CMRF-35-like molecule 3 preferentially promotes TLR9-triggered proinflammatory cytokine production in macrophages by enhancing TNF receptor-associated factor 6 ubiquitination

TLRs are critical innate immune sensors in the induction of proinflammatory cytokines to eliminate invading pathogens. However, the mechanisms for the full activation of TLR-triggered innate immune response need to be fully understood. The murine CMRF-35-like molecule (CLM)-3 is a representative of CLM family belonging to the Ig superfamily. Considering that CLM-3 is selectively expressed in macrophages and the roles of CLM members in innate immune response remain unclear, in this study we investigated the role of CLM-3 in the regulation of TLR-triggered innate response. We found that CLM-3 was an endosome/lysosome-localized molecule, and was downregulated in macrophages by stimulation with TLR9 ligand, but not TLR4 and TLR3 ligands. Interestingly, CLM-3 selectively promoted production of TNF-α and IL-6 in macrophages triggered by TLR9, but not TLR4 or TLR3. CLM-3 enhanced activation of MAPKs and NF-κB pathways in TLR9-triggered macrophages. Furthermore, CLM-3-transgenic mice were generated, and CLM-3 expression was confirmed by mAb against CLM-3 that we prepared. Accordingly, the macrophages derived from CLM-3-transgenic mice were more sensitive to TLR9 ligand stimulation, with more pronounced production of TNF-α, IL-6, and increased activation of MAPKs and NF-κB pathways. Moreover, ubiquitination of TNFR-associated factor 6, a crucial signaling transducer of TLR-triggered MAPKs and NF-κB activation, was found to be significantly promoted by CLM-3 in macrophages. Collectively, the endosome/lysosome-localized CLM-3 can promote full activation of TLR9-triggered innate responses by enhancing TNFR-associated factor 6 ubiquitination and subsequently activating MAPKs and NF-κB.     

Enhanced inhibitory effects of a novel CpG motif on osteoclast differentiation via TREM-2 down-regulation

Recognition of oligodeoxynucleotides containing CpG motifs (CpG-ODNs) by toll-like receptor 9 (TLR9) inhibits RANKL-induced osteoclastogenesis from precursors. This inhibitory effect suggests the possibility of using this strategy to block pathological bone loss. However, the enhancing effect of CpG-ODNs on OC formation from RANKL-primed pre-osteoclasts (pOCs) has hampered their clinical use. In this report, we developed a CpG-KSK13 oligonucleotide with an alternative CpG motif, and tested its effect on osteoclastogenesis in comparison with previously used murine CpG motif (CpG-1826) or human CpG motif (CpG-2006) oligonucleotides. Murine CpG-1826 inhibited RANKL-induced OC formation from BMMs but not from RANKL-primed pOCs, while CpG-KSK13 treatment strongly inhibited OC formation from both BMM and primed pOC cells. CpG-KSK13 also showed a potent inhibitory effect on human OC differentiation using peripheral blood mononuclear cells (PBMCs), which was in contrast to the species-specific response of murine CpG-1826 or human CpG-2006. Moreover, CpG-KSK13 effectively inhibited NFATc1 activity, but not NF-κB or AP-1 activity, and decreased TREM-2 promoter activity and subsequent surface expression of the TREM-2 protein induced by M-CSF and RANKL. These results demonstrate that the recognition of CpG-KSK13 via TLR9 inhibits osteoclastogenesis by down-regulating TREM-2 expression. Thus, our findings provide evidence for the potential use of CpG-KSK13 as an anti-osteoclastogenic agent for human and for pre-clinical animals.     

Abnormal trafficking and degradation of TLR4 underlie the elevated inflammatory response in cystic fibrosis

Morbidity and mortality in cystic fibrosis (CF) are due not only to abnormal epithelial cell function, but also to an abnormal immune response. We have shown previously that macrophages lacking CF transmembrane conductance regulator (CFTR), the gene mutated in CF, contribute significantly to the hyperinflammatory response observed in CF. In this study, we show that lack of functional CFTR in murine macrophages causes abnormal TLR4 subcellular localization. Upon LPS stimulation, CFTR macrophages have prolonged TLR4 retention in the early endosome and reduced translocation into the lysosomal compartment. This abnormal TLR4 trafficking leads to increased LPS-induced activation of the NF-κB, MAPK, and IFN regulatory factor-3 pathways and decreased TLR4 degradation, which affects downregulation of the proinflammatory state. In addition to primary murine cells, mononuclear cells isolated from CF patients demonstrate similar defects in response to LPS. Moreover, specific inhibition of CFTR function induces abnormal TLR4 trafficking and enhances the inflammatory response of wild-type murine cells to LPS. Thus, functional CFTR in macrophages influences TLR4 spatial and temporal localization and perturbs LPS-mediated signaling in both murine CF models and patients with CF.     

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.     

HMGB1 protein promotes glomerular mesangial matrix deposition via TLR2 in lupus nephritis

Lupus nephritis (LN) is the most common complication of systemic lupus erythematosus. Patients with LN mostly die of sclerosing glomerulonephritis and renal failure. The inhibition of glomerular mesangial matrix deposition is an efficient method to restrict the progress of renal injury. By recognizing and binding extracellular and intracellular ligands, Toll-like receptor 2 (TLR2) contributes to the pathogenesis of most immune diseases. However, the relationship between TLR2 and LN is still unknown. Our previous studies confirmed that high-mobility group box 1 (HMGB1), an important ligand of TLR2, promotes the progression of LN by inducing the proliferation of glomerular mesangial cells. However, whether or not HMGB1 participates in the pathogenesis of glomerular mesangial matrix deposition in LN remains unknown. In this study, we observed the upregulated expression of TLR2 in the glomeruli of LN patients and MRL/lpr mice. The inhibition of either TLR2 or HMGB1 inhibited the release of fibronectin and the activation of the MyD88/NF-κB pathway in mesangial cells cultured with LN plasma. In addition, both TLR2- and HMGB1-deficient mice showed reduced 24 hr urine protein levels and improved glomerular histological changes and sclerosis levels. These results indicate that TLR2 regulates glomerular mesangial matrix deposition in LN through the activation of the MyD88/NF-κB pathway by binding to HMGB1.     

CpG-ODNs induces up-regulated expression of chemokine CCL9 in mouse macrophages and microglia

Unmethylated CpG oligodeoxynucleotides (CpG-ODNs) interact with Toll-like receptor (TLR) 9 to activate macrophage/microglia in central nervous system (CNS). Here, we investigated the potential involvement of the chemokine CCL9 and its receptor CCR1 in the effects of CpG-ODNs on macrophage/microglial cells. CpG-ODNs enhanced the expression of TLR9 mRNA of RAW264.7 macrophage and BV2 microglia cells time dependently. The expression of CCL9 of macrophages/microglia showed different responsiveness upon stimulation with a variety of CpG-ODN sequences. The CpG-ODNs-mediated induction of CCL9 was TLR9/MyD88 dependent and associated with activation of stress kinases, particularly ERK, p38 MAPK and PI3K. The expression of CCR1 was also significantly increased by CpG-ODNs that increased CCL9 expression. These results reveal the potential involvement of CCL9 and CCR1 in regulation of macrophage and microglial cells by CpG-ODNs and may help improving our understanding about the role of the chemokine/chemokine receptor pairs in macrophage/microglia under physiologic and pathologic conditions.     

Glucan phosphate attenuates myocardial HMGB1 translocation in severe sepsis through inhibiting NF-κB activation

Myocardial dysfunction is a major consequence of septic shock and contributes to the high mortality of sepsis. High-mobility group box 1 (HMGB1) serves as a late mediator of lethality in sepsis. We have reported that glucan phosphate (GP) attenuates cardiac dysfunction and increases survival in cecal ligation and puncture (CLP)-induced septic mice. In the present study, we examined the effect of GP on HMGB1 translocation from the nucleus to the cytoplasm in the myocardium of septic mice. GP was administered to mice 1 h before induction of CLP. Sham-operated mice served as control. The levels of HMGB1, Toll-like receptor 4 (TLR4), and NF-κB binding activity were examined. In an in vitro study, H9C2 cardiomyoblasts were treated with lipopolysaccharide (LPS) in the presence or absence of GP. H9C2 cells were also transfected with Ad5-IκBα mutant, a super repressor of NF-κB activity, before LPS stimulation. CLP significantly increased the levels of HMGB1, TLR4, and NF-κB binding activity in the myocardium. In contrast, GP administration attenuated CLP-induced HMGB1 translocation from the nucleus to the cytoplasm and reduced CLP-induced increases in TLR4 and NF-κB activity in the myocardium. In vitro studies showed that GP prevented LPS-induced HMGB1 translocation and NF-κB binding activity. Blocking NF-κB binding activity by Ad5-IκBα attenuated LPS-induced HMGB1 translocation. GP administration also reduced the LPS-stimulated interaction of HMGB1 with TLR4. These data suggest that attenuation of HMGB1 translocation by GP is mediated through inhibition of NF-κB activation in CLP-induced sepsis and that activation of NF-κB is required for HMGB1 translocation.     

A GpC-Rich Oligonucleotide Acts on Plasmacytoid Dendritic Cells To Promote Immune Suppression

Short synthetic oligodeoxynucleotides (ODNs) rich in CpG or GpG motifs have been considered as potential modulators of immunity in clinical settings. In this study, we show that a synthetic GpC-ODN conferred highly suppressive activity on mouse splenic plasmacytoid dendritic cells, demonstrable in vivo in a skin test assay. The underlying mechanism involved signaling by noncanonical NF-κB family members and TGF-β-dependent expression of the immunoregulatory enzyme IDO. Unlike CpG-ODNs, the effects of GpC-ODN required TLR7/TRIF-mediated but not TLR9/MyD88-mediated events, as do sensing of viral ssRNA and the drug imiquimod. Induction of IDO by a GpC-containing ODN could also be demonstrated in human dendritic cells, allowing those cells to assist FOXP3(+) T cell generation in vitro. Among potentially therapeutic ODNs, this study identifies GpC-rich sequences as novel activators of TLR7-mediated, IDO-dependent regulatory responses.     

Characterization of sparstolonin B, a Chinese herb-derived compound, as a selective Toll-like receptor antagonist with potent anti-inflammatory properties

Blockade of excessive Toll-like receptor (TLR) signaling is a therapeutic approach being actively pursued for many inflammatory diseases. Here we report a Chinese herb-derived compound, sparstolonin B (SsnB), which selectively blocks TLR2- and TLR4-mediated inflammatory signaling. SsnB was isolated from a Chinese herb, Spaganium stoloniferum; its structure was determined by NMR spectroscopy and x-ray crystallography. SsnB effectively inhibited inflammatory cytokine expression in mouse macrophages induced by lipopolysaccharide (LPS, a TLR4 ligand), Pam3CSK4 (a TLR1/TLR2 ligand), and Fsl-1 (a TLR2/TLR6 ligand) but not that by poly(I:C) (a TLR3 ligand) or ODN1668 (a TLR9 ligand). It suppressed LPS-induced cytokine secretion from macrophages and diminished phosphorylation of Erk1/2, p38a, IκBα, and JNK in these cells. In THP-1 cells expressing a chimeric receptor CD4-TLR4, which triggers constitutive NF-κB activation, SsnB effectively blunted the NF-κB activity. Co-immunoprecipitation showed that SsnB reduced the association of MyD88 with TLR4 and TLR2, but not that with TLR9, in HEK293T cells and THP-1 cells overexpressing MyD88 and TLRs. Furthermore, administration of SsnB suppressed splenocyte inflammatory cytokine expression in mice challenged with LPS. These results demonstrate that SsnB acts as a selective TLR2 and TLR4 antagonist by blocking the early intracellular events in the TLR2 and TLR4 signaling. Thus, SssB may serve as a promising lead for the development of selective TLR antagonistic agents for inflammatory diseases.     

NLRC5 negatively regulates LTA-induced inflammation via TLR2/NF-κB and participates in TLR2-mediated allergic airway inflammation

NLRC5, the largest member of the Nod-like receptor (NLR) family, has been reported to play a pivotal role in regulating inflammatory responses. Recent evidence suggests that NLRC5 participates in Toll-like receptor (TLR) signaling pathways and negatively modulates nuclear factor-κB (NF-κB) activation. In this study, we investigated the interaction between NLRC5 and TLR2 in the NF-κB inflammatory signaling pathway and the involvement of NLRC5 in TLR2-mediated allergic airway inflammation. We knocked down TLR2 and NLRC5, respectively in the RAW264.7 macrophage cell line by small interfering RNA (siRNA) and then stimulated the knockdown cells with lipoteichoic acid (LTA). In comparison with the negative siRNA group, the level of NLRC5 expression was lower in the TLR2 siRNA group, with a reduction in the NF-κB-related inflammatory response. Conversely, in the NLRC5 knockdown cells, after LTA-treated the level of TLR2 expression did not change but the expression levels of both NF-κB pp65 and NLRP3 increased remarkably. Thus, we hypothesize that NLRC5 participates in the LTA-induced inflammatory signaling pathway and regulates the inflammation via TLR2/NF-κB. Similarly, in subsequent in vivo experiments, we demonstrated that the expression level of NLRC5 was significantly increased in the ovalbumin-induced allergic airway inflammation. However, this effect disappeared in TLR2-deficient (TLR2 ^−/−) mice and was accompanied by reduced levels of NF-κB expression and airway inflammation. In conclusion, NLRC5 negatively regulates LTA-induced inflammatory response via a TLR2/NF-κB pathway in macrophages and also participates in TLR2-mediated allergic airway inflammation.     

Macrophage iron retention aggravates atherosclerosis: Evidence for the role of autocrine formation of hepcidin in plaque macrophages

Iron accumulation has been frequently found in atherosclerotic lesions, especially in macrophages/foam cells, but the exact mechanisms by which hepcidin induces iron retention in plaque macrophages and its roles in atherogenesis remain unknown. Double immunofluorescence staining showed colocalization of hepcidin-positive macrophages with ox-LDL, TLR4, p-p65 and ferritin light chain (ferritin-L) both in human and murine atherosclerotic lesions. RAW264.7 macrophages incubated with ox-LDL showed elevated expression of TLR4, p-p65, hepcidin, ferritin-L/H, CYP27A1, CD36, PPARγ, liver X receptor α (LXRα), and ATP binding cassette transporter A1/G1 (ABCA1/G1), as well as increased intracellular labile iron pool level and lipid accumulation. Ox-LDL-induced iron retention and lipid accumulation were aggravated by lipopolysaccharide but blocked by TAK-242, an antagonist of TLR4. Moreover, macrophage TLR4/NF-κB pathway activation and foaming triggered by ox-LDL was enhanced by ferric ammonium citrate or exogenous hepcidin but attenuated by hepcidin silencing or the use of iron chelator. Meanwhile, the addition of hepcidin stimulated CD36-mediated Dil-labeled-ox-LDL uptake and inhibited the LXRα-ABCA1/G1 pathway-dependent cholesterol efflux in macrophages, which was significantly reversed by 27-hydroxycholesterol but further exacerbated by cyclosporin A, a selective inhibitor of CYP27A1. Our study provided the evidence that iron trapped in atherosclerosis plaque macrophages contributes to cholesterol disequilibrium-initiated foam cell formation, which is provoked by the unique but largely unknown autocrine formation of hepcidin in plaque macrophages via activating the TLR4/NF-κB pathway when exposed to ox-LDL. Such findings, considering the intricate vicious cycle between macrophage hepcidin autocrine-triggered iron retention and cholesterol disequilibrium, may shed new light on the “iron hypothesis” of atherosclerosis.     

Dectin-1 stimulation induces suppressor of cytokine signaling 1, thereby modulating TLR signaling and T cell responses

Suppressor of cytokine signaling (SOCS) proteins serve as negative regulators of cytokine receptor signaling. However, SOCS proteins are not only induced via the JAK/STAT pathway, but are also transcribed on triggering of pattern recognition receptors such as TLRs. We now show that SOCS1 can also be induced by the non-TLR pattern recognition receptor Dectin-1 in murine bone marrow-derived dendritic cells and macrophages (BMMs). The C-type lectin Dectin-1 binds to yeasts and signals either in an autonomous manner or can be triggered in combination with TLRs. In our study, SOCS1 was expressed independently of any TLR engagement as a direct target gene of the Dectin-1 ligand Zymosan. Induction of SOCS1 was mediated by a novel pathway encompassing the tyrosine kinases Src and Syk that activated the downstream kinase proline-rich tyrosine kinase 2. Proline-rich tyrosine kinase 2, in turn, caused activation of the MAPK ERK, thereby triggering SOCS1 induction. SOCS1 did not modulate Dectin-1 signaling but affected TLR signaling, leading to decreased and abbreviated NF-κB activation in BMMs triggered by TLR9. Furthermore, IL-12 and IL-10 secretion were inhibited by SOCS1. We additionally observed that IL-17-producing Th cells were clearly increased by SOCS1 in BMMs. Our results show that SOCS1 is expressed via a new, NF-κB-independent pathway in Dectin-1-triggered murine BMMs and influences TLR cross talk and T cell priming.     

miR-146a suppresses the sensitivity to interferon-α in hepatocellular carcinoma cells

Previous studies have demonstrated expression of Toll-like receptors (TLRs) in the surface epithelium of normal ovaries (OSE) and in epithelial ovarian tumors. Most notably, OSE-derived cancers express TLR4, which activates the nuclear factor-kappa B (NF-κB) signaling cascade as a mediator of inflammatory response. Currently, there is considerable interest in elucidating the role of TLR-mediated signaling in cancers. Nevertheless, the expression of TLRs in granulosa cell tumors (GCTs) of the ovary, and the extent to which GCT expression of TLRs may influence cell-signaling pathways and/or modulate the efficacy of chemotherapeutics, has yet to be determined. In the present study, human GCT lines (COV434 and KGN) were utilized to evaluate expression of functional TLR4. TLR4 is expressed in GCT cell lines and ligation of TLR4 with bacterial lipopolysaccharide (LPS) led to IκB degradation and activation of NF-κB. NF-κB activation was confirmed by nuclear localization of NF-κB p65 following treatment with LPS and the naturally occurring ligand, HSP60. Notably, immunoneutralization of TLR4 blocked nuclear localization, and inhibition of NF-κB signaling attenuated LPS-induced TNFα plus increased doubling time in both cell lines. Contradictory to reports using human OSE cell lines, inhibition of NF-κB signaling failed to sensitize GCT lines to TRAIL or cisplatin. In summary, findings herein are the first to demonstrate a functional TLR-signaling pathway specifically in GCTs, and indicate that in contrast to OSE-derived cancers, inhibition of NF-κB does not sensitize GCTs to TRAIL or cisplatin.