signaling TLR2/TLR4-MyD88 activation. The expressions of TLR2, TLR4, NLRP3, caspase-1, IL-1ß, α-SMA were increased and the expression of ERRα was decreased by Sirtinol, indicated that LUT might mediate SIRT1 to regulate TLR4 expression and further alleviate inflammation and fibrosis. LUT could regulate SIRT1-mediated TLR4 and ECM in HSCs was reduced, when HSCs were cultured with conditioned medium. Hence, LUT could decrease the expressions of fibrosis markers, reduce the releases of inflammatory cytokines in activated HSCs or macrophages. In conclusion, LUT might be a promising candidate that regulating SIRT1-TLR2/TLR4 signaling in macrophages interacting with HSCs during hepatic fibrosis.

[Protective effects of Lycium ruthenicum Murr. juice on alcoholic liver injury in rats].

Objective: To study the protective effects of Lycium ruthenicum Murr. juice on alcoholic liver injury in rats and explore the regulatory mechanism of toll-like receptors 4 (TLR4)/p38 mitogen-activated protein kinase (p38 MAPK) signaling pathway in this process. Methods: Sixty male SD rats were randomly divided into control group (C), model group (M), low-dose Lycium ruthenicum Murr. juice group (LLM), medium-dose Lycium ruthenicum Murr. juice group (MLM) and high-dose Lycium ruthenicum Murr. juice group (HLM), 12 rats in each group. The group M, LLM, MLM and HLM were treated with 20 ml/kg (8 g/(kg·d)) ethanol (400 g/L) intragastrically and the gavage was divided into two sessions, group C was treated with an equal volume of distilled water at the same time point. Four hours before the first alcohol gavage session, rats in each dose group of Lycium ruthenicum Murr. juice were administered with 2.4, 4.8, 9.6 ml/(kg·d) Lycium ruthenicum Murr. juice respectively, and the other groups were given equal volume of distilled water at the corresponding time points. Four weeks later, the rats were sacrificed 24 hours after the end of the last experiment, blood and liver were collected. The liver index was calculated. The morphology of the liver was observed by HE staining. The expressions of hepatic TLR4, p38 MAPK and phosphorylated p38 mitogen-activated protein kinase (p-p38 MAPK) were detected by immunohistochemistry. The activities of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were detected by colorimetry. The levels of hepatic tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), interleukin-10 (IL-10) and interleukin-18 (IL-18) were detected by enzyme linked immunosorbent assay. Results: Compared with group C, the alcoholic liver injury model was established successfully in Group M. Compared with group M, related indicators in each dose group of Lycium ruthenicum Murr. juice were improved, the improvement of hepatic morphology in group HLM was the most significant, the liver index, the levels of serum ALT, AST and hepatic TLR4, p38 MAPK/p-p38 MAPK ratio, TNF-α, IL-1ß, IL-18 were decreased (P＜ 0.05 or P＜0.01), while the level of hepatic IL-10 was increased (P＜0.01). Comparison among the dose groups of Lycium ruthenicum Murr. juice, the levels of liver index, serum AST and hepatic TLR4, p38 MAPK/p-p38 MAPK ratio, TNF-α, IL-18 in HLM were lower than those in LLM (P＜0.05 or P＜0.01); the level of hepatic IL-10 in HLM was higher than that in LLM and MLM (P＜0.05 or P＜0.01); the other indicators in each dose group had no statistical difference (P＞0.05). Conclusion: Lycium ruthenicum Murr. juice can improve the inflammatory stress by regulating TLR4/p38 MAPK signaling pathway, relieve alcoholic liver injury in rats, and the effect of high-dose group is better than the others.

Persistent TLR4 Activation Promotes Hepatocellular Carcinoma Growth through Positive Feedback Regulation by LIN28A/Let-7g miRNA.

Chronic inflammation caused by liver damage or infection plays an important role in the development and progression of hepatocellular carcinoma (HCC). The activation of Toll-like receptors 4 (TLR4) is involved in HCC tumorigenesis. Moreover, high TLR4 expression in HCC has been linked to poor prognosis. Although the expression of TLR4 in HCC is relatively low compared to hematopoietic cells, it is important to explore the molecular mechanism leading to the elevation of TLR4 in HCC. In this study, we aimed to investigate the positive regulating loop for TLR4 expression in HCC in response to chronic inflammation. Our results confirm that the mRNA expression of TLR4 and proinflammatory cytokines, including interleukin 6 (IL6) and C-C motif chemokine ligand 2 (CCL2), positively correlate in human HCC samples. High TLR4 expression in HCC is more susceptible to lipopolysaccharide (LPS); TLR4 activation in HCC provides growth and survival advantages and thus promotes tumorigenesis. It has been shown that the LIN28/let-7 microRNA (miRNA) axis is a downstream effector of the TLR4 signal pathway, and let-7 miRNA is a potential post-transcriptional regulator for TLR4. Thus, we investigated the correlation between TLR4 and LIN28A mRNA and let-7g miRNA in HCC clinical samples and found that the expression of TLR4 was positively correlated with LIN28A and negatively correlated with let-7g miRNA. Moreover, by culturing PLC/PRF5 (PLC5) HCC cells in low-dose LPS-containing medium to mimic chronic inflammation for persistent TLR4 activation, the mRNA and protein levels of TLR4 and LIN28A were elevated, and let-7g miRNA was decreased. Furthermore, the 3' untranslated region (3'UTR) of TLR4 mRNA was shown to be the target of let-7g miRNA, suggesting that inhibition of let-7g miRNA is able to increase TLR4 mRNA. While parental PLC5 cells have a low susceptibility to LPS-induced cell growth, long-term LPS exposure for PLC5 cells leads to increased proliferation, cytokine expression and stemness properties. In conclusion, our studies demonstrate positive feedback regulation for chronic TLR4 activation in the modulation of TLR4 expression level through the LIN28A/let-7g pathway in HCC and suggest a connection between chronic inflammation and TLR4 expression level in HCC for promoting tumorigenesis.

Hydrogen sulfide attenuates cigarette smoke­induced pyroptosis through the TLR4/NF­κB signaling pathway.

Pyroptosis, a type of programmed cell death mediated by caspases­1 or ­11, may play an important role in airway epithelial injury and airway remodeling, thereby promoting the occurrence of asthma and chronic obstructive pulmonary disease (COPD). Studies have suggested that hydrogen sulfide (H2S) plays a protective role against COPD by inhibiting the activation of the NLR family pyrin domain containing 3 (NLRP3) inflammasome. The present study established a rat model of cigarette smoke (CS)­induced COPD to observe the effects of H2S on cell pyroptosis. A 16HBE cell model was also used to further examine the effects of H2S on the Toll­like receptor 4 (TLR4)/NF­κB signaling pathway is affected by, and to determine the underlying mechanisms. The results revealed that cell pyroptosis was significantly promoted in the model of CS­induced COPD. The cellular experiments also revealed that CS induced the pyroptosis of the cells in a NLRP3/gasdermin D (GSDMD)­dependent manner. In addition, H2S significantly attenuated the effects of CS extract (CSE) on pyroptosis, cell viability and the expression levels of pyroptosis­related proteins, indicating that H2S inhibited pyroptosis by decreasing NLRP3 expression and promoting GSDMD activation. It was also identified that CSE activated TLR4 protein in 16HBE cells, while this was inhibited by H2S. Furthermore, TLR4 and NF­κB overexpression significantly abolished the effects of H2S on cell pyroptosis. On the whole, the findings of the present study demonstrate the role of pyroptosis in the development of COPD and provide an experimental basis for the use of H2S and drugs targeting the TLR4/NF­κB pathway to exert protective effects against COPD.

Ligustilide inhibits the proliferation of human osteoblastoma MG63 cells through the TLR4-ERK pathway.

OBJECTIVE: To study the proapoptotic effect of ligustilide on osteoblastoma (OS) and the relative related molecular mechanism. METHODS AND MATERIALS: An MTT was used to examine the proliferation of OS cells, and Flow cytometry was used to analyze apoptosis and the cell cycle. Western blotting was used to detect the signaling pathway of apoptosis, and immunohistochemical (IH) staining was used to detect the apoptosis status of OS cells. A TLR4 inhibitor was used to study the effect of ligustilide on OS. RESULTS: Ligustilide inhibited OS cell proliferation but had no inhibitory effect on normal bone marrow cells. Flow cytometry results showed that ligustilide induced apoptosis in OS cells, and the cell cycle was arrested at the M/G2 phase. Western blot results showed that ERK, P53, P21, Caspase 9, Caspase 8 and Caspase 3 were all activated; cytochrome C and Bax increased; and Bcl-2 decreased when OS was treated with ligustilide. When an ERK or Caspase inhibitor was added to the culture medium, the apoptosis of OS cells decreased to some degree. When OS cells were pretreated with CLI-095, which is a TLR4 inhibitor, the percentage of apoptotic cells and cell cycle arrest were both reversed. IH results also showed that ligustilide induced apoptosis in OS cells, and the effect was blocked by the TLR4 inhibitor. CONCLUSION: Ligustilide selectively inhibited the proliferation of OS cells by inducing apoptosis, which possibly included endogenous and exogenous apoptosis through TLR4.

Influence of herb-partitioned moxibustion at Qihai (CV6) and bilateral Tianshu (ST25) and Shangjuxu (ST37) acupoints on toll-like receptors 4 signaling pathways in patients with ulcerative coliti.

OBJECTIVE: To evaluate the efficacy of herb-partitioned moxibustion (HPM) at Qihai (CV6), Tianshu (ST25) and Shangjuxu (ST37) acupoints in relieving symptoms and the immune regulation of HPM on the toll-like receptors 4 (TLR4) signaling pathway in ulcerative colitis (UC) patients. METHODS: A randomized, single-blind study was conducted 63 patients to receive HPM or sham HPM treatment. The efficacy outcomes included scores of the Mayo, Baron, inflammatory bowel disease questionnaire (IBDQ), self-rating depression scale (SDS), self-rating anxiety scale (SAS). HE staining was used to observe the histopathological changes of the colon. The expression of inflammatory cytokines and TLR4 signaling pathway related molecules were determined by enzyme-linked immunosorbent assay and immunohistochemistry. RESULTS: Baron, SDS, SAS scores were significantly decreased in moxibustion group (P < 0.05), IBDQ score was significantly greater in the moxibustion group than in the sham moxibustion group (P < 0.05). Histopathology of mucosal biopsies showed that both two groups improved in mucosa after treatment. The expression levels of tumor necrosis factor-α, interleukin-2, interleukin-12, interferon-γ, and TLR4, lipopolysaccharide, myeloid differentiation factor 88, interleukin receptor associated kinase, tumor necrosis factor receptor associated factor 6 and nuclear factor kappa-B p65 were significantly lower in the moxibustion group than in the sham moxibustion group (P < 0.05). CONCLUSION: This study showed that HPM at Qihai?(CV6),?Tianshu?(ST25) and?Shangjuxu (ST37) acupoints is effective to relieve symptoms, anxiety, depression and improving life quality in UC patients, which may be related to the immune regulation of HPM on TLR4 signaling pathway.

Is Toll-like receptor 4 involved in the severity of COVID-19 pathology in patients with cardiometabolic comorbidities?

The severe form of COVID-19 is marked by an abnormal and exacerbated immunological host response favoring to a poor outcome in a significant number of patients, especially those with obesity, diabetes, hypertension, and atherosclerosis. The chronic inflammatory process found in these cardiometabolic comorbidities is marked by the overexpression of pro-inflammatory cytokines such as interleukin-6 (IL-6) and tumoral necrosis factor-alpha (TNF-α), which are products of the Toll-Like receptors 4 (TLR4) pathway. The SARS-CoV-2 initially infects cells in the upper respiratory tract and, in some patients, spread very quickly, needing respiratory support and systemically, causing collateral damage in tissues. We hypothesize that this happens because the SARS-CoV-2 spike protein interacts strongly with TLR4, causing an intensely exacerbated immune response in the host's lungs, culminating with the cytokine storm, accumulating secretions and hindering blood oxygenation, along with the immune system attacks the body, leading to multiple organ failure.

Critical role of TLR4 in uncovering the increased rewarding effects of cocaine and ethanol induced by social defeat in male mice.

BACKGROUND: Substance use disorders and social stress are currently associated with changes in the immune system response by which they induce a proinflammatory state in neurons and glial cells that eventually modulates the reward system. AIMS: The aim of the present work was to assess the role of the immune TLR4 (Toll-like receptors 4) and its signaling response in the increased contextual reinforcing effects of cocaine and reinforcing effects of ethanol (EtOH) induced by social defeat (SD) stress. METHODS: Adult male C57BL/6 J wild-type (WT) mice and mice deficient in TLR4 (TLR4-KO) were assigned to experimental groups according to stress condition (exploration or SD). Three weeks after the last SD, conditioned place preference (CPP) was induced by a subthreshold cocaine dose (1 mg/kg), while another set underwent EtOH 6% operant self-administration (SA). Several inflammatory molecules were analyzed in the hippocampus and the striatum. RESULTS: SD induced higher vulnerability to the conditioned rewarding effects of cocaine only in defeated WT mice. Similarly, defeated WT mice exhibited higher 6% EtOH consumption, an effect that was not observed in the defeated TLR4-KO group. However, the motivation to obtain the drug was observed in both genotypes of defeated animals. Notably, a significant upregulation of the protein proinflammatory markers NFkBp-p65, IL-1ß, IL-17 A and COX-2 were observed only in the defeated WT mice, but not in their defeated TLR4-KO counterparts. CONCLUSIONS: These results suggest that TLR4 receptors mediate the neuroinflammatory response underlying the increase in the rewarding effects of cocaine and EtOH induced by social stress.

Sevoflurane-induced cognitive decline in aged mice: Involvement of toll-like receptors 4.

Toll-like receptors 4 (TLR4) contributes to the pathogenesis of some neurodegenerative diseases. However, little is known about whether TLR4 is associated with sevoflurane-induced cognitive decline. This investigation aims to address the effect of global TLR4 gene knockout on cognitive decline following sevoflurane exposure to mice. Wild-type and TLR4-/- mice were exposed to 3% sevoflurane. Novel object recognition test and Y-maze test were used to analyze cognitive function. Tumor necrosis factor α (TNF-α), interleukin-1ß (IL-1ß) and interleukin-6 (IL-6) in plasma and hippocampus were measured by ELISA. Peripheral administration of recombinant TNF-α to TLR4-/- mice was used to observed the role of TNF-α in cognitive function following sevoflurane. Our results showed that, in contrast to wild-type mice, TLR4 deficiency protected against the cognitive function impairment following sevoflurane exposure, and abrogated IL-1ß, IL-6 and TNF-α response to sevoflurane in the system and the hippocampus. Subcutaneous administration of recombinant TNF-α elevated these cytokine levels in the hippocampus, and resulted in cognitive decline in TLR4-/- mice exposed to sevoflurane. Taken together, our results identify the crucial role of TLR4 in sevoflurane-induced cognitive decline, and showed that TLR4 mediated pro-inflammatory cytokine response to sevoflurane, and consequent cognitive decline in aged mice exposed to sevoflurane, and imply a novel target for improvement and therapy of sevoflurane-associated cognitive decline.

Toll-like receptors as diagnostic targets in pellucid marginal degeneration.

The main purpose of this study is to evaluate the diagnostic role of Toll-like receptors 2 (TLR2) and 4 (TLR4) expression in corneal and conjunctival epithelial cells of eyes with pellucid marginal degeneration (PMD) compared to keratoconus patients (KC) and control subjects. A prospective case-control study in 29 PMD eyes, 109 KC eyes and 72 healthy eyes was done. All participants were subjected to a clinical, topographic, aberrometric and tomographic exam with extraction of corneal and conjunctival epithelial cells through scraping. The TLR2 and TLR4 expression was measured with flow cytometry. Receiver operating characteristic (ROC) curve analysis was used to determine the most appropriate cutoff point for predicting the risk of PMD and KC. Correlations between TLR2/TLR4 expression and the severity of PMD/KC were evaluated. A TLRs follow-up review was made 19 ± 4 months after to the first review. As result, mean expression of TLR2 and TLR4 in both corneal and conjunctival epithelial cells was significantly higher in eyes with corneal ectasia (PMD and KC) than in control eyes (all p < 0.05). Conjunctival TLR4 expression showed the highest capacity to diagnose the existence of PMD (odd ratio 42.84; 95% confidence interval:6.20-296.20; p < 0.0001) after adjusting by eye rubbing and steeper corneal meridian. Moreover, we found an association between the TLR2/TLR4 overexpression with the severity of the PMD and KC measured by corneal topographic, aberrometric and tomographic quantitative parameters (all p < 0.05). Differences on TLR2/TLR4 expression between study groups were maintained during the follow-up period. In conclusion, the TLR2/TLR4 overexpression in corneal and conjunctival epithelial cells of PMD and KC patients compared to healthy control subjects have demonstrated their role as diagnostic target in both corneal ectatic disorders.

Acute glucose load induced islet ß cells dysfunction in TLR4 dependent manner in male mice.

Recent studies highlighted the significance of chronic inflammation, which is mediated in part by toll-like receptors 4 (TLR4), in islet ß cell dysfunction by high-glucose exposure. However, about it is unclear whether islet ß cell dysfunction in response to high glucose is associated with TLR4. This investigation was designed to address the effect of TLR4 deficiency on insulin secretion in mice in response to acute intravenous glucose load. Hyperglycemic clamp was used to impair insulin secretion, and intraperitoneal glucose tolerance test was carried out to analyze insulin secretion function of islet ß cells. Our results showed that TLR4 deficiency repressed insulin secretion impairment in response to acute intravenous glucose load. Compared to wild-type mice, TLR4-/- mice did not exhibit increase of IL-1ß and TNF-α level in plasma and pancreatic tissue in response to acute intravenous load of high glucose. However, recombinant IL-1ß or TNF-α administration restored insulin secretion impairment induced by high glucose in TLR4-/- mice. Taken together, our results demonstrated that TLR4 activation and subsequent IL-1ß and TNF-α production contribute to islet ß cell dysfunction in mice in response to acute intravenous load of high glucose, which may provide a theoretical basis for diabetes complication improvement by physical exercise.

Effects of lime (Citrus aurantifolia) peel to the expression of mRNA toll-like receptors 4 in balb/c mice-infected Salmonella typhi.

Lime peel contains metabolic compounds that have lethal effects of bacterial cells, but its effect as an antibacterial modulate innate immunity pathways, especially toll-like receptor 4 (TLR-4) signaling pathway, is unclear. This study examined the effects of lime peel extract (LPE) on the activity of TLR 4 in Balb/c mice induced by Salmonella typhi. Mice were induced intraperitoneally and then 3 days after induction, LPE was given orally on two doses (510 and 750 mg/kg BW). The number of bacterial colonization was counted using peritoneal fluid samples by the method of plate count agar. Intervention LPE for 5 days can degrade TLR-4 and the number of colonies of S. typhi. On day 3 after was induced S. typhi, TLR-4 gene expression of Balb/c mice is increased. Postintervention LPE for 5 days, the expression of TLR-4 gene decreased, significantly different at a dose of 750 mg/kg BW (P = 0.04). There was a positive correlation between the expression of TLR-4 gene by the number of bacterial colonization, decreasing gene expression of TLR-4, the number of bacterial colonization is also getting smaller (P = 0.013, r = 0.408). LPE can modulate the TLR-4 signaling pathway in host immunity so that the gene TLR-4 is expressed fewer in numbers. This mechanism causes the bacterial colony number to decrease, not even growth.