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.     

Inhibition of the NF-kappaB pathway in human intestinal epithelial cells by commensal Streptococcus salivarius

Streptococcus salivarius exhibited an anti-inflammatory effect on intestinal epithelial cells (IECs) and monocytes. Strains were screened using a reporter clone, HT-29/kB-luc-E, induced by tumor necrosis factor alpha (TNF-α). Supernatant from each strain downregulated NF-κB activation. The two most efficient strains produced an active metabolite (<3 kDa) which was able to downregulate the secretion of the proinflammatory chemokine interleukin-8 (IL-8).     

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.     

Mitochondrial DNA plays an important role in lung injury induced by sepsis

The effects and mechanisms of mitochondrial DNA (mtDNA) in the development of sepsis-induced lung injury is not well understood. In our present study, we studied the mtDNA effects in sepsis-induced lung injury model, in vitro and in vivo. Compared with the Normal group, the lung histopathological score, the number of positive apoptosis cell, wet/dry (W/D) ratio and TNF-α, IL-1β, and IL-6 concentrations of lipopolysaccharides (LPSs) and mtDNA groups were significantly increased (P < 0.001, respectively). Meanwhile, the lung histopathological score, positive W/D ratio, number of apoptosis cell and tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-6 concentrations of LPS + mtDNA and small interfering RNA (siRNA)-NC + LPS + mtDNA groups were significantly upregulated compared with those of LPS group (P < 0.05, respectively). However, the lung histopathological score, the number of positive apoptosis cell, W/D ratio and TNF-α, IL-1β, and IL-6 concentrations were significantly improved within the toll-like receptor (TLR9)siRNA + LPS + mtDNA group compared with the LPS group (P < 0.01, respectively). The TLR9, MyD88, and NF-κB proteins or gene expressions of the LPS group and mtDNA group were significantly upregulated compared with those of Normal group by Western blot analysis or immunohistochemistry assay (P < 0.01, respectively), and the TLR9, MyD88, and NF-κB proteins or gene expressions of LPS + mtDNA and siRNA-NC + LPS + mtDNA groups were significantly enhanced compared with those of LPS group (P < 0.05, respectively). However, the TLR9, MyD88, and NF-κB proteins or gene expressions of TLR9siRNA + LPS + mtDNA group were significantly suppressed compared with those of the LPS group (P < 0.01, respectively). In conclusion, mtDNA could provoke lung injury induced by sepsis via regulation of TLR9/MyD88/NF-κB pathway in vitro and in vivo.     

Adenosine is a negative regulator of NF-kappaB and MAPK signaling in human intestinal epithelial cells

Previous studies suggest that adenosine possesses anti-inflammatory properties, however, the mechanisms by which adenosine affects immune function remain unclear, particularly in the intestine. In this study, we hypothesized that adenosine directly affects pro-inflammatory gene expression in intestinal epithelial cells through modulation of NF-kappaB signaling. HT-29 cells were treated with adenosine prior to incubation with various stimuli and pro-inflammatory gene expression and signal transduction analyzed. Adenosine pretreatment resulted in a reduction in IL-8 expression and secretion in response to TNF-alpha, IL-1, LPS, and PMA. This effect was paralleled by inhibition of kappaB-driven luciferase expression and a reduction in recruitment of NF-kappaB to the IL-8 promoter. Pretreatment of HT-29 cells also resulted in reduced ERK, p38, and JNK MAPK phosphorylation, following TNF-alpha treatment. The observed effects in this study occurred independently of known surface adenosine receptors. This study identifies adenosine as a potent negative regulator of pro-inflammatory signaling in intestinal epithelial cells.     

Clostridium butyricum activates TLR2-mediated MyD88-independent signaling pathway in HT-29 cells

Oral administration of Clostridium butyricum as probiotic is increasingly gaining importance in the treatment of diarrhea and the improvement of animal performance. However, the mechanisms of host cell receptor recognition of C. butyricum and the downstream immune signaling pathways leading to these benefits remain unclear. The objective of this study was to analyze the mechanisms involved in C. butyricum induction of the toll-like receptor (TLR) signaling. Knockdown of myeloid differentiation primary response protein 88 (MyD88) expression using small interfering RNA in this manner did not affect C. butyricum-induced elevated levels of nuclear factor κB (NF-κB), interleukin-8 (IL-8), IL-6, and tumor necrosis factor alpha (TNF-α), suggesting a MyD88-independent route to TLR signaling transduction. However, a significant reduction in the levels of NF-κB, IL-8, IL-6, and TNF-α was evident in the absence of TLR2 expression, implying the need for TLR2 in C. butyricum recognition. Hence, C. butyricum activates TLR2-mediated MyD88-independent signaling pathway in human epithelial cells, which adds to our understanding of the molecular mechanisms of this probiotic action on gut epithelium.     

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.     

Ginkgolides and bilobalide protect BV2 microglia cells against OGD/reoxygenation injury by inhibiting TLR2/4 signaling pathways

Ginkgolide and bilobalide are major trilactone constituent of Ginkgo biloba leaves and have been shown to exert powerful neuroprotective properties. The aims of this study were to observe the inhibitory effects of ginkgolide and bilobalide on the activation of microglial cells induced by oxygen–glucose deprivation and reoxygenation (OGD/R) and the specific mechanisms by which these effects are mediated. For detecting whether ginkgolide and bilobalide increased cell viability in a dose-dependent manner, BV2 cells were subjected to oxygen–glucose deprivation for 4 h followed by 3 h reoxygenation with various concentrations of drugs (6.25, 12.5, 25, 50, and 100 μg/ml). The extent of apoptosis effect of OGD/R with or without ginkgolide and bilobalide treatment were also measured by Annexin V-FITC/PI staining. Similarly, the levels of pro-inflammatory cytokines TNF-α, IL-1β, IL-6, IL-8, and IL-10 were detected using a specific Bio-Plex Pro? Reagent Kit. The effects of ginkgolide and bilobalide on protein levels of TLR2/4, MyD88, p-TAK1, p-IKKβ, p-IkBα, NF-κB p65, Bcl-2, Bax, Bak, RIP3, cleaved-Caspase-3, cleaved PARP-1 and cellular localization of NF-κB p65 were evaluated by Western blot and double-labeled immunofluorescence staining, respectively. OGD/R significantly decreased the cell viability and increased the release of IL-1β, IL-6, IL-8, IL-10, TNF-α in BV2 microglia cells; these effects were suppressed by ginkgolide and bilobalide. Meanwhile, ginkgolide and bilobalide also attenuated the OGD/R-induced increases in TLR2, TLR4, MyD88, Bak, RIP3 levels and reversed cleaved caspase-3/caspase-3, Bax/Bcl-2 and cleaved PARP-1/PARP-1 ratio. Furthermore, ginkgolide and bilobalide also downregulated p-TAK1, p-IkBα, and p-IKKβ and inhibited the OGD/R-induced transfer of NF-κB p65 from cytoplasm to nucleus in BV2 microglia cells. The results showed that ginkgolide and bilobalide can inhibit OGD/R-induced production of inflammatory factors in BV2 microglia cells by regulating the TLRs/MyD88/NF-κB signaling pathways and attenuating inflammatory response. The possible mechanism of anti-inflammatory and neuroprotective effects of ginkgolides results from the synergistic reaction among each monomer constituents.     

Lobaric acid and pseudodepsidones inhibit NF-κB signaling pathway by activation of PPAR-γ

Herein we report the anti-inflammatory activity of lobaric acid and pseudodepsidones isolated from the nordic lichen Stereocaulon paschale. Lobaric acid (1) and three compounds (2, 7 and 9) were found to inhibit the NF-κB activation and the secretion of pro-inflammatory cytokines (IL-1β and TNF-α) in LPS-stimulated macrophages. Inhibition and docking simulation experiments provided evidence that lobaric acid and pseudodepsidones bind to PPAR-γ between helix H3 and the beta sheet, similarly to partial PPAR-γ agonists. These findings suggest that lobaric acid and pseudodepsidones reduce the expression of pro-inflammatory cytokines by blocking the NF-κB pathway via the activation of PPAR-γ.     

Casuarinin suppresses TNF-α-induced ICAM-1 expression via blockade of NF-κB activation in HaCaT cells

Hippophae rhamnoides has been extensively used in oriental traditional medicines for treatment of asthma, skin diseases, gastric ulcers, and lung disorders. In this study, we isolated casuarinin from the leaves of H.rhamnoides and examined the effect of casuarinin on the TNF-α-induced ICAM-1 expression in a human keratinocytes cell line HaCaT. Pretreatment with casuarinin inhibited TNF-α-induced protein and mRNA expression of ICAM-1 and subsequent monocyte adhesiveness in HaCaT cells. Casuarinin significantly inhibited TNF-α-induced NF-κB activation. In addition, casuarinin inhibited activation of ERK and p38 MAPK in a dose-dependent manner. Furthermore, pretreatment with casuarinin decreased TNF-α-induced pro-inflammatory mediators, such as IL-1β, IL-6, IL-8, and MCP-1. These results demonstrated that casuarinin exerts its anti-inflammatory activity by suppressing TNF-α-induced expression of ICAM-1 and pro-inflammatory cytokines/chemokines via blockage of activation of NF-κB and ERK/p38 MAPK and can be used as a therapeutic agent against inflammatory skin diseases.     

Activation of the MyD88 signaling pathway inhibits ischemia-reperfusion injury in the small intestine

Toll-like receptors (TLRs) recognize microbial components and trigger the signaling cascade that activates innate and adaptive immunity. Recent studies have shown that the activation of TLR-dependent signaling pathways plays important roles in the pathogenesis of ischemia-reperfusion (I/R) injuries in many organs. All TLRs, except TLR3, use a common adaptor protein, MyD88, to transduce activation signals. We investigated the role of MyD88 in I/R injury of the small intestine. MyD88 and cyclooxygenase-2 (COX-2) knockout and wild-type mice were subjected to intestinal I/R injury. I/R-induced small intestinal injury was characterized by infiltration of inflammatory cells, disruption of the mucosal epithelium, destruction of villi, and increases in myeloperoxidase activity and mRNA levels of TNF-α and the IL-8 homolog KC. MyD88 deficiency worsened the severity of I/R injury, as assessed using the histological grading system, measuring luminal contents of hemoglobin (a marker of intestinal bleeding), and counting apoptotic epithelial cells, while it inhibited the increase in mRNA expression of TNF-α and KC. I/R significantly enhanced COX-2 expression and increased PGE(2) concentration in the small intestine of wild-type mice, which were markedly inhibited by MyD88 deficiency. COX-2 knockout mice were also highly susceptible to intestinal I/R injury. Exogenous PGE(2) reduced the severity of injury in both MyD88 and COX-2 knockout mice to the level of wild-type mice. These findings suggest that the MyD88 signaling pathway may inhibit I/R injury in the small intestine by inducing COX-2 expression.     

Saikosaponin a inhibits lipopolysaccharide-oxidative stress and inflammation in Human umbilical vein endothelial cells via preventing TLR4 translocation into lipid rafts

Saikosaponin a (SSa), the major triterpenoid saponin derivatives from Radix bupleuri (RB), has been reported to have anti-inflammatory effects. The aim of this study was to investigate the effects of SSa on lipopolysaccharide (LPS)-induced oxidative stress and inflammatory response in human umbilical vein endothelial cells (HUVECs). HUVECs were stimulated with LPS in the presence or absence of SSa. The levels of TNF-α and IL-8 were detected by ELISA. The expression of COX-2 and iNOS, NF-κB and IκB protein were determined by Western blotting. To investigate the protective mechanisms of SSa, TLR4 expression was detected by Western blotting and membrane lipid rafts were separated by density gradient ultracentrifugation and analyzed by immunoblotting with anti-TLR4 antibody. The results showed that SSa dose-dependently inhibited the production of ROS, TNF-α, IL-8, COX-2 and iNOS in LPS-stimulated HUVECs. Western blot analysis showed that SSa suppressed LPS-induced NF-κB activation. SSa did not affect the expression of TLR4 induced by LPS. However, translocation of TLR4 into lipid rafts and oligomerization of TLR4 induce by LPS was inhibited by SSa. Furthermore, SSa disrupted the formation of lipid rafts by depleting cholesterol. Moreover, SSa activated LXRα-ABCA1 signaling pathway, which could induce cholesterol efflux from lipid rafts. Knockdown of LXRα abrogated the anti-inflammatory effects of SSa. In conclusion, the effects of SSa is associated with activating LXRα-ABCA1 signaling pathway which results in disrupting lipid rafts by depleting cholesterol and reducing translocation of TLR4 to lipid rafts and oligomerization of TLR4, thereby attenuating LPS mediated oxidative and inflammatory responses.     

Activation of MyD88 signaling upon staphylococcal enterotoxin binding to MHC class II molecules

Ligands binding to Toll-like receptor (TLR), interleukin 1 receptor (IL-1R), or IFN-γR1 are known to trigger MyD88-mediated signaling, which activates pro-inflammatory cytokine responses. Recently we reported that staphylococcal enterotoxins (SEA or SEB), which bind to MHC class II molecules on APCs and cross link T cell receptors, activate MyD88- mediated pro-inflammatory cytokine responses. We also reported that MyD88(-/-) mice were resistant to SE- induced toxic shock and had reduced levels of serum cytokines. In this study, we investigated whether MHC class II- SE interaction by itself is sufficient to activate MyD88 in MHC class II(+) cells and induce downstream pro-inflammatory signaling and production of cytokines such as TNF-α and IL-1β. Here we report that human monocytes treated with SEA, SEB, or anti-MHC class II monoclonal antibodies up regulated MyD88 expression, induced activation of NF-kB, and increased expression of IL-1R1 accessory protein, TNF-α and IL-1β. MyD88 immunoprecipitated from cell extracts after SEB stimulation showed a greater proportion of MyD88 phosphorylation compared to unstimulated cells indicating that MyD88 was a component of intracellular signaling. MyD88 downstream proteins such as IRAK4 and TRAF6 were also up regulated in monocytes after SEB stimulation. In addition to monocytes, primary B cells up regulated MyD88 in response to SEA or SEB stimulation. Importantly, in contrast to primary B cells, MHC class II deficient T2 cells had no change of MyD88 after SEA or SEB stimulation, whereas MHC class II-independent activation of MyD88 was elicited by CpG or LPS. Collectively, these results demonstrate that MHC class II utilizes a MyD88-mediated signaling mechanism when in contact with ligands such as SEs to induce pro-inflammatory cytokines.     

Carabrol suppresses LPS-induced nitric oxide synthase expression by inactivation of p38 and JNK via inhibition of I-κBα degradation in RAW 264.7 cells

Carabrol, isolated from Carpesium macrocephalum, showed anti-inflammatory potential in LPS-induced RAW 264.7 murine macrophages. In present study, carabrol demonstrated the inhibitory activity on pro-inflammatory cytokines such as IL-1β, IL-6 and TNF-α. In addition, mRNA and protein levels of iNOS and COX-2 were reduced by carabrol. Molecular analysis revealed that these suppressive effects were correlated with the inactivation of p38 and JNK via inhibition of NF-κB activation. Immunoblotting showed that carabrol suppressed LPS-induced degradation of I-κBα and decreased nuclear translocation of p65. Taken together, these results suggest that carabrol can be a modulator of pro-inflammatory signal transduction pathway in RAW 264.7 cells.     

Kir3.1 channel is functionally involved in TLR4-mediated signaling

We aimed to study the involvement of Kir3.1 channel in TLR4-mediated signaling. LPS stimulation induced the recruitment of TLR4 and Kir3.1 into the lipid raft in THP-1 cells. Treatment with Tertiapin-Q, an inhibitor of Kir3.1, markedly abolished the recruitment of TLR4 into the lipid raft and inhibited the LPS-induced NF-κB activation, resulting in decreased production of TNF-α, IL-1β, and IL-6. To verify the specific role of the Kir3.1 channel, we generated Kir3.1-knockdown THP-1 cells. The Kir3.1^KD THP-1 cells exhibited inhibition of NF-κB activation and production of these pro-inflammatory cytokines in response to TLR4 stimulation. Taken together, our results demonstrate that the Kir3.1 channel is involved in the TLR4-mediated signal at an early event by facilitating the recruitment of TLR4 into lipid raft.