TGF-β, together with IL-6 and IL-21, promotes Th17 cell development. IL-6 and IL-21 induce activation of STAT3, which is crucial for Th17 cell differentiation, as well as the expression of suppressor of cytokine signaling (SOCS)3, a major negative feedback regulator of STAT3-activating cytokines that negatively regulates Th17 cells. However, it is still largely unclear how TGF-β regulates Th17 cell development and which TGF-β signaling pathway is involved in Th17 cell development. In this report, we demonstrate that TGF-β inhibits IL-6- and IL-21-induced SOCS3 expression, thus enhancing as well as prolonging STAT3 activation in naive CD4⁺CD25⁻ T cells. TGF-β inhibits IL-6-induced SOCS3 promoter activity in T cells. Also, SOCS3 small interfering RNA knockdown partially compensates for the action of TGF-β on Th17 cell development. In mice with a dominant-negative form of TGF-β receptor II and impaired TGF-β signaling, IL-6-induced CD4⁺ T cell expression of SOCS3 is higher whereas STAT3 activation is lower compared with wild-type B6 CD4⁺ T cells. The addition of a TGF-β receptor I kinase inhibitor that blocks Smad-dependent TGF-β signaling greatly, but not completely, abrogates the effect of TGF-β on Th17 cell differentiation. Our data indicate that inhibition of SOCS3 and, thus, enhancement of STAT3 activation is at least one of the mechanisms of TGF-β promotion of Th17 cell development.

Immunological activation of macrophages/microglia within the CNS leads to the production of cytokines and chemokines that ultimately impact on glial and neuronal function. Suppressor of cytokine signaling (SOCS) proteins are negative regulators of adaptive and innate immune responses. Our previous studies demonstrated that SOCS-3 attenuates macrophage/microglial activation in vitro, suggesting that SOCS-3 may exert beneficial effects for immune-mediated CNS diseases in vivo. In this study, we describe LPS as a potent inducer of SOCS-3 transcription and expression in macrophages/microglia. An analysis of the SOCS-3 promoter indicates that AP-1 and IFN-γ activation sequence (GAS) elements are involved in LPS-induced SOCS-3 transcription. LPS-induced SOCS-3 expression was diminished in IL-10-deficient macrophages at later time points, indicating the involvement of endogenous IL-10 in this response. Blocking STAT-3 expression and activation using STAT-3 small interfering RNA reduced LPS-induced SOCS-3 gene expression. LPS activated the MAPK-ERK1/2, JNK, and p38 pathways that, in addition to STAT-3, were also involved in LPS-induced SOCS-3 expression. LPS treatment of cells led to the acetylation of histones H3 and H4 on the SOCS-3 promoter and the recruitment of STAT-3, c-Jun, c-Fos, CREB-binding protein, p300, and RNA polymerase II to the endogenous SOCS-3 promoter in a time-dependent manner. These results indicate that LPS-induced MAPK activation, the production of endogenous IL-10, and STAT-3 activation play critical roles in SOCS-3 expression, which provides for feedback attenuation of cytokine-induced immune and inflammatory responses in macrophages and microglia.

Astrocytes play a number of important physiological roles in CNS homeostasis. Inflammation stimulates astrocytes to secrete cytokines and chemokines that guide macrophages/microglia and T cells to sites of injury/inflammation. Herein, we describe how these processes are controlled by the suppressor of cytokine signaling (SOCS) proteins, a family of proteins that negatively regulate adaptive and innate immune responses. In this study, we describe that the immunomodulatory cytokine IFN-β induces SOCS-1 and SOCS-3 expression in primary astrocytes at the transcriptional level. SOCS-1 and SOCS-3 transcriptional activity is induced by IFN-β through IFN-γ activation site (GAS) elements within their promoters. Studies in STAT-1α-deficient astrocytes indicate that STAT-1α is required for IFN-β-induced SOCS-1 expression, while STAT-3 small interfering RNA studies demonstrate that IFN-β-induced SOCS-3 expression relies on STAT-3 activation. Specific small interfering RNA inhibition of IFN-β-inducible SOCS-1 and SOCS-3 in astrocytes enhances their proinflammatory responses to IFN-β stimulation, such as heightened expression of the chemokines CCL2 (MCP-1), CCL3 (MIP-1α), CCL4 (MIP-1β), CCL5 (RANTES), and CXCL10 (IP-10), and promoting chemotaxis of macrophages and CD4⁺ T cells. These results indicate that IFN-β induces SOCS-1 and SOCS-3 in primary astrocytes to attenuate its own chemokine-related inflammation in the CNS.

Key Points: Genetically accurate xenografts of CMML are achievable with near 100% frequency in NSGS mice. Robust human engraftment and overt phenotypes of CMML and JMML xenografts here facilitate preclinical therapeutic evaluation in vivo.