Aberrant RNA splicing is the major pathogenic effect in a knock-in mouse model of the dominantly inherited c.1430A>G human RPE65 mutation.

Human RPE65 mutations cause a spectrum of retinal dystrophies that result in blindness. While RPE65 mutations have been almost invariably recessively inherited, a c.1430A>G (p.(D477G)) mutation has been reported to cause autosomal dominant retinitis pigmentosa (adRP). To study the pathogenesis of this human mutation, we have replicated the mutation in a knock-in (KI) mouse model using CRISPR/Cas9-mediated genome editing. Significantly, in contrast to human patients, heterozygous KI mice do not exhibit any phenotypes in visual function tests. When raised in regular vivarium conditions, homozygous KI mice display relatively undisturbed visual functions with minimal retinal structural changes. However, KI/KI mouse retinae are more sensitive to light exposure and exhibit signs of degenerative features when subjected to light stress. We find that instead of merely producing a missense mutant protein, the A>G nucleotide substitution greatly affects appropriate splicing of Rpe65 mRNA by generating an ectopic splice site in comparable context to the canonical one, thereby disrupting RPE65 protein expression. Similar splicing defects were also confirmed for the human RPE65 c.1430G mutant in an in vitro Exontrap assay. Our data demonstrate that a splicing defect is associated with c.1430G pathogenesis, and therefore provide insights in the therapeutic strategy for human patients.

Topical administration of a suppressor of cytokine signaling-1 (SOCS1) mimetic peptide inhibits ocular inflammation and mitigates ocular pathology during mouse uveitis.

Uveitis is a diverse group of potentially sight-threatening intraocular inflammatory diseases and pathology derives from sustained production of pro-inflammatory cytokines in the optical axis. Although topical or systemic steroids are effective therapies, their adverse effects preclude prolonged usage and are impetus for seeking alternative immunosuppressive agents, particularly for patients with refractory uveitis. In this study, we synthesized a 16 amino acid membrane-penetrating lipophilic suppressor of cytokine signaling 1 peptide (SOCS1-KIR) that inhibits JAK/STAT signaling pathways and show that it suppresses and ameliorates experimental autoimmune uveitis (EAU), the mouse model of human uveitis. Fundus images, histological and optical coherence tomography analysis of eyes showed significant suppression of clinical disease, with average clinical score of 0.5 compared to 2.0 observed in control mice treated with scrambled peptide. We further show that SOCS1-KIR conferred protection from ocular pathology by inhibiting the expansion of pathogenic Th17 cells and inhibiting trafficking of inflammatory cells into the neuroretina during EAU. Dark-adapted scotopic and photopic electroretinograms further reveal that SOCS1-KIR prevented decrement of retinal function, underscoring potential neuroprotective effects of SOCS1-KIR in uveitis. Importantly, SOCS1-KIR is non-toxic, suggesting that topical administration of SOCS1-Mimetics can be exploited as a non-invasive treatment for uveitis and for limiting cytokine-mediated pathology in other ocular inflammatory diseases including scleritis.

SOCS3 deletion in T lymphocytes suppresses development of chronic ocular inflammation via upregulation of CTLA-4 and expansion of regulatory T cells.

Suppressors of cytokine signaling (SOCS) proteins are negative-feedback regulators of the JAK/STAT pathway, and SOCS3 contributes to host immunity by regulating the intensity and duration of cytokine signals and inflammatory responses. Mice with Socs3 deletion in myeloid cells exhibit enhanced STAT3 signaling, expansion of Th1 and Th17 cells, and develop severe experimental autoimmune encephalomyelitis. Interestingly, development of the unique IL-17/IFN-γ double-producing (Th17/IFN-γ and Tc17/IFN-γ) subsets that exhibit strong cytotoxic activities and are associated with pathogenesis of several autoimmune diseases has recently been shown to depend on epigenetic suppression of SOCS3 expression, further suggesting involvement of SOCS3 in autoimmunity and tumor immunity. In this study, we generated mice with Socs3 deletion in the CD4 T cell compartment (CD4-SOCS3 knockout [KO]) to determine in vivo effects of the loss of Socs3 in the T cell-mediated autoimmune disease, experimental autoimmune uveitis (EAU). In contrast to the exacerbation of experimental autoimmune encephalomyelitis in myeloid-specific SOCS3-deleted mice, CD4-SOCS3KO mice were protected from acute and chronic uveitis. Protection from EAU correlated with enhanced expression of CTLA-4 and expansion of IL-10-producing regulatory T cells with augmented suppressive activities. We further show that SOCS3 interacts with CTLA-4 and negatively regulates CTLA-4 levels in T cells, providing a mechanistic explanation for the expansion of regulatory T cells in CD4-SOCS3 during EAU. Contrary to in vitro epigenetic studies, Th17/IFN-γ and Tc17/IFN-γ populations were markedly reduced in CD4-SOCS3KO, suggesting that SOCS3 promotes expansion of the Th17/IFN-γ subset associated with development of severe uveitis. Thus, SOCS3 is a potential therapeutic target in uveitis and other autoinflammatory diseases.

TH17 cells contribute to uveitis and scleritis and are expanded by IL-2 and inhibited by IL-27/STAT1.

T-helper type 17 cells (T(H)17) are implicated in rodent models of immune-mediated diseases. Here we report their involvement in human uveitis and scleritis, and validate our findings in experimental autoimmune uveoretinitis (EAU), a model of uveitis. T(H)17 cells were present in human peripheral blood mononuclear cells (PBMC), and were expanded by interleukin (IL)-2 and inhibited by interferon (IFN)-gamma. Their numbers increased during active uveitis and scleritis and decreased following treatment. IL-17 was elevated in EAU and upregulated tumor necrosis factor (TNF)-alpha in retinal cells, suggesting a mechanism by which T(H)17 may contribute to ocular pathology. Furthermore, IL-27 was constitutively expressed in retinal ganglion and photoreceptor cells, was upregulated by IFN-gamma and inhibited proliferation of T(H)17. These findings suggest that T(H)1 cells may mitigate uveitis by antagonizing the T(H)17 phenotype through the IFN-gamma-mediated induction of IL-27 in target tissue. The finding that IL-2 promotes T(H)17 expansion provides explanations for the efficacy of IL-2R antibody therapy in uveitis, and suggests that antagonism of T(H)17 by IFN-gamma and/or IL-27 could be used for the treatment of chronic inflammation.

Novel IL27p28/IL12p40 cytokine suppressed experimental autoimmune uveitis by inhibiting autoreactive Th1/Th17 cells and promoting expansion of regulatory T cells.

IL-12 family cytokines are important in host immunity. Whereas some members (IL-12, IL-23) play crucial roles in pathogenesis of organ-specific autoimmune diseases by inducing the differentiation of Th1 and Th17 lymphocytes, others (IL-27 and IL-35) suppress inflammatory responses and limit tissue injury induced by these T cell subsets. In this study, we have genetically engineered a novel IL27p28/IL12p40 heterodimeric cytokine (p28/p40) that antagonizes signaling downstream of the gp130 receptor. We investigated whether p28/p40 can be used to ameliorate uveitis, a CNS inflammatory disease. Experimental autoimmune uveitis (EAU) is the mouse model of human uveitis and is mediated by Th1 and Th17 cells. We show here that p28/p40 suppressed EAU by inhibiting the differentiation and inflammatory responses of Th1 and Th17 cells while promoting expansion of IL-10(+)- and Foxp3(+)-expressing regulatory T cells. Lymph node cells from mice treated with p28/p40 blocked adoptive transfer of EAU to naïve syngeneic mice by immunopathogenic T cells and suppressive effects of p28/p40 derived in part from antagonizing STAT1 and STAT3 pathways induced by IL-27 and IL-6. Interestingly, IL27p28 also suppressed EAU, but to a lesser extent than p28/p40. The inhibition of uveitogenic lymphocyte proliferation and suppression of EAU by p28/p40 and IL27p28 establish efficacy of single chain and heterodimeric IL-12 family cytokines in treatment of a CNS autoimmune disease. Creation of the biologically active p28/p40 heterodimeric cytokine represents an important proof-of-concept experiment, suggesting that cytokines comprising unique IL-12 α- and ß-subunit pairing may exist in nature and may constitute a new class of therapeutic cytokines.

STAT3 protein promotes T-cell survival and inhibits interleukin-2 production through up-regulation of Class O Forkhead transcription factors.

Much is known about the role of STAT3 in regulating differentiation of interleukin-17-producing Th17 cells, but its function in other lymphocyte subsets is not well understood. In this report, we reveal wide-ranging functions of STAT3 in T-cells and provide evidence that STAT3 is convergence point for mechanisms that regulate lymphocyte quiescence and those controlling T-cell activation and survival. We show here that STAT3 inhibits T-lymphocyte proliferation by up-regulating the expression of Class-O Forkhead transcription factors, which play essential roles in maintaining T-cells in quiescent state. We further show that STAT3 binds directly to FoxO1 or FoxO3a promoter and that STAT3-deficiency resulted in down-regulation of the expression of FoxO1, FoxO3a and FoxO-target genes (IκB and p27Kip1). Compared with wild-type T-cells, STAT3-deficient T-cells produced more IL-2, due in part, to marked decrease in IκB-mediated sequestration of NF-κB in the cytoplasm and resultant enhancement of NF-κB activation. However, the high level of IL-2 production by STAT3-deficient T-cells was partially restored to normal levels by overexpressing FoxO1. It is notable that their exaggerated increase in IL-2 production rendered STAT3-deficient lymphocytes more susceptible to activation-induced cell death, suggesting that STAT3 might protect T-cells from apoptosis by limiting their production of IL-2 through up-regulation of FoxO1/FoxO3a expression. Moreover, we found that STAT3 enhanced survival of activated T-cells by up-regulating OX-40 and Bcl-2 while down-regulating FasL and Bad expression, suggesting that similar to role of FoxOs in regulating the lifespan of worms, STAT3 and FoxO pathways converge to regulate lifespan of T-lymphocytes.

Persistence of IL-2 expressing Th17 cells in healthy humans and experimental autoimmune uveitis.

Compared with other T-helper subsets, Th17 cell numbers are very low in human blood but become elevated in chronic inflammatory diseases. In this study, we investigated mechanisms that may explain the frequent involvement of Th17 cells in autoimmune diseases such as uveitis. We compared Th17 and Th1 subsets and found that Th17 cells expressed lower IL-2 levels during Ag-priming and this correlated with their decreased susceptibility to activation-induced cell death (AICD). However, complete depletion of IL-2 with IL-2 neutralizing antibodies rendered Th17 cells as susceptible to apoptosis as Th1 cells, suggesting that the low levels of IL-2 produced by Th17 cells conferred survival advantages to this subset. We describe here a Th17 subtype that constitutively produces very low levels of IL-2 (Th17-DP). The Th17-DP population increased dramatically in the blood and retina of mice during experimental autoimmune uveitis, indicating their potential involvement in the etiology of uveitis. We further show that the majority of the memory Th17 cells in human blood are Th17-DP and are targets of daclizumab, an IL-2R antibody used in treating recalcitrant uveitis. Thus, Th17 cells may persist in tissues and contribute to chronic inflammation by limiting IL-2 production to levels that cannot provoke IL-2-induced AICD yet are sufficient to promote Th17 homeostatic expansion.

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.

Interferon Regulator Factor 8 (IRF8) Limits Ocular Pathology during HSV-1 Infection by Restraining the Activation and Expansion of CD8+ T Cells.

Interferon Regulatory Factor-8 (IRF8) is constitutively expressed in monocytes and B cell lineages and plays important roles in immunity to pathogens and cancer. Although IRF8 expression is induced in activated T cells, the functional relevance of IRF8 in T cell-mediated immunity is not well understood. In this study, we used mice with targeted deletion of Irf8 in T-cells (IRF8KO) to investigate the role of IRF8 in T cell-mediated responses during herpes simplex virus 1 (HSV-1) infection of the eye. In contrast to wild type mice, HSV-1-infected IRF8KO mice mounted a more robust anti-HSV-1 immune response, which included marked expansion of HSV-1-specific CD8+ T cells, increased infiltration of inflammatory cells into the cornea and trigeminal ganglia (TG) and enhanced elimination of virus within the trigeminal ganglion. However, the consequence of the enhanced immunological response was the development of ocular inflammation, limbitis, and neutrophilic infiltration into the cornea of HSV-1-infected IRF8KO mice. Surprisingly, we observed a marked increase in virus-specific memory precursor effector cells (MPEC) in IRF8KO mice, suggesting that IRF8 might play a role in regulating the differentiation of effector CD8+ T cells to the memory phenotype. Together, our data suggest that IRF8 might play a role in restraining excess lymphocyte proliferation. Thus, modulating IRF8 levels in T cells can be exploited therapeutically to prevent immune-mediated ocular pathology during autoimmune and infectious diseases of the eye.

Suppressor of cytokine signaling 1 (SOCS1) mitigates anterior uveitis and confers protection against ocular HSV-1 infection.

Immunological responses to pathogens are stringently regulated in the eye to prevent excessive inflammation that damage ocular tissues and compromise vision. Suppressors of cytokine signaling (SOCS) regulate intensity/duration of inflammatory responses. We have used SOCS1-deficient mice and retina-specific SOCS1 transgenic rats to investigate roles of SOCS1 in ocular herpes simplex virus (HSV-1) infection and non-infectious uveitis. We also genetically engineered cell-penetrating SOCS proteins (membrane-translocating sequence (MTS)-SOCS1, MTS-SOCS3) and examined whether they can be used to inhibit inflammatory cytokines. Overexpression of SOCS1 in transgenic rat eyes attenuated ocular HSV-1 infection while SOCS1-deficient mice developed severe non-infectious anterior uveitis, suggesting that SOCS1 may contribute to mechanism of ocular immune privilege by regulating trafficking of inflammatory cells into ocular tissues. Furthermore, MTS-SOCS1 inhibited IFN-γ-induced signal transducers and activators of transcription 1 (STAT1) activation by macrophages while MTS-SOCS3 suppressed expansion of pathogenic Th17 cells that mediate uveitis, indicating that MTS-SOCS proteins maybe used to treat ocular inflammatory diseases of infectious or autoimmune etiology.

Interleukin-35 induces regulatory B cells that suppress autoimmune disease.

Interleukin-10 (IL-10)-producing regulatory B (Breg) cells suppress autoimmune disease, and increased numbers of Breg cells prevent host defense to infection and promote tumor growth and metastasis by converting resting CD4(+) T cells to regulatory T (Treg) cells. The mechanisms mediating the induction and development of Breg cells remain unclear. Here we show that IL-35 induces Breg cells and promotes their conversion to a Breg subset that produces IL-35 as well as IL-10. Treatment of mice with IL-35 conferred protection from experimental autoimmune uveitis (EAU), and mice lacking IL-35 (p35 knockout (KO) mice) or defective in IL-35 signaling (IL-12Rß2 KO mice) produced less Breg cells endogenously or after treatment with IL-35 and developed severe uveitis. Adoptive transfer of Breg cells induced by recombinant IL-35 suppressed EAU when transferred to mice with established disease, inhibiting pathogenic T helper type 17 (TH17) and TH1 cells while promoting Treg cell expansion. In B cells, IL-35 activates STAT1 and STAT3 through the IL-35 receptor comprising the IL-12Rß2 and IL-27Rα subunits. As IL-35 also induced the conversion of human B cells into Breg cells, these findings suggest that IL-35 may be used to induce autologous Breg and IL-35(+) Breg cells and treat autoimmune and inflammatory disease.

Therapeutic targeting of STAT3 (signal transducers and activators of transcription 3) pathway inhibits experimental autoimmune uveitis.

Mice with targeted deletion of STAT3 in CD4(+) T-cells do not develop experimental autoimmune uveitis (EAU) or experimental autoimmune encephalomyelitis (EAE), in part, because they cannot generate pathogenic Th17 cells. In this study, we have used ORLL-NIH001, a small synthetic compound that inhibits transcriptional activity of STAT3, to ameliorate EAU, an animal model of human posterior uveitis. We show that by attenuating inflammatory properties of uveitogenic lymphocytes, ORLL-NIH001 inhibited the recruitment of inflammatory cells into the retina during EAU and prevented the massive destruction of the neuroretina caused by pro-inflammatory cytokines produced by the autoreactive lymphocytes. Decrease in disease severity observed in ORLL-NIH001-treated mice, correlated with the down-regulation of α4ß1 and α4ß7 integrin activation and marked reduction of CCR6 and CXCR3 expression, providing a mechanism by which ORLL-NIH001 mitigated EAU. Furthermore, we show that ORLL-NIH001 inhibited the expansion of human Th17 cells, underscoring its potential as a drug for the treatment of human uveitis. Two synthetic molecules that target the Th17 lineage transcription factors, RORγt and RORα, have recently been suggested as potential drugs for inhibiting Th17 development and treating CNS inflammatory diseases. However, inhibiting STAT3 pathways completely blocks Th17 development, as well as, prevents trafficking of inflammatory cells into CNS tissues, making STAT3 a more attractive therapeutic target. Thus, use of ORLL-NIH001 to target the STAT3 transcription factor, thereby antagonizing Th17 expansion and expression of proteins that mediate T cell chemotaxis, provides an attractive new therapeutic approach for treatment of posterior uveitis and other CNS autoimmune diseases mediated by Th17 cells.

Suppressor of cytokine signaling-1 (SOCS1) inhibits lymphocyte recruitment into the retina and protects SOCS1 transgenic rats and mice from ocular inflammation.

PURPOSE: Suppressors of cytokine signaling (SOCS) proteins regulate the intensity and duration of cytokine signals and defective expression of SOCS1 and SOCS3 has been reported in a number of human diseases. The purpose of this study was to investigate the role of SOCS1 in intraocular inflammatory diseases (uveitis) and whether SOCS1 expression is defective in patients with ocular inflammatory diseases. METHODS: Blood from patients with scleritis or healthy human volunteers was analyzed for SOCS expression by RNase protection assay and RT-PCR. The authors generated SOCS1 transgenic rats and mice (SOCS1-Tg), induced experimental autoimmune uveoretinitis (EAU) by active immunization with interphotoreceptor retinal binding protein or adoptive transfer of uveitogenic T cells, and investigated effects of SOCS1 overexpression on EAU. SOCS1-mediated protection of retinal cells from apoptosis was assessed by annexin V staining. RESULTS: Induction of cytokine-induced SH2 protein was comparable between patients and volunteers, whereas 80% of lymphocytes from patients with scleritis failed to induce SOCS1 in response to IL-2. Compared with wild-type littermates, SOCS1-Tg rats/mice developed less severe EAU. Constitutive overexpression of SOCS1 in retina inhibited expression of chemokines (CCL17, CCL20, CXCL9, CXCL10), reduced Th17/Th1 expansion, and inhibited recruitment of inflammatory cells into the retina. The authors also show that SOCS1 protected retinal cells from staurosporine as well as H2O2-induced apoptosis. CONCLUSIONS: Defective expression of SOCS1 in patients with scleritis, taken together with SOCS1-mediated protection of neuroretinal cells from apoptosis, suggest that SOCS1 has neuroprotective function in the retina, implying that administration of SOCS1 mimetic peptides may be useful in treating uveitis or scleritis.

Hydrodynamic vaccination with DNA encoding an immunologically privileged retinal antigen protects from autoimmunity through induction of regulatory T cells.

The eye is an immunologically privileged organ whose Ags serve as targets for experimental autoimmune uveitis (EAU), a model for human uveitis. We used a hydrodynamic i.v. injection of naked DNA to express the uveitogenic retinal Ag interphotoreceptor retinoid-binding protein (IRBP) in the periphery, thus revoking its immune-privileged status. IRBP was expressed in the liver within hours of administration of as little as 10 microg of IRBP-DNA. Vaccinated mice were highly protected from EAU induced by immunization with IRBP for at least 10 wk after vaccination. Protection was partial in a reversal protocol. Mechanistic studies revealed specific hyporesponsiveness to IRBP without immune deviation, no evidence for apoptosis either by the Fas- or Bcl-2-regulated (mitochondrial) pathway and apparent lack of dependence on CD8(+) cells, IL-10, or TGF-beta. In contrast, depletion of CD25(+) cells after vaccination and before challenge markedly abrogated protection. IRBP-specific CD4(+)CD25(high) T cells could be cultured from vaccinated mice and transferred protection to unvaccinated, EAU-challenged recipients. In vitro characterization of these cells revealed that they are Ag specific, anergic, express FoxP3, CTLA-4, and glucocorticoid-induced TNFR, and suppress by contact. Thus, expression of IRBP in the periphery by DNA vaccination results in tolerance that acts at least in part through induction of IRBP-specific, FoxP3(+)CD4(+)CD25(+) regulatory T cells. DNA vaccination may offer a new approach to Ag-specific therapy of uveitis.

Thymic expression of peripheral tissue antigens in humans: a remarkable variability among individuals.

The majority of maturing T lymphocytes that recognize self-antigens is eliminated in the thymus upon exposure to their target antigens. This physiological process of negative selection requires that tissue-specific antigens be expressed by thymic cells, a phenomenon that has been well studied in experimental animals. Here, we have examined the expression in human thymi of four retinal antigens, that are capable of inducing autoimmune ocular disease retinal S-antigen (S-Ag), recoverin, RPE65 and inter-photoreceptor retinoid-binding protein (IRBP)], as well as four melanocyte-specific antigens, two of which are used as targets for melanoma immunotherapy [gp100, melanoma antigen recognized by T cells 1, tyrosinase-related protein (TRP)-1 and TRP-2]. Using reverse transcription (RT)-PCR, we found that all thymic samples from the 18 donors expressed mRNA transcripts of most or all the eight tested tissue antigens. Yet, the expression of the transcripts varied remarkably among the individual thymic samples. In addition, S-Ag, RPE65 and IRBP were detected by immunostaining in rare cells in sections of human thymi by antibodies against these proteins. Quantitative real-time RT-PCR analysis revealed that the retinal antigen transcripts in the human thymus are present at trace levels, that are lower by approximately five orders of magnitude than those in the retina. Our observations thus support the notions that thymic expression is a common feature for all tissue-specific antigens and that the levels of expression play a role in determining the susceptibility to autoimmunity against these molecules.

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.

Suppressors of cytokine signaling proteins are differentially expressed in Th1 and Th2 cells: implications for Th cell lineage commitment and maintenance.

Positive regulatory factors induced by IL-12/STAT4 and IL-4/STAT6 signaling during T cell development contribute to polarized patterns of cytokine expression manifested by differentiated Th cells. These two critical and antagonistic signaling pathways are under negative feedback regulation by a multimember family of intracellular proteins called suppressor of cytokine signaling (SOCS). However, it is not known whether these negative regulatory factors also modulate Th1/Th2 lineage commitment and maintenance. We show here that CD4(+) naive T cells constitutively express low levels of SOCS1, SOCS2, and SOCS3 mRNAs. These mRNAs and their proteins increase significantly in nonpolarized Th cells after activation by TCR signaling. We further show that differentiation into Th1 or Th2 phenotype is accompanied by preferential expression of distinct SOCS mRNA transcripts and proteins. SOCS1 expression is 5-fold higher in Th1 than in Th2 cells, whereas Th2 cells contain 23-fold higher levels of SOCS3. We also demonstrate that IL-12-induced STAT4 activation is inhibited in Th2 cells that express high levels of SOCS3 whereas IL-4/STAT6 signaling is constitutively activated in Th2 cells, but not Th1 cells, with high SOCS1 expression. These results suggest that mutually exclusive use of STAT4 and STAT6 signaling pathways by differentiated Th cells may derive in part, from SOCS3- or SOCS1-mediated repression of IL-12/STAT4- or IL-4/STAT6 signaling in Th2 and Th1 cells, respectively. Given the strong correlation between distinct patterns of SOCS expression and differentiation into the Th1 or Th2 phenotype, SOCS1 and SOCS3 proteins are therefore Th lineage markers that can serve as therapeutic targets for immune modulation therapy.

Suppressor of cytokine signaling 3 regulates proliferation and activation of T-helper cells.

Suppressors of cytokine signaling (SOCS) have been implicated in regulation of T-cell activation and cytokine-mediated differentiation of T-helper cells. In this study we have characterized the pattern of SOCS expression in naïve and activated primary T-helper cells, examined whether expression of SOCS genes is regulated by cytokine or T-cell receptor signaling, and analyzed the function of SOCS in differentiated T-cells. We show that SOCS1, SOCS2, SOCS3, CIS (cytokine-induced SH2 protein) genes are constitutively expressed in naïve T-helper cells, with SOCS3 being the most abundant. Antigen stimulation of naïve T-helper cells down-regulates SOCS3 expression and concomitantly up-regulates SOCS1, SOCS2, and CIS gene transcription, suggesting that SOCS genes are regulated differentially by T-cell activation. Down-regulation of SOCS3 expression is subsequently followed by gradual increase in SOCS3 level and corresponding decline in interleukin 2 (IL-2) secretion. In fact, SOCS3 mRNA levels are inversely correlated with the amount of IL-2 secretion and proliferative responses of differentiating T-helper cells, suggesting mutually antagonistic effects of SOCS3 and IL-2 and feedback regulation of T-cell activation by SOCS3. Furthermore, the degree of SOCS3 inhibition is antigen concentration-dependent and is mediated in part by growth factor independence-1, a T-cell transcription factor that regulates S-phase entry in T-cells. Forced overexpression of SOCS3 inhibits proliferation of T-helper cells, whereas depletion of endogenous SOCS3 by antisense SOCS3 cDNA enhances T-cell receptor- and cytokine-induced proliferation. Taken together, these results suggest a role for SOCS3 in maintaining T-helper cells in a quiescent state. Transient inhibition of SOCS3 by antigen stimulation may therefore be essential in allowing activation of resting T-cells.

Cell proliferation and STAT6 pathways are negatively regulated in T cells by STAT1 and suppressors of cytokine signaling.

Suppressor of cytokine signaling (SOCS) proteins have emerged as important regulators of cytokine signals in lymphocytes. In this study, we have investigated regulation of SOCS expression and their role in Th cell growth and differentiation. We show that SOCS genes are constitutively expressed in naive Th cells, albeit at low levels, and are differentially induced by Ag and Th-polarizing cytokines. Whereas cytokines up-regulate expression of SOCS1, SOCS2, SOCS3, and cytokine-induced Src homology 2 protein, Ags induce down-regulation of SOCS3 within 48 h of Th cell activation and concomitantly up-regulate SOCS1, SOCS2, and cytokine-induced Src homology 2 protein expression. We further show that STAT1 signals play major roles in inducing SOCS expression in Th cells and that induction of SOCS expression by IL-4, IL-12, or IFN-gamma is compromised in STAT1-deficient primary Th cells. Surprisingly, IL-4 is a potent inducer of STAT1 activation in Th2 but not Th1 cells, and SOCS1 or SOCS3 expression is dramatically reduced in STAT1(-/-) Th2 cells. To our knowledge, this is the first report of IL-4-induced STAT1 activation in Th cells, and suggests that its induction of SOCS, may in part, regulate IL-4 functions in Th2 cells. In fact, overexpression of SOCS1 in Th2 cells represses STAT6 activation and profoundly inhibits IL-4-induced proliferation, while depletion of SOCS1 by an anti-sense SOCS1 cDNA construct enhances cell proliferation and induces constitutive activation of STAT6 in Th2 cells. These results are consistent with a model where IL-4 has dual effects on differentiating T cells: it simulates proliferation/differentiation through STAT6 and autoregulates its effects on Th2 growth and effector functions via STAT1-dependent up-regulation of SOCS proteins.