Lipoproteins, not lipopolysaccharide, are the key mediators of the proinflammatory response elicited by heat-killed Brucella abortus

Inflammation is a hallmark of brucellosis. Although Brucella abortus, one of the disease's etiologic agents, possesses cytokine-stimulatory properties, the mechanism by which this bacterium triggers a proinflammatory response is not known. We examined the mechanism whereby heat-killed B. abortus (HKBA), as well as its LPS, induces production of inflammatory cytokines in monocytes/macrophages. Polymyxin B, a specific inhibitor of LPS activity, did not inhibit the production of TNF-alpha- and IL-6-induced HKBA in the human monocytic cell line THP-1. HKBA induced the production of these cytokines in peritoneal macrophages of both C3H/HeJ and C3H/HeN mice, whereas B. abortus LPS only stimulated cells from C3H/HeN mice. Anti-TLR2 Ab, but not anti-TLR4 Ab, blocked HKBA-mediated TNF-alpha and IL-6 production in THP-1 cells. Because bacterial lipoproteins, a TLR2 ligand, have potent inherent stimulatory properties, we investigated the capacity of two B. abortus lipoproteins, outer membrane protein 19 (Omp19) and Omp16, to elicit a proinflammatory response. Lipidated (L)-Omp16 and L-Omp19, but not their unlipidated forms, induced the secretion of TNF-alpha, IL-6, IL-10, and IL-12 in a time- and dose-dependent fashion. Preincubation of THP-1 cells with anti-TLR2 Ab blocked L-Omp19-mediated TNF-alpha and IL-6 production. Together, these results entail a mechanism whereby B. abortus can stimulate cells from the innate immune system and induce cytokine-mediated inflammation in brucellosis. We submit that LPS is not the cause of inflammation in brucellosis; rather, lipoproteins of this organism trigger the production of proinflammatory cytokines, and TLR2 is involved in this process.     

IRF-8/interferon (IFN) consensus sequence-binding protein is involved in Toll-like receptor (TLR) signaling and contributes to the cross-talk between TLR and IFN-gamma signaling pathways

Toll-like receptor (TLR) and interferon-gamma (IFN-gamma) signaling pathways are important for both innate and adaptive immune responses. However, the cross-talk between these two signaling pathways is incompletely understood. Here we show that IFN-gamma and LPS synergistically induce the expression of proinflammatory factors, including interleukin-1 (IL-1), IL-6, IL-12, NO, and tumor necrosis factor-alpha (TNF-alpha). Comparable synergism was observed between IFN-gamma and peptidoglycan (PGN; a TLR2 ligand) and poly(I:C) (a TLR3 ligand) in the induction of IL-12 promoter activity. IFN-gamma enhanced lipopolysaccharide (LPS)-induced ERK and JNK phosphorylation but had no effect on LPS-induced NF-kappaB activation. Interestingly, we found that IRF-8-/- macrophages were impaired in the activation of LPS-induced ERK and JNK and the production of proinflammatory cytokines induced by LPS or IFN-gamma plus LPS. Retroviral transduction of IRF-8 into IRF-8-/- macrophages rescued ERK and JNK activation. Furthermore, co-immunoprecipitation experiments show that IRF-8 physically interacts with TRAF6 at a binding site between amino acid residues 356 and 305 of IRF-8. Transfection of IRF-8 enhanced TRAF6 ubiquitination, which is consistent with a physical interaction of IRF-8 with TRAF6. Taken together, the results suggest that the interaction of IRF-8 with TRAF6 modulates TLR signaling and may contribute to the cross-talk between IFN-gamma and TLR signal pathways.     

Lipopolysaccharide promotes the survival of osteoclasts via Toll-like receptor 4, but cytokine production of osteoclasts in response to lipopolysaccharide is different from that of macrophages

Lipopolysaccharide is a pathogen that causes inflammatory bone loss. Monocytes and macrophages produce proinflammatory cytokines such as IL-1, TNF-alpha, and IL-6 in response to LPS. We examined the effects of LPS on the function of osteoclasts formed in vitro in comparison with its effect on bone marrow macrophages, osteoclast precursors. Both osteoclasts and bone marrow macrophages expressed mRNA of Toll-like receptor 4 (TLR4) and CD14, components of the LPS receptor system. LPS induced rapid degradation of I-kappaB in osteoclasts, and stimulated the survival of osteoclasts. LPS failed to support the survival of osteoclasts derived from C3H/HeJ mice, which possess a missense mutation in the TLR4 gene. The LPS-promoted survival of osteoclasts was not mediated by any of the cytokines known to prolong the survival of osteoclasts, such as IL-1beta, TNF-alpha, and receptor activator of NF-kappaB ligand. LPS stimulated the production of proinflammatory cytokines such as IL-1beta, TNF-alpha, and IL-6 in bone marrow macrophages and peritoneal macrophages, but not in osteoclasts. These results indicate that osteoclasts respond to LPS through TLR4, but the characteristics of osteoclasts are quite different from those of their precursors, macrophages, in terms of proinflammatory cytokine production in response to LPS.     

Tobacco smoking inhibits expression of proinflammatory cytokines and activation of IL-1R-associated kinase, p38, and NF-kappaB in alveolar macrophages stimulated with TLR2 and TLR4 agonists

Tobacco smoking has been associated with impaired pulmonary functions and increased incidence of infections; however, mechanisms that underlie these phenomena are poorly understood. In this study, we examined whether smokers' alveolar macrophages (AM) exhibit impaired sensing of bacterial components via TLR2 and TLR4 and determined the effect of smoking on expression levels of TLR2, TLR4 and coreceptors, and activation of signaling intermediates. Smokers' AMs exhibited reduced gene expression and secretion of proinflammatory cytokines (TNF-alpha, IL-1beta, IL-6) and chemokines (RANTES and IL-8) upon stimulation with TLR2 and TLR4 agonists, S-[2,3-bis(palmitoyloxy)-(2-RS)-propyl]-N-palmitoyl-(R)-Cys-(S)-Ser-Lys4-OH trihydrochloride (Pam(3)Cys), and LPS, whereas expression of anti-inflammatory cytokines (IL-10 and IL-1 receptor antagonist) was not affected. TLR3 activation with polyinosinic-polycytidylic acid led to comparable or even higher cytokine responses in smokers' AMs, indicating that smoking-induced suppression does not affect all TLRs. Comparable expression of cytokines and chemokines was detected in PBMC and purified monocytes obtained from smokers and nonsmokers, demonstrating that the suppressive effect of smoking is restricted to the lung. TLR2/4-inducible IL-1R-associated kinase-1 (IRAK-1) and p38 phosphorylation and NF-kappaB activation was suppressed in smokers' AMs, whereas TLR2, TLR4, CD14, MD-2 mRNA levels, and TLR4 protein expression were not altered. These data suggest that changes in expression and/or activities of signaling intermediates at the postreceptor level account for smoking-induced immunosuppression. Thus, exposure of AMs to tobacco smoke induces a hyporesponsive state similar to endotoxin tolerance as manifested by inhibited TLR2/4-induced expression of proinflammatory cytokines, chemokines, and impaired activation of IRAK-1, p38, and NF-kappaB, resulting in suppressed expression of proinflammatory mediators.     

PECAM-1 ligation negatively regulates TLR4 signaling in macrophages

Uncontrolled TLR4 signaling may induce excessive production of proinflammatory cytokines and lead to harmful inflammation; therefore, negative regulation of TLR4 signaling attracts much attention now. PECAM-1, a member of Ig-ITIM family, can mediate inhibitory signals in T cells and B cells. However, the role and the mechanisms of PECAM-1 in the regulation of TLR4-mediated LPS response in macrophages remain unclear. In this study, we demonstrate that PECAM-1 ligation with CD38-Fc fusion protein negatively regulates LPS-induced proinflammatory cytokine TNF-alpha, IL-6, and IFN-beta production by inhibiting JNK, NF-kappaB, and IFN regulatory factor 3 activation in macrophages. In addition, PECAM-1 ligation-recruited Src homology region 2 domain-containing phosphatase 1 (SHP-1) and Src homology region 2 domain-containing phosphatase 2 (SHP-2) may be involved in the inhibitory effect of PECAM-1 on TLR4 signaling. Consistently, silencing of PECAM-1 enhances the macrophage response to LPS stimulation. Taken together with the data that PECAM-1 is constitutively expressed in macrophages and its expression is up-regulated by LPS stimulation, PECAM-1 might function as a feedback negative regulator of LPS inflammatory response in macrophages. This study may provide a potential target for intervention of inflammatory diseases.     

Induction of in vitro reprogramming by Toll-like receptor (TLR)2 and TLR4 agonists in murine macrophages: effects of TLR "homotolerance" versus "heterotolerance" on NF-kappa B signaling pathway components

In this study, tolerance induction by preexposure of murine macrophages to Toll-like receptor (TLR)2 and TLR4 agonists was revisited, focusing on the major signaling components associated with NF-kappaB activation. Pretreatment of macrophages with a pure TLR4 agonist (protein-free Escherichia coli (Ec) LPS) or with TLR2 agonists (Porphyromonas gingivalis LPS or synthetic lipoprotein Pam3Cys) led to suppression of TNF-alpha secretion, IL-1R-associated kinase-1, and IkappaB kinase (IKK) kinase activities, c-jun N-terminal kinase, and extracellular signal-regulated kinase phosphorylation, and to suppression of NF-kappaB DNA binding and transactivation upon challenge with the same agonist (TLR4 or TLR2 "homotolerance," respectively). Despite inhibited NF-kappaB DNA binding, increased levels of nuclear NF-kappaB were detected in agonist-pretreated macrophages. For all the intermediate signaling elements, heterotolerance was weaker than TLR4 or TLR2 homotolerance with the exception of IKK kinase activity. IKK kinase activity was unperturbed in heterotolerance. TNF-alpha secretion was also suppressed in P. gingivalis LPS-pretreated, Ec LPS-challenged cells, but not vice versa, while Pam3Cys and Ec LPS did not induce a state of cross-tolerance at the level of TNF-alpha. Experiments designed to elucidate novel mechanisms of NF-kappaB inhibition in tolerized cells revealed the potential contribution of IkappaBepsilon and IkappaBxi inhibitory proteins and the necessity of TLR4 engagement for induction of tolerance to Toll receptor-IL-1R domain-containing adapter protein/MyD88-adapter-like-dependent gene expression. Collectively, these data demonstrate that induction of homotolerance affects a broader spectrum of signaling components than in heterotolerance, with selective modulation of specific elements within the NF-kappaB signaling pathway.     

In vivo ethanol exposure down-regulates TLR2-, TLR4-, and TLR9-mediated macrophage inflammatory response by limiting p38 and ERK1/2 activation

Ethanol is known to increase susceptibility to infections, in part, by suppressing macrophage function. Through TLRs, macrophages recognize pathogens and initiate inflammatory responses. In this study, we investigated the effect of acute ethanol exposure on murine macrophage activation mediated via TLR2, TLR4, and TLR9. Specifically, the study focused on the proinflammatory cytokines IL-6 and TNF-alpha and activation of p38 and ERK1/2 MAPKs after a single in vivo exposure to physiologically relevant level of ethanol followed by ex vivo stimulation with specific TLR ligands. Acute ethanol treatment inhibited IL-6 and TNF-alpha synthesis and impaired p38 and ERK1/2 activation induced by TLR2, TLR4, and TLR9 ligands. We also addressed the question of whether ethanol treatment modified activities of serine/threonine-specific, tyrosine-specific phosphatases, and MAPK phosphatase type 1. Inhibitors of three families of protein phosphatases did not restore ethanol-impaired proinflammatory cytokine production nor p38 and ERK1/2 activation. However, inhibitors of serine/threonine protein phosphatase type 1 and type 2A significantly increased IL-6 and TNF-alpha levels, and prolonged activation of p38 and ERK1/2 when triggered by TLR4 and TLR9 ligands. In contrast, with TLR2 ligand stimulation, TNF-alpha production was reduced, whereas IL-6 levels, and p38 and ERK1/2 activation were not affected. In conclusion, acute ethanol exposure impaired macrophage responsiveness to multiple TLR agonists by inhibiting IL-6 and TNF-alpha production. Mechanism responsible for ethanol-induced suppression involved inhibition of p38 and ERK1/2 activation. Furthermore, different TLR ligands stimulated IL-6 and TNF-alpha production via signaling pathways, which showed unique characteristics.     

Oxidatively modified low density lipoprotein (LDL) inhibits TLR2 and TLR4 cytokine responses in human monocytes but not in macrophages

Inflammation characterized by the expression and release of cytokines and chemokines is implicated in the development and progression of atherosclerosis. Oxidatively modified low density lipoproteins, central to the formation of atherosclerotic plaques, have been reported to signal through Toll-like receptors (TLRs), TLR4 and TLR2, in concert with scavenger receptors to regulate the inflammatory microenvironment in atherosclerosis. This study evaluates the role of low density lipoproteins (LDL) and oxidatively modified LDL (oxmLDL) in the expression and release of proinflammatory mediators IκBζ, IL-6, IL-1β, TNFα, and IL-8 in human monocytes and macrophages. Although standard LDL preparations induced IκBζ along with IL-6 and IL-8 production, this inflammatory effect was eliminated when LDL was isolated under endotoxin-restricted conditions. However, when added with TLR4 and TLR2 ligands, this low endotoxin preparation of oxmLDL suppressed the expression and release of IL-1β, IL-6, and TNFα but surprisingly spared IL-8 production. The suppressive effect of oxmLDL was specific to monocytes as it did not inhibit LPS-induced proinflammatory cytokines in human macrophages. Thus, TLR ligand contamination of LDL/oxmLDL preparations can complicate interpretations of inflammatory responses to these modified lipoproteins. In contrast to providing a proinflammatory function, oxmLDL suppresses the expression and release of selected proinflammatory mediators.     

Dectin-1 synergizes with TLR2 and TLR4 for cytokine production in human primary monocytes and macrophages

The beta-glucan receptor dectin-1 and Toll-like receptors TLR2 and TLR4 are the main receptors for recognition of Candida albicans by the innate immune system. It has been reported that dectin-1 amplifies TLR2-dependent induction of cytokines in mouse models. In the present study we hypothesized that dectin-1 has potent synergistic effects with both TLR2 and TLR4 in human PBMCs and macrophages. Human PBMCs and monocyte-derived macrophages were stimulated with curdlan, a linear beta-1,3-glucan-polymer derived from Alcaligenes faecalis with specific ligand affinity for dectin-1, in combination with the synthetic TLR2 ligand Pam3Cys and the ultrapure TLR4 ligand LPS. TNF-alpha and IL-10 production was measured in the supernatants with ELISA. Curdlan is a specific dectin-1 ligand without TLR2- or TLR4-stimulating properties. Human primary monocytes and macrophages express dectin-1 on the cell membrane. Stimulation of human PBMCs with curdlan in combination with Pam3Cys or LPS leads to synergistic increase in TNF-alpha production that was inhibited by GE2, a neutralizing dectin-1 antibody. Dectin-1-dependent synergy between curdlan and TLR agonists was also apparent in human monocyte-derived macrophages. Conclusively, dectin-1 synergizes with both TLR2 and TLR4 pathways for the production of TNF-alpha in human primary PBMCs and in monocyte-derived macrophages.     

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.     

Functional genomics of silencing TREM-1 on TLR4 signaling in macrophages

Triggering receptor expressed on myeloid cells 1 (TREM-1) is a recently discovered molecule that is expressed on the cell surface of monocytes and neutrophils. Engagement of TREM-1 triggers synthesis of proinflammatory cytokines in response to microbes, but the extent and mechanism by which TREM-1 modulates the inflammatory response is poorly defined. In the present study, we investigated the functional effects of blocking TREM-1 on the Toll-like receptor (TLR)4-mediated signaling pathway in macrophages. By transfecting cells with small hairpin interfering RNA molecules to TREM-1 (shRNA), we confirmed that TREM-1 mRNA and protein expression was greatly attenuated in RAW cells in response to treatment with LPS. PCR array for genes related to or activated by the TLR pathway revealed that although the expression of TLR4 itself was not significantly altered by silencing of TREM-1, expression of several genes, including MyD88, CD14, IkappaBalpha, IL-1beta, MCP-1, and IL-10 was significantly attenuated in the TREM-1 knockdown cells in response to treatment with LPS. These data indicate that expression of TREM-1 modulates the TLR signaling in macrophages by altering the expression of both adaptor and effector proteins that are critical to the endotoxin response.     

Disparities in TLR5 expression and responsiveness to flagellin in equine neutrophils and mononuclear phagocytes

As sentinel cells of the innate immune system, neutrophils and mononuclear phagocytes use specific TLRs to recognize the conserved molecular patterns that characterize microbes. This study was performed to compare the responses of equine neutrophils and mononuclear phagocytes to LPS and flagellin, components of bacteria that are recognized by TLR4 and TLR5, respectively. Neutrophils and mononuclear phagocytes isolated from healthy horses were incubated in vitro with LPS, flagellin, or pronase-inactivated flagellin in the presence or absence of polymyxin B. Production of reactive oxygen species and expression of mRNA for proinflammatory cytokines were used as readouts for activation of neutrophils; production of TNF-α was used for the mononuclear cells. Western blot analysis and flow cytometry were used to detect TLR5 protein in both cell types. Although the neutrophils responded to both LPS and flagellin by producing reactive oxygen species and expressing mRNA for proinflammatory cytokines, flagellin had no stimulatory effect on monocytes or macrophages. Although both neutrophils and monocytes expressed mRNA for TLR5, it appeared to be translated into protein only by the neutrophils. Incubation with neither LPS nor IFN-γ altered TLR5 expression by the monocytes. These findings indicate that flagellin has disparate effects on neutrophils and mononuclear phagocytes isolated from horses, a species that is exquisitely sensitive to the TLR4 ligand, LPS, and that equine mononuclear phagocytes, unlike corresponding cells of other mammalian species, lack surface expression of TLR5 and do not respond to flagellin.     

Lung-restricted macrophage activation in the pearl mouse model of Hermansky-Pudlak syndrome

Pulmonary inflammation, abnormalities in alveolar type II cell and macrophage morphology, and pulmonary fibrosis are features of Hermansky-Pudlak Syndrome (HPS). We used the naturally occurring "pearl" HPS2 mouse model to investigate the mechanisms of lung inflammation observed in HPS. Although baseline bronchoalveolar lavage (BAL) cell counts and differentials were similar in pearl and strain-matched wild-type (WT) mice, elevated levels of proinflammatory (MIP1gamma) and counterregulatory (IL-12p40, soluble TNFr1/2) factors, but not TNF-alpha, were detected in BAL from pearl mice. After intranasal LPS challenge, BAL levels of TNF-alpha, MIP1alpha, KC, and MCP-1 were 2- to 3-fold greater in pearl than WT mice. At baseline, cultured pearl alveolar macrophages (AMs) had markedly increased production of inflammatory cytokines. Furthermore, pearl AMs had exaggerated TNF-alpha responses to TLR4, TLR2, and TLR3 ligands, as well as increased IFN-gamma/LPS-induced NO production. After 24 h in culture, pearl AM LPS responses reverted to WT levels, and pearl AMs were appropriately refractory to continuous LPS exposure. In contrast, cultured pearl peritoneal macrophages and peripheral blood monocytes did not produce TNF-alpha at baseline and had LPS responses which were no different from WT controls. Exposure of WT AMs to heat- and protease-labile components of pearl BAL, but not WT BAL, resulted in robust TNF-alpha secretion. Similar abnormalities were identified in AMs and BAL from another HPS model, pale ear HPS1 mice. We conclude that the lungs of HPS mice exhibit hyperresponsiveness to LPS and constitutive and organ-specific macrophage activation.     

Synthetic TLR agonists reveal functional differences between human TLR7 and TLR8

Although TLR7 and TLR8 are phylogenetically and structurally related, their relative functions are largely unknown. The role of TLR7 has been established using TLR7-deficient mice and small molecule TLR7 agonists. The absence of TLR8-selective agonists has hampered our understanding of the role of TLR8. In this study TLR agonists selective for TLR7 or TLR8 were used to determine the repertoire of human innate immune cells that are activated through these TLRs. We found that TLR7 agonists directly activated purified plasmacytoid dendritic cells and, to a lesser extent, monocytes. Conversely, TLR8 agonists directly activated purified myeloid dendritic cells, monocytes, and monocyte-derived dendritic cells (GM-CSF/IL-4/TGF-beta). Accordingly, TLR7-selective agonists were more effective than TLR8-selective agonists at inducing IFN-alpha- and IFN-regulated chemokines such as IFN-inducible protein and IFN-inducible T cell alpha chemoattractant from human PBMC. In contrast, TLR8 agonists were more effective than TLR7 agonists at inducing proinflammatory cytokines and chemokines, such as TNF-alpha, IL-12, and MIP-1alpha. Thus, this study demonstrated that TLR7 and TLR8 agonists differ in their target cell selectivity and cytokine induction profile.     

IL-6 and maturation govern TLR2 and TLR4 induced TLR agonist tolerance and cross-tolerance in dendritic cells

Stimulation of naive mouse dendritic cells (DC) with LPS or Pam(3)CSK(4) (P3C) induces production of TNF-alpha via TLR4- or TLR2-signaling. Although tolerance in macrophages has been studied in detail, we investigated the role of TLR agonist concentration and IL-6 for tolerance in DC. P3C- or LPS-primed DC were nonresponsive to P3C or LPS restimulation in terms of TNF-alpha but not IL-6 production. The mechanisms involved in tolerance were dependent on the concentration of the TLR ligand used for DC priming. DC primed with LPS or P3C at high concentrations developed a maturation dependent, IL-6 independent tolerance associated with inhibition of TLR signaling upstream of IkappaB as indicated by decreased IkappaB degradation. In contrast, priming of DC with LPS or P3C at low concentrations resulted in IL-6-dependent tolerance, which was abolished in IL-6 deficient DC, and was not accompanied by maturation of DC or by down-regulation of TLR2 or TLR4. In homotolerogenic DC primed with LPS or P3C at high concentrations, degradation of IkappaB upon restimulation with LPS or P3C was inhibited suggesting tolerance mechanism(s) upstream of IkappaB; in contrast, cross-tolerance in DC primed with LPS or P3C at low concentrations was not associated with reduced IkappaB degradation suggesting tolerance mechanisms downstream of IkappaB. Our data indicate that in naive DC TLR4- and TLR2-stimulation results in homo- and cross-tolerance; the mechanisms involved in tolerance depend on the concentration of the TLR agonist used for DC priming and are governed by IL-6 and maturation.     

MNK kinases regulate multiple TLR pathways and innate proinflammatory cytokines in macrophages

The MNK kinases are downstream of both the p38 and ERK MAP kinase pathways and act to increase gene expression. MNK inhibition using the compound CGP57380 has recently been reported to inhibit tumor necrosis factor (TNF) production in macrophage cell lines stimulated with Escherichia coli lipopolysaccharide (LPS). However, the range of receptors that signal through the MNK kinases and the extent of the resultant cytokine response are not known. We found that TNF production was inhibited in RAW264.7 macrophage cells by CGP57380 in a dose-responsive manner with agonists for Toll-like receptor (TLR) 2 (HKLM), TLR4 (Salmonella LPS), TLR6/2 (FSL), TLR7 (imiquimod), and TLR9 (CpG DNA). CGP57380 also inhibited the peak of TNF mRNA production and increased the rate of TNF mRNA decay, effects not due to the destabilizing RNA binding protein tristetraprolin (TTP). Similar to its effects on TNF, CGP57380 caused dose-responsive inhibition of TTP production from stimulation with either LPS or CpG DNA. MNK inhibition also blocked IL-6 but permitted IL-10 production in response to LPS. Studies using bone marrow-derived macrophages (BMDM) isolated from a spontaneous mouse model of Crohn's disease-like ileitis (SAMP1/YitFc strain) revealed significant inhibition by CGP57380 of the proinflammatory cytokines TNF, IL-6, and monocyte chemoattractant protein-1 at 4 and 24 h after LPS stimulation. IL-10 production was higher in CGP53870-treated BMDM at 4 h but was similar to the controls by 24 h. Taken together, these data demonstrate that MNK kinases signal through a variety of TLR agonists and mediate a potent innate, proinflammatory cytokine response.     

Functional consequences of CD36 downregulation by TLR signals

TLR recognition activates the secretion of pro- and anti-inflammatory cytokines and it also modulates the expression of crucial molecules involved in phagocytosis and antimicrobial activity. Scavenger receptors can act as TLR co-receptors or facilitate antigen loading. However, it remains unknown whether TLR can modulate the expression of these scavenger receptors. We stimulated human peripheral blood mononuclear cells (PBMC) with TLR2 (Pam3CSK4 and FSL1) and TLR4 ligand lipopolysaccharide (LPS) and then analyzed CD36 expression on different monocyte subpopulations by flow cytometry. TLR2 and TLR4 ligands can downregulate CD36 on the surface of monocytes, guiding the protein to intracellular compartments. Even though TLR-activation induced TNFα, IL-10 and IL-6 production, only recombinant TNFα was able to downregulate CD36. Neutralizing anti-TNFα antibodies showed that the Pam3CSK4 and FSL1-induced downregulation was partially mediated by TNFα but not by IL-6 or IL-10. However, LPS-induced downregulation could have also been caused by direct TLR4 targeting and signaling, and/or mediated by other unknown factors. CD36 downregulation reduced the capability of monocytes to phagocyte apoptotic neutrophils. In conclusion, modulation of scavenger receptor expression by TLR targeting on monocytes has functional consequences. Characterization this complex regulation may help us to understand this innate response and develop specific therapeutic drugs for each mechanism.     

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.