Adenylate cyclase toxin from Bordetella pertussis synergizes with lipopolysaccharide to promote innate interleukin-10 production and enhances the induction of Th2 and regulatory T cells

Adenylate cyclase toxin (CyaA) from Bordetella pertussis can subvert host immune responses allowing bacterial colonization. Here we have examined its adjuvant and immunomodulatory properties and the possible contribution of lipopolysaccharide (LPS), known to be present in purified CyaA preparations. CyaA enhanced antigen-specific interleukin-5 (IL-5) and IL-10 production and immunoglobulin G1 antibodies to coadministered antigen in vivo. Antigen-specific CD4(+)-T-cell clones generated from mice immunized with antigen and CyaA had cytokine profiles characteristic of Th2 or type 1 regulatory T (Tr1) cells. Since innate immune cells direct the induction of T-cell subtypes, we examined the influence of CyaA on activation of dendritic cells (DC) and macrophages. CyaA significantly augmented LPS-induced IL-6 and IL-10 and inhibited LPS-driven tumor necrosis factor alpha and IL-12p70 production from bone marrow-derived DC and macrophages. CyaA also enhanced cell surface expression of CD80, CD86, and major histocompatibility class II on immature DC. The stimulatory activity of our CyaA preparation for IL-10 production and CD80, CD86, and major histocompatibility complex class II expression was attenuated following the addition of polymyxin B or with the use of DC from Toll-like receptor (TLR) 4-defective mice. However, treatment of DC with LPS alone at the concentration present in the CyaA preparation (0.2 ng/ml) failed to activate DC in vitro. Our findings demonstrate that activation of innate cells in vitro by CyaA is dependent on a second signal through a TLR and that CyaA can promote Th2/Tr1-cell responses by inhibiting IL-12 and promoting IL-10 production by DC and macrophages.     

IL-18 enhances IFN-gamma-induced production of CXCL9, CXCL10, and CXCL11 in human keratinocytes

IL-18 is involved in the pathogenesis of atopic dermatitis, psoriasis, and allergic contact dermatitis. CXCL9, CXCL10, and CXCL11 recruit type 1 T cells, and the production of these chemokines by keratinocytes is enhanced in these dermatoses. We examined the in vitro effects of IL-18 on IFN-gamma-induced CXCL9, CXCL10, and CXCL11 production in human keratinocytes. IL-18 enhanced the IFN-gamma-induced secretion and mRNA expression of CXCL9, CXCL10, and CXCL11 in parallel to the activation of NF-kappaB, STAT1, and IFN-regulatory factor (IRF)-1. Antisense oligonucleotides against NF-kappaB p50, p65, or STAT1 suppressed CXCL9, CXCL10, and CXCL11 production, and antisense IRF-1 suppressed CXCL11 production. Inhibitors of PI3 K, p38 MAPK, and MEK suppressed IL-18 plus IFN-gamma-induced CXCL9, CXCL10, and CXCL11 production and NF-kappaB, STAT1, and IRF-1 activities. IL-18 induced phosphorylation of ERK and Akt, while IFN-gamma induced phosphorylation of p38 MAPK. These results suggest that IL-18 may potentiate IFN-gamma-induced CXCL9, CXCL10, and CXCL11 production in keratinocytes by activating NF-kappaB, STAT1, or IRF-1 through PI3 K/Akt and MEK/ERK pathways. These effects of IL-18 may promote the infiltration of type 1 T cells into lesions with inflammatory dermatoses and amplify the skin inflammation. IL-18 may act as a pro-inflammatory cytokine in these dermatoses and thus is a candidate therapeutic target.     

Reduced expression of IL-12 p35 by SJL/J macrophages responding to Theiler's virus infection is associated with constitutive activation of IRF-3

Macrophages responding to viral infections may contribute to autoimmune demyelinating diseases (ADD). Macrophages from ADD-susceptible SJL/J mice responding to Theiler's Virus (TMEV) infection, the TLR7 agonist loxoribine, or the TLR4 agonist-LPS expressed less IL-12 p35 but more IL-12/23 p40 and IFN-beta than macrophages from ADD-resistant B10.S mice. While expression of IRF-1 and -7 was similar between B10.S and SJL/J TMEV-infected macrophages, SJL/J but not B10.S macrophages exhibited constitutively active IRF-3. In contrast to overexpressed IRF-1, IRF-5, and IRF-7, which stimulated p35 promoter reporter activity, overexpressed IRF-3 repressed p35 promoter activity in response to TMEV infection, loxoribine, IFN-gamma/LPS, but not IFN-gamma alone. IRF-3 lessened but did not eliminate IRF-1-stimulated p35 promoter activity. Repression by IRF-3 required bp -172 to -122 of the p35 promoter. The data suggest that pre-activated IRF-3 is a major factor in the differences in IL-12 production between B10.S and SJL/J macrophages responding to TMEV.     

Control of dual-specificity phosphatase-1 expression in activated macrophages by IL-10

Ligation of Toll-like receptors (TLR) on macrophages induces cytokines and mediators important for the control of pathogens. Macrophage activation has to be tightly controlled to prevent hyper-inflammation. Accordingly, the hallmarks of TLR-triggered signaling, nuclear translocation of NF-kappaB and phosphorylation of mitogen-activated protein kinases (MAPK), are transient events. We have mined microarray datasets for changes in the expression of phosphatases in resting and TLR-activated macrophages. Several members of the dual-specificity phosphatases (DUSP) were induced upon triggering TLR4 with LPS. Up-regulation of DUSP1 mRNA was transient after stimulation with LPS alone, but addition of the immunosuppressive cytokine IL-10 resulted in robust, continued DUSP1 expression. IL-10 also synergized with the anti-inflammatory glucocorticoid dexamethasone in the induction of DUSP1 mRNA expression in activated macrophages, as well as in the inhibition of IL-6 and IL-12 production. Increased expression of DUSP1 in IL-10-treated activated macrophages was correlated with a faster down-regulation of p38 MAPK activation. Thus, these data suggest an operational link between IL-10 and inhibition of p38 MAPK via sustained expression of DUSP1.     

Selective synergy in anti-inflammatory cytokine production upon cooperated signaling via TLR4 and TLR2 in murine conventional dendritic cells

Toll-like receptor (TLR) ligands, i.e. lipopolysaccharide (LPS), induce dendritic cell (DC) production of both inflammatory and anti-inflammatory cytokines including interleukin (IL)-12, tumor necrosis factor (TNF)-, and IL-10. The balance of inflammatory versus anti-inflammatory cytokines appears to be crucial to control immune homeostasis. In the present study, we investigated TLR-mediated regulation of inflammatory versus anti-inflammatory cytokine production using murine bone marrow derived conventional DCs. Standard LPS (sLPS) that contains lipoprotein, a TLR2 ligand, induced vigorous production of both IL-10 and IL-12 p40 by DCs. Highly purified LPS (ultra-pure LPS, upLPS) also induced vigorous production of IL-12 p40, but markedly low IL-10 production. Thus, signal deficiency through TLR2 appeared to result in marked reduction in DC production of IL-10 but not IL-12 p40 upon stimulation with upLPS. To examine this possibility, DCs were stimulated with Pam3CSK4, a synthetic ligand of TLR2, in addition to stimulation with upLPS. It was shown that Pam3CSK4 alone failed to induce IL-10 production. However, Pam3CSK4 synergistically enhanced upLPS-induced DC production of IL-10 but neither IL-12 p40 nor TNF-. Extracellular signal-regulated kinase (ERK)1/2, p38 mitogen-activated protein kinase (MAPK), and c-jun N-terminal kinase (JNK)1/2 in DCs were significantly activated by upLPS stimulation. The upLPS-induced activities of these MAPKs were considerably enhanced by additional stimulation with Pam3CSK4. Blocking either p38 MAPK or JNK1/2 pathway completely inhibited the synergistic enhancement of the IL-10 production by DCs upon upLPS and Pam3CSK4 stimulation. Thus, cooperated stimulation of these MAPKs via TLR4 and TLR2 appeared to induce selective synergy in anti-inflammatory cytokine production by murine conventional DCs.     

Polysaccharide purified from Ganoderma lucidum induced activation and maturation of human monocyte-derived dendritic cells by the NF-kappaB and p38 mitogen-activated protein kinase pathways

Ganoderma lucidum, a fungus native to China, has been widely used to promote health and longevity in the Chinese. The polysaccharide component with a branched (1-->6)-beta-D-glucan moiety of G. lucidum (PS-G) has been reported to exert anti-tumor activity and activation of natural killer cells. In this study, we investigated the effects of PS-G on human monocyte-derived dendritic cells (DC). Treatment of DC with PS-G resulted in the enhanced cell-surface expression of CD80, CD86, CD83, CD40, CD54, and human leukocyte antigen (HLA)-DR, as well as the enhanced production of interleukin (IL)-12p70, p40, and IL-10 and also IL-12p35, p40, and IL-10 mRNA expression, and the capacity for endocytosis was suppressed in DC. In addition, treatment of DC with PS-G resulted in enhanced T cell-stimulatory capacity and increased T cell secretion of interferon-gamma and IL-10. Neutralization with antibodies against Toll-like receptor (TLR)-4 inhibited the PS-G-induced production of IL-12 p40 and IL-10, suggesting a vital role for TLR-4 in signaling DC upon incubation with PS-G. Further study showed that PS-G was able to augment inhibitor of kappaB (IkappaB) kinase and nuclear factor (NF)-kappaB activity and also IkappaB alpha and p38 mitogen-activated protein kinase (MAPK) phosphorylation. Further, inhibition of NF-kappaB by helenalin and p38 MAPK by SB98059 prevented the effects of PS-G in the expression of CD80, CD86, CD83, CD40, CD54, and HLA-DR and production of IL-12p70, p40, and IL-10 in various degrees. Taken together, our data demonstrate that PS-G can effectively promote the activation and maturation of immature DC, suggesting that PS-G may possess a potential in regulating immune responses.     

Differential role of mitogen-activated protein kinases in CD40-mediated IL-12 production by immature and mature dendritic cells

Using a murine spleen-derived dendritic cell (DC) line (BC1) CD40-mediated interleukin (IL)-12 production was analyzed and compared between immature and mature DC. BC1 cells, immature DC (iDC), were maturated by treatment with lipopolysaccharide (LPS) or tumor necrosis factor (TNF)-alpha. IL-12 production of LPS-treated DC (LPS/DC) was markedly enhanced by treatment with an anti-CD40 monoclonal antibody (mAb). Although the anti-CD40 mAb also enhanced IL-12 productions of iDC and TNF-alpha-treated DC (TNF/DC), these production levels were considerably low compared with that of LPS/DC. CD40-mediated IL-12-productions by iDC and TNF/DC were significantly enhanced by treatment with PD98059, a specific inhibitor of extracellular signal-related kinase (ERK) pathway. In contrast, PD98059 showed no significant effects on CD40-mediated IL-12-production by LPS/DC. These results demonstrated that ERK pathway was involved in negative regulation of the IL-12 productions by iDC and TNF/DC but not by LPS/DC. On the other hand, SB203580, a specific inhibitor of p38 mitogen activated protein kinase (MAPK) pathway, completely inhibited CD40-mediated IL-12-production by iDC, while not affecting those of TNF/DC and LPS/DC. Thus, p38 MAPK pathway appeared to positively regulate the IL-12 production in iDC but not in mature DC. It seems that roles of ERK and p38 MAPK for IL-12 production are developmentally changed in murine DC.     

Up-regulation of TLR9 gene expression by LPS in mouse macrophages via activation of NF-kappaB, ERK and p38 MAPK signal pathways

Toll-like receptors (TLR) are critical in the activation of macrophages by bacterial products. It has been shown that TLR2 and TLR4 mediate lipopolysaccharide (LPS) and lipoproteins signal transduction, respectively. Regulation of TLR2 and TLR4 expression by LPS was considered to be one of the mechanisms to control the overall responses of immune cells to bacteria. However, little is known about whether the other members of TLR family are regulated by LPS. Recently, TLR9 was demonstrated to be essential for CpG DNA signaling. Given the effective immune modulation by CpG DNA, regulation of TLR9 expression might play important role in controlling the overall responses of immune cells to bacteria. In this study, regulation of TLR9 gene expression in mouse macrophage cell line RAW264.7 by LPS was investigated. Semiquantitative RT-PCR was performed to determine gene expression of TLR9. Following LPS stimulation, TLR9 gene expression was upregulated within 1 h and reached peak level at about 3 h. LPS stimulation activated NF-kappaB, ERK and p38 MAPK signal pathways. Pretreatment of macrophages with inhibitors of NF-kappaB, ERK and p38 MAPK signal pathways inhibited LPS-induced upregulation of TLR9 mRNA expression. Our results demonstrated that LPS stimulation could upregulate gene expression of TLR9 via NF-kappaB, ERK, and p38 MAPK signal pathways in macrophages, indicating that macrophages with increased TLR9 expression induced by LPS might respond to invading bacteria more effectively.     

CpG oligodeoxynucleotides induce IL-8 expression in CD34+ cells via mitogen-activated protein kinase-dependent and NF-kappaB-independent pathways

To elucidate the role of Toll-like receptor 9 (TLR9) activation along with the intracellular signaling pathways triggered by CpG DNA in CD34+ cells, we investigated whether synthetic oligodeoxynucleotides (ODNs), containing unmethylated CpG motifs, could induce IL-8 expression in CD34+ cells through mitogen-activated protein kinase (MAPK) or nuclear factor-kappaB (NF-kappaB) pathway. We demonstrated evidence for the first time that CD34+ cells constitutively expressed TLR9. Exposure of the cells to CpG ODN resulted in a time- and dose-dependent increase of IL-8 expression, and activation of phosphorylated ERK1/2 and phosphorylated p38. In addition, CpG ODN stimulated AP-1, but not NF-kappaB, signals. Moreover, inhibitors of MAPK (U0126 and SB203580) significantly reduced the IL-8 production, while the inhibition of NF-kappaB (pyrrolidinedithiocarbamate and retrovirus containing dominant-negative IkappaB alpha plasmid) did not affect the IL-8 expression increased by CpG ODN. Moreover, co-stimulation with LPS and CpG synergistically up-regulates IL-8 in CD34+ cells. These results suggest that CpG DNA, acting on TLR9, activates CD34+ cells to express IL-8 through MAPK-dependent and NF-kappaB-independent pathways.     

IRF-1 and NF-kappaB p50/cRel bind to distinct regions of the proximal murine IL-12 p35 promoter during costimulation with IFN-gamma and LPS

LPS and IFN-gamma, which activate NF-kappaB cRel/p50 and IFN regulatory factor-1 (IRF-1), respectively, costimulate expression of the IL-12 p35 subunit in macrophages. The murine p35 promoter proximal to exon 2 is active during costimulation with IFN-gamma and LPS because it contains kappaB and IRF elements (E) with significant homology to the human p35 promoter. IFN-gamma or LPS stimulate nuclear localization of IRF-1 or cRel/p50, respectively, in the RAW 264.7 macrophage cell line. EMSAs reveal that IFN-gamma/LPS stimulates within 2 h, in RAW 264.7 cells or peritoneal macrophages, nuclear localization of proteins that target nt -137/-93 of the p35 promoter. DNA affinity assays utilizing nuclear extracts from RAW 264.7 cells show that NF-kappaB cRel and p50 bind to the kappaB-E within nt -122 to -93 of the p35 exon 2 promoter while IRF-1 binds to the IRF-E within nt -157 to -113 but not the one within nt -122 to -93. In addition, p50/cRel attachment to the kappaB-E was not dependent upon IRF-1 association with the IRF-E, and vice versa. Chromosome immunoprecipitation assays confirm inducible recruitment of IRF-1 and cRel to the endogenous p35 exon 2 promoter in both RAW 264.7 and primary macrophages costimulated with IFN-gamma and LPS. IFN-gamma, IFNgamma/LPS, or overexpression of IRF-1 plus cRel activated the wild-type p35 promoter reporter but not the p35 promoter reporter mutated at nt -110/-101 or in the presence of IRF-1 siRNA. Thus, cRel with IRF-1 induce p35 expression through a small region of the p35 exon 2 promoter during IFN-gamma and LPS costimulation of macrophages.     

TLR9 cooperates with TLR4 to increase IL-12 release by murine dendritic cells

Toll-like receptors (TLR) are expressed on the surface or intracellularly by dendritic cells (DC) and recognize specifically different pathogen-associated molecular patterns (PAMPs). Increasing evidence suggests that TLR expressed by DC can cooperate to synergize their functions. Here, we describe the cooperation of TLR9 and TLR4 triggering of murine bone marrow derived DC by CpG oligonucleotides and LPS, respectively. The simultaneous DC stimulation of LPS and CpG showed additive effects on the production of IL-12 but not on other cytokines, such as TNF, IL-6 or IL-10. CpG pretreatment before LPS induced five times more IL-12p40 and IL-12p70 production by DC, whereas LPS pretreatment before CpG showed no effect. The optimal time interval between CpG and LPS treatment was 4h and the synergistic effects were dependent on myeloid differentiation factor 88 (MyD88) but independent from the DNA backbone and did not mediate by nucleosome remodeling. The stimulatory effect could be further enhanced by addition of IFN-gamma but not anti-CD40 antibodies. These data show, that TLR4 and TLR9 can cooperate to increase selectively IL-12 production by DC.     

TRAF6-dependent mitogen-activated protein kinase activation differentially regulates the production of interleukin-12 by macrophages in response to Toxoplasma gondii

The production of interleukin-12 (IL-12) is critical to the development of innate and adaptive immune responses required for the control of intracellular pathogens. Many microbial products signal through Toll-like receptors (TLR) and activate NF-kappaB family members that are required for the production of IL-12. Recent studies suggest that components of the TLR pathway are required for the production of IL-12 in response to the parasite Toxoplasma gondii; however, the production of IL-12 in response to this parasite is independent of NF-kappaB activation. The adaptor molecule TRAF6 is involved in TLR signaling pathways and associates with serine/threonine kinases involved in the activation of both NF-kappaB and mitogen-activated protein kinase (MAPK). To elucidate the intracellular signaling pathways involved in the production of IL-12 in response to soluble toxoplasma antigen (STAg), wild-type and TRAF6(-/-) mice were inoculated with STAg, and the production of IL-12(p40) was determined. TRAF6(-/-) mice failed to produce IL-12(p40) in response to STAg, and TRAF6(-/-) macrophages stimulated with STAg also failed to produce IL-12(p40). Studies using Western blot analysis of wild-type and TRAF6(-/-) macrophages revealed that stimulation with STAg resulted in the rapid TRAF6-dependent phosphorylation of p38 and extracellular signal-related kinase, which differentially regulated the production of IL-12(p40). The studies presented here demonstrate for the first time that the production of IL-12(p40) in response to toxoplasma is dependent upon TRAF6 and p38 MAPK.     

Aging negatively skews macrophage TLR2- and TLR4-mediated pro-inflammatory responses without affecting the IL-2-stimulated pathway

We recently reported that macrophages from aged mice produced less tumor necrosis factor (TNF)-alpha following lipopolysaccharide (LPS) stimulation than macrophages from young animals. This correlated with decreased levels of phosphorylated and total p38 and c-Jun N-terminal kinase (JNK) mitogen-activated protein kinases (MAPKs). Here, we went on to determine if age affects other Toll-like (TLR) and non-TLR signaling pathways. We found that LPS- and zymosan-stimulated TNF-alpha and IL-6 production is attenuated in splenic macrophages from aged mice compared to young. Conversely, LPS-stimulated, but not zymosan-stimulated, IL-10 production from the aged group was elevated over that of the young group. In contrast, IL-2-stimulated TNF-alpha and IL-6 production was not affected by age. The age-associated changes did not correlate with alterations in the cell-surface expression of TLR2, TLR4, or IL-2Rbeta. Macrophages from aged mice demonstrated lower p38 MAPK and MAPK-activated protein kinase (APK)-2 activation. Protein expression of p38, but not MAPK-APK-2, was reduced with age. Additionally, nuclear factor (NF)-kappaB activation was significantly decreased in macrophages from aged mice after exposure to LPS, but not IL-2. These data indicate that age-associated macrophage signaling alterations are pathway-specific and suggest that TLR-mediated pathways are impaired with age at the level of MAPK expression.     

IFN-beta modulates the response to TLR stimulation in human DC: involvement of IFN regulatory factor-1 (IRF-1) in IL-27 gene expression

Type I IFN are cytokines which play a central role in host resistance to viral or microbial infections and are important components linking innate and adaptive immunity. We and others have previously demonstrated that the production of IFN-beta by DC following bacterial infections or TLR triggering influences, in an autocrine manner, their maturation. In this study, we investigated whether IFN-beta release modulates the phenotype of the immature DC and their response to a subsequent TLR stimulation. The induction of CD86, HLA-DR, CD38 and B7H1 and the absence of CCR7 and CD83 expression upon IFN-beta treatment suggest that IFN-beta-primed DC remain at the site of infection acquiring an activated phenotype. These results prompted us to investigate the response of IFN-beta-primed DC to TLR stimulation. While IFN-beta pretreatment increases slightly the expression of maturation markers in TLR2- or TLR4-stimulated DC, it is able to modulate selectively the secretion of inflammatory and immuno-regulating cytokines. Interestingly, IL-27p28 subunit was induced by IFN-beta alone or during LPS-induced maturation of DC in a type I IFN-dependent manner through IFN regulatory factor-1 (IRF-1) activation. Taken together, our results shed light on the capacity of IFN-beta to finely tune DC response to invading pathogens.     

脂多糖和金黄色葡萄球菌对IL-23、IL-12表达的调控

目的：检测脂多糖（LPS）和金黄色葡萄球菌（SAC）对人外周血单个核细胞（PBMC）IL-23和IL-12各亚基表达的调节作用并探索其作用机制。方法：用LPS和SAC直接刺激人PBMC，或用CD14抗体或p38丝裂原活化蛋白激酶（MAPK）抑制剂Mastoparan预处理PBMC后再予LPS和SAC刺激，半定量RT-PCR方法检测IL-23p19、IL-12p35和IL-12p40亚基的基因表达变化。结果：人PBMC组成性表达p19和p35，不表达p40。LPS和SAC可上调各亚基的表达。LPS诱导的IL-23和IL-12各亚基表达均需通过CDl4；CDl4仅部分参与SAC诱导的IL-12p40和p35表达上调，而与p19上调无关。LPS和SAC诱导的IL-23和IL-12各亚基表达均需要p38MAPK通路。结论：LPS和SAC刺激下IL-23和IL-12亚基表达及信号传导通路既有相似之处又有不同点，为单独或同时调控这两种因子的表达提供线索。