骨髓间充质干细胞通过JAK／STAT3信号通路抑制树突状细胞成熟

骨髓间充质干细胞（bone marrow mesenchymal stem cells,bMSCs）具有自我更新、支持造血、多向分化和低免疫原性等特点,在调控树突状细胞（dendritic cells,DCs）成熟的过程中发挥重要作用。为了探讨bMSCs调控DCs成熟的机制,本研究通过分离培养正常捐献者bMSCs,并分离获取外周静脉血单个核细胞,诱导未成熟的树突状细胞（immature dendritic cells,imDCs）和成熟的树突状细胞（mature dendritic cells,mDCs）生成。根据Genebank中人STAT3全长基因序列,设计针对STAT3的siRNA。根据培养条件不同设计实验分组：正常bMSCs与imDCs共培养（阴性对照组）,转染siRNA的bMSCs与imDCs共培养（siRNA组）、加入JAK/STAT通路抑制剂AG490的bMSCs与imDCs共培养（AG490组）、加入TNF-α诱导的mDCs（阳性对照组）共4组,共培养72 h,流式细胞术分析DCs表型变化,ELISA检测培养液上清中IL-12水平变化。结果显示,阴性对照组不表达CD40、CD80、CD83、CD86和HLA DR标志树突细胞成熟的分子,而表达CD11b,其表型与imDCs一致;而siRNA组和AG490组的DCs表达CD40、CD80、CD83、CD86和HLA-DR等标志分子,而不表达CD11b,其表型与TNF-α诱导成熟的mDCs表型一致;siRNA组、AG490组和阳性对照组的IL-12水平较阴性对照组的IL-12水平显著升高（P＜0.05）,但siRNA组、AG490组和阳性对照组之间无明显差异（P＞0.05）。以上结果表明,通过siRNA和抑制剂AG490阻断bMSCs中JAK/STAT3通路促进了imDCs的成熟,提示bMSCs通过JAK/STAT3通路参与调控imDCs成熟。     

Dendritic cell maturation requires STAT1 and is under feedback regulation by suppressors of cytokine signaling

In this study we show that activation of STAT pathways is developmentally regulated and plays a role in dendritic cell (DC) differentiation and maturation. The STAT6 signaling pathway is constitutively activated in immature DC (iDC) and declines as iDCs differentiate into mature DCs (mDCs). However, down-regulation of this pathway during DC differentiation is accompanied by dramatic induction of suppressors of cytokine signaling 1 (SOCS1), SOCS2, SOCS3, and cytokine-induced Src homology 2-containing protein expression, suggesting that inhibition of STAT6 signaling may be required for DC maturation. In contrast, STAT1 signaling is most robust in mDCs and is not inhibited by the up-regulated SOCS proteins, indicating that STAT1 and STAT6 pathways are distinctly regulated in maturing DC. Furthermore, optimal activation of STAT1 during DC maturation requires both IL-4 and GM-CSF, suggesting that synergistic effects of both cytokines may in part provide the requisite STAT1 signaling intensity for DC maturation. Analyses of STAT1(-/-) DCs reveal a role for STAT1 in repressing CD86 expression in precursor DCs and up-regulating CD40, CD11c, and SOCS1 expression in mDCs. We further show that SOCS proteins are differentially induced by IL-4 and GM-CSF in DCs. SOCS1 is primarily induced by IL-4 through a STAT1-dependent mechanism, whereas SOCS3 is induced mainly by GM-CSF. Taken together, these results suggest that cytokine-induced maturation of DCs is under feedback regulation by SOCS proteins and that the switch from constitutive activation of the STAT6 pathway in iDCs to predominant use of STAT1 signals in mDC is mediated in part by STAT1-induced SOCS expression.     

IL-12 p35 silenced dendritic cells modulate immune responses by blocking IL-12 signaling through JAK-STAT pathway in T lymphocytes

Dendritic cells (DC) constitute a complex system of uniquely specialized antigen-presenting cells (APC) that play crucial roles in the initiation and regulation of immune responses. Recent studies have demonstrated that DC silenced by siRNA IL-12 p35 showed tolerogenic capacity in vitro. However, their mechanism of action is not fully understood. In this study, IL-12p35 siRNA was chemically synthesized and transfected into DCs. A coculture of T cells and DCs was performed. After 30 min coculture, T cells were harvested and analyzed. We showed that the IL-12 p35 silenced DCs decreased IL-12-induced T cell responses through blocking tyrosine phosphorylation of JAK2, TYK2, STAT3, and STAT4 proteins in T cells. These results demonstrate IL-12 p35 silenced DCs modulate immune responses by blocking IL-12 signaling through JAK-STAT pathway in T cells.     

Human activation-induced cytidine deaminase is induced by IL-4 and negatively regulated by CD45: implication of CD45 as a Janus kinase phosphatase in antibody diversification

Activation-induced cytidine deaminase (AID) plays critical roles in Ig class switch recombination and V(H) gene somatic hypermutation. We investigated the role of IL-4 in AID mRNA induction, the signaling transduction involved in IL-4-mediated AID induction, and the effect of CD45 on IL-4-dependent AID expression in human B cells. IL-4 was able to induce AID expression in human primary B cells and B cell lines, and IL-4-induced AID expression was further enhanced by CD40 signaling. IL-4-dependent AID induction was inhibited by a dominant-negative STAT6, indicating that IL-4 induced AID expression via the Janus kinase (JAK)/STAT6 signaling pathway. Moreover, triggering of CD45 with anti-CD45 Abs can inhibit IL-4-induced AID expression, and this CD45-mediated AID inhibition correlated with the ability of anti-CD45 to suppress IL-4-activated JAK1, JAK3, and STAT6 phosphorylations. Thus, in humans, IL-4 alone is sufficient to drive AID expression, and CD40 signaling is required for optimal AID production; IL-4-induced AID expression is mediated via the JAK/STAT signaling pathway, and can be negatively regulated by the JAK phosphatase activity of CD45. This study indicates that the JAK phosphatase activity of CD45 can be induced by anti-CD45 Ab treatment, and this principle may find clinical application in modulation of JAK activation in immune-mediated diseases.     

The JAK-STAT signaling pathway: input and output integration

Universal and essential to cytokine receptor signaling, the JAK-STAT pathway is one of the best understood signal transduction cascades. Almost 40 cytokine receptors signal through combinations of four JAK and seven STAT family members, suggesting commonality across the JAK-STAT signaling system. Despite intense study, there remain substantial gaps in understanding how the cascades are activated and regulated. Using the examples of the IL-6 and IL-10 receptors, I will discuss how diverse outcomes in gene expression result from regulatory events that effect the JAK1-STAT3 pathway, common to both receptors. I also consider receptor preferences by different STATs and interpretive problems in the use of STAT-deficient cells and mice. Finally, I consider how the suppressor of cytokine signaling (SOCS) proteins regulate the quality and quantity of STAT signals from cytokine receptors. New data suggests that SOCS proteins introduce additional diversity into the JAK-STAT pathway by adjusting the output of activated STATs that alters downstream gene activation.     

Ovarian carcinoma cells inhibit T cell proliferation: suppression of IL-2 receptor beta and gamma expression and their JAK-STAT signaling pathway

Deficient T cell immune function and intracellular signaling in cancer patients may result from effects of tumors or their products on lymphocytes. Recently, it was demonstrated that several ovarian carcinoma cell lines could produce soluble factors that inhibited T cell proliferation. The aim of this study is to assess the effect of supernatants from 3 ovarian carcinoma cell lines (OVCAR3, CAOV3, SKOV3) on signal transduction elements that are linked to the IL-2R and its JAK-STAT pathway. A profound inhibition of proliferation, lower level of IFN-gamma and higher level of IL-10 gene expression were observed when CD8+ T cells were co-cultured with supernatants from 3 ovarian carcinoma cell lines. Cell cycle studies on inhibited CD8+ T cells showed most of them were growth arrested in G0/G1 phase. Western blot analysis showed that tumor supernatants suppressed expression of JAK3 and tyrosine phosphorylation of STAT5. JAK1 was not altered and the inhibition of STAT3 only appeared in OVCAR3 cells. Tumor supernatants also partially blocked induction of IL-2R beta and gamma chains expression. These findings suggest that ovarian carcinoma cells may suppress T cell proliferation through inhibition IL-2 dependent signaling pathways, which may be a mechanism of ovarian carcinoma induced immunosuppression.     

CD86 and IL-12p70 Are Key Players for T Helper 1 Polarization and Natural Killer Cell Activation by Toll-Like Receptor-Induced Dendritic Cells

Background

Dendritic cells (DCs) determine the activation and polarization of T cells via expression of costimulatory molecules and secretion of cytokines. The function of DCs derived from monocytes ex vivo strongly depends on the composition of the maturation cocktail used.

Methodology/Principal Findings

We analyzed the effect of costimulatory molecule expression and cytokine secretion by DCs on T and natural killer (NK) cell activation by conducting a head-to-head comparison of a Toll-like receptor (TLR) agonist-based cocktail with the standard combination of proinflammatory cytokines or IL-10 alone. We could show that TLR-induced DCs are characterized by a predominance of costimulatory over coinhibitory molecules and by high secretion of IL-12p70, but not IL-10. Functionally, these signals translated into an increase in IFN-γ secreting Th1 cells and a decrease in regulatory T cells. T cell activation and polarization were dependent on IL-12p70 and CD86, but remarkably not on CD80 signaling. By means of IL-12p70 secretion, only TLR-induced DCs activated NK cells.

Conclusions/Significance

TLR-matured DCs are highly suitable for application in immunotherapeutic strategies that rely on strong type 1 polarization and NK cell activation. Their effects particularly depend on high CD86 expression and IL-12p70 secretion.     

Human mesenchymal stem cells and renal tubular epithelial cells differentially influence monocyte-derived dendritic cell differentiation and maturation

Mesenchymal stem cells (MSCs) possess immunosuppressive properties. But also fully differentiated human renal tubular epithelial cells (RTECs) are able to modulate T-cell proliferation in vitro. In this study we compared two MSC populations, human adipose derived stem cells (ASCs) and human amniotic mesenchymal stromal cells (hAMSCs), and RTECs regarding their potential to inhibit monocyte-derived dendritic cell (DC) differentiation and maturation in indirect co-culture.In the presence of hAMSCs and RTECs, monocytes stimulated to undergo DC differentiation were inhibited to acquire surface phenotype of immature and mature DCs. In contrast, ASCs showed only limited suppressive capacity. Secretion of IL-12p70 was suppressed in hAMSC co-cultures and high IL-10 levels were detected in all co-cultures. Prostaglandin E₂ was found in ASC and hAMSC co-cultures, whereas soluble human leukocyte antigen-G was highly elevated only in RTEC co-cultures. Thus, inhibition of DC generation by MSCs and RTECs might be mediated by different soluble factors.     

Selective regulation of interleukin-10 production via Janus kinase pathway in murine conventional dendritic cells

In this study, we examined the role of JAKs in regulation of inflammatory versus anti-inflammatory cytokine balance in murine conventional dendritic cells (DCs). Highly purified lipopolysaccharide (upLPS) combined with imiquimod (IQ) synergistically induced IL-10 production by DCs, while each ligand alone showed a slight effect on the IL-10 production. Marked phosphorylation of JAK2, STAT1 and STAT3 was detected in DCs following upLPS plus IQ stimulation. Blocking the JAK pathway by JAK inhibitor I (JAKi) resulted in significant inhibition of IL-10 production by the DCs. However, JAKi showed negligible effect on the DC production of IL-12, IL-6 and TNF-α. JAKi completely blocked the TLR-mediated STATs activation, and attenuated the activation of Akt, a downstream effector of PI3K, in DCs stimulated by upLPS plus IQ. LY294002, a specific inhibitor of PI3K, markedly inhibited the DC production of IL-10. Thus, JAK-PI3K axis appeared to be responsible for the IL-10 production by DCs.     

Dendritic cells matured in the presence of the lycopodium alkaloid annotine direct T cell responses toward a Th2/Treg phenotype

Annotine is a lycopodane-type alkaloid isolated from the Icelandic club moss Lycopodium annotinum ssp. alpestre. Annotine does not inhibit acetylcholinesterase, as some other lycopodium alkaloids do, and other bioactivities have not been reported. The aim of this study was to determine the effects of annotine on maturation of dendritic cells (DCs) and their ability to activate allogeneic CD4⁺ T cells. Human monocyte-derived DCs were matured in the absence or presence of annotine at a concentration of 1, 10 or 100 μg/ml. The effect of the annotine on maturation of the DCs was determined by measuring concentration of cytokines in culture supernatant by ELISA and expression of surface molecules by flow cytometry. DCs matured in the absence or presence of annotine at 100 µg/ml were also co-cultured with allogeneic CD4⁺ T cells and concentration of cytokines in supernatants determined by ELISA and expression of surface molecules by flow cytometry. When cultured alone, DCs matured in the presence of annotine secreted less of the pro-inflammatory cytokines IL-6 and IL-23 and had a tendency toward less secretion of IL-12p40 than DCs matured in the absence of annotine. However, when DCs were matured in the presence of annotine and then co-cultured with allogeneic CD4⁺ T cells they secreted more IL-12p40 and had a tendency toward secreting more IL-6 than DCs matured in the absence of annotine and then co-cultured with T cells. Allogeneic CD4⁺ T cells co-cultured with DCs matured in the presence of annotine secreted more IL-13 than T cells co-cultured with DCs matured in the absence of annotine, but stimulating the DCs in the presence of annotine did not affect T cell secretion of IFN-γ and IL-17. There was also more IL-10 in co-cultures of T cells and DCs matured in the presence of annotine than in co-cultures of T cells and DCs matured in the absence of annotine. These results show that annotine increases the ability of DCs to direct the differentiation of allogeneic CD4⁺ T cells toward a Th2/Treg phenotype, which may be of interest in the development of new treatments for Th1- and/or Th17-mediated inflammatory diseases.