Regulation of IL-21 signaling by suppressor of cytokine signaling-1 (SOCS1) in CD8(+) T lymphocytes

Mice lacking the gene for suppressor of cytokine signaling 1 (SOCS1) show defective homeostasis of T lymphocytes due to accumulation of CD8⁺ T cells, resulting at least partly from dysregulated IL-15 signaling. IL-15 alone does not stimulate proliferation of naïve CD8 T cells, but can synergize with IL-21 to induce proliferation, suggesting a potential role for IL-21 in the defective homeostasis of CD8⁺ T lymphocytes in SOCS1^−/− mice. Since IL-21 strongly induced SOCS1 mRNA in CD8⁺ T cells, we investigated whether SOCS1 regulates their response to IL-21. CD8⁺ T cells isolated from SOCS1-deficient mice proliferated vigorously in response to IL-21 + IL-15. In CD8⁺ T lymphocytes expressing transgenic TCR, IL-21 + IL-7 provided a stronger stimulus to naïve cells whereas IL-15 + IL-21 potently stimulated memory cells. Compared to truly naïve or memory cells, SOCS1^−/− H-Y TCR⁺ CD8⁺ T cells displayed CD44^loLy6C^hiCD122^intCD127^lo partial memory phenotype and exhibited stronger response to IL-15 + IL-21 than truly naïve cells. In SOCS1^−/− CD8⁺ T cells, IL-21 caused greater reduction in IL-15 threshold for activation in a dose-dependent manner. SOCS1 deficiency did not modulate IL-21Rα expression or sensitivity to IL-21, but delayed the loss of IL-21-induced phospho-STAT3 signal. These results show that SOCS1 is a critical regulator of IL-21 signaling in CD8⁺ T cells, and support the notion that sustained IL-21 signaling might also contribute to the aberrant T cell homeostasis in SOCS1-deficient mice.     

IL-7/STAT5 cytokine signaling pathway is essential but insufficient for maintenance of naive CD4 T cell survival in peripheral lymphoid organs

Constitutive expression of suppressors of cytokine signaling (SOCS)1 in T lineage in vivo attenuated cytokine signaling and resulted in a dramatic reduction in the number of naive CD44(low)CD62L(high) CD4 T cells in the spleen. After adoptive transfer of thymocytes from SOCS1 transgenic mice into normal recipients, naive CD4 T cells rapidly disappeared from the spleen within 1 wk. Likewise, T cell-specific deletion of STAT5a/b in vivo resulted in a similar phenotype characterized by loss of naive CD4 T cells. Thus, STAT5-mediated signaling is crucial for promoting naive T cell survival. However, forced expression of constitutively active STAT5 failed to rescue CD4 T cells in SOCS1 transgenic mice, implying that STAT5 activation is necessary but not sufficient for naive CD4 T cell survival. Although blockade of the IL-7R, a SOCS1 target, resulted in clear inhibition of naive T cell survival, the effect occurred 3 wk after anti-IL-7R Ab treatment, but not at earlier time points. These results suggest that IL-7-mediated STAT5 activation is essential for long-term survival of naive CD4 cells after export from thymus, and that another SOCS1-sensitive cytokine is critical for short-term naive T cell survival.     

Suppressor of cytokine signaling 1 regulates IL-15 receptor signaling in CD8+CD44high memory T lymphocytes

T lymphocyte survival, proliferation, and death in the periphery are dependent on several cytokines. Many of these cytokines induce the expression of suppressor of cytokine signaling-1 (SOCS1), a feedback inhibitor of JAK kinases. However, it is unclear whether the cytokines that regulate T lymphocyte homeostasis are critically regulated by SOCS1 in vivo. Using SOCS1(-/-)IFN-gamma(-/-) mice we show that SOCS1 deficiency causes a lymphoproliferative disorder characterized by decreased CD4/CD8 ratio due to chronic accumulation of CD8+CD44(high) memory phenotype T cells. SOCS1-deficient CD8+ T cells express elevated levels of IL-2Rbeta, show increased proliferative response to IL-15 and IL-2 in vitro, and undergo increased bystander proliferation and vigorous homeostatic expansion in vivo. Sorted CD8+CD44(high) T cells from SOCS1(-/-)IFN-gamma(-/-) mice respond 5 times more strongly than control cells, indicating that SOCS1 is a critical regulator of IL-15R signaling. Consistent with this idea, IL-15 stimulates sustained STAT5 phosphorylation in SOCS1-deficient CD8+ T cells. IL-15 strongly induces TNF-alpha production in SOCS1-deficient CD8+ T cells, indicating that SOCS1 is also a critical regulator of CD8+ T cell activation by IL-15. However, IL-15 and IL-2 induce comparable levels of Bcl-2 and Bcl-x(L) in SOCS1-deficient and SOCS1-sufficient CD8+ T cells, suggesting that cytokine receptor signals required for inducing proliferation and cell survival signals are not identical. These results show that SOCS1 differentially regulates common gamma-chain cytokine signaling in CD8+ T cells and suggest that CD8+ T cell homeostasis is maintained by distinct mechanisms that control cytokine-mediated survival and proliferation signals.     

High glucose induces mitochondrial dysfunction and apoptosis in human retinal pigment epithelium cells via promoting SOCS1 and Fas/FasL signaling

Diabetic retinopathy (DR) is one of the most serious complications of diabetes mellitus (DM), however, the contribution of high glucose (HG) or hyperglycemia to DR is far from fully understanding. In the present study, we examined the expression of Fas/FasL signaling and suppressors of cytokine signaling (SOCS)1 and 3 in HG-induced human retinal pigment epithelium cells (ARPE-19 cells). And then we investigated the regulatory role of both Fas and SOCS1 in HG-induced mitochondrial dysfunction and apoptosis. Results demonstrated that HG with more than 40 mM induced mitochondrial dysfunction via reducing mitochondrial membrane potential (MMP) and via inhibiting the Bcl-2 level, which is the upstream signaling of mitochondria in ARPE-19 cells. HG also upreuglated the Fas signaling and SOCS levels probably via promoting JAK/STAT signaling in ARPE-19 cells. Moreover, the exogenous Fas or entogenous overexpressed SOCS1 accentuated the HG-induced mitochondrial dysfunction and apoptosis, whereas the knockdown of either Fas or SOCS1 reduced the HG-induced mitochondria dysfunction and apoptosis. Thus, the present study confirmed that both Fas/FasL signaling and SOCS1 promoted the HG-induced mitochondrial dysfunction and apoptosis. These results implies the key regulatory role of Fas signaling and SOCS in DR.     

Suppressor of cytokine signaling 1 inhibits IL-10-mediated immune responses

IL-10 has proved to be a key cytokine in regulating inflammatory responses by controlling the production and function of various other cytokines. The suppressor of cytokine signaling (SOCS) gene products are a family of cytoplasmic molecules that are essential mediators for negatively regulating cytokine signaling. It has been previously shown that IL-10 induced SOCS3 expression and that forced constitutive expression of SOCS3 inhibits IL-10/STAT3 activation and LPS-induced macrophage activation. In this report, we show that, in addition to SOCS3 expression, IL-10 induces SOCS1 up-regulation in all cell lines tested, including Ba/F3 pro-B cells, MC/9 mast cells, M1 leukemia cells, U3A human fibroblasts, and primary mouse CD4(+) T cells. Induction of SOCS molecules is dependent on STAT3 activation by IL-10R1. Cell lines constitutively overexpressing SOCS proteins demonstrated that SOCS1 and SOCS3, but not SOCS2, are able to partially inhibit IL-10-mediated STAT3 activation and proliferative responses. Pretreatment of M1 cells with IFN-gamma resulted in SOCS1 induction and a reduction of IL-10-mediated STAT3 activation and cell growth inhibition. IL-10-induced SOCS is associated with the inhibition of IFN-gamma signaling in various cell types, and this inhibition is independent of C-terminal serine residues of the IL-10R, previously shown to be required for other anti-inflammatory responses. Thus, the present results show that both SOCS1 and SOCS3 are induced by IL-10 and may be important inhibitors of both IL-10 and IFN-gamma signaling. IL-10-induced SOCS1 may directly inhibit IL-10 IFN-gamma signaling, while inhibition of other proinflammatory cytokine responses may use additional IL-10R1-mediated mechanisms.     

IL-21 can supplement suboptimal Lck-independent MAPK activation in a STAT-3-dependent manner in human CD8(+) T cells

Although both MHC class II/CD8α double-knockout and CD8β null mice show a defect in the development of MHC class I-restricted CD8(+) T cells in the thymus, they possess low numbers of high-avidity peripheral CTL with limited clonality and are able to contain acute and chronic infections. These in vivo data suggest that the CD8 coreceptor is not absolutely necessary for the generation of Ag-specific CTL. Lack of CD8 association causes partial TCR signaling because of the absence of CD8/Lck recruitment to the proximity of the MHC/TCR complex, resulting in suboptimal MAPK activation. Therefore, there should exist a signaling mechanism that can supplement partial TCR activation caused by the lack of CD8 association. In this human study, we have shown that CD8-independent stimulation of Ag-specific CTL previously primed in the presence of CD8 coligation, either in vivo or in vitro, induced severely impaired in vitro proliferation. When naive CD8(+) T cells were primed in the absence of CD8 binding and subsequently restimulated in the presence of CD8 coligation, the proliferation of Ag-specific CTL was also severely hampered. However, when CD8-independent T cell priming and restimulation were supplemented with IL-21, Ag-specific CD8(+) CTL expanded in two of six individuals tested. We found that IL-21 rescued partial MAPK activation in a STAT3- but not STAT1-dependent manner. These results suggest that CD8 coligation is critical for the expansion of postthymic peripheral Ag-specific CTL in humans. However, STAT3-mediated IL-21 signaling can supplement partial TCR signaling caused by the lack of CD8 association.     

IL-6, in synergy with IL-7 or IL-15, stimulates TCR-independent proliferation and functional differentiation of CD8+ T lymphocytes

Recent reports have shown that IL-21, in synergy with IL-15, stimulates proliferation of CD8(+) T lymphocytes in the absence of signaling via the TCR. In this study, we show that IL-6, which induces phosphorylation of STAT3 similarly to IL-21, also can stimulate proliferation of CD8(+) T cells in synergy with IL-7 or IL-15. IL-6 displays a stronger synergy with IL-7 than with IL-15 to stimulate naive CD8(+) T cells. Concomitant stimulation by IL-6 or IL-21 augments phosphorylation and DNA-binding activity of STAT5 induced by IL-7 or IL-15. Like IL-21, IL-6 reduces the TCR signaling threshold required to stimulate CD8(+) T cells. Prior culture of P14 TCR transgenic CD8 T cells with IL-6 or IL-21 in the presence of IL-7 or IL-15 augments their proliferation and cytolytic activity upon subsequent stimulation by Ag. Furthermore, cytokine stimulation induces quantitatively and qualitatively distinct phenotypic changes on CD8(+) T cells compared with those induced by TCR signaling. We propose that the ability of IL-6 to induce TCR-independent activation of CD8(+) T cells in synergy with IL-7 or IL-15 may play an important role in the transition from innate to adaptive immunity.     

IL-27 suppresses CD28-mediated [correction of medicated] IL-2 production through suppressor of cytokine signaling 3

IL-27 is a novel IL-6/IL-12 family cytokine that not only plays a role in the early regulation of Th1 differentiation, but also exerts an inhibitory effect on immune responses, including the suppression of proinflammatory cytokine production. However, the molecular mechanism by which IL-27 exerts the inhibitory effect remains unclear. In this study we demonstrate that IL-27 inhibits CD28-mediated IL-2 production and that suppressor of cytokine signaling 3 (SOCS3) plays a critical role in the inhibitory effect. Although IL-27 enhanced IFN-gamma production from naive CD4+ T cells stimulated with plate-coated anti-CD3 and anti-CD28 in the presence of IL-12, IL-27 simultaneously inhibited CD28-mediated IL-2 production. Correlated with the inhibition, IL-27 was shown to augment SOCS3 expression. Analyses using various mice lacking a signaling molecule revealed that the inhibition of IL-2 production was dependent on STAT1, but not on STAT3, STAT4, and T-bet, and was highly correlated with the induction of SOCS3 expression. Similar inhibition of CD28-mediated IL-2 production and augmentation of SOCS3 expression by IL-27 were observed in a T cell hybridoma cell line, 2B4. Forced expression of antisense SOCS3 or dominant negative SOCS3 in the T cell line blocked the IL-27-inudced inhibition of CD28-mediated IL-2 production. Furthermore, pretreatment with IL-27 inhibited IL-2-mediated cell proliferation and STAT5 activation, although IL-27 hardly affected the induction level of CD25 expression. These results suggest that IL-27 inhibits CD28-mediated IL-2 production and also IL-2 responses, and that SOCS3, whose expression is induced by IL-27, plays a critical role in the inhibitory effect in a negative feedback mechanism.     

Tec kinases Itk and Rlk are required for CD8+ T cell responses to virus infection independent of their role in CD4+ T cell help

Itk and Rlk are members of the Tec kinase family of nonreceptor protein tyrosine kinases that are expressed in T cells, NK cells, and mast cells. These proteins are involved in the regulation of signaling processes downstream of the TCR in CD4(+) T cells, particularly in the phosphorylation of phospholipase C-gamma1 after TCR activation; furthermore, both Itk and Rlk are important in CD4(+) T cell development, differentiation, function, and homeostasis. However, few studies have addressed the roles of these kinases in CD8(+) T cell signaling and function. Using Itk(-/-) and Itk(-/-)Rlk(-/-) mice, we examined the roles of these Tec family kinases in CD8(+) T cells, both in vitro and in vivo. These studies demonstrate that the loss of Itk and Rlk impairs TCR-dependent signaling, causing defects in phospholipase C-gamma1, p38, and ERK activation as well as defects in calcium flux and cytokine production in vitro and expansion and effector cytokine production by CD8(+) T cells in response to viral infection. These defects cannot be rescued by providing virus-specific CD4(+) T cell help, thereby substantiating the important role of Tec kinases in CD8(+) T cell signaling.     

Angoline: A selective IL-6/STAT3 signaling pathway inhibitor isolated from Zanthoxylum nitidum

STAT3 signaling pathway is an important target for human cancer therapy. Thus, the identification of small-molecules that target STAT3 signaling will be of great interests in the development of anticancer agents. The aim of this study was to identify novel inhibitors of STAT3 pathway from the roots of Zanthoxylum nitidum (Roxb.) DC. The bioassay-guided fractionation of MeOH extract of Z. nitidum using a STAT3-responsive gene reporter assay led to the isolation of angoline (1) as a potent and selective inhibitor of the STAT3 signaling pathway (IC₅₀ = 11.56 μM). Angoline inhibited STAT3 phosphorylation and its target gene expression and consequently induced growth inhibition of human cancer cells with constitutively activated STAT3 (IC₅₀ = 3.14–4.72 μM). This work provided a novel lead for the development of anti-cancer agents targeting the STAT3 signaling pathway.     

Suppressor of cytokine signaling-1 has IFN-gamma-independent actions in T cell homeostasis

Suppressor of cytokine signaling (SOCS)-1 is a member of a family of proteins that negatively regulate cytokine signaling pathways. We have previously established that SOCS-1 is a key regulator of IFN-gamma signaling and that IFN-gamma is responsible for the complex inflammatory disease that leads to the death of SOCS-1-deficient mice. In this study, we provide evidence that SOCS-1 is also a critical regulator of IFN-gamma-independent immunoregulatory factors. Mice lacking both SOCS-1 and IFN-gamma, although outwardly healthy, have clear abnormalities in their immune system, including a reduced ratio of CD4:CD8 T cells in lymphoid tissues and increased expression of T cell activation markers. To examine the contribution of TCR Ag specificity to these immune defects, we have generated two lines of SOCS-1-deficient mice expressing a transgenic TCR specific for an exogenous Ag, OVA (OT-I and OT-II). Although TCR transgenic SOCS-1(-/-) mice have a longer lifespan than nontransgenic SOCS-1(-/-) mice, they still die as young adults with inflammatory disease and the TCR transgenic SOCS-1(-/-) T cells appear activated despite the absence of OVA. This suggests that both Ag-dependent and -independent mechanisms contribute to the disease in SOCS-1-deficient mice. Thus, SOCS-1 is a critical regulator of T cell activation and homeostasis, and its influence extends beyond regulating IFN-gamma signaling.     

IL-22-mediated liver cell regeneration is abrogated by SOCS-1/3 overexpression in vitro

The IL-10-like cytokine IL-22 is produced by activated T cells. In this study, we analyzed the role of this cytokine system in hepatic cells. Expression studies were performed by RT-PCR and quantitative PCR. Signal transduction was analyzed by Western blot experiments and ELISA. Cell proliferation was measured by MTS and [(3)H]thymidine incorporation assays. Hepatocyte regeneration was studied in in vitro restitution assays. Binding of IL-22 to its receptor complex expressed on human hepatic cells and primary human hepatocytes resulted in the activation of MAPKs, Akt, and STAT proteins. IL-22 stimulated cell proliferation and migration, which were both significantly inhibited by the phosphatidylinositol 3-kinase inhibitor wortmannin. IL-22 increased the mRNA expression of suppressor of cytokine signaling (SOCS)-3 and the proinflammatory cytokines IL-6, IL-8, and TNF-alpha. SOCS-1/3 overexpression abrogated IL-22-induced STAT activation and decreased IL-22-mediated liver cell regeneration. Hepatic IL-22 mRNA expression was detectable in different forms of human hepatitis, and hepatic IL-22 mRNA levels were increased in murine T cell-mediated hepatitis in vivo following cytomegalovirus infection, whereas no significant differences were seen in an in vivo model of ischemia-reperfusion injury. In conclusion, IL-22 promotes liver cell regeneration by increasing hepatic cell proliferation and hepatocyte migration through the activation of Akt and STAT signaling, which is abrogated by SOCS-1/3 overexpression.     

SOCS-1 can suppress CD3zeta- and Syk-mediated NF-AT activation in a non-lymphoid cell line

To elucidate T cell antigen receptor (TCR) signaling leading to activation nuclear factor of activated T cells (NF-AT), we reconstituted TCR signaling to activate NF-AT in a non-lymphoid cell line, 293T. We demonstrated that co-expression of CD8/zeta and Syk were necessary for NF-AT activation in 293T. This NF-AT response was completely inhibited by the addition of cyclosporin A or FK506, but markedly enhanced by the additional expression of Tec protein tyrosine kinase. We also show that the cytokine signaling suppressor, suppressor of cytokine signaling 1, potently inhibited this response by interacting with Syk and immunoreceptor tyrosine-based activation motifs in CD8/zeta. These results imply that this novel system may provide a useful tool to delineate or identify the regulatory molecules for CD3zeta/Syk-mediated NF-AT activation.     

SOCS1 regulates CCR7 expression and migration of CD4+ T cells into peripheral tissues

Suppressors of cytokine signaling (SOCS) proteins control many aspects of lymphocyte function through regulation of STAT pathways. SOCS1-deficient mice develop severe skin and eye diseases that result from massive infiltration of inflammatory cells into these tissues. In this study, we have used SOCS1-, STAT1-, or STAT6-deficient mice, as well as, T cells with stable overexpression or deletion of SOCS1, to examine whether SOCS1 is involved in regulating lymphocyte trafficking to peripheral tissues. We show that SOCS1-deficient mice have increased numbers of T cells with characteristics of effector memory cells and expression of CCR7, a protein that promotes retention of T cells in lymphoid tissues, is markedly reduced in these cells. The decrease in CCR7 expression correlates with hyperactivation of STAT6, suggesting that aberrant recruitment of T cells into SOCS1-deficient mouse skin or eye results from abrogation of negative feedback regulation of STAT6 activation and CCR7 expression. Consistent with in vivo regulation of CCR7 expression and lymphocyte migration by SOCS1, forced overexpression of SOCS1 in T cells up-regulates CCR7 expression and enhances chemotaxis toward CCL19 or CCL21. CCR6 and CXCR3 are also up-regulated on SOCS1-deficient T cells and in situ analysis of the cornea or retina further reveal that these cells may mediate the chronic skin and eye inflammation through recruitment of Th1 and Th17 cells into these tissues. Collectively, these results suggest that SOCS1 regulates steady-state levels of chemokine receptors through its inhibitory effects on STAT pathways and this may underscore its role in regulating recruitment and retention of effector cells into nonlymphoid tissues.