RelB/p50 regulates TNF production in LPS-stimulated dendritic cells and macrophages.

Dendritic cells (DCs) and macrophages are effective antigen-presenting cells, and DCs, once matured, have the ability to potently activate naïve T cells. While the canonical p65/p50 NF-κB pathway seems to have an important role during LPS-stimulation of these cells, the specific contribution of the non-canonical RelB/p50 subunits is not clear yet. We aimed to investigate the relevance of this pathway in DCs and macrophages by using replication-deficient adenoviruses overexpressing RelB and p50 subunits to test their effect on cytokine production. In both cells, after LPS treatment, overexpression of RelB and p50 inhibited the production of some pro-inflammatory cytokines e.g., TNF, but not of others e.g. IL6. Anti-inflammatory IL10 was not affected. Moreover, when overexpressing p50 alone, IL10 was increased in LPS-activated macrophages. We thus demonstrated that the dimer RelB/p50 rather than the p50/p50 complex inhibits TNF production in LPS-stimulated DCs and macrophages. This implies that the non-canonical RelB/p50 could modulate the canonical p65/p50 pathway.

Tumor necrosis factor-alpha triggers a cytokine cascade yielding postoperative cognitive decline.

Cognitive decline following surgery in older individuals is a major clinical problem of uncertain mechanism; a similar cognitive decline also follows severe infection, chemotherapy, or trauma and is currently without effective therapy. A variety of mechanisms have been proposed, and exploring the role of inflammation, we recently reported the role of IL-1ß in the hippocampus after surgery in mice with postoperative cognitive dysfunction. Here, we show that TNF-α is upstream of IL-1 and provokes its production in the brain. Peripheral blockade of TNF-α is able to limit the release of IL-1 and prevent neuroinflammation and cognitive decline in a mouse model of surgery-induced cognitive decline. TNF-α appears to synergize with MyD88, the IL-1/TLR superfamily common signaling pathway, to sustain postoperative cognitive decline. Taken together, our results suggest a unique therapeutic potential for preemptive treatment with anti-TNF antibody to prevent surgery-induced cognitive decline.

Nonsteroidal anti-inflammatory drugs increase TNF production in rheumatoid synovial membrane cultures and whole blood.

Nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit cyclooxygenase activity and hence PG production. However, the ability of NSAIDs to ameliorate pain and tenderness does not prevent disease progression in rheumatoid arthritis, a disease whose pathogenesis is linked to the presence of proinflammatory cytokines, such as TNF-alpha. To understand this observation, we have examined the effect of NSAIDs on the production of clinically validated proinflammatory cytokines. We show that a variety of NSAIDs superinduce production of TNF from human peripheral blood monocytes and rheumatoid synovial membrane cultures. A randomized, double-blinded, crossover, placebo-controlled trial in healthy human volunteers also revealed that the NSAID drug celecoxib increased LPS-induced TNF production in whole blood. NSAID-mediated increases in TNF are reversed by either the addition of exogenous PGE(2) or by a PGE(2) EP2 receptor agonist, revealing that PGE(2) signaling via its EP2 receptor provides a valuable mechanism for controlling excess TNF production. Thus, by reducing the level of PGE(2), NSAIDs can increase TNF production and may exacerbate the proinflammatory environment both within the rheumatoid arthritis joint and the systemic environment.

Inhibitors of p38 suppress cytokine production in rheumatoid arthritis synovial membranes: does variable inhibition of interleukin-6 production limit effectiveness in vivo?

OBJECTIVE: The activity of p38 MAPK regulates lipopolysaccharide (LPS)-stimulated production of key proinflammatory cytokines such as tumor necrosis factor α (TNFα). Consequently, p38 MAPK inhibitors have attracted considerable interest as potential treatments of rheumatoid arthritis (RA), and studies in murine models of arthritis have yielded promising results. However, the performance of several compounds in human clinical trials has been disappointing. At present, the reason for this poor performance is unclear. The aim of this study was to examine the effects of p38 inhibitors on both diseased and normal human tissue and cells, in order to test whether this kinase still plays a critical role in cytokine production under conditions of chronic inflammation. METHODS: Proinflammatory and antiinflammatory cytokine production was monitored after treatment of primary human monocytes, macrophages, and RA synovial membrane cultures with p38 MAPK inhibitor compounds. The following 3 inhibitors were used in these studies: SB-203580 (inhibits the α and ß isoforms), BIRB-796 (inhibits the α, ß, γ, and δ isoforms), and a novel, structurally distinct p38 MAPK inhibitor, SB-731445 (inhibits the α and ß isoforms). RESULTS: SB-731445 and SB-203580 produced profound inhibition of spontaneous production of proinflammatory cytokines (TNFα and interleukin-1 [IL-1]) in both RA membrane cultures and LPS-stimulated primary human monocytes. However, this and other p38 MAPK inhibitors produced a significant increase in IL-6 production by LPS-stimulated primary human macrophages and a decrease in IL-10 production by all cell types examined. CONCLUSION: The potentially proinflammatory consequences of these activities (decreased IL-10 production and increased IL-6 production) may offer some explanation for the inability of p38 MAPK inhibitors to provide the therapeutic benefit that had been hoped for in RA.

SIGIRR/TIR-8 is an inhibitor of Toll-like receptor signaling in primary human cells and regulates inflammation in models of rheumatoid arthritis.

OBJECTIVE: Single-immunoglobulin interleukin-1 receptor-related (SIGIRR), which is also known as Toll/interleukin-1 receptor 8 (TIR-8), is a member of the TIR domain-containing family of receptors and was first characterized as an inhibitor of interleukin-1 receptor (IL-1R) and Toll-like receptor (TLR) signaling. In the Dextran sulfate sodium-induced colitis model, SIGIRR(-/-) mice were shown to have increased inflammation and to be more susceptible to endotoxin challenge. Increasing evidence implicates TLR and IL-1R signaling in the pathology of rheumatoid arthritis (RA). Therefore, the purpose of this study was to investigate the involvement of SIGIRR in regulating inflammation in disease-relevant models. METHODS: Primary human monocyte-derived macrophages and dendritic cells (DCs) were used to overexpress SIGIRR as well as to knock down endogenously expressed SIGIRR using small interfering RNAs. SIGIRR was also overexpressed in synovial cells derived from RA patients. To investigate the role of SIGIRR in vivo, zymosan-induced arthritis (ZIA) and collagen antibody-induced arthritis (CAIA) were induced in SIGIRR-knockout mice. RESULTS: SIGIRR overexpression inhibited TLR-induced cytokine production in macrophages and DCs, while SIGIRR knockdown resulted in increased cytokine production following TLR stimulation. Moreover, SIGIRR overexpression inhibited the spontaneous release of cytokines by human RA synovial cells. The role of SIGIRR as an inhibitor of inflammation was confirmed in vivo, since SIGIRR(-/-) mice developed a more severe disease in both the ZIA and CAIA models. CONCLUSION: Our study is the first to show the expression pattern and function of SIGIRR in primary human cells. Furthermore, this investigation defines the role of SIGIRR in disease-relevant cell types and demonstrates that SIGIRR is a potential therapeutic target for RA.

Oral activated charcoal prevents experimental cerebral malaria in mice and in a randomized controlled clinical trial in man did not interfere with the pharmacokinetics of parenteral artesunate.

BACKGROUND: Safe, cheap and effective adjunct therapies preventing the development of, or reducing the mortality from, severe malaria could have considerable and rapid public health impact. Oral activated charcoal (oAC) is a safe and well tolerated treatment for acute poisoning, more recently shown to have significant immunomodulatory effects in man. In preparation for possible efficacy trials in human malaria, we sought to determine whether oAC would i) reduce mortality due to experimental cerebral malaria (ECM) in mice, ii) modulate immune and inflammatory responses associated with ECM, and iii) affect the pharmacokinetics of parenteral artesunate in human volunteers. METHODS/PRINCIPAL FINDINGS: We found that oAC provided significant protection against P. berghei ANKA-induced ECM, increasing overall survival time compared to untreated mice (p<0.0001; hazard ratio 16.4; 95% CI 6.73 to 40.1). Protection from ECM by oAC was associated with reduced numbers of splenic TNF(+) CD4(+) T cells and multifunctional IFNgamma(+)TNF(+) CD4(+) and CD8(+) T cells. Furthermore, we identified a whole blood gene expression signature (68 genes) associated with protection from ECM. To evaluate whether oAC might affect current best available anti-malarial treatment, we conducted a randomized controlled open label trial in 52 human volunteers (ISRCTN NR. 64793756), administering artesunate (AS) in the presence or absence of oAC. We demonstrated that co-administration of oAC was safe and well-tolerated. In the 26 subjects further analyzed, we found no interference with the pharmacokinetics of parenteral AS or its pharmacologically active metabolite dihydroartemisinin. CONCLUSIONS/SIGNIFICANCE: oAC protects against ECM in mice, and does not interfere with the pharmacokinetics of parenteral artesunate. If future studies succeed in establishing the efficacy of oAC in human malaria, then the characteristics of being inexpensive, well-tolerated at high doses and requiring no sophisticated storage would make oAC a relevant candidate for adjunct therapy to reduce mortality from severe malaria, or for immediate treatment of suspected severe malaria in a rural setting. TRIAL REGISTRATION: Controlled-Trials.com ISRCTN64793756.

The role of toll-like receptors in chronic inflammation.

The role of Toll-like receptors (TLRs) in innate immunity and their ability to recognise microbial products has been well characterised. TLRs are also able to recognise endogenous molecules which are released upon cell damage and necrosis and have been shown to be present in numerous autoimmune diseases. Therefore, the release of endogenous TLR ligands during inflammation and consequently the activation of TLR signalling pathways may be one mechanism initiating and driving autoimmune diseases. An increasing body of circumstantial evidence implicates a role of TLR signalling in systemic lupus erythematosus (SLE), atherosclerosis, asthma, type 1 diabetes, multiple sclerosis, bowl inflammation and rheumatoid arthritis (RA). Although at present their involvement is not comprehensively defined. However, future therapies targeting individual TLRs or their signalling transducers may provide a more specific way of treating inflammatory diseases without global suppression of the immune system.

Toll-like receptor-2 mediates inflammation and matrix degradation in human atherosclerosis.

BACKGROUND: Inflammation and matrix degradation are the hallmarks of high-risk atherosclerosis that leads to myocardial infarction and stroke. Toll-like receptors (TLRs), key players in innate immunity, are upregulated in atherosclerotic lesions, but their functional role in human atherosclerosis is unknown. We explored the effects of blocking TLR-2, TLR-4, and myeloid differentiation primary response gene 88 (MyD88), a signaling adaptor shared by most TLRs and interleukin-1 receptor (IL-1R), in an in vitro model of human atherosclerosis. METHODS AND RESULTS: Carotid endarterectomies were obtained from patients with symptomatic carotid disease. Cells were isolated via enzymatic tissue dissociation and cultured in the presence or absence of TLR signaling blockers. A dominant-negative form of MyD88 (MyD88(DN)) decreased the production of monocyte chemotactic protein-1/CCL2 (P=0.000), IL-8/CXCL8 (P=0.006), IL-6 (P=0.002), matrix metalloproteinase-1 (MMP-1; P=0.002), and MMP-3 (P=0.000), as well as nuclear factor-kappaB activation (P<0.05) in atheroma cell cultures. IL-1R antagonist, TLR-4 blocking antibodies, or overexpression of a dominant-negative form of the TLR-4 signaling adaptor TRIF-related adaptor molecule reduced nuclear factor-kappaB activity but did not have a broad impact on the production of the mediators studied. In contrast, TLR-2 neutralizing antibodies inhibited nuclear factor-kappaB activation (P<0.05) and significantly reduced monocyte chemotactic protein-1/CCL2 (P=0.000), IL-8/CXCL8 (P=0.009), IL-6 (P=0.000), and MMP-1 (P=0.000), MMP-2 (P=0.004), MMP-3 (P=0.000), and MMP-9 (P=0.006) production. CONCLUSIONS: Our data indicate that TLR-2 signaling through MyD88 plays a predominant role in inflammation and matrix degradation in human atherosclerosis. TLR-2 blockade may represent a therapeutic strategy for atherosclerosis and its complications.

TREM-1 expression is increased in the synovium of rheumatoid arthritis patients and induces the expression of pro-inflammatory cytokines.

OBJECTIVES: To investigate the expression and function of triggering receptor expressed on myeloid cells-1 (TREM-1) in the synovium of human RA patients as well as the level of soluble TREM-1 in the plasma of RA patients. METHODS: Twenty-four RA synovial samples were analysed by gene expression oligonucleotide microarrays. Expression levels of TREM-1 mRNA in murine CIA paws were determined by quantitative PCR (qPCR). TREM-1 protein expression was detected by immunohistochemistry in five RA synovial samples and two OA synovial samples. TREM-1-positive cells from five RA synovial tissues were analysed by FACS staining to determine the cell type. Activation of TREM-1 was tested in five RA synovial samples. Soluble TREM-1 was measured in serum from 32 RA patients. RESULTS: The expression of TREM-1 mRNA was found to increase 6.5-fold in RA synovial samples, whereas it was increased 132-fold in CIA paws. Increased numbers of TREM-1-positive cells were seen in RA synovium sections and these cells co-expressed CD14. Using a TREM-1-activating cross-linking antibody in RA synovial cultures, multiple pro-inflammatory cytokines were induced. The average amount of soluble TREM-1 in plasma from RA patients was found to be higher than that in plasma from healthy volunteers. CONCLUSIONS: These findings suggest that the presence of high levels of functionally active TREM-1 in RA synovium may contribute to the development or maintenance of RA, or both. Inhibiting TREM-1 activity may, therefore, have a therapeutic effect on RA. High levels of soluble TREM-1 in the plasma of RA patients compared with healthy volunteers may indicate disease activity.

RelB/p50 regulates CCL19 production, but fails to promote human DC maturation.

DC, when fully matured are the APC best able to activate naïve T cells. Recently, we demonstrated using adenoviruses overexpressing IkappaBalpha and proteosome inhibitors that NF-kappaB is involved in DC activation, but the role of the individual subunits is still not clear. We investigated the role of the NF-kappaB subunits RelB and p50 in human DC activation using adenoviral vectors expressing RelB or p50. Nuclear RelB, in the form of RelB/p50, was active only in DC infected with both viruses, this induced the production of the soluble homeostatic chemokine CCL19, but not other homeostatic chemokines, particularly in LPS-matured DC. However, RelB/p50 did not affect the expression of costimulatory and antigen-presenting molecules, and increased the allogeneic mixed lymphocyte reaction only in LPS-matured DC. This enhanced mixed lymphocyte reaction is most likely due to enhanced CCL19 production, which sustains the interaction between mature DC and naïve T cells. In conclusion, we demonstrated that RelB/p50 was active only in DC expressing both RelB and p50, and induced CCL19 production, but not DC maturation.

Disordered macrophage cytokine secretion underlies impaired acute inflammation and bacterial clearance in Crohn's disease.

The cause of Crohn's disease (CD) remains poorly understood. Counterintuitively, these patients possess an impaired acute inflammatory response, which could result in delayed clearance of bacteria penetrating the lining of the bowel and predispose to granuloma formation and chronicity. We tested this hypothesis in human subjects by monitoring responses to killed Escherichia coli injected subcutaneously into the forearm. Accumulation of (111)In-labeled neutrophils at these sites and clearance of (32)P-labeled bacteria from them were markedly impaired in CD. Locally increased blood flow and bacterial clearance were dependent on the numbers of bacteria injected. Secretion of proinflammatory cytokines by CD macrophages was grossly impaired in response to E. coli or specific Toll-like receptor agonists. Despite normal levels and stability of cytokine messenger RNA, intracellular levels of tumor necrosis factor (TNF) were abnormally low in CD macrophages. Coupled with reduced secretion, these findings indicate accelerated intracellular breakdown. Differential transcription profiles identified disease-specific genes, notably including those encoding proteins involved in vesicle trafficking. Intracellular destruction of TNF was decreased by inhibitors of lysosomal function. Together, our findings suggest that in CD macrophages, an abnormal proportion of cytokines are routed to lysosomes and degraded rather than being released through the normal secretory pathway.

The role of transposable elements in the regulation of IFN-lambda1 gene expression.

IFNs lambda1, lambda2, and lambda3, or type III IFNs, are recently identified cytokines distantly related to type I IFNs. Despite an early evolutionary divergence, the 2 types of IFNs display similar antiviral activities, and both are produced primarily in dendritic cells. Although virus induction of the type I IFN-beta gene had served as a paradigm of gene regulation, relatively little is known about the regulation of IFN-lambda gene expression. Studies of virus induction of IFN-lambda1 identified an essential role of IFN regulatory factors (IRF) 3 and 7, which bind to a regulatory DNA sequence near the start site of transcription. Here, we report that the proximal promoter region of the IFN-lambda1 regulatory region is not sufficient for maximal gene induction in response to bacterial LPS, and we identify an essential cluster of homotypic NF-kappaB binding sites. Remarkably, these sites, which bind efficiently to NF-kappaB and function independently of the IRF3/7 binding sites, originate as transposable elements of the Alu and LTR families. We also show that depletion of the NF-kappaB RelA protein significantly reduces the level of the IFN-lambda1 gene expression. We conclude that IFN-lambda1 gene expression requires NF-kappaB, and we propose a model for IFN-lambda1 gene regulation, in which IRF and NF-kappaB activate gene expression independently via spatially separated promoter elements. These observations provide insights into the independent evolution of the IFN-lambda1 and IFN-beta promoters and directly implicate transposable elements in the regulation of the IFN-lambda1 gene by NF-kappaB.

Tenascin-C is an endogenous activator of Toll-like receptor 4 that is essential for maintaining inflammation in arthritic joint disease.

Although there have been major advances in the treatment of rheumatoid arthritis with the advent of biological agents, the mechanisms that drive cytokine production and sustain disease chronicity remain unknown. Tenascin-C (encoded by Tnc) is an extracellular matrix glycoprotein specifically expressed at areas of inflammation and tissue damage in inflamed rheumatoid joints. Here we show that mice that do not express tenascin-C show rapid resolution of acute joint inflammation and are protected from erosive arthritis. Intra-articular injection of tenascin-C promotes joint inflammation in vivo in mice, and addition of exogenous tenascin-C induces cytokine synthesis in explant cultures from inflamed synovia of individuals with rheumatoid arthritis. Moreover, in human macrophages and fibroblasts from synovia of individuals with rheumatoid arthritis, tenascin-C induces synthesis of proinflammatory cytokines via activation of Toll-like receptor 4 (TLR4). Thus, we have identified tenascin-C as a novel endogenous activator of TLR4-mediated immunity that mediates persistent synovial inflammation and tissue destruction in arthritic joint disease.

Mycolactone inhibits monocyte cytokine production by a posttranscriptional mechanism.

The virulence and immunosuppressive activity of Mycobacterium ulcerans is attributed to mycolactone, a macrolide toxin synthesized by the bacteria. We have explored the consequence and mechanism of mycolactone pretreatment of primary human monocytes activated by a wide range of TLR ligands. The production of cytokines (TNF, IL-1beta, IL-6, IL-10, and IFN-gamma-inducible protein-10), chemokines (IL-8), and intracellular effector molecules (exemplified by cyclooxygenase-2) was found to be powerfully and dose dependently inhibited by mycolactone, irrespective of the stimulating ligand. However, mycolactone had no effect on the activation of signaling pathways that are known to be important in inducing these genes, including the MAPK and NF-kappaB pathways. Unexpectedly, LPS-dependent transcription of TNF, IL-6, and cyclooxygenase-2 mRNA was found not to be inhibited, implying that mycolactone has a novel mechanism of action and must function posttranscriptionally. We propose that mycolactone mediates its effects by inhibiting the translation of a specific subset of proteins in primary human monocytes. This mechanism is distinct from rapamycin, another naturally occurring immunosuppressive lactone. The current findings also suggest that monocyte-derived cytokine transcript and protein levels may not correlate in Buruli ulcer lesions, and urge caution in the interpretation of RT-PCR data obtained from patient biopsy samples.

Tyrosine kinases and inflammatory signalling.

The activity of tyrosine kinases is central to many cellular processes, and accumulating evidence suggests that their role in inflammation is no less profound. Three main tyrosine kinase families, the Src, Tec and Syk kinase families are intimately involved in TLR signalling, the critical first step in cellular recognition of invading pathogens and tissue damage. Their activity results in changes in gene expression in affected cells. Key amongst these genes are the cytokines, which orchestrate both the duration and extent of inflammation. Tyrosine kinases also play important roles in cytokine function, and are implicated in signalling through both pro- and anti-inflammatory cytokines such as TNF, IL-6 and IL-10. Thus, strategies to modulate tyrosine kinase activity have significant therapeutic potential in combating the chronic inflammatory state that is typical of many major health issues that face us today, including Rheumatoid Arthritis, Cardiovascular disease and cancer. Here we review current knowledge of the role of tyrosine kinases in inflammation with particular emphasis on their role in TLR signalling.

Tolerance induced via TLR2 and TLR4 in human dendritic cells: role of IRAK-1.

BACKGROUND: While dendritic cells (DCs) can induce tolerance in T cells, little is known about tolerance induction in DCs themselves. We have analysed tolerance induced in human in-vitro generated DCs by repeated stimulation with ligands for TLR4 and TLR2. RESULTS: DCs stimulated with the TLR4 ligand LPS did show a rapid and pronounced expression of TNF mRNA and protein. When DCs were pre-cultured for 2 days with 5 ng LPS/ml then the subsequent response to stimulation with a high dose of LPS (500 ng/ml) was strongly reduced for both TNF mRNA and protein. At the promoter level there was a reduced transactivation by the -1173 bp TNF promoter and by a construct with a tetrameric NF-kappaB motif. Within the signalling cascade leading to NF-kappaB activation we found an ablation of the IRAK-1 adaptor protein in LPS-tolerant DCs. Pre-culture of DCs with the TLR2 ligand Pam3Cys also led to tolerance with respect to TNF gene expression and IRAK-1 protein was ablated in such tolerant cells as well, while IRAK-4 protein levels were unchanged. CONCLUSION: These data show that TLR-ligands can render DCs tolerant with respect to TNF gene expression by a mechanism that likely involves blockade of signal transduction at the level of IRAK-1.

Inhibitors of TLR8 reduce TNF production from human rheumatoid synovial membrane cultures.

The advent of anti-TNF biologicals has been a seminal advance in the treatment of rheumatoid arthritis (RA) and has confirmed the important role of TNF in disease pathogenesis. However, it is unknown what sustains the chronic production of TNF. In this study, we have investigated the anti-inflammatory properties of mianserin, a serotonin receptor antagonist. We discovered mianserin was able to inhibit the endosomal TLRs 3, 7, 8, and 9 in primary human cells and inhibited the spontaneous release of TNF and IL-6 from RA synovial membrane cultures. This suggested a role for these TLRs in production of TNF and IL-6 from RA which was supported by data from chloroquine, an inhibitor of endosomal acidification (a prerequisite for TLRs 3, 7, 8, and 9 activation) which also inhibited production of these cytokines from RA synovial cultures. Only stimulation of TLR 3 or 8 induced TNF from these cultures, indicating that TLR7 and TLR9 were of less consequence in this model. The key observation that indicated the importance of TLR8 was the inhibition of spontaneous TNF production by imiquimod, which we discovered to be an inhibitor of TLR8. Together, these data suggest that TLR8 may play a role in driving TNF production in RA. Because this receptor can be inhibited by small m.w. molecules, it may prove to be an important therapeutic target.

Cell signalling in macrophages, the principal innate immune effector cells of rheumatoid arthritis.

Rheumatoid arthritis is a multisystemic auto-inflammatory disease affecting up to 1% of the population and leading to the destruction of the joints. Evidence exists for the involvement of the innate as well as the adaptive immune systems in the pathology of the disease. The success of anti-tumour necrosis factor-alpha indicates the importance of pro-inflammatory mediators produced by innate immune cells in rheumatoid arthritis progression. Therefore, considerable efforts have been made in elucidating the signalling pathways leading to the expression of those mediators. This review will concentrate on the role of signalling pathways in innate immune cells in the context of rheumatoid arthritis.

The scaffold MyD88 acts to couple protein kinase Cepsilon to Toll-like receptors.

Mice lacking protein kinase Cepsilon (PKCepsilon) are hypersensitive to both Gram-positive and Gram-negative bacterial infections; however, the mechanism of PKCepsilon coupling to the Toll-like receptors (TLRs), responsible for pathogen detection, is poorly understood. Here we sought to investigate the mechanism of PKCepsilon involvement in TLR signaling and found that PKCepsilon is recruited to TLR4 and phosphorylated on two recently identified sites in response to lipopolysaccharide (LPS) stimulation. Phosphorylation at both of these sites (Ser-346 and Ser-368) resulted in PKCepsilon binding to 14-3-3beta. LPS-induced PKCepsilon phosphorylation, 14-3-3beta binding, and recruitment to TLR4 were all dependent on expression of the scaffold protein MyD88. In mouse embryo fibroblasts and activated macrophages from MyD88 knock-out mice, LPS-stimulated PKCepsilon phosphorylation was reduced compared with wild type cells. Acute knockdown of MyD88 in LPS-responsive 293 cells also resulted in complete loss of Ser-346 phosphorylation and TLR4/PKCepsilon association. By contrast, MyD88 overexpression in 293 cells resulted in constitutive phosphorylation of PKCepsilon. A general role for MyD88 was evidenced by the finding that phosphorylation of PKCepsilon was induced by the activation of all TLRs tested that signal through MyD88 (i.e. all except TLR3) both in RAW cells and in primary human macrophages. Functionally, it is established that phosphorylation of PKCepsilon at these two sites is required for TLR4- and TLR2-induced NFkappaB reporter activation and IkappaB degradation in reconstituted PKCepsilon(-/-) cells. This study therefore identifies the scaffold protein MyD88 as the link coupling TLRs to PKCepsilon recruitment, phosphorylation, and downstream signaling.

Role of STAT3 in glucocorticoid-induced expression of the human IL-10 gene.

In the present report we have determined the molecular mechanisms, which govern the expression of the human IL-10 gene when induced by the glucocorticoid Methyl-Prednisolone (MP). Treatment of cells with MP at 10(-6) M will readily induce IL-10 in CD19+ primary B cells and in a human B cell line. Analysis of the IL-10 promoter showed a robust 18-fold induction and demonstrated that a potential GRE motif was not required, while mutation of the -120 STAT-motif strongly reduced MP-induced trans-activation. A strong induction was also seen with a trimeric STAT-motif and over-expression of dominant-negative STAT3 could block MP induction of IL-10 mRNA. Finally, MP treatment induced binding of STAT3 to the promoter as shown by gelshift, supershift and by chromatin-immunoprecipitation. These data show that glucocorticoid-induced expression of the IL-10 gene is mediated by the transcription factor STAT3.