Mycobacterium tuberculosis lipoarabinomannan-mediated IRAK-M induction negatively regulates Toll-like receptor-dependent interleukin-12 p40 production in macrophages

Mannose-capped lipoarabinomannans (Man-LAMs) are members of the repertoire of Mycobacterium tuberculosis modulins that the bacillus uses to subvert the host innate immune response. Interleukin-12 (IL-12) production is critical for mounting an effective immune response by the host against M. tuberculosis. We demonstrate that Man-LAM inhibits IL-12 p40 production mediated by subsequent challenge with lipopolysaccharide (LPS). Man-LAM inhibits LPS-induced IL-12 p40 expression in an IL-10-independent manner. It attenuates LPS-induced NF-kappaB-driven luciferase gene expression, suggesting that its effects are likely directly related to inhibition of NF-kappaB. This is probably because of dampening of the Toll-like receptor signaling. Man-LAM inhibits IL-1 receptor-associated kinase (IRAK)-TRAF6 interaction as well as IkappaB-alpha phosphorylation. It directly attenuates nuclear translocation and DNA binding of c-Rel and p50. Man-LAM exerts these effects by inducing the expression of Irak-M, a negative regulator of TLR signaling. Knockdown of Irak-M expression by RNA interference reinstates LPS-induced IL-12 production in Man-LAM-pretreated cells. The fact that Irak-M expression could be elicited by yeast mannan suggested that ligation of the mannose receptor by the mannooligosaccharide caps of LAM was the probable trigger for IRAK-M induction.     

IL-10 inhibits lipopolysaccharide-induced CD40 gene expression through induction of suppressor of cytokine signaling-3

Costimulation between T cells and APCs is required for adaptive immune responses. CD40, an important costimulatory molecule, is expressed on a variety of cell types, including macrophages and microglia. The aberrant expression of CD40 is implicated in diseases including multiple sclerosis, rheumatoid arthritis, and Alzheimer's disease, and inhibition of CD40 signaling has beneficial effects in a number of animal models of autoimmune diseases. In this study, we discovered that IL-10, a cytokine with anti-inflammatory properties, inhibits LPS-induced CD40 gene expression. We previously demonstrated that LPS induction of CD40 in macrophages/microglia involves both NF-kappaB activation and LPS-induced production of IFN-beta, which subsequently activates STAT-1alpha. IL-10 inhibits LPS-induced IFN-beta gene expression and subsequent STAT-1alpha activation, but does not affect NF-kappaB activation. Our results also demonstrate that IL-10 inhibits LPS-induced recruitment of STAT-1alpha, RNA polymerase II, and the coactivators CREB binding protein and p300 to the CD40 promoter, as well as inhibiting permissive histone H3 acetylation (AcH3). IL-10 and LPS synergize to induce suppressor of cytokine signaling (SOCS)-3 gene expression in macrophages and microglia. Ectopic expression of SOCS-3 attenuates LPS-induced STAT activation, and inhibits LPS-induced CD40 gene expression, comparable to that seen by IL-10. These results indicate that SOCS-3 plays an important role in the negative regulation of LPS-induced CD40 gene expression by IL-10.     

Activation of the murine interleukin-12 p40 promoter by functional interactions between NFAT and ICSBP

Interleukin (IL)-12 is a heterodimeric cytokine that is critical for the development of a T-helper-1 immune response and immunity against intracellular pathogens. The IL-12 p40 gene product, expressed specifically in macrophages and dendritic cells, heterodimerizes with p35 to form bioactive IL-12, and heterodimerizes with p19 to comprise the cytokine IL-23. Regulation of the murine IL-12 p40 promoter is complex. Multiple cis-acting elements have been characterized that are involved in activation by bacterial products. However, molecular mechanisms through which interferon (IFN)-gamma and bacterial products synergistically activate IL-12 p40 gene expression are less clear. In this study, a composite NFAT/ICSBP binding site at -68 to -54 is identified that is functionally important for p40 promoter activation by lipopolysaccharide (LPS) and LPS plus IFN-gamma. DNA binding of NFAT and ICSBP is demonstrated on the endogenous promoter by chromatin immunoprecipitation. NFAT is required for ICSBP binding to this region. Overexpression of NFAT and ICSBP synergistically activates the p40 promoter. A dominant negative NFAT molecule attenuates LPS- and IFN-gamma-activated endogenous IL-12 p40 mRNA expression. A physical association between NFAT and ICSBP in the absence of DNA is detected by co-immunoprecipitation of endogenous proteins. Three NFAT domains are required for ICSBP interaction. Finally, in LPS- and IFN-gamma-activated RAW-264.7 cells, the association between NFAT and ICSBP is abrogated by IL-10 priming.     

Promoting effect of IFN-γ on the expression of LPS-induced IL-12 p40 and p35 mRNA in murine suppressor macrophages

We detected the expression of IL-12 p40/p35 mRNA by semi-quantitative RT-PCR and silver staining, and studied the molecular interaction between the IL-12 expression and the NF-kB activation induced by LPS and IFN-γ/LPS in murine peritoneal suppressor macrophages (MPSMs). It was found that IFN-γ strongly enhanced the LPS-induced IL-12 p40 and p35 mRNA expression. Both p40 and p35 mRNA levels were approximately equal. IFN-γ also greatly promoted the LPS-induced secretion of IL-12 p70 in MPSMs. The Proteasome Inhibitor I (PSI) could block the expres-sion of IL-12 p40 and p35 mRNA, and the degradation of IkBα induced by LPS or LPS/IFN-γ. EM-SA showed that LPS could augment the NF-kB binding activity to p40 promoter DNA. However, IFN-γ could neither enhance the LPS-induced NF-kB activity nor promote the degradation of IkBα. Taken together, the data suggest: (i) IFN-γ/LPS could strongly induce the expression of IL-12 p40 and p35 mRNA; both the expression levels were equal; this phenomenon coincided with the high-level secretion of IL-12 p70 induced by IFN-γ/LPS; (ii) NF-kB signal pathway is essential for IFN-γ/LPS to induce IL-12 mRNA expression; (iii) by blocking the degradation of IkB, the PSI sup-presses the IL-12 p40/p35 mRNA expression induced by LPS and IFN-γ/LPS; (iv) NF-kB signal may not be involved in the mechanism by which IFN-γ enhanced the expression of the LPS-induced IL-12 p40/p35 mRNA.     

Inhibition of IFN-gamma-induced janus kinase-1-STAT1 activation in macrophages by vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide

The vasoactive intestinal peptide (VIP) and the pituitary adenylate cyclase-activating polypeptide (PACAP), two immunomodulatory neuropeptides that affect both innate and acquired immunity, down-regulate IL-12 p40 and inducible NO synthase expression in LPS/IFN-gamma-stimulated macrophages. We showed previously that VIP/PACAP inhibit NF-kappaB nuclear translocation through the stabilization of IkappaB and reduce IFN regulatory factor-1 (IRF-1) binding to the regulatory elements found in the IL-12 p40 and inducible NO synthase promoters. In this paper we studied the molecular mechanisms involved in the VIP/PACAP regulation of IRF-1 transactivating activity. Our studies indicate that the inhibition in IRF-1 binding correlates with a reduction in IRF-1 protein and mRNA in IFN-gamma-treated Raw 264.7 macrophages. In agreement with the described Janus kinase (Jak)1/Jak2/STAT1/IRF-1 activation pathway, VIP/PACAP inhibit Jak1/Jak2, STAT1 phosphorylation, and the binding of STAT1 to the GAS sequence motif in the IRF-1 promoter. The effects of VIP/PACAP are mediated through the specific VIP/PACAP receptor-1 and the cAMP/protein kinase A (PKA) transduction pathway, but not through the induction of suppressor of cytokine signaling-1 or suppressor of cytokine signaling-3. Because IFN-gamma is a major stimulator of innate immune responses in vivo, the down-regulation of IFN-gamma-induced gene expression by VIP and PACAP could represent a significant element in the regulation of the inflammatory response by endogenous neuropeptides.     

Mercury inhibits nitric oxide production but activates proinflammatory cytokine expression in murine macrophage: differential modulation of NF-kappaB and p38 MAPK signaling pathways.

Mercury is well known to adversely affect the immune system; however, little is known regarding its molecular mechanisms. Macrophages are major producers of nitric oxide (NO) and this signaling molecule is important in the regulation of immune responses. The present study was designed to determine the impact of mercury on NO and cytokine production and to investigate the signaling pathways involved. The murine macrophage cell line J774A.1 was used to study the effects of low-dose inorganic mercury on the production of NO and proinflammatory cytokines. Cells were treated with mercury in the presence or absence of lipopolysaccharide (LPS). Mercury (5-20 microM) dose-dependently decreased the production of NO in LPS-stimulated cells. Concomitant decreases in the expression of inducible nitric oxide synthase (iNOS) mRNA and protein were detected. Treatment of J774A.1 cells with mercury alone did not affect the production of NO nor the expression of iNOS mRNA or protein. Interestingly, mercury alone stimulated the expression of tumor necrosis factor alpha (TNFalpha), and increased LPS-induced TNFalpha and interleukin-6 mRNA expression. Mercury inhibited LPS-induced nuclear translocation of nuclear factor kappaB (NF-kappaB) but had no effect alone. In contrast, mercury activated p38 mitogen-activated protein kinase (p38 MAPK) and additively increased LPS-induced p38 MAPK phosphorylation. These results indicate that mercury suppresses NO synthesis by inhibition of the NF-kappaB pathway and modulates cytokine expression by p38 MAPK activation in J774A.1 macrophage cells.     

NF-kappa B regulates the LPS-induced expression of interleukin 12 p40 in murine peritoneal macrophages: roles of PKC, PKA, ERK, p38 MAPK, and proteasome

NF-kappa B plays a critical role in coordinating the control of gene expression during monocyte/macrophage activation. In this report we describe our investigation of the mechanisms of LPS-induced NF-kappa B activation and IL-12 expression in murine peritoneal suppressor macrophages. Treatment of these macrophages with LPS induced I kappa B alpha degradation and NF-kappa B activation. EMSAs demonstrated that NF-kappa B bound to a cis-acting element located in the murine IL-12 p40 promoter. LPS signal transduction has been shown to involve a variety of signal pathways. The results in this paper indicate that LPS-induced NF-kappa B binding activity was independent of PKC, PKA, ERK, and p38 MAPK, but was regulated by proteasome. Furthermore, Proteasome Inhibitor I abolished the LPS-induced mRNA expression of IL-12 p35 and p40, and SB203580 reduced these mRNA levels, whereas the blockade of PKC, PKA, and ERK had little effect. These data demonstrate that the LPS-induced activation of proteasome. I kappa B. NF-kappa B and p38 MAPK signal pathways regulate the IL-12 expression in murine peritoneal suppressor macrophages.     

Enhanced induction of LPS-induced fibroblast MCP-1 by interferon-gamma: involvement of JNK and MAPK phosphatase-1

IFN-gamma has significant immunoregulatory activity and plays an important role in both innate and adaptive immunity. Additive effects of IFN-gamma and the Toll-like receptor ligand LPS has been investigated in macrophages, but in fibroblasts is incompletely understood. IFN-gamma and LPS synergistically induced MCP-1 and NO release in primary murine dermal fibroblasts. IFN-gamma enhanced LPS-induced JNK and p38 MAPK phosphorylation but had no effect on NF-kappaB activity. The induction of both MCP-1 and NO was attenuated by inhibition of JNK but not p38 MAPK. Serine 727 STAT1 phosphorylation by IFN-gamma was increased by LPS, and this was also attenuated by inhibition of JNK but not p38 MAPK. IFN-gamma inhibited the basal expression of MAPK phosphatase-1, a negative regulator of MAPK signaling pathway. These results suggest that enhancement of LPS-induced JNK activation by IFN-gamma associated with inhibition of MAPK phosphatase-1 may be one of the mechanisms of additive effects between IFN-gamma and LPS in fibroblasts.     

Role of phosphatidylinositol-3 kinase in transcriptional regulation of TLR-induced IL-12 and IL-10 by Fc gamma receptor ligation in murine macrophages

Ligation of FcgammaR concurrent with LPS stimulation of murine macrophages results in decreased IL-12 and increased IL-10 production. Because PI3K deficiency has been associated with increased IL-12, we hypothesized that PI3K was central to the anti-inflammatory effect of FcgammaR ligation on TLR-induced IL-12. FcgammaR ligation of macrophages increased pAKT, a correlate of PI3K activity, above levels induced by TLR4 or TLR2 agonists. This increase was blocked by PI3K inhibitors, wortmannin or LY294002, as was the effect of FcgammaR ligation on TLR-induced IL-12 and IL-10. LPS-induced binding of NF-kappaB to the IL-12 p40 promoter NF-kappaB-binding site was not affected by FcgammaR ligation at 1 h; however, by 4 h, NF-kappaB binding was markedly inhibited, confirmed in situ by chromatin immunoprecipitation analysis. This effect was wortmannin sensitive. Although TLR-induced IkappaBalpha degradation was not affected by FcgammaR ligation, IkappaBalpha accumulated in the nuclei of cells treated with LPS and FcgammaR ligation for 4 h, and was blocked by PI3K inhibitors. LPS-induced IFN regulatory factor-8/IFN consensus sequence-binding protein mRNA, and an IFN regulatory factor-8-dependent gene, Nos2, were inhibited by concurrent FcgammaR ligation, and this was also reversed by wortmannin. Thus, FcgammaR ligation modulates LPS-induced IL-12 via multiple PI3K-sensitive pathways that affect production, accumulation, and binding of key DNA-binding proteins required for IL-12 induction.