Charles E. Egwuagu

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

T-helper type 17 cells (TH17) 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. TH17 cells were present in human peripheral blood mononuclear cells (PBMC), and were expanded by interleukin (IL)-2 and inhibited by interferon (IFN)-γ. Their numbers increased during active uveitis and scleritis and decreased following treatment. IL-17 was elevated in EAU and upregulated tumor necrosis factor (TNF)-α in retinal cells, suggesting a mechanism by which TH17 may contribute to ocular pathology. Furthermore, IL-27 was constitutively expressed in retinal ganglion and photoreceptor cells, was upregulated by IFN-γ and inhibited proliferation of TH17. These findings suggest that TH1 cells may mitigate uveitis by antagonizing the TH17 phenotype through the IFN-γ–mediated induction of IL-27 in target tissue. The finding that IL-2 promotes TH17 expansion provides explanations for the efficacy of IL-2R antibody therapy in uveitis, and suggests that antagonism of TH17 by IFN-γ and/or IL-27 could be used for the treatment of chronic inflammation.

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-12Rβ2 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-12Rβ2 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.

Loss of STAT3 in CD4+ T Cells Prevents Development of Experimental Autoimmune Diseases

Th17 cells are implicated in CNS autoimmune diseases. We show that mice with targeted-deletion of Stat3 in CD4+ T cells (CD4Stat3−/−) do not develop experimental autoimmune uveoretinitis (EAU) or experimental autoimmune encephalomyelitis. Defective Th17 differentiation noted in CD4Stat3−/− mice is compensated by exaggerated increases in Foxp3-, IL-10-, IL-4-, and IFN-γ-expressing T cells, suggesting critical roles of STAT3 in shaping Ag-specific CD4+ T cell repertoire. In mice with EAU, a high percentage of IL-17-expressing T cells in their peripheral lymphoid organs also secrete IFN-γ while these double-expressors are absent in CD4Stat3−/− and wild-type mice without EAU, raising the intriguing possibility that uveitis maybe mediated by Th17 and IL-17-expressing Th1 cells. Resistance of Stat3-deficient mice to EAU derives in part from an inability of uveitogenic Th17 and Th1 cells to enter eyes or brain of the CD4Stat3−/− mouse because of the reduction in the expression of activated α4/β1 integrins on CD4Stat3−/− T cells. Adoptive transfer of activated interphotoreceptor retinoid-binding protein-specific uveitogenic T cells induced in CD4Stat3−/− mice a severe EAU characterized by development of retinal folds, infiltration of inflammatory cells into the retina, and destruction of retinal architecture, underscoring our contention that the loss of STAT3 in CD4+ T cells results in an intrinsic developmental defect that renders CD4Stat3−/− resistant to CNS inflammatory diseases. STAT3 requirement for IL-17 production by Th17, generation of double positive T cells expressing IL-17 and IFN-γ, and for T cell trafficking into CNS tissues suggests that STAT3 may be a therapeutic target for modulating uveitis, sceritis, or multiple sclerosis.

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.

STAT3 in CD4+ T helper cell differentiation and inflammatory diseases

Jak/STAT pathways influence cell-fate decisions made by differentiating naïve T cells, regulate the intensity and duration of inflammatory responses and are implicated in pathogenic mechanisms of a number of chronic inflammatory diseases. Among the STATs, the STAT3 protein has emerged as an important determinant of whether the naïve T cell differentiates into regulatory (Treg) or an inflammatory (Th17) T cell lineage. STAT3 also has potent anti-inflammatory effects and regulates critical cellular processes such as, cell growth, apoptosis and transcription of inflammatory genes. Dysregulation of STAT3 pathway has therefore been implicated in the development of chronic inflammatory diseases, as well as, a number of malignant and neurodegenerative diseases. This review focuses on recent findings regarding the role of STAT3 in immunity, with particular emphasis on T cell lineage specification and disease etiology. New insights from animal models of uveitis, multiple sclerosis and inflammatory bowel diseases are discussed as exemplars of critical roles that STAT3 pathways play in inflammatory diseases and on how inhibiting STAT3 can be exploited to mitigate pathogenic autoimmunity.

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-γ 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.

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.

T Cell Tolerance to a Neo-Self Antigen Expressed by Thymic Epithelial Cells: The Soluble Form Is More Effective Than the Membrane-Bound Form

We have previously shown that transgenic (Tg) mice expressing either soluble or membrane-bound hen egg lysozyme (sHEL or mHEL, respectively) under control of the αA-crystallin promoter develop tolerance due to thymic expression of minuscule amounts of HEL. To further address the mechanisms by which this tolerance develops, we mated these two lines of Tg mice with the 3A9 line of HEL-specific TCR Tg mice, to produce double-Tg mice. Both lines of double-Tg mice showed deletion of HEL-specific T cells, demonstrated by reduction in numbers of these cells in the thymus and periphery, as well as by reduced proliferative response to HEL in vitro. In addition, the actual deletional process in thymi of the double-Tg mice was visualized in situ by the TUNEL assay and measured by binding of Annexin V. Notably, the apoptosis localized mainly in the thymic medulla, in line with the finding that the populations showing deletion and increased Annexin V binding consisted mainly of single- and double-positive thymocytes. Interestingly, the thymic deletional effect of sHEL was superior to that of mHEL in contrast to the opposite differential tolerogenic effects of these HEL forms on B cells specific to this Ag. Analysis of bone marrow chimeras indicates that both forms of HEL are produced by irradiation-resistant thymic stromal cells and the data suggest that sHEL is more effective in deleting 3A9 T cells due mainly to its higher accessibility to cross-presentation by dendritic APC.

Identification of Toxoplasma gondii in Paraffin-Embedded Sections by the Polymerase Chain Reaction

We used the polymerase chain reaction to amplify DNA fragments specific to Toxoplasma gondii. The sensitivity of the technique allowed for the detection of as few as ten cultured T. gondii tachyzoites. We applied the same amplification technique to deparaffinized ocular sections from two cases of ocular toxoplasmosis. Although toxoplasmic cysts could only be seen in one eye by optical microscopy, polymerase chain reaction allowed the identification of the parasite in both cases. Our study indicates the feasibility of a sensitive DNA-based assay to complement pathologic studies of an ocular parasitic disease.

Retinal cells suppress intraocular inflammation (uveitis) through production of interleukin-27 and interleukin-10

Neuronal or photoreceptor deficit observed in uveitis and multiple sclerosis derives in part from inability to control inflammatory responses in neuroretina or brain. Recently, IL‐27 was found to play a role in suppressing experimental autoimmune uveitis and experimental autoimmune encephalomyelitis, two animal models that share essential pathological features of human uveitis and multiple sclerosis, respectively. However, the mechanism by which interleukin‐27 (IL‐27) inhibits central nervous system (CNS) inflammation is not clear. In this study we have investigated mechanisms that mitigate or curtail intraocular inflammation (uveitis) and examined whether inhibitory effects of IL‐27 are mediated locally by neuroretinal cells or by regulatory T cells. We show here that microglia cells in the neuroretina constitutively secrete IL‐27 and its expression is up‐regulated during uveitis. We further show that photoreceptors constitutively express IL‐27 receptor and respond to IL‐27 signalling by producing anti‐inflammatory molecules, IL‐10 and suppressor of cytokine signalling 1 (SOCS1) through signal transducer and activator of transcription 1 (STAT1) ‐dependent mechanisms. Moreover, STAT1‐deficient mice produced reduced amounts of IL‐27, IL‐10 and SOCS1 and developed more severe uveitis. Surprisingly, IL‐10‐producing regulatory T cells had marginal roles in suppressing uveitis. These results suggest that suppression of intraocular inflammation might be mediated through endogenous production of IL‐27 and IL‐10 by retinal cells, whereas SOCS proteins induced by IL‐27 during uveitis may function to protect the neuroretinal cells from the toxic effects of pro‐inflammatory cytokines. Targeted delivery of IL‐27 into immune privileged tissues of the CNS may therefore be beneficial in the treatment of CNS inflammatory diseases, such as uveitis and multiple sclerosis.