TLR2 signaling depletes IRAK1 and inhibits induction of type I IFN by TLR7/9

Pathogens may signal through multiple TLRs with synergistic or antagonistic effects on the induction of cytokines, including type I IFN (IFN-I). IFN-I is typically induced by TLR9, but not TLR2. Moreover, we previously reported that TLR2 signaling by Mycobacterium tuberculosis or other TLR2 agonists inhibited TLR9 induction of IFN-I and IFN-I-dependent MHC-I Ag cross processing. The current studies revealed that lipopeptide-induced TLR2 signaling inhibited induction of first-wave IFN-α and IFN-β mRNA by TLR9, whereas induction of second-wave IFN-I mRNA was not inhibited. TLR2 also inhibited induction of IFN-I by TLR7, another MyD88-dependent IFN-I-inducing receptor, but did not inhibit IFN-I induction by TLR3 or TLR4 (both Toll/IL-1R domain-containing adapter-inducing IFN-β dependent, MyD88 independent). The inhibitory effect of TLR2 was not dependent on new protein synthesis or intercellular signaling. IL-1R-associated kinase 1 (IRAK1) was depleted rapidly (within 10 min) by TLR2 agonist, but not until later (e.g., 2 h) by TLR9 agonist. Because IRAK1 is required for TLR7/9-induced IFN-I production, we propose that TLR2 signaling induces rapid depletion of IRAK1, which impairs IFN-I induction by TLR7/9. This novel mechanism, whereby TLR2 inhibits IFN-I induction by TLR7/9, may shape immune responses to microbes that express ligands for both TLR2 and TLR7/TLR9, or responses to bacteria/virus coinfection.     

Early production of IL-22 but not IL-17 by peripheral blood mononuclear cells exposed to live Borrelia burgdorferi: the role of monocytes and interleukin-1

If insufficiently treated, Lyme borreliosis can evolve into an inflammatory disorder affecting skin, joints, and the CNS. Early innate immunity may determine host responses targeting infection. Thus, we sought to characterize the immediate cytokine storm associated with exposure of PBMC to moderate levels of live Borrelia burgdorferi. Since Th17 cytokines are connected to host defense against extracellular bacteria, we focused on interleukin (IL)-17 and IL-22. Here, we report that, despite induction of inflammatory cytokines including IL-23, IL-17 remained barely detectable in response to B. burgdorferi. In contrast, T cell-dependent expression of IL-22 became evident within 10 h of exposure to the spirochetes. This dichotomy was unrelated to interferon-γ but to a large part dependent on caspase-1 and IL-1 bioactivity derived from monocytes. In fact, IL-1β as a single stimulus induced IL-22 but not IL-17. Neutrophils display antibacterial activity against B. burgdorferi, particularly when opsonized by antibodies. Since neutrophilic inflammation, indicative of IL-17 bioactivity, is scarcely observed in Erythema migrans, a manifestation of skin inflammation after infection, protective and antibacterial properties of IL-22 may close this gap and serve essential functions in the initial phase of spirochete infection.     

Intracellular IL-1Ra type 1 inhibits IL-1-induced IL-6 and IL-8 production in Caco-2 intestinal epithelial cells through inhibition of p38 mitogen-activated protein kinase and NF-kappaB pathways

Interleukin-1 (IL-1) plays a pivotal role in the pathogenesis of inflammatory bowel disease (IBD). IL-1 action is regulated in part by its naturally occurring inhibitor, the IL-1 receptor antagonist (IL-1Ra). Four splice variants of IL-1Ra gene product have been described, one secreted (sIL-1Ra) and three intracellular (icIL-1Ra1, 2, 3). Although sIL-1Ra and icIL-1Ra1 bind to type I IL-1 receptor with equal affinity, icIL-1Ra1 may carry out unique functions inside cells. The goal of this study was to determine the role of icIL-1Ra1 in regulation of cytokine-induced IL-6 and IL-8 production in Caco-2 intestinal epithelial cells. icIL-1Ra1 inhibited IL-1-induced IL-6 and IL-8 production. IL-1 activated all three mitogen-activated protein (MAP) kinase family members: p38 MAP kinase, extracellular-regulated kinases (ERK), and c-Jun amino-terminal kinases (JNK). Specific inhibitors of each MAP kinase pathway decreased IL-1-induced IL-6 and IL-8 production. Overexpression of icIL-1Ra1 inhibited p38 MAP kinase phosphorylation, but had no effect on ERK and JNK phosphorylation. In addition, icIL-1Ra1 inhibited nuclear translocation of NF-kappaB after IL-1 stimulation. In conclusion, these data indicate that icIL-1Ra1, acting in the cytoplasm of Caco-2 cells, decreased IL-1-induced IL-6 and IL-8 production. This intracellular anti-inflammatory activity of icIL-1Ra1 was mediated through inhibition of p38 MAP kinase and NF-kappaB signal transduction pathways.     

Novel role of AMP-activated protein kinase signaling in cigarette smoke induction of IL-8 in human lung epithelial cells and lung inflammation in mice

Cigarette smoke (CS) increases chemokine production in lung epithelial cells (LECs), but the pathways involved are not completely understood. AMP-activated protein kinase (AMPK), a crucial regulator of energy homeostasis, may modulate inflammation. Here, we show that cigarette smoke extract sequentially activated NADPH oxidase; increased intracellular reactive oxygen species (ROS) level; activated AMPK, NF-κB, and STAT3; and induced interleukin 8 (IL-8) in human LECs. Inhibition of NADPH oxidase activation by apocynin or siRNA targeting p47(phox) (a subunit of NADPH oxidase) attenuated the increased intracellular ROS level, AMPK activation, and IL-8 induction. Removal of intracellular ROS by N-acetylcysteine reduced the AMPK activation and IL-8 induction. Prevention of AMPK activation by Compound C or AMPK siRNA lessened the activation of both NF-κB and STAT3 and the induction of IL-8. Abrogation of the activation of NF-κB and STAT3 by BAY11-7085 and AG490, respectively, attenuated the IL-8 induction. We additionally show that chronic CS exposure in mice promoted AMPK phosphorylation and expression of MIP-2α (an IL-8 homolog) in LECs and lungs, as well as lung inflammation, all of which were reduced by Compound C treatment. Thus, a novel NADPH oxidase-dependent, ROS-sensitive AMPK signaling is important for CS-induced IL-8 production in LECs and possibly lung inflammation.     

Damping excessive inflammation and tissue damage in Mycobacterium tuberculosis infection by Toll IL-1 receptor 8/single Ig IL-1-related receptor, a negative regulator of IL-1/TLR signaling

Toll IL-1R 8/single Ig IL-1-related receptor (TIR8/SIGIRR) is a member of the IL-1R family, expressed by epithelial tissues and immature dendritic cells, and is regarded as a negative regulator of TLR/IL-1R signaling. Tir8-deficient mice were rapidly killed by intranasal administration of low doses of Mycobacterium tuberculosis, despite controlling efficiently the number of viable bacilli in different organs. Tir8(-/-)-infected mice showed an increased number of neutrophils and macrophages in the lungs; however, mycobacteria-specific CD4 and CD8 T cells were similar in Tir8(-/-) and Tir8(+/+) mice. Exaggerated mortality of Tir8(-/-) mice was due to massive liver necrosis and was accompanied by increased levels of IL-1beta and TNF-alpha in lung mononuclear cells and serum, as well as by increased production of IL-1beta and TNF-alpha by M. tuberculosis-infected dendritic cells in vitro. Accordingly, blocking IL-1beta and TNF-alpha with a mix of anti-cytokine Abs, significantly prolonged survival of Tir8(-/-) mice. Thus, TIR8/SIGIRR plays a key role in damping inflammation and tissue damage in M. tuberculosis infection.     

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.     

Eicosapentaenoic acid and docosahexaenoic acid modulate MAP kinase (ERK1/ERK2) signaling in human T cells.

This study was conducted on human Jurkat T cell lines to elucidate the role of EPA and DHA, n-3 PUFA, in the modulation of two mitogen-activated protein (MAP) kinases, that is, extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2). The n-3 PUFA alone failed to induce phosphorylation of ERK1/ERK2. We stimulated the MAP kinase pathway with anti-CD3 antibodies and phorbol 12-myristate 13-acetate (PMA), which act upstream of the MAP kinase (MAPK)/ERK kinase (MEK) as U0126, an MEK inhibitor, abolished the actions of these two agents on MAP kinase activation. EPA and DHA diminished the PMA- and anti-CD3-induced phosphorylation of ERK1/ERK2 in Jurkat T cells. In the present study, PMA acts mainly via protein kinase C (PKC) whereas anti-CD3 antibodies act via PKC-dependent and -independent mechanisms. Furthermore, DHA and EPA inhibited PMA-stimulated PKC enzyme activity. EPA and DHA also significantly curtailed PMA- and ionomycin-stimulated T cell blastogenesis. Together these results suggest that EPA and DHA modulate ERK1/ERK2 activation upstream of MEK via PKC-dependent and -independent pathways and that these actions may be implicated in n-3 PUFA-induced immunosuppression.     

IL-1-mediated inhibition of IL-6-induced STAT3 activation is modulated by IL-4, MAP kinase inhibitors and redox state of HepG2 cells

It is known that during acute phase reaction IL-6 activates STAT3 in hepatoma cells and IL-1 downregulates this response. We found that the inhibitory properties of IL-1 on STAT signalling cascade in human hepatoma HepG2 cells are considerably decreased not only in the presence of MAP kinase inhibitors SB203580 and PD98059 but also by some antioxidants (N-acetyl cysteine and pyrrolidine dithiocarbamate) and by anti-inflammatory cytokine IL-4. It appears that cytokine crosstalk between IL-6 and IL-1 includes a direct pathway sensitive to antioxidants and MAP kinase inhibitors, whereas the indirect prolonged response depends probably on synthesis of suppressors of cytokine signalling (SOCS).     

Thrombin induces NF-kappaB activation and IL-8/CXCL8 expression in lung epithelial cells by a Rac1-dependent PI3K/Akt pathway

We previously showed that thrombin induces interleukin (IL)-8/CXCL8 expression via the protein kinase C (PKC)α/c-Src-dependent IκB kinase α/β (IKKα/β)/NF-κB signaling pathway in human lung epithelial cells. In this study, we further investigated the roles of Rac1, phosphoinositide 3-kinase (PI3K), and Akt in thrombin-induced NF-κB activation and IL-8/CXCL8 expression. Thrombin-induced IL-8/CXCL8 release and IL-8/CXCL8-luciferase activity were attenuated by a PI3K inhibitor (LY294002), an Akt inhibitor (1-L-6-hydroxymethyl-chiro-inositol-2-((R)-2-O-methyl-3-O-octadecylcarbonate)), and the dominant negative mutants of Rac1 (RacN17) and Akt (AktDN). Treatment of cells with thrombin caused activation of Rac and Akt. The thrombin-induced increase in Akt activation was inhibited by RacN17 and LY294002. Stimulation of cells with thrombin resulted in increases in IKKα/β activation and κB-luciferase activity; these effects were inhibited by RacN17, LY294002, an Akt inhibitor, and AktDN. Treatment of cells with thrombin induced Gβγ, p85α, and Rac1 complex formation in a time-dependent manner. These results imply that thrombin activates the Rac1/PI3K/Akt pathway through formation of the Gβγ, Rac1, and p85α complex to induce IKKα/β activation, NF-κB transactivation, and IL-8/CXCL8 expression in human lung epithelial cells.     

ERK/Egr-1 signaling pathway is involved in CysLT2 receptor-mediated IL-8 production in HEK293 cells

The CysLT₂ receptor is involved in myocardial ischemia/reperfusion injury, differentiation of colorectal cancers, bleomycin-induced pulmonary inflammation and fibrosis. However, the signal transduction of cysteinyl leukotriene receptor 2 (CysLT₂) in inflammatory responses remains to be clarified. In HEK293 cells stably expressing hCysLT₁, hCysLT₂ and rGPR17, we determined the signaling pathways for interleukin-8 (IL-8) production after CysLT₂ receptor activation. HEK293 cells were stably transfected with the recombinant plasmids of pcDNA3.1(+)-hCysLT₁, pcDNA3.1(+)-hCysLT₂ and pcDNA3.1-rGPR17. Leukotriene C₄ (LTC₄) and LTD₄ were used as the agonists to induce IL-8 production and the related changes in signal molecules. We found that LTC₄ and LTD₄ significantly induced IL-8 promoter activation in the HEK293 cells stably expressing hCysLT₂, but not in those expressing hCysLT₁ and rGPR17. In hCysLT₂-HEK293 cells, LTC₄ induced elevation of intracellular calcium, ERK1/2 phosphorylation and Egr-1 expression, and stimulated IL-8 expression and release. These responses were blocked by the selective CysLT₂ receptor antagonist HAMI3379. The ERK1/2 inhibitor U0126 inhibited Egr-1 and IL-8 expression as well as IL-8 release, but the JNK and p38 inhibitors did not have the inhibitory effects. Down-regulation of Egr-1 by RNA interference with its siRNA inhibited the LTC₄-induced IL-8 expression and release. In conclusion, these findings indicate the ERK-Egr-1 pathway of CysLT₂ receptors mediates IL-8 production induced by the pro-inflammatory mediators LTC₄ and LTD₄.     

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.     

Urokinase-type plasminogen activator receptor crosslinking in an NK cell line increases integrin surface expression by the MAP kinase/ERK 1/2 signaling pathway

Urokinase-type plasminogen activator receptor (uPAR) is attached to cell membranes by a glycosylphosphatidylinositol (GPI) anchor, and as such is devoid of an intracellular domain, but is nevertheless able to initiate signal transduction. Herein, we report a relationship between integrins and uPAR on the surface of the human NK cell line, YT. Our data reveals that crosslinking uPAR, which mimics uPAR clustering at focal adhesion sites, causes increases in expression of the alpha(M), alpha(V), and beta(2) integrins on the surface of YT cells. Activation of the MEK/ERK signaling cascade occurs following uPAR crosslinking, as phosphorylation of both MEK 1/2 and ERK 1/2 results from receptor clustering. The MEK-specific inhibitors PD98059 and U0126 blocked MAP kinase phosphorylation; furthermore, PD98059 inhibited the increase in integrin expression induced by uPAR clustering. This study suggests that uPAR is a signaling receptor and regulator of integrins in NK cells and may impact NK cell function, including the potential for their accumulation within tumor metastases following adoptive transfer.