Cooperation of Toll-like receptor 2 and 6 for cellular activation by soluble tuberculosis factor and Borrelia burgdorferi outer surface protein A lipoprotein: role of Toll-interacting protein and IL-1 receptor signaling molecules in Toll-like receptor 2 signaling.

Toll-like receptor 2 (TLR2) and TLR4 play important roles in innate immune responses to various microbial agents. We have previously shown that human dermal endothelial cells (HMEC) express TLR4, but very little TLR2, and respond to LPS, but not to Mycobacterium tuberculosis 19-kDa lipoprotein, unless transfected with TLR2. Here we report that HMEC are unresponsive to several additional biologically relevant TLR2 ligands, including, phenol-soluble modulin (PSM), a complex of three small secreted polypeptides from the skin commensal Staphylococcus epidermidis, soluble tuberculosis factor (STF), and Borrelia burgdorferi outer surface protein A lipoprotein (OspA-L). Expression of TLR2 renders HMEC responsive to all these ligands. We further characterized the signaling pathway in response to STF, OspA-L, and PSM in TLR2-transfected HMEC. The TLR2 signaling pathway for NF-kappaB trans-activation shares the IL-1R signaling molecules. Dominant negative constructs of TLR2 or TLR6 inhibit the responses of STF and OspA-L as well as PSM in TLR2-transfected HMEC, supporting the concept of functional cooperation between TLR2 and TLR6 for all these TLR2 ligands. Moreover, we show that Toll-interacting protein (Tollip) coimmunoprecipitates with TLR2 and TLR4 using HEK 293 cells, and overexpression of Tollip inhibits NF-kappaB activation in response to TLR2 and TLR4 signaling. Collectively, these findings suggest that there is functional interaction between TLR2 and TLR6 in the cellular response to STF and OspA-L in addition to S. epidermidis (PSM) Ags, and that engagement of TLR2 triggers a signaling cascade, which shares the IL-1R signaling molecules, similar to the TLR4-LPS signaling cascade. Our data also suggest that Tollip may be an important constituent of both the TLR2 and TLR4 signaling pathways.     

Involvement of TLR4/type I IL-1 receptor signaling in the induction of inflammatory mediators and cell death induced by ethanol in cultured astrocytes

Activated astroglial cells are implicated in neuropathogenesis of many infectious and inflammatory diseases of the brain. A number of inflammatory mediators and cytokines have been proposed to play a key role in glial cell-related brain damage. Cytokine production seems to be initiated by signaling through TLR4/type I IL-1R (IL-1RI) in response to their ligands, LPS and IL-1beta, playing vital roles in innate host defense against infections, inflammation, injury, and stress. We have shown that glial cells are stimulated by ethanol, up-regulating cytokines and inflammatory mediators associated with TLR4 and IL-1RI signaling pathways in brain, suggesting that ethanol may contribute to brain damage via inflammation. We explore the possibility that ethanol, in the absence of LPS or IL-1beta, triggers signaling pathways and inflammatory mediators through TLR4 and/or IL-1RI activation in astrocytes. We show in this study that ethanol, at physiologically relevant concentrations, is capable of inducing rapid phosphorylation within 10 min of IL-1R-associated kinase, ERK1/2, stress-activated protein kinase/JNK, and p38 MAPK in astrocytes. Then an activation of NF-kappaB and AP-1 occurs after 30 min of ethanol treatment along with an up-regulation of inducible NO synthase and cyclooxygenase-2 expression. Finally, we note an increase in cell death after 3 h of treatment. Furthermore, by using either anti-TLR4- or anti-IL-1RI-neutralizing Abs, before and during ethanol treatment, we inhibit ethanol-induced signaling events, including NF-kappaB and AP-1 activation, inducible NO synthase, and cyclooxygenase-2 up-regulation and astrocyte death. In summary, these findings indicate that both TLR4 and IL-1RI activation occur upon ethanol treatment, and suggest that signaling through these receptors mediates ethanol-induced inflammatory events in astrocytes and brain.     

Toll-like receptor-mediated responses of primary intestinal epithelial cells during the development of colitis

The interleukin-2-deficient (IL-2(-/-)) mouse model of ulcerative colitis was used to test the hypothesis that colonic epithelial cells (CEC) directly respond to bacterial antigens and that alterations in Toll-like receptor (TLR)-mediated signaling may occur during the development of colitis. TLR expression and activation of TLR-mediated signaling pathways in primary CEC of healthy animals was compared with CEC in IL-2(-/-) mice during the development of colitis. In healthy animals, CEC expressed functional TLR, and in response to the TLR4 ligand LPS, proliferated and secreted the cytokines IL-6 and monocyte chemoattractant protein-1 (MCP-1). However, the TLR-responsiveness of CEC in IL-2(-/-) mice was different with decreased TLR4 responsiveness and augmented TLR2 responses that result in IL-6 and MCP-1 secretion. TLR signaling in CEC did not involve NF-kappaB (p65) activation with the inhibitory p50 form of NF-kappaB predominating in CEC in both the healthy and inflamed colon. Development of colitis was, however, associated with the activation of MAPK family members and upregulation of MyD88-independent signaling pathways characterized by increased caspase-1 activity and IL-18 production. These findings identify changes in TLR expression and signaling during the development of colitis that may contribute to changes in the host response to bacterial antigens seen in colitis.     

Decreased expression of Toll-like receptor-4 and MD-2 correlates with intestinal epithelial cell protection against dysregulated proinflammatory gene expression in response to bacterial lipopolysaccharide

The lumenal surface of the colonic epithelium is continually exposed to Gram-negative commensal bacteria and LPS. Recognition of LPS by Toll-like receptor (TLR)-4 results in proinflammatory gene expression in diverse cell types. Normally, however, commensal bacteria and their components do not elicit an inflammatory response from intestinal epithelial cells (IEC). The aim of this study is to understand the molecular mechanisms by which IEC limit chronic activation in the presence of LPS. Three IEC lines (Caco-2, T84, HT-29) were tested for their ability to activate an NF-kappaB reporter gene in response to purified, protein-free LPS. No IEC line responded to LPS, whereas human dermal microvessel endothelial cells (HMEC) did respond to LPS. IEC responded vigorously to IL-1beta in this assay, demonstrating that the IL-1 receptor signaling pathway shared by TLRs was intact. To determine the reason for LPS hyporesponsiveness in IEC, we examined the expression of TLR4 and MD-2, a critical coreceptor for TLR4 signaling. IEC expressed low levels of TLR4 compared with HMEC and none expressed MD-2. To determine whether the low level of TLR4 expression or absent MD-2 was responsible for the LPS signaling defect in IEC, the TLR4 or MD-2 gene was transiently expressed in IEC lines. Transient transfection of either gene individually was not sufficient to restore LPS signaling, but cotransfection of TLR4 and MD-2 in IEC led to synergistic activation of NF-kappaB and IL-8 reporter genes in response to LPS. We conclude that IEC limit dysregulated LPS signaling by down-regulating expression of MD-2 and TLR4. The remainder of the intracellular LPS signaling pathway is functionally intact.     

The role of MAPK in Kupffer cell toll-like receptor (TLR) 2-, TLR4-, and TLR9-mediated signaling following trauma-hemorrhage

Severe injury deranges immune function and increases the risk of sepsis and multiple organ failure. Kupffer cells play a major role in mediating posttraumatic immune responses, in part via different Toll-like receptors (TLR). Although mitogen-activated protein kinases (MAPK) are key elements in the TLR signaling pathway, it remains unclear whether the activation of different MAPK are TLR specific. Male C3H/HeN mice underwent midline laparotomy (i.e., soft tissue injury), hemorrhagic shock (MAP approximately 35 mm Hg for 90 min), and resuscitation. Kupffer cells were isolated 2 h thereafter, lysed and immunoblotted with antibodies to p38, ERK1/2, or JNK proteins. In addition, cells were preincubated with specific inhibitors of p38, ERK1/2, or JNK MAPK followed by stimulation with the TLR2 agonist, zymosan; the TLR4 agonist, LPS; or the TLR9 agonist, CpG DNA. Cytokine (TNF-alpha, interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), and KC) production was determined by cytometric bead array after 24 h in culture. MAPK activity as well as TNF-alpha, MCP-1, and KC production by Kupffer cells were significantly increased following trauma-hemorrhage. TLR4 activation by LPS stimulation increased the levels of all measured cytokines. CpG-stimulated TLR9 signaling increased TNF-alpha and IL-6 levels; however, it had no effect on chemokine production. Selective MAPK inhibition demonstrated that chemokine production was mediated via p38 and JNK MAPK activation in TLR2, -4, and -9 signaling. In contrast, TNF-alpha and IL-6 production was differentially regulated by MAPK depending on the TLR pathway stimulated. Thus, Kupffer cell TLR signaling employs different MAPK pathways in eliciting cytokine and chemokine responses following trauma-hemorrhage.     

Bacterial lipopolysaccharide activates nuclear factor-kappaB through interleukin-1 signaling mediators in cultured human dermal endothelial cells and mononuclear phagocytes

Bacterial lipopolysaccharide (LPS)-mediated immune responses, including activation of monocytes, macrophages, and endothelial cells, play an important role in the pathogenesis of Gram-negative bacteria-induced sepsis syndrome. Activation of NF-kappaB is thought to be required for cytokine release from LPS-responsive cells, a critical step for endotoxic effects. Here we investigated the role and involvement of interleukin-1 (IL-1) and tumor necrosis factor (TNF-alpha) signal transducer molecules in LPS signaling in human dermal microvessel endothelial cells (HDMEC) and THP-1 monocytic cells. LPS stimulation of HDMEC and THP-1 cells initiated an IL-1 receptor-like NF-kappaB signaling cascade. In transient cotransfection experiments, dominant negative mutants of the IL-1 signaling pathway, including MyD88, IRAK, IRAK2, and TRAF6 inhibited both IL-1- and LPS-induced NF-kappaB-luciferase activity. LPS-induced NF-kappaB activation was not inhibited by a dominant negative mutant of TRAF2 that is involved in TNF signaling. LPS-induced activation of NF-kappaB-responsive reporter gene was not inhibited by IL-1 receptor antagonist. TLR2 and TLR4 were expressed on the cell surface of HDMEC and THP-1 cells. These findings suggest that a signal transduction molecule in the LPS receptor complex may belong to the IL-1 receptor/toll-like receptor (TLR) super family, and the LPS signaling cascade uses an analogous molecular framework for signaling as IL-1 in mononuclear phagocytes and endothelial cells.     

Negative regulation of toll-like receptor-mediated signaling by Tollip.

Toll-like receptor (TLR)-mediated recognition of pathogens represents one of the most important mechanisms of innate immunity and disease resistance. The adaptor protein Tollip was identified initially as an intermediate in interleukin (IL)-1 signaling. Here we report that Tollip also associates directly with TLR2 and TLR4 and plays an inhibitory role in TLR-mediated cell activation. Inhibition by Tollip is mediated through its ability to potently suppress the activity of IL-1 receptor-associated kinase (IRAK) after TLR activation. In addition, we show for the first time that Tollip is a bona fide substrate for IRAK and is phosphorylated by IRAK upon stimulation with lipopolysaccharide or IL-1. Negative regulation of TLR signaling by Tollip may therefore serve to limit the production of proinflammatory mediators during inflammation and infection.     

Lipopolysaccharide- and lipoteichoic acid-induced tolerance and cross-tolerance: distinct alterations in IL-1 receptor-associated kinase

Human Toll-like receptor (TLR) 4 and TLR2 receptors recognize LPS or lipoteichoic acid (LTA), respectively. Prolonged exposure of human macrophages/monocytes to bacterial LPS induces a state of adaptation/tolerance to subsequent LPS challenge. Inflammatory gene expressions such as IL-1beta and TNF-alpha are selectively repressed, while certain anti-inflammatory genes such as secretory IL-1R antagonist are still induced in LPS-adapted/tolerant cells. In this report, we demonstrate that LPS-tolerized human promonocytic THP-1 cells develop cross-tolerance and no longer respond to LTA-induced IL-1beta/TNF-alpha production, indicating that disruption of common intracellular signaling is responsible for the decreased IL-1beta/TNF-alpha production. We observe that down-regulation of IL-1R-associated kinase (IRAK) protein level and kinase activity closely correlates with the development of cross-tolerance. IRAK protein levels and kinase activities in LPS-tolerized cells remain low and hyporesponsive to subsequent LPS or LTA challenges. We also demonstrate that THP-1 cells with prolonged LTA treatment develop LTA tolerance and do not express IL-1beta/TNF-alpha upon further LTA challenge. Strikingly, cells tolerized with LTA are only refractory to subsequent LTA challenge and can still respond to LPS stimulation. Correspondingly, stimulation of TLR2 by LTA, although activating IRAK, does not cause IRAK degradation. IRAK from LTA-tolerized cells can be subsequently activated and degraded by further LPS challenge, but not LTA treatment. Our studies reveal that LTA-induced tolerance is distinct compared with that of LPS tolerance, and is likely due to disruption of unique TLR2 signaling components upstream of MyD88/IRAK.     

Mastoparan, a G protein agonist peptide, differentially modulates TLR4- and TLR2-mediated signaling in human endothelial cells and murine macrophages

Previous studies have implicated a role for heterotrimeric G protein-coupled signaling in B cells, monocytes, and macrophages stimulated with LPS and have shown that G proteins coimmunoprecipitate with membrane-bound CD14. In this study, we have extended these observations in human dermal microvessel endothelial cells (HMEC) that lack membrane-bound CD14 and in murine macrophages to define further the role of heterotrimeric G proteins in TLR signaling. Using the wasp venom-derived peptide, mastoparan, to disrupt G protein-coupled signaling, we identified a G protein-dependent signaling pathway in HMEC stimulated with TLR4 agonists that is necessary for the activation of p38 phosphorylation and kinase activity, NF-kappaB and IL-6 transactivation, and IL-6 secretion. In contrast, HMEC activation by TLR2 agonists, TNF-alpha, or IL-1beta was insensitive to mastoparan. In the murine macrophage cell line, RAW 264.7, and in primary murine macrophages, G protein dysregulation by mastoparan resulted in significant inhibition of LPS-induced signaling leading to both MyD88-dependent and MyD88-independent gene expression, while TLR2-mediated gene expression was not significantly inhibited. In addition to inhibition of TLR4-mediated MAPK phosphorylation in macrophages, mastoparan blunted IL-1R-associated kinase-1 kinase activity induced by LPS, but not by TLR2 agonists, yet failed to affect phosphorylation of Akt by phosphoinositol-3-kinase induced by either TLR2- or TLR4-mediated signaling. These data confirm the importance of heterotrimeric G proteins in TLR4-mediated responses in cells that use either soluble or membrane-associated CD14 and reveal a level of TLR and signaling pathway specificity not previously appreciated.     

Role of human CD36 in bacterial recognition, phagocytosis, and pathogen-induced JNK-mediated signaling

Scavenger receptor CD36 mediates Staphylococcus aureus phagocytosis and initiates TLR2/6 signaling. We analyzed the role of CD36 in the uptake and TLR-independent signaling of various bacterium, including Escherichia coli, Klebsiella pneumoniae, Salmonella typhimurium, S. aureus, and Enterococcus faecalis. Expression of human CD36 in HeLa cells increased the uptake of both gram-positive and gram-negative bacteria compared with the control mock-transfected cells. Bacterial adhesion was associated with pathogen phagocytosis. Upon CD36 transfection, HEK293 cells, which demonstrate no TLR2/4 expression, acquired LPS responsiveness as assessed by IL-8 production. The cells demonstrated a marked 5- to 15-fold increase in cytokine release upon exposure to gram-negative bacteria, while the increase was much smaller (1.5- to 3-fold) with gram-positive bacteria and lipoteichoic acid. CD36 down-regulation utilizing CD36 small interfering RNA reduced cytokine release by 40-50% in human fibroblasts induced by both gram-negative and gram-positive bacteria as well as LPS. Of all MAPK signaling cascade inhibitors tested, only the inhibitor of JNK, a stress-activated protein kinase, potently blocked E. coli/LPS-stimulated cytokine production. NF-kappaB inhibitors were ineffective, indicating direct TLR-independent signaling. JNK activation was confirmed by Western blot analyses of phosphorylated JKN1/2 products. Synthetic amphipathic peptides with an alpha-helical motif were shown to be efficient inhibitors of E. coli- and LPS-induced IL-8 secretion as well as JNK1/2 activation/phosphorylation in CD36-overexpressing cells. These results indicate that CD36 functions as a phagocytic receptor for a variety of bacteria and mediates signaling induced by gram-negative bacteria and LPS via a JNK-mediated signaling pathway in a TLR2/4-independent manner.     

C-36 peptide, a degradation product of alpha1-antitrypsin, modulates human monocyte activation through LPS signaling pathways

alpha1-Antitrypsin (AAT), a major endogenous inhibitor of serine proteases, plays an important role in minimizing proteolytic injury to host tissue at sites of infection and inflammation. There is now increasing evidence that AAT undergoes post-translational modifications to yield by-products with novel biological activity. One such molecule, the C-terminal fragment of AAT, corresponding to residues 359-394 (C-36 peptide) has been reported to stimulate significant pro-inflammatory activity in monocytes and neutrophils in vitro. In this study we showed that C-36 peptide is present in human lung tissue and mimics the effects of lipopolysaccharide (LPS), albeit with lower magnitude, by inducing monocyte cytokine (TNFalpha, IL-1beta) and chemokine (IL-8) release in conjunction with the activation of nuclear factor-kappaB (NF-kappaB). Using receptor blocking antibodies and protein kinase inhibitors, we further demonstrated that C-36, like LPS, utilizes CD14 and Toll-like receptor 4 (TLR4) receptors and enzymes of the mitogen-activated protein kinase (MAPK) signaling pathways to stimulate monocyte TNFalpha release. The specificity of C-36 effects were demonstrated by failure of a shorter peptide (C-20) to elicit biological activity and the failure of C-36 to inhibit CD3/CD28-stimulated IL-2 receptor expression or proliferation in T-cells which lack TLR4 and CD14. We suggest that C-36 mediates its effects though the activation of LPS signaling pathways.     

Cutting Edge: Differential inhibition of TLR signaling pathways by cell-permeable peptides representing BB loops of TLRs

We designed cell-penetrating peptides comprised of the translocating segment of Drosophila antennapedia homeodomain fused with BB loop sequences of TLR2, TLR4, and TLR1/6. TLR2- and TLR4-BB peptides (BBPs) inhibited NF-kappaB translocation and early IL-1beta mRNA expression induced by LPS, and the lipopeptides S-[2,3-bis(palmitoyloxy)-(2-RS)-propyl]-N-palmitoyl-(R)-Cys-Ser-Lys(4)-OH (P3C) and S-[2,3-bis(palmitoyloxy)-(2-RS)-propyl]-Cys-Ser-Lys(4)-OH (P2C). TLR4- and TLR2-BBPs also strongly inhibited LPS-induced activation of ERK. Only TLR2-BBP significantly inhibited ERK activation induced by P3C, which acts via TLR2/1 heterodimers. BBPs did not inhibit activation of ERK induced by P2C, a TLR2/6 agonist. The TLR2-BBP induced weak activation of p38, but not ERK or cytokine mRNA. The TLR1/6-BBP failed to inhibit NF-kappaB or MAPK activation induced by any agonist. Our results suggest that the receptor BBPs selectively affect different TLR signaling pathways, and that the BB loops of TLR1/6 and TLR2 play distinct roles in formation of receptor heterodimers and recruitment of adaptor proteins.     

Bruton's tyrosine kinase is required for TLR2 and TLR4-induced TNF, but not IL-6, production

Bruton's tyrosine kinase (Btk), the gene mutated in the human immunodeficiency X-linked agammaglobulinemia, is activated by LPS and is required for LPS-induced TNF production. In this study, we have investigated the role of Btk both in signaling via another TLR (TLR2) and in the production of other proinflammatory cytokines such as IL-1beta, IL-6, and IL-8. Our data show that in X-linked agammaglobulinemia PBMCs, stimulation with TLR4 (LPS) or TLR2 (N-palmitoyl-S-[2, 3-bis(palmitoyloxy)-(2R)-propyl]-(R)-cysteine) ligands produces significantly less TNF and IL-1beta than in normal controls. In contrast, a lack of Btk has no impact on the production of IL-6, IL-8, or the anti-inflammatory cytokine, IL-10. Our previous data suggested that Btk lies within a p38-dependent pathway that stabilizes TNF mRNA. Accordingly, TaqMan quantitative PCR analysis of actinomycin D time courses presented in this work shows that overexpression of Btk is able to stabilize TNF, but not IL-6 mRNA. Furthermore, using the p38 inhibitor SB203580, we show that the TLR4-induced production of TNF, but not IL-6, requires the activity of p38 MAPK. These data provide evidence for a common requirement for Btk in TLR2- and TLR4-mediated induction of two important proinflammatory cytokines, TNF and IL-1beta, and reveal important differences in the TLR-mediated signals required for the production of IL-6, IL-8, and IL-10.     

Signaling events induced by lipopolysaccharide-activated toll-like receptor 2.

Human Toll-like receptor 2 (TLR2) is a signaling receptor that responds to LPS and activates NF-kappaB. Here, we investigate further the events triggered by TLR2 in response to LPS. We show that TLR2 associates with the high-affinity LPS binding protein membrane CD14 to serve as an LPS receptor complex, and that LPS treatment enhances the oligomerization of TLR2. Concomitant with receptor oligomerization, the IL-1R-associated kinase (IRAK) is recruited to the TLR2 complex. Intracellular deletion variants of TLR2 lacking C-terminal 13 or 141 aa fail to recruit IRAK, which is consistent with the inability of these mutants to transmit LPS cellular signaling. Moreover, both deletion mutants could still form complexes with wild-type TLR2 and act in a dominant-negative (DN) fashion to block TLR2-mediated signal transduction. DN constructs of myeloid differentiation protein, IRAK, TNF receptor-associated factor 6, and NF-kappaB-inducing kinase, when coexpressed with TLR2, abrogate TLR2-mediated NF-kappaB activation. These results reveal a conserved signaling pathway for TLR2 and IL-1Rs and suggest a molecular mechanism for the inhibition of TLR2 by DN variants.     

Involvement of protein tyrosine kinase in Toll-like receptor 4-mediated NF-kappa B activation in human peripheral blood monocytes

Bacterial lipopolysaccharide (LPS) is a powerful activator of the innate immune system. Exposure to LPS induces an inflammatory reaction in the lung mediated primarily by human blood monocytes and alveolar macrophages, which release an array of inflammatory chemokines and cytokines including IL-8, TNF-alpha, IL-1beta, and IL-6. The signaling mechanisms utilized by LPS to stimulate the release of cytokines and chemokines are still incompletely understood. Pretreatment with the protein tyrosine kinase-specific inhibitors genistein and herbimycin A effectively blocked LPS-induced NF-kappaB activation as well as IL-8 gene expression in human peripheral blood monocytes. However, when genistein was added 2 min after the addition of LPS, no inhibition was observed. Utilizing a coimmunoprecipitation assay, we further showed that LPS-stimulated tyrosine phosphorylation of Toll-like receptor 4 (TLR4) may be involved in downstream signaling events induced by LPS. These findings provide evidence that LPS-induced NF-kappaB activation and IL-8 gene expression use a signaling pathway requiring protein tyrosine kinase and that such regulation may occur through tyrosine phosphorylation of TLR4.