Bacterial superantigens enhance the in vitro proinflammatory response and in vivo lethality of the TLR2 agonist bacterial lipoprotein

Bacterial superantigens are Gram-positive exotoxins that induce proinflammatory cytokine release in vitro, cause lethal shock in vivo, and can be detected in the bloodstream of critically ill patients. They also have a powerful priming effect on the TLR4 agonist LPS. The aim of this study was to investigate the relationship between superantigens and the TLR2 agonist bacterial lipoprotein (BLP). Priming of human monocytes or PBMCs with superantigens significantly enhanced proinflammatory cytokine TNF-α and IL-6 release in response to BLP stimulation. The priming effect of superantigens could be blocked by inhibiting p38 MAPK during the priming phase as opposed to NF-κB or ERK inhibition. This was consistent with higher expression of the phosphorylated p38 after superantigen priming and BLP or LPS stimulation. C57BL/6 mice with superantigen priming (10 μg/mouse) when challenged with BLP (600 μg/mouse) exhibited substantially higher mortality (100%) compared with mice without superantigen priming (zero). Mice given superantigen alone did not demonstrate any signs of illness. Mice challenged with both superantigen and BLP had significantly higher levels of serum TNF-α and IL-6 compared with those of mice challenged with either agent alone. Depletion of the monocyte/macrophage subpopulation significantly reduced the mortality rate from 100 to 20% in superantigen-primed, BLP-challenged C57BL/6 mice, with a 5- to 10-fold decrease in serum TNF-α and IL-6. Our results demonstrate that bacterial superantigens enhance the in vitro proinflammatory cytokine release and in vivo lethality of BLP. This novel finding may help to explain the massive proinflammatory cytokine release seen in superantigen-mediated septic shock.     

MicroRNA-146a Is Upregulated by and Negatively Regulates TLR2 Signaling

TLR signaling is a crucial component of the innate immune response to infection. MicroRNAs (miRNAs) have been shown to be upregulated during TLR signaling. Specifically, microRNA-146a (miR-146a) plays a key role in endotoxin tolerance by downregulating interleukin-1 receptor-associated kinase 1 (IRAK-1). The aim of this study was to assess the role of miR-146a in the TLR2 signaling and development of bacterial lipoprotein (BLP) self-tolerance and cross-tolerance to bacteria. Expression of miR-146a increased in a dose- and time-dependent manner in BLP-stimulated human THP-1 promonocytic cells. In BLP-tolerised cells miR-146a was even further upregulated in response to BLP re-stimulation (p<0.001). Re-stimulation of BLP-tolerised cells with heat-killed gram-negative Salmonella typhimurium (S. typhimurium), but not gram-positive Staphylococcus aureus (S. aureus), led to significant overexpression of miR-146a (p<0.05). Transfection of naive cells with a miR-146a mimic substantially suppressed TNF-α production (p<0.05). Furthermore, overexpression of miR-146a resulted in strong reduction in IRAK-1 and phosphorylated IκBα expression in naive and S. typhimurium-stimulated THP-1 cells. Collectively, miR-146a is upregulated in response to BLP and bacterial stimulation in both naive and BLP-tolerised cells. Overexpression of miR-146a induces a state analogous to tolerance in BLP-stimulated cells and therefore may represent a future target for exogenous modulation of tolerance during microbial infection and sepsis.     

乳酸调控大鼠肠黏膜微血管内皮细胞的NF-κB信号通路

目的探讨内毒素（LPS）刺激大鼠肠黏膜微血管内皮细胞（RIMMVECs）后,乳酸（LA）调控NF-κB信号通路中磷酸化IκBα和NF-κB p65蛋白表达情况,肿瘤坏死因子α（TNF-α）和白细胞介素6（IL-6）mRNA表达情况,阐明乳酸发挥作用的最佳时间及其调控NF-κB信号通路的部位。方法提取RIMMVECs总蛋白和总RNA,用Western blotting检测NF-κB p65、IκBα及p-IκBα蛋白表达水平,用real-time PCR对TNF-α和IL-6 mRNA进行定量检测。结果乳酸能降低LPS诱导RIMMVECs分泌的TNF-α和IL-6 mRNA表达水平,并分别于24 h和3 h下调效果最明显;乳酸能抑制IκBα磷酸化及NF-κB转录活性,并于4～8 h达到最佳效果;乳酸发挥作用部位是抑制信号通路中IκBα磷酸化。结论乳酸通过抑制IκBα磷酸化而阻断NF-κB的激活,抑制下游炎性因子表达,进而发挥出很好的预防炎症效果。     

B7-H3 participates in the development of experimental pneumococcal meningitis by augmentation of the inflammatory response via a TLR2-dependent mechanism

In addition to a well-documented role in regulating T cell-mediated immune responses, B7-H3, a newly discovered member of the B7 superfamily, has been recently identified as a costimulator in the innate immunity-mediated inflammatory response. In this study, we further report that B7-H3 participates in the development of pneumococcal meningitis in a murine model. Exogenous administration of B7-H3 strongly amplified the inflammatory response, exacerbated blood-brain barrier disruption, and aggravated the clinical disease status in Streptococcus pneumoniae-infected C3H/HeN wild-type mice. Consistent with the in vivo findings, B7-H3 substantially augmented proinflammatory cytokine and chemokine production, upregulated NF-κB p65 and MAPK p38 phosphorylation, and enhanced the nuclear transactivation of NF-κB p65 at both TNF-α and IL-6 promoters in S. pneumoniae-stimulated primary murine microglia cells. These B7-H3-associated in vitro and in vivo effects appeared to be dependent on TLR2 signaling, as B7-H3 almost completely lost its amplifying actions in both TLR2-deficient microglial cells and TLR2-deficient mice. Furthermore, administration of the anti-B7-H3 mAb (MIH35) attenuated the inflammatory response and ameliorated blood-brain barrier disruption in S. pneumoniae-infected wild-type mice. Collectively, our results indicate that B7-H3 plays a contributory role in the development of S. pneumoniae infection-induced bacterial meningitis.     

Interleukin-10 activates Toll-like receptor 4 and requires MyD88 for cardiomyocyte survival

Toll-like receptors (TLRs) are important in a variety of inflammatory diseases including acute cardiac disorders. TLR4 innate signaling regulates the synthesis of anti-inflammatory cytokine, interleukin-10 (IL-10) upon TLR4 agonists’ re-stimulation. Anti-apoptotic action of IL-10 in cardiac dysfunction is generally accepted but its protective mechanism through TLR4 is not yet understood. We studied the effect of IL-10 in the activation of TLR4 downstream signals leading to cardiomyocytes survival. IL-10 caused a significant increase in the expression of CD14, MyD88 and TLR4. TLR4 activation led to the translocation of the interferon regulatory factor 3 (IRF3) into the nucleus. Phosphorylation of IRF3 enhanced mRNA synthesis for IL-1β but not TNF-α and was elevated even after removal of IL-10 stimulation. Furthermore, degradation of inhibitory kappa B (IκB) kinase (Ikk) suggested that IκBβ was the main activating kinase for IRF3-regulated NF-κB activation and phosphorylation of p65. Phosphorylated NF-κB p65 was translocated into the nucleus. Concomitantly, an increase in Bcl-xL activity inhibited Bax and the proteolytic activity of caspase 3 as well as a decrease in PARP cleavage. An inhibition of MyD88, modulated the above listed responses to IL-10 as there was a decrease in TLR4 and IRF3 and an increase in TNF-α mRNA. This was associated with a decrease in NF-κB p65, Bcl-xL mRNA and protein levels as well as there was an activation of Bax and PARP cleavage independent of caspase 3 activation. These data in cardiomyocytes suggest that IL-10 induced anti-apoptotic signaling involves upregulation of TLR4 through MyD88 activation.     

Induction of bacterial lipoprotein tolerance is associated with suppression of toll-like receptor 2 expression

Tolerance to bacterial cell wall components including lipopolysaccharide (LPS) may represent an essential regulatory mechanism during bacterial infection. Two members of the Toll-like receptor (TLR) family, TLR2 and TLR4, recognize the specific pattern of bacterial cell wall components. TLR4 has been found to be responsible for LPS tolerance. However, the role of TLR2 in bacterial lipoprotein (BLP) tolerance and LPS tolerance is unclear. Pretreatment of human THP-1 monocytic cells with a synthetic bacterial lipopeptide induced tolerance to a second BLP challenge with diminished tumor necrosis factor-alpha and interleukin-6 production, termed BLP tolerance. Furthermore, BLP-tolerized THP-1 cells no longer responded to LPS stimulation, indicating a cross-tolerance to LPS. Induction of BLP tolerance was CD14-independent, as THP-1 cells that lack membrane-bound CD14 developed tolerance both in serum-free conditions and in the presence of a specific CD14 blocking monoclonal antibody (MEM-18). Pre-exposure of THP-1 cells to BLP suppressed mitogen-activated protein kinase phosphorylation and nuclear factor-kappaB activation in response to subsequent BLP and LPS stimulation, which is comparable with that found in LPS-tolerized cells, indicating that BLP tolerance and LPS tolerance may share similar intracellular pathways. However, BLP strongly enhanced TLR2 expression in non-tolerized THP-1 cells, whereas LPS stimulation had no effect. Furthermore, a specific TLR2 blocking monoclonal antibody (2392) attenuated BLP-induced, but not LPS-induced, tumor necrosis factor-alpha and interleukin-6 production, indicating BLP rather than LPS as a ligand for TLR2 engagement and activation. More importantly, pretreatment of THP-1 cells with BLP strongly inhibited TLR2 activation in response to subsequent BLP stimulation. In contrast, LPS tolerance did not prevent BLP-induced TLR2 overexpression. These results demonstrate that BLP tolerance develops through down-regulation of TLR2 expression.     

Sophocarpine exert protective effect against ox-LDL-induced endothelial damage via regulating NF-κB signaling pathway

ABSTRACT

Oxidized low-density lipoprotein (ox-LDL) was known to induce endothelial cell injury to the progression of atherosclerosis (AS). Sophocarpine (SPC), a compound of sophora alkaloids isolated from the plant Sophora alopecuroides, has been shown to exhibit various pharmacological activities. This study was designed to investigate the protective effect of SPC on ox-LDL-induced endothelial cells and explored its underlying mechanism. Our results show that SPC pre-incubation ameliorated ox-LDL-mediated HAECs cytotoxicity, DNA fragmentation, and apoptosis in a dose-dependent manner. Moreover, SPC significantly downregulated the mRNA or protein expression level of pro-inflammatory mediators (TGF-β, IL-6, IL-1β, TNF-α) and pro-inflammatory vascular adhesion molecules (VCAM-1, ICAM-1, and E-selectin). Mechanistically, SPC pre-treatment downregulated IκBα expression and inhibited translocation of NF-κB in ox-LDL-mediated HAECs, overexpression of NF-κB p65 counteracted the cytoprotective and anti-apoptotic effect of SPC, suggesting that its action is dependent on NF-κB signaling pathway. Collectively, SPC suppresses ox-LDL-induced HAECs injury by inhibiting the NF-κB signaling pathway.     

Inhibitory effects of dynorphin 3-14 on the lipopolysaccharide-induced toll-like receptor 4 signalling pathway

Dynorphin 1-17 (DYN 1-17) is biotransformed rapidly to a range of fragments in rodent inflamed tissue with dynorphin 3-14 (DYN 3-14) being the most stable and prevalent. DYN 1-17 has been shown previously to be involved in the regulation of inflammatory response following tissue injury, in which the biotransformation fragments of DYN 1-17 may possess similar features. This study investigated the effects of DYN 3-14 on lipopolysaccharide (LPS)-induced nuclear factor-kappaB/p65 (NF-κB/p65) nuclear translocation and the release of pro-inflammatory cytokines interleukin-1beta (IL-1β) and tumor necrosis factor-alpha (TNF-α) in differentiated THP-1 cells. Treatment with DYN 3-14 (10 nM) resulted in 35% inhibition of the LPS-induced nuclear translocation of NF-κB/p65. Furthermore, DYN 3-14 modulated both IL-1β and TNF-α release; inhibiting IL-1β and paradoxically augmenting TNF-α release in a concentration-independent manner. A number of opioids have been implicated in the modulation of the toll-like receptor 4 (TLR4), highlighting the complexity of their immunomodulatory effects. To determine whether DYN 3-14 modulates TLR4, HEK-Blue™⁻hTLR4 cells were stimulated with LPS in the presence of DYN 3-14. DYN 3-14 (10 μM) inhibited TLR4 activation in a concentration-dependent fashion by suppressing the LPS signals around 300-fold lower than LPS-RS, a potent TLR4 antagonist. These findings indicate that DYN 3-14 is a potential TLR4 antagonist that alters cellular signaling in response to LPS and cytokine release, implicating a role for biotransformed endogenous opioid peptides in immunomodulation.     

TLR2–MyD88–NF-κB pathway is involved in tubulointerstitial inflammation caused by proteinuria

Proteinuria is an important risk factor for chronic kidney diseases (CKD). Several studies have suggested that proteinuria initiates tubulointerstitial inflammation, while the mechanisms have not been fully understood. In this study, we hypothesized whether the activation of the TLR2–MyD88–NF-κB pathway is involved in tubulointerstitial inflammation induced by proteinuria. We observed expression of TLR2, MyD88, NF-κB, as well as TNF-α and IL-6 detected by immunohistostaining, Western blotting and real-time PCR in albumin-overloaded (AO) nephropathy rats. In vitro, we observed these markers in HK-2 cells stimulated by albumin. We used TLR2 siRNA or the NF-κB inhibitor BAY 11-7082 to observe the influence of TNF-α and IL-6 expression caused by albumin overload. Finally, we studied these markers in non-IgA mesangioproliferative glomerulonephritis (MsPGN) patients with different levels of proteinuria. It was demonstrated that expression of TLR2, MyD88 and NF-κB were significantly increased in AO rats and in non-IgA MsPGN patients with high levels of proteinuria, and TNF-α and IL-6 expressions were increased after NF-κB activation. Furthermore, TNF-α and IL-6 expression was positively correlated with the level of proteinuria. Albumin-overload induced TNF-α and IL-6 secretions by the TLR2–MyD88–NF-κB pathway activation, which could be attenuated by the TLR2 siRNA or BAY 11-7082 in HK-2 cells. In summary, we demonstrated that proteinuria may exhibit an endogenous danger-associated molecular pattern (DAMP) that induces tubulointerstitial inflammation via the TLR2–MyD88–NF-κB pathway activation.     

The molecular mechanisms of the effect of Dexamethasone and Cyclosporin A on TLR4 /NF-κB signaling pathway activation in oral lichen planus

Toll-like receptors (TLRs) and the nuclear factor-kappa B (NF-κB) signaling transduction pathway play important roles in the pathogenesis of several chronic inflammatory diseases, but its function in oral lichen planus (OLP) remains unclear. In this study, we examined the expression of TLR4 and NF-κB-p65 and inflammatory cytokines TNF-α and IL-1β by immunohistochemistry in OLP tissues, and found that TLR4 and NF-κB-p65 were significantly upregulated in OLP compared to normal oral mucosa (P<0.05). We used keratinocytes HaCaT stimulated with lipopolysaccharide (LPS) to simulate the local OLP immune environment to some extent. RT-PCR and immunoblotting analyses showed significant activation of TLR4 and NF-κB-p65 in the circumstance of LPS-induced inflammatory response. The high expression of TLR4 and NF-κB-p65 are correlated with expression of cytokines TNF-α and IL-1β (P<0.05). We further showed that NF-κB could act as an anti-apoptotic molecule in OLP. We conclude that TLR4 and the NF-κB signaling pathway may interact with the perpetuation of OLP. Steroids and cyclosporine are effective in the treatment of symptomatic OLP. However, there was some weak evidence for the mechanism over Dexamethasone (DeX) and Cyclosporine A (CsA) for the palliation of symptomatic OLP. In the present study, we found that Dexamethasone and Cyclosporine A negatively regulated NF-κB signaling pathway under LPS simulation in HaCaT cells by inhibiting TLR4 expression, on the other hand, Cyclosporine A could inhibit HaCaT cell proliferation by the induction of the apoptosis of HaCaT cells to protect OLP from the destruction of epidermal cells effectively.     

CpG oligonucleotides induce an immune response of odontoblasts through the TLR9, MyD88 and NF-κB pathways

Odontoblasts are the first-line defense cells against invading microorganisms. Toll-like receptors (TLRs) play a crucial role in innate immunity, and TLR9 is involved in the recognition of microbial DNA. This study aimed to investigate whether odontoblasts can respond to CpG DNA and to determine the intracellular signaling pathways triggered by CpG DNA. We found that the mouse odontoblast-like cell line MDPC-23 constitutively expressed TLR9. Exposure to CpG ODN induced a potent proinflammatory response based on an increase of IL-6 and TNF-α expression. Pretreatment with an inhibitory MyD88 peptide or a specific inhibitor for TLR9, NF-κB or IκBα markedly inhibited CpG ODN-induced IL-6 and TNF-α expression. Moreover, the CpG ODN-mediated increase of κB-luciferase activity in MDPC-23 cells was suppressed by the overexpression of dominant negative mutants of TLR9, MyD88 and IκBα, but not by the dominant negative mutant of TLR4. This result suggests a possible role for the CpG DNA-mediated immune response in odontoblasts and indicates that TLR9, MyD88 and NF-κB are involved in this process.     

Requirement of Rab21 in LPS-induced TLR4 signaling and pro-inflammatory responses in macrophages and monocytes

Lipopolysaccharide (LPS) induces macrophage/monocyte activation and pro-inflammatory cytokines production by activating Toll-like receptor 4 (TLR-4) signaling. Rab GTPase 21 (Rab21) is a member of the Rab GTPase subfamily. In the present study, we show that LPS induced TLR4 and Rab21 association and endosomal translocation in murine bone marrow–derived macrophages (BMDMs) and primary human peripheral blood mononuclear cells (PBMCs). In BMDMs, shRNA-mediated stable knockdown of Rab21 inhibited LPS-induced expression and production of pro-inflammatory cytokines (IL-1β, IL-6 and TNF-α). Conversely, forced overexpression of Rab21 by an adenovirus construct potentiated LPS-induced IL-1β, IL-6 and TNF-α production in BMDMs. Further studies show that LPS-induced TLR4 endosomal traffic and downstream c-Jun and NFκB (nuclear factor-kappa B) activation were significantly inhibited by Rab21 shRNA, but intensified with Rab21 overexpression in BMDMs. Finally, in the primary human PBMCs, siRNA-induced knockdown of Rab21 significantly inhibited LPS-induced IL-1β, IL-6 and TNF-α production. Taken together, we suggest that Rab21 regulates LPS-induced pro-inflammatory responses by promoting TLR4 endosomal traffic and downstream signaling activation.     

血红素加氧酶-1对急性重症胰腺炎相关肺损伤TLR4/NF-κB通路的影响

目的:探讨血红素加氧酶-1(HO-1)对急性重症胰腺炎相关肺损伤(PALI)Toll样受体-4(TLR4)/核因子-κB(NF-κB)信号传导通路的影响。方法:32只SD大鼠随机分为Sham组、PALI组、HO-1促进剂组、HO-1抑制剂组,每组8只。PALI组经胆胰管注入牛磺胆酸钠制备急性重症胰腺炎(ANP)动物模型。Sham组胆胰管内不注入牛磺胆酸钠,其余操作同PALI组。HO-1促进剂组于造模后30 min经腹腔注射牛血晶素75μg/kg;HO-1抑制剂组于造模后30 min经腹腔注射锌-原卟啉20μmol/kg。PALI组和Sham组均于造模后30 min经腹腔注射等量生理盐水。各组大鼠术后24 h,进行肺损伤学评分,统计肺湿/干重比值。检测大鼠术后24 h血清淀粉酶、TNF-α、IL-6、NGAL水平。检测大鼠术后24 h肺组织中TLR4、NF-κB p65蛋白表达。结果:PALI组肺损伤学评分、肺湿/干重比值、淀粉酶、TNF-α、IL-6、NGAL、TLR4、NF-κB p65明显高于Sham组;HO-1促进剂组肺损伤学评分、肺湿/干重比值、淀粉酶、TNF-α、IL-6、NGAL、TLR4、NF-κB p65明显低于PALI组;HO-1抑制剂组肺损伤学评分、肺湿/干重比值、淀粉酶、TNF-α、IL-6、NGAL、TLR4、NF-κBp65明显高于PALI组;差异均有统计学意义(P<0.05)。结论:HO-1能够通过抑制TLR4/NF-κB信号通路的激活,下调TNF-α、IL-6、NGAL等炎症因子的释放,从而发挥减轻急性重症胰腺炎相关肺损伤的作用。