TLR2 and TLR4 Mediate Differential Responses to Limb Ischemia through MyD88-Dependent and Independent Pathways

Introduction

The danger signal HMGB1 is released from ischemic myocytes, and mediates angiogenesis in the setting of hindlimb ischemia. HMGB1 is a ligand for innate immune receptors TLR2 and TLR4. While both TLR2 and TLR4 signal through myeloid differentiation factor 88 (MyD88), TLR4 also uniquely signals through TIR-domain-containing adapter-inducing interferon-β (TRIF). We hypothesize that TLR2 and TLR4 mediate ischemic myocyte regeneration and angiogenesis in a manner that is dependent on MyD88 signaling.

Methods

Mice deficient in TLR2, TLR4, MyD88 and TRIF underwent femoral artery ligation in the right hindlimb. Laser Doppler perfusion imaging was used to assess the initial degree of ischemia and the extent of perfusion recovery. Muscle regeneration, necrosis and fat replacement at 2 weeks post-ligation were assessed histologically and vascular density was quantified by immunostaining. In vitro, endothelial tube formation was evaluated in matrigel in the setting of TLR2 and TLR4 antagonism.

Results

While control and TLR4 KO mice demonstrated prominent muscle regeneration, both TLR2 KO and TRIF KO mice exhibited marked necrosis with significant inflammatory cell infiltrate. However, MyD88 KO mice had a minimal response to the ischemic insult with little evidence of injury. This observation could not be explained by differences in perfusion recovery which was similar at two weeks in all the strains of mice. TLR2 KO mice demonstrated abnormal vessel morphology compared to other strains and impaired tube formation in vitro.

Discussion

TLR2 and TRIF signaling are necessary for muscle regeneration after ischemia while MyD88 may instead mediate muscle injury. The absence of TLR4 did not affect muscle responses to ischemia. TLR4 may mediate inflammatory responses through MyD88 that are exaggerated in the absence of TLR2. Additionally, the actions of TLR4 through TRIF may promote regenerative responses that are required for recovery from muscle ischemia.     

MICAL2 is expressed in cancer associated neo-angiogenic capillary endothelia and it is required for endothelial cell viability,motility and VEGF response

The capacity of inducing angiogenesis is a recognized hallmark of cancer cells. The cancer microenvironment, characterized by hypoxia and inflammatory signals, promotes proliferation, migration and activation of quiescent endothelial cells (EC) from surrounding vascular network. Current anti-angiogenic drugs present side effects, temporary efficacy, and issues of primary resistance, thereby calling for the identification of new therapeutic targets.MICALs are a unique family of redox enzymes that destabilize F-actin in cytoskeletal dynamics. MICAL2 mediates Semaphorin3A-NRP2 response to VEGFR1 in rat ECs. MICAL2 also enters the p130Cas interactome in response to VEGF in HUVEC. Previously, we showed that MICAL2 is overexpressed in metastatic cancer. A small-molecule inhibitor of MICAL2 exists (CCG-1423).Here we report that 1) MICAL2 is expressed in neo-angiogenic ECs in human solid tumors (kidney and breast carcinoma, glioblastoma and cardiac myxoma, n = 67, were analyzed with immunohistochemistry) and in animal models of ischemia/inflammation neo-angiogenesis, but not in normal capillary bed; 2) MICAL2 protein pharmacological inhibition (CCG-1423) or gene KD reduce EC viability and functional performance; 3) MICAL2 KD disables ECs response to VEGF in vitro. Whole-genome gene expression profiling reveals MICAL2 involvement in angiogenesis and vascular development pathways.Based on these results, we propose that MICAL2 expression in ECs participates to inflammation-induced neo-angiogenesis and that MICAL2 inhibition should be tested in cancer- and noncancer-associated neo-angiogenesis, where chronic inflammation represents a relevant pathophysiological mechanism.     

TLR4 signaling induced TLR2 expression in the process of mimic cerebral ischemia/reperfusion <Emphasis Type="Italic">in vitro</Emphasis>

Both TLR4 and TLR2 participated in the mediation of the inflammatory injury in the process of partial cerebral ischemia/reperfusion. However, it still remains unclear whether a crosstalk exists between TLR2 and TLR4 in ischemic cerebral damage. In the present study, we investigated the effect of TLR4 signaling on TLR2 expression during mimic cerebral I/R in vitro. BV-2 cells were cultured and treated with ischemia/reperfusion, then transfected with the plasmid pEGFP-H1/TLR4-siRNA, the plasmid pEGFP-H1/control sequence-siRNA and the blank plasmid, respectively. Interestingly, the expression of TLR2 and TLR4 mRNA and protein, NF-κB p65 mRNA and supernatant TNF-α level were significantly higher in ischemia/reperfusion treated cells than those lack of ischemia/reperfusion treatment, and as compared with those in ischemia/reperfusion treated cells without transfection, no significant differences about the above mentioned gene and protein expression were found in the blank plasmid tranfected cells and the plasmid pEGFP-H1/control sequence-siRNA transfected cells respectively, while the expression levels in the plasmid pEGFP-H1/TLR4-siRNA transfected cells were significantly lower. Additionally, in order to determine the effects of pyrrolidinediethyldithiocarbamate (PDTC), an NF-κB inhibitor, on the TLR4-induced TLR2 expression in BV-2 cells treated with ischemia/reperfusion, it was found that TLR4 and TLR2 mRNA expressions in PDTC pretreated cells were significantly lower in comparison with normal saline pretreated cells and non-pretreated cells. The data suggested that TLR2 activation, signaled by TLR4 and regulated by NF-κB, might be directly involved play an important role in ischemia/reperfusion induced brain damage.     

Activation of endothelial TLR2 by bacterial lipoprotein upregulates proteins specific for the neutrophil response

The vascular endothelium is integrally involved in the host response to infection and in organ failure during acute inflammatory disorders such as sepsis. Gram-negative and Gram-positive bacterial lipoproteins circulate in sepsis and can directly activate the endothelium by binding to endothelial cell (EC) TLR2. In this report, we perform the most comprehensive analysis to date of the immune-related genes regulated after activation of endothelial TLR2 by bacterial di- and triacylated lipopeptides. We found that TLR2 activation specifically induces the expression of the genes IL-6, IL-8, CSF2, CSF3, ICAM1 and SELE by human umbilical vein ECs and human lung microvascular ECs. These proteins participate in neutrophil recruitment, adherence and activation at sites of inflammation. Significantly, our studies demonstrate that TLR2-mediated EC responses are specifically geared towards recruitment, activation, and survival of neutrophils and not mononuclear leukocytes, that ECs do not require priming by other inflammatory stimuli to respond to bacterial lipopeptides and, unlike mononuclear leukocytes, TLR2 agonists do not induce ECs to secrete TNF-α. This study suggests that endothelial TLR2 may be an important regulator of neutrophil trafficking to sites of infection in general, and that direct activation of lung endothelial TLR2 may contribute to acute lung injury during sepsis.     

Tetraspanin CD63 Promotes Vascular Endothelial Growth Factor Receptor 2-β1 Integrin Complex Formation,Thereby Regulating Activation and Downstream Signaling in Endothelial Cells in Vitro and in Vivo

CD63 is a member of the transmembrane-4 glycoprotein superfamily (tetraspanins) implicated in the regulation of membrane protein trafficking, leukocyte recruitment, and adhesion processes. We have investigated the involvement of CD63 in endothelial cell (EC) signaling downstream of β1 integrin and VEGF. We report that silencing of CD63 in primary ECs arrested capillary sprouting and tube formation in vitro because of impaired adhesion and migration of ECs. Mechanistically, CD63 associated with both β1 integrin and the main VEGF receptor on ECs, VEGFR2. Our data suggest that CD63 serves to bridge between β1 integrin and VEGFR2 because CD63 silencing disrupted VEGFR2-β1 integrin complex formation identified using proximity ligation assays. Signaling downstream of β1 integrin and VEGFR2 was attenuated in CD63-silenced cells, although their cell surface expression levels remained unaffected. CD63 was furthermore required for efficient internalization of VEGFR2 in response to VEGF. Importantly, systemic delivery of VEGF failed to potently induce VEGFR2 phosphorylation and downstream signaling in CD63-deficient mouse lungs. Taken together, our findings demonstrate a previously unrecognized role for CD63 in coordinated integrin and receptor tyrosine kinase signaling in vitro and in vivo.     

Profibrotic Effect of Interleukin-18 in HK-2 Cells Is Dependent on Stimulation of the Toll-like Receptor 4 (TLR4) Promoter and Increased TLR4 Expression

IL-18 is an important mediator of obstruction-induced renal fibrosis and tubular epithelial cell injury independent of TGF-β1 activity. We sought to determine whether the profibrotic effect of IL-18 is mediated through Toll-like receptor 4 (TLR4). Male C57BL6 wild type and mice transgenic for human IL-18-binding protein were subjected to left unilateral ureteral obstruction versus sham operation. The kidneys were harvested 1 week postoperatively and analyzed for IL-18 production and TLR4 expression. In a separate arm, renal tubular epithelial cells (HK-2) were directly stimulated with IL-18 in the presence or absence of a TLR4 agonist, TLR4 antagonist, or TLR4 siRNA knockdown. Cell lysates were analyzed for TLR4, α-smooth muscle actin, and E-cadherin expression. TLR4 promotor activity, as well as AP-1 activation and the effect of AP-1 knockdown on TLR4 expression, was evaluated in HK-2 cells in response to IL-18 stimulation. The results demonstrate that IL-18 induces TLR4 expression during unilateral ureteral obstruction and induces TLR4 expression in HK-2 cells via AP-1 activation. Inhibition of TLR4 or knockdown of TLR4 gene expression in turn prevents IL-18-induced profibrotic changes in HK-2 cells. These results suggest that IL-18 induces profibrotic changes in tubular epithelial cells via increased TLR4 expression/signaling.     

Retinal pigment epithelium-derived transforming growth factor-β2 inhibits the angiogenic response of endothelial cells by decreasing vascular endothelial growth factor receptor-2 expression

Transforming growth factor-β (TGF-β) is a multifunctional cytokine that is known to modulate various aspects of endothelial cell (EC) biology. Retinal pigment epithelium (RPE) is important for regulating angiogenesis of choriocapillaris and one of the main cell sources of TGF-β secretion, particularly TGF-β2. However, it is largely unclear whether and how TGF-β2 affects angiogenic responses of ECs. In the current study, we demonstrated that TGF-β2 reduces vascular endothelial growth factor receptor-2 (VEGFR-2) expression in ECs and thereby inhibits vascular endothelial growth factor (VEGF) signaling and VEGF-induced angiogenic responses such as EC migration and tube formation. We also demonstrated that the reduction of VEGFR-2 expression by TGF-β2 is due to the suppression of JNK signaling. In coculture of RPE cells and ECs, RPE cells decreased VEGFR-2 levels in ECs and EC migration. In addition, we showed that TGF-β2 derived from RPE cells is involved in the reduction of VEGFR-2 expression and inhibition of EC migration. These results suggest that TGF-β2 plays an important role in inhibiting the angiogenic responses of ECs during the interaction between RPE cells and ECs and that angiogenic responses of ECs may be amplified by a decrease in TGF-β2 expression in RPE cells under pathologic conditions.     

TLR4-MyD88/Mal-NF-kB Axis Is Involved in Infection of HSV-2 in Human Cervical Epithelial Cells

We have established an in vitro HSV-2 acute infection model with Human cervical epithelial (HCE cells, the primary target and natural host cells for HSV-2) to investigate the role of TLRs-mediated innate immune response to HSV-2. In current study, we found that HSV-2 infection induced activity of NF-kB reporter and expression of cytokines are TLR4-dependent using approaches with shRNA and TLR4 antagonist. Knockdown experiments demonstrated that the adaptor molecules MyD88 and Mal of the TLRs signaling pathway are required in the HSV-2 induced TLR4-dependent NF-kB activation in HCE cells. Western blot assay suggested that knockdown of TLR4 decreased the phosphorylation of IRAK1 and inhibitor of NF-kB (IkB-α) upon HSV-2 infection. Finally, decreased expression of either TLR4 or MyD88/Mal alone or both significantly abolished productions of IL-6 and IFN-β by ELISA analysis. Taken together, our results from the in vitro infection model reveal for the first time that there exists the pathway via TLR4-Mal/MyD88-IRAK1-NF-kB axis in human cervical epithelial cells in response to HSV-2 infection.     

TLR2 and TLR7 mediate distinct immunopathological and antiviral plasmacytoid dendritic cell responses to SARS‐CoV‐2 infection

Understanding the molecular pathways driving the acute antiviral and inflammatory response to SARS‐CoV‐2 infection is critical for developing treatments for severe COVID‐19. Here, we find decreasing number of circulating plasmacytoid dendritic cells (pDCs) in COVID‐19 patients early after symptom onset, correlating with disease severity. pDC depletion is transient and coincides with decreased expression of antiviral type I IFNα and of systemic inflammatory cytokines CXCL10 and IL‐6. Using an in vitro stem cell‐based human pDC model, we further demonstrate that pDCs, while not supporting SARS‐CoV‐2 replication, directly sense the virus and in response produce multiple antiviral (interferons: IFNα and IFNλ1) and inflammatory (IL‐6, IL‐8, CXCL10) cytokines that protect epithelial cells from de novo SARS‐CoV‐2 infection. Via targeted deletion of virus‐recognition innate immune pathways, we identify TLR7‐MyD88 signaling as crucial for production of antiviral interferons (IFNs), whereas Toll‐like receptor (TLR)2 is responsible for the inflammatory IL‐6 response. We further show that SARS‐CoV‐2 engages the receptor neuropilin‐1 on pDCs to selectively mitigate the antiviral interferon response, but not the IL‐6 response, suggesting neuropilin‐1 as potential therapeutic target for stimulation of TLR7‐mediated antiviral protection.     

RP105 involved in activation of mouse macrophages via TLR2 and TLR4 signaling

RP105 is a member of the toll-like receptor family of proteins that transmits an activation signal in B cells, playing a role in regulation of B cell growth and death; in macrophages and dendritic cells, RP105 is a specific inhibitor of TLR4 signaling. RP105 is uniquely important for regulating TLR4-dependent signaling. It also proved that RP105 is closely related to TLR2 in macrophage activation by Mycobacterium tuberculosis lipoproteins. The aim of our study is to investigate the role of RP105 in mouse macrophages activation of TLR4 and TLR2 signaling by lipopolysaccharides (LPS) and Pam3CysSerLys4 (Pam3CSK4) alone or in combination, and the interaction between TLR2 and TLR4 signaling through RP105. Our results indicate that besides exhibiting negative regulation of TNF-α and IL12-p40 secretion in macrophage activated by LPS, RP105 is also involved in macrophages activation by Pam3CSK4 through TLR2 signaling and exhibited regulation to IL-10 and RANTES production by mouse peritoneal macrophage activated by Pam3CSK4. In macrophages activation by LPS and Pam3CSK4 in combination, TLR2 signaling can overcome RP105-mediated regulation of TLR4 signaling. Thus, our data demonstrate that not only TLR4 signaling, but also RP105 appears to be an essential accessory for immune responses through TLR2 signaling. The function of TLR2 and TLR4 in response to TLR ligands could be associated with each other by RP105. These results can help us understanding the unique role of RP105 in macrophages response to TLR ligands.     

Toll-like receptor 2 induced angiogenesis and invasion is mediated through the Tie2 signalling pathway in rheumatoid arthritis

Background

Angiogenesis is a critical early event in inflammatory arthritis, facilitating leukocyte migration into the synovium resulting in invasion and destruction of articular cartilage and bone. This study investigates the effect of TLR2 on angiogenesis, EC adhesion and invasion using microvascular endothelial cells and RA whole tissue synovial explants ex-vivo.

Methods

Microvascular endothelial cells (HMVEC) and RA synovial explants ex vivo were cultured with the TLR2 ligand, Pam3CSK4 (1 µg/ml). Angiopoietin 2 (Ang2), Tie2 and TLR2 expression in RA synovial tissue was assessed by immunohistology. HMVEC tube formation was assessed using Matrigel matrix assays. Ang2 was measured by ELISA. ICAM-1 cell surface expression was assessed by flow cytometry. Cell migration was assessed by wound repair scratch assays. ECM invasion, MMP-2 and -9 expression were assessed using transwell invasion chambers and zymography. To examine if the angiopoietin/Tie2 signalling pathway mediates TLR2 induced EC tube formation, invasion and migration assays were performed in the presence of a specific neutralising anti-Tie2mAb (10 ug/ml) and matched IgG isotype control Ab (10 ug/ml).

Results

Ang2 and Tie2 were localised to RA synovial blood vessels, and TLR2 was localised to RA synovial blood vessels, sub-lining infiltrates and the lining layer. Pam3CSK4 significantly increased angiogenenic tube formation (p<0.05), and upregulated Ang2 production in HMVEC (p<0.05) and RA synovial explants (p<0.05). Pam3CSK4 induced cell surface expression of ICAM-1, from basal level of 149±54 (MFI) to 617±103 (p<0.01). TLR-2 activation induced an 8.8±2.8 fold increase in cell invasion compared to control (p<0.05). Pam3CSK4 also induced HMVEC cell migration and induced MMP-2 and -9 from RA synovial explants. Neutralisation of the Ang2 receptor, Tie2 significantly inhibited Pam3CSK4-induced EC tube formation and invasion (p<0.05).

Conclusion

TLR2 activation promotes angiogenesis, cell adhesion and invasion, effects that are in part mediated through the Tie2 signalling pathway, key mechanisms involved in the pathogenesis of RA.     

Acetylbritannilactone Modulates Vascular Endothelial Growth Factor Signaling and Regulates Angiogenesis in Endothelial Cells

The present study was conducted to determine the effects of 1-O-acetylbritannilactone (ABL), a compound extracted from Inula britannica L., on vascular endothelial growth factor (VEGF) signaling and angiogenesis in endothelial cells (ECs). We showed that ABL promotes VEGF-induced cell proliferation, growth, migration, and tube formation in cultured human ECs. Furthermore, the modulatory effect of ABL on VEGF-induced Akt, MAPK p42/44, and p38 phosphorylation, as well as on upstream VEGFR-2 phosphorylation, were associated with VEGF-dependent Matrigel angiogenesis in vivo. In addition, animals treated with ABL (26 mg/kg/day) recovered blood flow significantly earlier than control animals, suggesting that ABL affects ischemia-mediated angiogenesis and arteriogenesis in vivo. Finally, we demonstrated that ABL strongly reduced the levels of VEGFR-2 on the cell surface, enhanced VEGFR-2 endocytosis, which consistent with inhibited VE-cadherin, a negative regulator of VEGF signaling associated with VEGFR-2 complex formation, but did not alter VE-cadherin or VEGFR-2 expression in ECs. Our results suggest that ABL may serve as a novel therapeutic intervention for various cardiovascular diseases, including chronic ischemia, by regulating VEGF signaling and modulating angiogenesis.     

Salvianolic acid A alleviated inflammatory response mediated by microglia through inhibiting the activation of TLR2/4 in acute cerebral ischemia-reperfusion

BackgroundToll-like receptor 2 and Toll-like receptor 4 (TLR2/4) on microglia have been found as important regulators in the inflammatory response during cerebral ischemia/reperfusion (I/R). In China, traditional Chinese medicine Salvia miltiorrhiza (danshen) and its some components are considered to be effective in rescuing cerebral I/R injury through clinical practice.Hypothesis/PurposeHere we examined the effect of Salvianolic acid A (SAA), a monomer compound in the water extract of Salvia miltiorrhiza, on TLR2/4 of microglia and its mediated inflammatory injury during cerebral I/R in vivo and in vitro.Study DesignFor exploring the effect of SAA on cerebral I/R and TLR2/4, classic middle cerebral artery occlusion (MCAO) model and oxygen glucose deprivation / reoxygenation (OGD/R) model of co-culture with primary hippocampal neurons and microglia in vitro were used. Signal pathway research and gene knockout have been applied to further explain its mechanism.MethodsThe evaluation indexes of I/R injury included infarct size, edema degree and pathology as well as primary hippocampal neurons and microglia culture, ELISA, western, RT-PCR, HE staining, immunofluorescence, flow cytometry, siRNA gene knockout were also employed.ResultsSAA significantly improved the degree of brain edema and ischemic area in I/R rats accompanied by decreases in levels of interleukin-1β (IL-1β) and tumor necrosis factor-alpha (TNF-α). Pathological staining revealed that SAA could reduce inflammatory cell infiltration and mcirogila activation after reperfusion. Both protein and gene expression of TLR2 and TLR4 in ischemic hemisphere were obviously inhibited by SAA treatment while changes were not found in the non-ischemic hemisphere. In order to further study its mechanism, OGD/R model was used to mimic inflammatory damage of ischemic tissue by co-culturing primary rat hippocampal neurons and microglial cells. It was found that SAA also inhibited the protein and gene expression of TLR2 and TLR4 after OGD/R injury in microglia. After TLR2/4 knockout, the inhibitory effect of SAA on IL-1β and TNF-α levels in cell supernatant and neuron apoptosis were significantly weakened in each dose group. Moreover, expression levels of myeloid differentiation factor 88 (MyD88), NFκB, IL-1β and IL-6 in TLR2/4 mediated inflammatory pathway were reduced with SAA treatment.ConclusionSAA could significantly reduce the inflammatory response and injury in cerebral ischemia-reperfusion in vivo and in vitro, and its mechanism may be through the inhibition of TLR2/4 and its related signal pathway.     

TLR2 Directing PD-L2 Expression Inhibit T Cells Response in Schistosoma japonicum Infection

Toll-like receptor 2 (TLR2) was shown to be an important immune receptor involved in the recognition of schistosome antigens, especially soluble egg antigen (SEA). In mice models with Schistosoma japonicum acute infection, we observed enhanced T cell-mediated immune responses in TLR2 knock out (TLR2^−/−) mice compared with B6 mice. In Schistosoma japonicum chronic infection models, programmed death ligand 1 (PD-L1) and programmed death ligand 2 (PD-L2) expression as well as TLR2 expression gradually increased in B6 mice, while only PD-L2 expression significantly decreased in TLR2^−/− mice. Meanwhile, Programmed Death 1(PD-1) expression on CD4⁺T cells was down-regulated in TLR2^−/− mice after a large number of egg appeared. We also found that stimulation with schistosome antigens, especially SEA, could up-regulate PD-L2 expression on BMDCs in a TLR2-dependent manner in vitro. Schistosome antigens primed-BMDCs with impaired expression of TLR2 or PD-L2 could induce CD4⁺T cells to produce low level of IL-10 or high level of IFN-γ. Our results indicated that TLR2 signaling can direct PD-L2 expression on DCs, which binds to PD-1 mainly on CD4⁺T cells, to help inhibit T cells response in Schistosoma japonicum infection.     

The HIV-1 Envelope Transmembrane Domain Binds TLR2 through a Distinct Dimerization Motif and Inhibits TLR2-Mediated Responses

HIV-1 uses a number of means to manipulate the immune system, to avoid recognition and to highjack signaling pathways. HIV-1 infected cells show limited Toll-Like Receptor (TLR) responsiveness via as yet unknown mechanisms. Using biochemical and biophysical approaches, we demonstrate that the trans-membrane domain (TMD) of the HIV-1 envelope (ENV) directly interacts with TLR2 TMD within the membrane milieu. This interaction attenuates TNFα, IL-6 and MCP-1 secretion in macrophages, induced by natural ligands of TLR2 both in in vitro and in vivo models. This was associated with decreased levels of ERK phosphorylation. Furthermore, mutagenesis demonstrated the importance of a conserved GxxxG motif in driving this interaction within the membrane milieu. The administration of the ENV TMD in vivo to lipotechoic acid (LTA)/Galactosamine-mediated septic mice resulted in a significant decrease in mortality and in tissue damage, due to the weakening of systemic macrophage activation. Our findings suggest that the TMD of ENV is involved in modulation of the innate immune response during HIV infection. Furthermore, due to the high functional homology of viral ENV proteins this function may be a general character of viral-induced immune modulation.     

Statins attenuate high mobility group box-1 protein induced vascular endothelial activation : a key role for TLR4/NF-κB signaling pathway

High mobility group box-1 (HMGB1) has recently been implicated as a proinflammatory cytokine that plays critical roles in endothelial dysfunction and atherosclerosis. Atorvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, exerts anti-inflammatory effects in the cardiovascular system beyond its cholesterol-lowering property. The aim of our study was to investigate whether atorvastatin inhibits HMGB1-induced vascular endothelial activation, and elucidate the underlying molecular mechanism. In this study, we found that atorvastatin, at concentrations ranging from 0.1 to 10 μM, effectively and in a dose-dependent manner inhibited HMGB1-induced endothelial cells (ECs) activation. Incubation of ECs with 10 μM atorvastatin reduced adhesion molecules (ICAM-1 and E-selectin) expression concomitant with a significant inhibition in HMGB1-stimulated leukocyte-endothelial adhesion. Further experiments showed that atorvastatin markedly suppressed HMGB1-induced Toll like receptor 4 (TLR4) expression, Nuclear factor kappaB (NF-κB) nuclear translocation and DNA binding activity in ECs. Similar effects were also observed in ECs pretreated with the TLR4- specific inhibitor CLI-095, suggesting an important role of TLR4/NF-κB pathway. These findings indicate that atorvastatin attenuates HMGB1-induced vascular endothelial activation. The underlying mechanism involves, at least in part, inhibition of TLR4/NF-κB-dependent signaling pathway, which provied the new evidence for therapeutic application of statins to target inflammatory processes in cardiovascular disease.     

Role of TLR2- and TLR4-mediated signaling in Mycobacterium tuberculosis-induced macrophage death

Infection of macrophages with Mycobacterium tuberculosis (Mtb) induces cell death by apoptosis or necrosis. TLRs 2 and 4 recognition of mycobacterial ligands has been independently associated to apoptosis induction. To try to understand the particular contribution of these receptors to apoptotic or necrotic signaling upon infection with live Mtb H37Rv, we used macrophage lines derived from wild-type or TLR2-, TLR4-, and MyD88-deficient mouse strains. Mtb-infection triggered apoptosis depending on a TLR2/TLR4/MyD88/p38/ERK/PI-3K/NF-kB pathway; however, necrosis was favored in absence of TLR4 signaling independently of p38, ERK1/2, PI-3K or NF-κB activity. In conclusion, our results indicate that cooperation between TLR2- and TLR4-dependent mediated signals play a critical role in macrophage apoptosis induced by Mtb and the TLR4-mediated signaling has important role in the maintenance of the balance between apoptotic vs. necrotic cell death induced by macrophage infection with Mtb.     

Toll-like Receptor 4 (TLR4) Antagonist Eritoran Tetrasodium Attenuates Liver Ischemia and Reperfusion Injury through Inhibition of High-Mobility Group Box Protein B1 (HMGB1) Signaling

Toll-like receptor 4 (TLR4) is ubiquitously expressed on parenchymal and immune cells of the liver and is the most studied TLR responsible for the activation of proinflammatory signaling cascades in liver ischemia and reperfusion (I/R). Since pharmacological inhibition of TLR4 during the sterile inflammatory response of I/R has not been studied, we sought to determine whether eritoran, a TLR4 antagonist trialed in sepsis, could block hepatic TLR4-mediated inflammation and end organ damage. When C57BL/6 mice were pretreated with eritoran and subjected to warm liver I/R, there was significantly less hepatocellular injury compared to control counterparts. Additionally, we found that eritoran is protective in liver I/R through inhibition of high-mobility group box protein B1 (HMGB1)-mediated inflammatory signaling. When eritoran was administered in conjunction with recombinant HMGB1 during liver I/R, there was significantly less injury, suggesting that eritoran blocks the HMGB1–TLR4 interaction. Not only does eritoran attenuate TLR4-dependent HMGB1 release in vivo, but this TLR4 antagonist also dampened HMGB1’s release from hypoxic hepatocytes in vitro and thereby weakened HMGB1’s activation of innate immune cells. HMGB1 signaling through TLR4 makes an important contribution to the inflammatory response seen after liver I/R. This study demonstrates that novel blockade of HMGB1 by the TLR4 antagonist eritoran leads to the amelioration of liver injury.