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.     

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.     

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.     

Adenylate cycalse toxin of Bordetella pertussis inhibits TLR-induced IRF-1 and IRF-8 activation and IL-12 production and enhances IL-10 through MAPK activation in dendritic cells

Adenylate cyclase toxin (CyaA) of Bordetella pertussis binds to CD11b/CD18 on macrophages and dendritic cells (DC) and confers virulence to the bacteria by subverting innate immune responses of the host. We have previously demonstrated that CyaA promotes the induction of IL-10-secreting regulatory T cells in vivo by modulating DC activation. Here, we examine the mechanism of immune subversion, specifically, the modulation of TLR signaling pathways in DC. We found that CyaA synergized with LPS to induce IL-10 mRNA and protein expression in DC but significantly inhibited IL-12p70 production. CyaA enhanced LPS-induced phosphorylation of p38 MAPK and ERK in DC, and inhibitors of p38 MAPK, MEK, or NF-kappaB suppressed IL-10 production in response to LPS and CyaA. However, inhibition of p38 MAPK, MEK, and NF-kappaB did not reverse the inhibitory effect of CyaA on TLR agonist-induced IL-12 production. Furthermore, CyaA suppression of IL-12 was independent of IL-10. In contrast, CyaA suppressed LPS- and IFN-gamma-induced IFN-regulatory factor-1 (IRF-1) and IRF-8 expression in DC. The modulatory effects of CyaA were dependent on adenylate cyclase activity and induction of intracellular cAMP, as an enzyme-inactive mutant of CyaA failed to modulate TLR-induced signaling in DC, whereas the effects of the wild-type toxin were mimicked by stimulation of the DC with PGE2. Our findings demonstrate that CyaA modulates TLR agonist-induced IL-10 and IL-12p70 production in DC by, respectively, enhancing MAPK phosphorylation and inhibiting IRF-1 and IRF-8 expression and that this is mediated by elevation of intercellular cAMP concentrations.     

Lipopolysaccharide and IL-1β coordinate a synergy on cytokine production by upregulating MyD88 expression in human gingival fibroblasts

Both lipopolysaccharide (LPS) and interleukin (IL)-1β activate the MyD88-dependent signaling pathways to stimulate proinflammatory cytokine expression. However, it remains unknown how LPS and IL-1β interact with each other to coordinate the stimulation. In this study, we sought to investigate the interaction between LPS and IL-1β on MyD88-dependent signaling pathways in human gingival fibroblasts (HGFs). Results showed that LPS derived from Porphyromonas gingivalis (Pg LPS) and IL-1β cooperatively stimulated mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NFκB) signaling pathways, and subsequent expression of proinflammatory cytokine expression. Furthermore, our results showed that Pg LPS and IL-1β exerted a synergy on MyD88 expression and knockdown of MyD88 expression by small interfering RNA diminished the synergistic effect of Pg LPS and IL-1β on IL-6 expression, suggesting that upregulation of MyD88 is involved in the coordinated stimulation by Pg LPS and IL-1β of proinflammatory cytokine expression. Finally, our results showed that pharmacological inhibitors for MAPK and NFκB significantly reduced IL-6 secretion stimulated by Pg LPS and IL-1β, indicating that the MyD88-dependent MAPK and NFκB signaling pathways are essential for the upregulation of proinflammatory cytokine expression by Pg LPS and IL-1β. Taken together, this study showed that LPS and IL-1β coordinate a synergy on cytokine production by upregulating MyD88 expression in HGFs.     

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.     

p38 MAPK 和STAT3参与CCK-8抑制LPS诱导的大鼠促炎症细胞因子生成

 目的：观察八肽胆囊收缩素（CCK-8）是否改善脂多糖（LPS）引起的大鼠细胞因子的变化,并探讨p38丝裂原活化蛋白激酶(p38 MAPK)和信号转导子及转录激活子3(STAT3)的信号转导作用,以及CCK受体（CCK-R）的作用。方法：4组大鼠尾静脉分别注入生理盐水（对照）、LPS (8 mg/kg)、CCK-8(40 μg/kg)和CCK-8(40 μg/kg)+LPS (8 mg/kg), 酶联免疫吸附法（ELISA）检测血清、肺脏及脾脏中肿瘤坏死因子α（TNF-α）、白细胞介素1β（IL-1β）和IL-6的变化,Western blotting和免疫荧光双标激光共聚焦显微镜检测肺脏和脾脏磷酸化p38 MAPK和磷酸化STAT3的表达,RT-PCR检测脾脏CCK-R亚型的mRNA表达。结果：CCK-8可显著抑制LPS诱导的TNF-α、IL-1β和IL-6的增加。CCK-8可增加LPS诱导的大鼠肺脏和脾脏磷酸化p38 MAPK和磷酸化STAT3的表达。LPS有诱导CCK-AR及CCK-BR mRNA表达量增加的作用。结论：CCK-8对LPS刺激的大鼠促炎症细胞因子过量产生有抑制作用,p38 MAPK和STAT3可能参与了其信号转导机制。LPS刺激时,CCK-R受体发生正向调节,CCK-8有可能用于治疗全身性感染及其它的炎症性疾病。     

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.     

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。     

Involvement of multiple P2Y receptors and signaling pathways in the action of adenine nucleotides diphosphates on human monocyte-derived dendritic cells

Adenosine 5'-triphosphate (ATP), which is released from necrotic cells, induces a semimaturation state of dendritic cells (DC), characterized by the up-regulation of costimulatory molecules and the inhibition of proinflammatory cytokines. This action is mediated by cyclic adenosine monophosphate (cAMP) and involves the P2Y11 receptor. As DC express the ecto-enzyme CD39, which converts ATP into adenosine 5'-diphosphate (ADP), the effects of adenine nucleotides diphosphates on molecular signaling [intracellular calcium ([Ca2+]i), cAMP, extracellular signal-regulated kinase 1 (ERK1)], costimulatory molecule expression (CD83), and cytokine production [interleukin (IL)-12, tumor necrosis factor alpha (TNF-alpha), IL-10] were investigated in human monocyte-derived DC. ADP, 2-methylthio-ADP, and ADPbetaS had no effect on cAMP, increased [Ca2+]i, and stimulated the phosphorylation of ERK1. The effect on ERK1 was inhibited by AR-C69931MX, a P2Y12 and P2Y13 antagonist. On the contrary the effect on [Ca2+]i was neither inhibited by AR-C69931MX or by the P2Y1 antagonist MRS-2179. Both effects were inhibited by pertussis toxin. ADPbetaS alone was less potent for up-regulation of CD83 than ATPgammaS and did not increase the CD83 expression by DC stimulated with lipopolysaccharide (LPS). Similar to ATPgammaS, ADPbetaS inhibited the release of IL-12p40, IL-12p70, and TNF-alpha stimulated by LPS (1-100 ng/ml). The inhibitory effect of ADPbetaS on IL-12 release was neither reversed by AR-C69931MX or by MRS-2179. The two nucleotides had opposite effects on IL-10 production: inhibition by ADPbetaS and potentiation by ATPgammaS. In conclusion, ATP can modulate the function of DC, directly via a cAMP increase mediated by the P2Y11 receptor and indirectly via its degradation into ADP, which acts via Gi-coupled receptors coupled to ERK activation and calcium mobilization. These distinct mechanisms converge on the inhibition of inflammatory cytokine production, particularly IL-12, but have a differential effect on IL-10.     

Effect of p38 mitogen-activate protein kinase on MUC5AC protein expression of bile duct epithelial cells in hepatolithiasis patients

Primary hepatolithiasis is a common bile duct disease with benign nature but complicated mechanisms. Current studies have revealed its correlation with cytokine release by chronic inflammation, which also increased mucin (MUC) synthesis. This study investigated the role of p38 mitogen-activated protein kinase (MAPK) in regulating cytokine release and mucin synthesis, in an attempt to elucidate the role of p38 signaling molecule in the pathogenesis of hepatolithiasis. In human intrahepatic bile duct endothelial cells (HIBECs), lipoprotein (LPS) was used to induce the high expression of MUC. Small interference RNA (siRNA) was then used to silencing p38 gene expression. Cytokines including interleukin (IL)-1β and tumor necrosis factor (TNF)-α were measured, along with MUC5AC protein and mRNA expression assay. The interference of p38 gene expression inhibited the release of IL-1β and TNF-α in cultured cells. It also depressed both mRNA and protein levels of MUC5A. P38 MAPK signal pathway may be involved in the formation and progression of hepatolithiasis. This study provides potential new strategy for treating hepatolithiasis using p38 MAPK signal pathway as the drug target.     

Mycophenolic acid differentially affects dendritic cell maturation induced by tumor necrosis factor-α and lipopolysaccharide through a different modulation of MAPK signaling

Mycophenolic acid (MPA) is an immunosuppressive drug which induces resistance to several maturation signals in human dendritic cells (DC) by unknown mechanisms. As mitogen-activated protein kinases (MAPK) are involved in the maturation process, we studied whether MPA affected p38MAPK and extracellular signal-regulated kinase (ERK1/2) in human DC. We first showed that MPA reduced TNFα-induced phenotype maturation, whereas it had no effect after LPS activation, suggesting that MPA preferentially affects the signaling pathway used by TNFα. We found that TNFα preferentially used p38MAPK to induce phenotype maturation in DC, whereas LPS preferentially activated NF-κB. Importantly, we showed that MPA more strongly inhibited p38MAPK phosphorylation induced by TNFα than by LPS. This difference in inhibition may therefore explain its different effect on DC phenotype. Interestingly, MPA inhibited the inflammatory cytokine synthesis and allostimulatory capacity induced by both stimuli. Exogenous guanosine antagonized the effect of MPA on the phenotype of TNFα-matured-DC as well as the IL-12p70 and IFNγ secretion induced by both stimuli, without affecting p38MAPK phosphorylation. The action of MPA on human DC phenotype maturation appears mainly to be due to its ability to inhibit p38MAPK. Furthermore, the difference between LPS and TNFα emphasizes that the DC microenvironment strongly influences DC sensitivity to MPA.