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.

Uveitisis a group of sight-threatening intraocular inflammatory diseases that includes Behçet’s disease, birdshot retinochoroidopathy, Vogt-Koyanagi-Harada’s, sympathetic ophthalmia, and ocular sarcoidosis (1). Studies of experimental autoimmune uveoretinitis (EAU),2 the model of uveitis (1), led to the conclusion that Th1 cells are the etiologic agent of uveitis because IFN-γ levels are elevated in the retina during uveitis and IL12p40 is required for EAU induction (2). However, subsequent studies revealed that IL-12 down-regulates EAU (3), and treatment of mice with EAU with anti-IFN-γ Abs was found to exacerbate the disease (4), casting doubts on the role of this T cell subset as the etiologic agent of uveitis. Recent reports implicating Th17 cells in pathogenesis of human uveitis and scleritis has further complicated our understanding of the role of different T cell subsets in uveitis (5). Besides Th1 and Th17 cells, Th2 and T regulatory (Treg) cells are also detected in the eye during uveitis (6, 7), and exact roles of these T cell subtypes in the immunopathogenic process are largely unknown. Thus, in context of treating uveitis, it is important to note that presence of T cells in the retina compromises vision, and preventing entry of all T cell types or limiting their expansion in the eye is of utmost importance (8).

Accordingly, identifying molecular pathways amendable to therapeutic targeting has attracted attention as a potential strategy for limiting expansion of uveitogenic T cells in the eye. Recent reports indicating requirement of STAT3 for commitment of naive T cells toward the Th17 developmental pathway (9, 10) suggest a potential involvement of STAT3 pathway in mediating CNS inflammatory diseases. In this study, we have generated mice with targeted deletion of Stat3 in the CD4+ T cell compartment (CD4Stat3−/−) and used them to examine whether STAT3 pathways are required for development of EAU, as well as, experimental autoimmune encephalomyelitis (EAE), another CNS disease that shares essential immunopathologic features as EAU (11). In this study, we have shown that unlike the partial protection conferred by IL-17 blockage with IL-17 Abs (5), CD4stat3−/− mice are completely resistant to EAU or EAE, and this dramatic outcome derives from combinatory mechanisms that include: IL-17 blockade; altered T cell homeostasis that favor expansion of anti-inflammatory responses; and inhibition of T cell entry to CNS tissues. Resistance of Stat3 conditional knockout mice to development of EAE and EAU suggests that the STAT3 pathway is a potential therapeutic target for modulating these CNS inflammatory diseases.

Mice with conditional deletion of Stat3 in CD4+ T cells (CD4Stat3−/−) were derived by breeding Stat3fl/fl with CD4-Cre mice (Taconic Farms). Littermate Stat3fl/fl mice, in C57BL/6 background, were used as wild-type (WT) controls. Animal care and experimentation conformed to National Institutes of Health guidelines.

Mice were immunized with 150 μg interphotoreceptor retinoid-binding protein (IRBP) and 300 μg of human IRBP peptide (1–20) in 0.2 ml emulsion 1:1 v/v with CFA containing Mycobacterium tuberculosis strain H37RA (2.5 mg/ml). The mice also received Bordetella pertussis toxin (0.2 μg/mouse) concurrent with immunization, and clinical disease was established by fundoscopy as described previously (12). Eyes for histological EAU evaluation were harvested 0, 7, 10, 14, and 21 days postimmunization, fixed in 10% buffered formalin, embedded in methacrylate, and stained with H&E.

EAE was induced by immunization with 200 μg myelin oligodendrocyte glycoprotein (MOG) peptide (35–55) (Sigma-Aldrich) in CFA emulsion, containing 2.5 mg/ml heat killed, pulverized Mycobacterium tuberculosis strain H37RA, by s.c. injection into the tail base. The mice also received 200 ng Bordetella pertussis toxin concurrent with immunization. Spinal cord and brain were harvested 21 days postimmunization and stained with H&E as described above. Clinical signs of EAE were graded according to the following scale: 0, No clinical symptoms; 1, clumsiness, incontinence or atonic bladder, flaccid tail; 2, mild paraparesis (trouble initiating movement); 3, moderate paraparesis (hind limb weakness); 4, complete front and hind limb paralysis; and 5, moribund state. CNS infiltrates were collected from the brain and spinal code at day 21 postimmunization and lymphocytes/mononuclear cells were isolated by percoll gradient.

EAU was induced in WT C57BL/6 by immunization with IRBP in CFA, and disease was confirmed by fundoscopy as described above. Donor mice were sacrificed, and CD4+ T cells isolated from drawing lymph nodes and spleen were stimulated for 3 days with IRBP (20 μg/ml) and irradiated APC. The IRBP-specific T cells were then transferred i.v. into naive CD4Stat3−/− recipient mice at 10 × 106 cells/mouse. Ten days after the T cell transfer, disease was assessed by fundoscopy and the CD4Stat3−/− recipient mice were then euthanized. Eyes were enucleated, fixed in 10% buffered formalin, and sectioned for histopathological examination.

CD4+ T cells were purified from peripheral blood, thymus, spleens, or lymph nodes and stimulated with 10× irradiated syngeneic splenocytes (as APC) in the presence of 20 μg/ml MOG peptide or IRBP. Some cultures were activated in plate-bound anti-CD3 Ab (10 μg/ml) (BD Biosciences) and anti-CD28 Ab (5 μg/ml) in complete medium. For propagation under Th1 condition, medium was supplemented with anti-IL-4 Ab (10 μg/ml) and IL-12 (10 ng/ml) (PeproTech). For Th2 condition, the medium contained IL-4 (10 ng/ml), anti-IFN-γ Ab (10 μg/ml), and anti-IL-12 Ab (10 μg/ml), whereas Th17 polarization medium contained TGF-β (10 ng/ml), IL-6 (10 ng/ml), anti-IFN-γ Ab (10 μg/ml), and anti-IL-4 Ab (10 μg/ml). Most cultures were stimulated for 4 days and some were subsequently expanded for 4 days in IL-2 before analysis. FACS analysis using anti-CD3, CD4, CD8, CD44, IFN-γ, IL-4, IL-10, IL-17, Foxp3, α4-integrin, or β1-integrin mAbs and corresponding isotype control Abs (Pharmingen) was performed on Becton Dickinson FACSCalibur (BD Biosciences) as previously described (5).

Draining lymph nodes were collected 14 days after immunization with IRBP. Cells were stimulated with PPD (5 μg/ml), PHA (10 μg/ml), or IRBP (0.1, 1.0 or 10 μg/ml) in the presence or absence of exogenous cytokine. After 60 h, cultures were pulsed with [3H]thymidine (0.5 μCi/10 μl/well) for 12 additional hours. The presented data are mean cpm ± SE of responses of triplicate cultures. ELISA was performed using Pierce SearchLight technology (Pierce). For intracellular cytokine detection, freshly isolated or cultured CD4+ T cells were restimulated for 5 h with PMA (20 ng/ml)/ionomycin (1 μm) in the presence of Golgistop at the recommended concentrations (BD Pharmingen). The cells were surface stained with labeled Abs, fixed, permeabilized, and stained with the requisite Abs using the BD Biosciences Cytofix/Cytoperm kit according to the manufacturer’s instructions.

All RNA samples were DNA free. cDNA was generated as described previously (13); each gene-specific primer pair used for RT-PCR analysis spans at least an intron. Quantitative RT-PCR analysis was performed as previously described (14) using primers and probes from Applied Biosystems. The mRNA expression levels were normalized to the levels of ACTB (encoding β-actin) and GAPDH housekeeping genes.

Preparation of whole cell lysates was as described (12). Blots were probed with rabbit polyclonal STAT3 or β-actin specific Abs (Santa Cruz Biotechnology). Preimmune serum was used in parallel as controls, and signals were detected with HRP conjugated-secondary F(ab′)2 Abs (Ab) (Zymed Laboratories) using the ECL system (Amersham Biosciences).

All experiments were performed at least twice and were highly reproducible. Figures show data from representative experiments or from combined experiments as indicated. The Student’s t test was performed on the data.

Th17 cells are implicated in pathogenic mechanisms of scleritis and uveitis (5), and recent reports have suggested that STAT3 is required for development of naive T cells to the Th17 phenotype (9, 10). To investigate potential involvement of STAT3 pathway in mediating CNS inflammatory diseases, we generated mice with targeted deletion of Stat3 in their CD4+ T cells. Western blot analysis of purified CD4+ T cells confirms the absence of Stat3 expression in CD4Stat3−/− T cells (Fig. 1,A). The CD4Stat3−/− mice harbor fewer CD4+ T cells with slightly elevated numbers of CD8+ T cells in peripheral lymphoid tissues (data not shown). In line with other Stat3 conditional mice generated using CD4-Cre mice (9), CD4Stat3−/− appeared healthy and normal. EAU was induced in WT or CD4Stat3−/− by immunization with IRBP in CFA, and consistent with previous studies, all WT mice developed severe EAU characterized by targeted destruction of photoreceptor cells (Fig. 1,B) and very high clinical EAU scores (Fig. 1,C). In contrast, CD4Stat3−/− mice do not develop EAU; in three separate experiments involving >24 mice, none of the mice developed EAU (Fig. 1, B and C). WT and CD4Stat3−/− T cells respond to PPD, PHA, or IRBP in a dose-dependent manner (Fig. 1,D), suggesting that resistance of CD4Stat3−/− to EAU cannot be solely attributed to inability to respond to the autoantigen. It is of note that the higher proliferative response of WT cells is observed only in which Ag-experienced T cells are stimulated with IRBP but not if naive T cells from these mouse strains are stimulated with anti-CD3 and anti-CD28 Abs (data not shown). This suggests that the higher proliferative responses of the WT cells is not due to an intrinsic developmental defect that renders the CD4 T cells less responsive to Ag (see ahead). Similar results were obtained in delayed type hypersensitivity assays (Fig. 1 E), underscoring the fact that CD4Stat3−/− T cells recognize and respond to IRBP but do not develop EAU.

During EAU in mice, both Th1 and Th17 cells are expanded and recruited into the retina, and their levels vary during the course of the disease (12). In WT mice, initial clinical signs of EAU is observed 7–12 days after immunization with full-blown clinical disease characterized by substantial increase in proinflammatory cytokines occurring by postimmunization days 12–14 (Fig. 1, B and C) (12). As Th17 cells have recently been implicated in uveitis, we examined whether the resistance of CD4Stat3−/− to development of EAU derives from defects in generation of Th17 cells. Freshly isolated cells from the lymph node and spleen of IRBP-immunized WT or CD4Stat3−/− mice on day 0, 10, and 21 postimmunization were therefore analyzed (without stimulation) to determine the relative abundance of Th17 and Th1 cells. In line with a published report (5), onset of EAU pathology is temporally correlated with an increase of Th17 and Th1 in these peripheral lymphoid tissues (Fig. 2,A). However, there are notable differences between WT and CD4Stat3−/− in their T cell population dynamics elicited by immunization with IRBP. Compared with WT, numbers of Th17 in spleen and lymph nodes of IRBP-immunized CD4Stat3−/− is markedly reduced and remained low at all time points analyzed. Unlike the Th17 population, we observe progressive increase in the percentage of IFN-γ-expressing CD4Stat3−/− T cells (Fig. 2,A), suggesting that Th1 cells do not cause EAU. We also analyzed PBMC (Fig. 2,B) and spleen/lymph node CD4+ T cells (Fig. 2,C) from both mouse strains, and across the board, we detected a substantially lower percentage of Th17 cells in these tissues of the CD4Stat3−/− mouse compared with the WT strain. It is also remarkable that pathology in the WT is associated with significant increase in the numbers of cells expressing both IFN-γ and IL-17 (referred to here as double positive or DP cells). Interestingly, expansion of the purified lymph node cells by IL-2 preferentially increased the percentage of the DP cells (Fig. 2,D), and consistent with previous reports (5), the cells that are predominantly expanded by IL-2 are memory cells (Fig. 2,E). It is also of note that a majority of the CD4Stat3−/− cells exhibit an activated T cell phenotype, suggesting that the defect in Th17 developmental pathway does not interfere with the capacity of the CD4Stat3−/− T cells to respond to activation signals. In contrast, to the increase in IL-17-expressing and DP cells in WT lymph node and spleen, the percentage of Foxp3-expressing cells are increased in CD4Stat3−/− compared with WT cells (Fig. 2,F). Lymph node and spleen cells isolated from mice 7 or 14 days postimmunization were stimulated with IRBP, and analysis of cytokine secretion by ELISA reveals marked increase in IL-17, TNF-α, IL-23, and IL-1α in the WT but not the CD4Stat3−/− cells (Fig. 2 G).

In vivo intracellular cytokine-staining assays of T cell populations of the PBMC, lymph nodes, and spleen elicited by immunization of mice with the IRBP autoantigen reveal a marked diminution of IL-17-expressing T cells in CD4Stat3−/− mice (Fig. 2). To further characterize the function of STAT3, purified naive CD4+ T cells isolated from lymph nodes and spleen were stimulated with anti-CD3 and anti-CD28 Abs under Th1, Th2, or Th17 polarization conditions, RNA from the cells was analyzed by RT-PCR, and patterns of cytokine expression of cells from the two mouse strains were determined by intracellular cytokine-staining assays and ELISA. Substantial reduction in IL-23R and RORγt expression (Fig. 2,A) further underscores the Th17-developmental defects in CD4Stat3−/− mice (9, 10). Similar to results of analysis of freshly isolated lymph node and spleen cells (Fig. 2,F), Foxp3-expressing CD4+ T cells are markedly elevated in the CD4Stat3−/− mouse (Fig. 3,B). Consistent with in vivo results obtained in freshly isolated tissues of mice immunized with IRBP, the percentage of IL-17-expressing CD4+ T cells is also much higher in in vitro stimulated T cells of the WT compared with CD4Stat3−/− mice (Fig. 3,C). Interestingly, we observed an important distinction between T cells that are specific to the IRBP autoantigen and those that were nonspecifically stimulated with anti-CD3 and anti-CD28 Abs. In >3 independent studies, we consistently observe significant numbers of DP cells in cultures that were stimulated with IRBP while all cultures stimulated by anti-CD3 and anti-CD28 Abs contain relatively low numbers of the DP cells. Interestingly, similar increase in DP cells has also been observed in EAE (15, 16). Surprisingly, intracellular cytokine analysis of the purified CD4+ T cells reveal tremendous increase in IL-4- and IL-10-expressing cells in CD4Stat3−/− lymph nodes and spleen (Fig. 3,D). Stimulation of the cells under Th1, Th2, or Th17 polarizing condition further confirm that the secretion of IL-4 is markedly increased in CD4Stat3−/− (Fig. 3 E), suggesting that deletion of STAT3 in CD4+ cells may induce a compensatory increase in Th1, Th2, and Treg subsets.

Essential immunopathogenic features of EAU are very similar to those observed in EAE, and in both diseases, Th17 and Th1 are implicated (17). We show here that targeted deletion of Stat3 in CD4+ T cells also confers protection from developing EAE (Fig. 4). Representative tissue sections of spinal cord from mice immunized with MOG reveal presence of inflammatory cell infiltrates in the white matter and perivascular lesions of WT (Fig. 4, A–C) but not CD4Stat3−/− mice (Fig. 4, D–F); in two separate experiments, 16 WT mice developed classic symptoms of EAE ranging from hind limb weakness, ascending paralysis, to complete front and hind limb paralysis. In contrast, all 16 CD4Stat3−/− mice remain normal after 1 mo of observation (Fig. 4,G) with no sign of CNS inflammation. Similar to EAU, development of EAE correlates with increase in IL-17-expressing T cells while the MOG-specific immune response of the CD4Stat3−/− mouse strain is dominated by IFN-γ-expressing T cells (Fig. 4 H). These results further underscore the role of STAT3 pathways in shaping the T cell repertoire during CNS inflammatory diseases and suggest that, whereas activation of STAT3 in vivo is required for generation of Th17 lineage, it limits expansion of Th1 and possibly other CD4+ subsets.

In addition to its roles in skewing differentiation of naive T cells toward the Th17 developmental pathway, STAT3 pathways also regulate expression of adhesion molecules and may therefore have critical roles in regulating trafficking of uveitogenic or encephalitogenic T cells into the CNS during EAU or EAE. As indicated on Fig. 4, B and C, there is significant infiltration of the spinal cord of WT mice with EAE but not in CD4Stat3−/− mice immunized with MOG (Fig. 4, E and F). Analysis of the eyes of both mouse strains 21 days postimmunization reveal the presence of Th17 and Th1 cells in retina of WT mice. In contrast, T cells are not detectable in the retina of CD4Stat3−/− mice immunized with IRBP. We further show that the inability to recruit uveitogenic Th1 and Th17 cells into the eyes of CD4Stat3−/− mice following immunization with IRBP correlates with substantial decrease in retinal expression of genes coding for VLA-4 or α4 integrin (CD49d) and β1 integrin (CD29) (Fig. 5,B); proteins that promote trans-endothelial migration of leukocytes into the CNS and entry of activated lymphocytes into the retina (18, 19). To further confirm this observation at the protein level, freshly isolated PBMC or spleen cells at days 0, 7, or 14 postimmunization with IRBP were immediately analyzed for the expression of activated α4 integrin (CD49d) and β1 integrin (CD29). In PBMC (Fig. 5,C), as well as spleen cells (Fig. 5 D), there is substantial elevation of percentage of cells coexpressing α4 and β1 integrins in WT compared with reduced levels in CD4Stat3−/− mice. These results suggest that decreased levels of activated integrins resulting from STAT3 deficiency may inhibit trans-endothelial migration of leukocytes and recruitment of T cells into the retina of CD4Stat3−/− mice. However, because expression of these integrins is not completely extinguished, the inhibition of these integrins may not be solely responsible for inability of CD4Stat3−/− T cells to enter the eye.

We performed adoptive transfer experiments to further confirm that the resistance of CD4Stat3−/− mice to EAU is a direct consequence of the absence of STAT3 expression by CD4+ T cells. Activated IRBP-specific CD4+ T cells from WT mice with EAU were adoptively transferred into CD4Stat3−/− mice. The mice exhibited signs of EAU 12 days after adoptive transfer of the WT cells, as determined by fundoscopy and histology (Fig. 6, A and B). Similar to immunized WT mice, EAU in CD4Stat3−/− mice is characterized by development of retinal folds, disruption of the retinal architecture, and infiltration of inflammatory cells into the retina. In addition, disease development in the CD4Stat3−/− mice correlates with detection of substantial percent of donor Th17 cells, and substantial percentage of PBMC are DP T cells expressing IL-17 and IFN-γ (Fig. 6 C). It is also of note that we also adoptively transferred STAT3-deficient immune cells to WT mice, but these mice did not develop EAU (data not shown), further underscoring the involvement of STAT3 pathways in development of EAU.

In this study, we have provided direct experimental evidence that expression of STAT3 in CD4+ T cells is essential for the development of two CNS inflammatory diseases, EAU and EAE. In contrast to mice with conditional deletion of STAT3 in their T cell CD4 compartment (CD4Stat3−/−) that are completely resistant to either EAU or EAE (Figs. 1 and 4), WT (Fig. 1,B) and CD4Stat3−/− mice injected with activated IRBP-specific uveitogenic T cells from WT mice with EAU (Fig. 6) developed EAU characterized by massive infiltration of inflammatory cells into the retina and targeted destruction of photoreceptor cells. WT mice also developed severe EAE characterized by massive infiltration of inflammatory cells into the spinal cord and developed classic symptoms of EAE ranging from hind limb weakness, ascending paralysis, to complete front and hind limb paralysis. In contrast to the WT mouse strain where onset of CNS pathology is temporally correlated with increase of Th17 in peripheral lymphoid tissues, numbers of Th17 in spleen and lymph nodes of IRBP- or MOG-immunized CD4Stat3−/− mice is markedly reduced due to inability of naive Stat3-deficient T cells to differentiate into Th17 phenotype. Of particular note is our finding that the level of cells expressing IFN-γ is markedly elevated in the CD4Stat3−/− compared with WT mice, suggesting that paucity of Th17 cells is compensated for by substantial increases in Th1 cells (Fig. 2, D and E) and that etiology of EAU or EAE cannot be attributed to Th1 cells alone. Consistent with the role of TNF-α and IL-1α production by Th17 cells in EAU pathology (5), we observe significant secretion of TNF-α, IL-17, and IL-1α by WT lymph node T cells of mice with EAU but not T cells of CD4Stat3−/− mice immunized with IRBP. The inherent defect in Th17 differentiation pathway of CD4Stat3−/− T cells does not appear to derive from failure to respond to T cell activation signals, as CD4Stat3−/− T cells exhibit a highly activated T cell phenotype compared with WT cells (Fig. 2,E) and respond to the autoantigen (Fig. 1, D and E). Interestingly, Ag-experienced WT T cells exhibit much higher in vitro proliferative responses to IRBP (Fig. 1,D), whereas naive WT and CD4Stat3−/− T cells are similar in their proliferative responses when stimulated with anti-CD3 and anti-CD28 Abs (data not shown). Thus, the higher proliferative responses of the WT cells to IRBP (Fig. 1, D and E) may derive, in part, from the fact that IRBP-specific immune responses in WT mice are dominated by Th17, Th1, and DP (IL-17- and IFN-γ-expressing) T cells, whereas responses of CD4stat3−/− T cells is characterized by the absence of Th17 and DP cells and increase in cells expressing IL-10 and Foxp3.

Stimulated CD4Stat3−/− T cells secrete exaggerated levels of IFN-γ and IL-4 (Fig. 3, C–E), suggesting that whereas STAT3 positively regulates IL-17 expression, it negatively regulates cytokines that antagonize the development and functions of the Th17 subset. Similarly, IL-10- and Foxp3-expressing cells are higher in CD4Stat3−/− compared with WT mice (Fig. 3, B and D), suggesting that compensatory increase in Tr1 and inducible Treg cells may represent another mechanism that restrains development of pathogenic CNS autoimmune diseases in CD4Stat3−/− mice. Decrease in Treg subsets and marked increase in Th17 signature cytokines during EAU in WT mice (Fig. 2,G) may provide a mechanistic link between activation of STAT3 pathways and development of pathogenic CNS autoimmune diseases. It is, however, paradoxical that STAT3 has recently been shown to induce rapid expression of anti-inflammatory genes (20, 21) and is also required for differentiation and expansion of Th17 cells that mediate a number of human inflammatory diseases (5). The role of STAT3 pathways of CD4+ T cells in mediating CNS inflammatory diseases appears to extend beyond promoting Th17 developmental pathway, as STAT3 pathways also regulate expression of adhesion molecules that regulate trafficking of uveitogenic or encephalitogenic T cells into the CNS during EAU and EAE. Although we detect Th17 and Th1 cells in WT eyes, they are not detectable in retina of CD4Stat3−/− mice immunized with IRBP (Fig. 5,A). We show that inability to recruit uveitogenic T cells into CD4Stat3−/− retina following immunization with IRBP correlates with substantial decrease in the number of cells expressing activated α4/β1-integrin (Fig. 5, B–D). In contrast, enhanced expression and expression of these adhesion molecules correlates with entry of activated lymphocytes into the retina and brain (Fig. 5 A) (22, 23).

With regards to uveitis and other ocular inflammatory diseases, there is compelling evidence for involvement of both Th1 and Th17 cell types. Th17 are most abundant in retina at early stages of the disease, increase during active uveitis/scleritis, and decrease following treatment (5). In contrast, greatest numbers of Th1 in the retina coincides with recovery phase of EAU, and together these observations have led to the suggestion that Th1 may confer protection, whereas Th17 may promulgate EAU pathology (5). It is, however, premature to surmise that Th1 cells are not etiologic agents of uveitis because adoptive transfer of an IRBP-specific T cell line, depleted of IL-17-expressing T cells, induces EAU (24). Moreover, IL-17−/− mice are partially protected from EAU (27), and only partial remediation of EAU is achieved by neutralizing IL-17 with anti-IL-17 Abs (5). These and other studies underscore involvement of other etiologic mechanisms and continue to fuel debate over the exact role of Th1 and Th17 subtypes in uveitis. In fact, in a very recent report published while our manuscript was in revision, STAT3 signaling was found to be required in the development of Th17-dependent autoimmunity, and consistent with data noted in this study, ablation of STAT3 signaling in CD4 cells increased Th1 responses, suggesting that STAT3 signaling may skew Th responses away from the Th1 pathway and toward the Th17 pathway (25). In our study, analysis of IRBP-specific T cells from lymph node of mice with EAU further revealed presence of substantial numbers of T cells coexpressing IL-17 and IFN-γ (Fig. 2,D and 6C), and recent reports have identified these double-expressors as Th1-like cells (26). Considering that evidence linking Th17 subset to etiology of CNS inflammatory diseases are largely based on correlations between IL-17 expression in CNS target tissue and initiation of CNS disease, our results raise the intriguing possibility that uveitis may indeed be mediated not only by Th17 but also by DP cells expressing IFN-γ and IL-17. Similar analysis of IRBP-immunized CD4Stat3−/− mice revealed a virtual absence of the DP cells (Fig. 2 D) and establishes for the first time that STAT3 may be required for their generation. As many inflammatory cytokines activate STAT3 pathways in lymphocytes, we cannot rule out the possibility that generation of DP cells expressing both IL-17 and IFN-γ does occur in situ at target sites of inflammation and may therefore contribute to the disease process.

Identifying pathways that mediate pathogenic autoimmune diseases of the CNS is potentially applicable to other organ-specific autoimmune diseases and of practical importance in development of organ-specific anti-inflammatory therapy. In context of ocular inflammatory diseases, it should be emphasized that all major T cell subsets (Th1, Th2, Th17, Tr1, and Treg) enter the eye during uveitis. Regardless of whether these cells confer protection or induce uveitis, prolonged presence of any T cell type in the eye compromises vision by interfering with light refraction and is therefore undesirable. Thus, the therapeutic goal is to inhibit or limit expansion of T cells that enter the eye during ocular inflammation; knowing the T cell subset that initiates the disease process is irrelevant in the context of restoring normal vision. In this study, we have shown that in contrast to the partial protection conferred by IL-17 blockage with IL-17 Abs, CD4stat3−/− mice are completely resistant to EAU or EAE. This dramatic outcome derives from combinatory mechanisms that include: IL-17 blockade; altered T cell homeostasis that favor expansion of anti-inflammatory responses; and inhibition of T cell entry to CNS tissues. Although recent studies suggest that cytokines such as IL-21 that promote Th17 development may be effective targets for modulating CNS disease (15, 16), we believe that STAT3 is a more attractive target for therapeutic modulation of uveitis and possibly other CNS inflammatory diseases; its deletion impacts not only Th17 but also IL-17-expressing Th1 cells and prevents access of T cells into immunologically privileged tissues of the CNS.

We thank P. Silver (National Eye Institute, National Institutes of Health) for assistance with the EAU studies, D. Levy (New York University) for providing the Stat3fl/fl mice, and Igal Gery (National Eye Institute, National Institutes of Health) for assistance with the EAU studies and for reading the manuscript.

The authors have no financial conflict of interest.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


Abbreviations used in this paper: EAU, experimental autoimmune uveoretinitis; Treg, T regulatory cell; EAE, experimental autoimmune encephalomyelitis; IRBP, interphotoreceptor retinoid-binding protein; WT, wild type; MOG, myelin oligodendrocyte glycoprotein; PPD, purified protein derivative; DP, double positive.

Nussenblatt, R. B..
. Proctor lecture: experimental autoimmune uveitis: mechanisms of disease and clinical therapeutic indications.
Invest. Ophthalmol. Visual Sci.
Tarrant, T. K., P. B. Silver, C. C. Chan, B. Wiggert, R. R. Caspi.
. Endogenous IL-12 is required for induction and expression of experimental autoimmune uveitis.
J. Immunol.
Tarrant, T. K., P. B. Silver, J. L. Wahlsten, L. V. Rizzo, C. C. Chan, B. Wiggert, R. R. Caspi.
. Interleukin 12 protects from a T helper type 1-mediated autoimmune disease, experimental autoimmune uveitis, through a mechanism involving interferon γ, nitric oxide, and apoptosis.
J. Exp. Med.
Caspi, R. R., P. B. Silver, C. C. Chan, B. Sun, R. K. Agarwal, J. Wells, S. Oddo, Y. Fujino, F. Najafian, R. L. Wilder.
. Genetic susceptibility to experimental autoimmune uveoretinitis in the rat is associated with an elevated Th1 response.
J. Immunol.
Amadi-Obi, A., C. R. Yu, X. Liu, R. M. Mahdi, G. L. Clarke, R. B. Nussenblatt, I. Gery, Y. S. Lee, C. E. Egwuagu.
. T(H)17 cells contribute to uveitis and scleritis and are expanded by IL-2 and inhibited by IL-27/STAT1.
Nat. Med.
Dullforce, P. A., G. W. Seitz, K. L. Garman, J. A. Michael, S. M. Crespo, R. J. Fleischman, S. R. Planck, D. C. Parker, J. T. Rosenbaum.
. Antigen-specific accumulation of naive, memory, and effector CD4 T cells during anterior uveitis monitored by intravital microscopy.
Cell. Immunol.
Crane, I. J., J. V. Forrester.
. Th1 and Th2 lymphocytes in autoimmune disease.
Crit. Rev. Immunol.
Nussenblatt, R. B..
. Bench to bedside: new approaches to the immunotherapy of uveitic disease.
Int. Rev. Immunol.
Mathur, A. N., H. C. Chang, D. G. Zisoulis, G. L. Stritesky, Q. Yu, J. T. O’Malley, R. Kapur, D. E. Levy, G. S. Kansas, M. H. Kaplan.
. Stat3 and Stat4 direct development of IL-17-secreting Th cells.
J. Immunol.
Yang, X. O., A. D. Panopoulos, R. Nurieva, S. H. Chang, D. Wang, S. S. Watowich, C. Dong.
. STAT3 regulates cytokine-mediated generation of inflammatory helper T cells.
J. Biol. Chem.
Calder, V. L., S. L. Lightman.
. Experimental autoimmune uveoretinitis (EAU) versus experimental allergic encephalomyelitis (EAE): a comparison of T cell-mediated mechanisms.
Clin. Exp. Immunol.
Takase, H., C. R. Yu, X. Liu, C. Fujimoto, I. Gery, C. E. Egwuagu.
. Induction of suppressors of cytokine signaling (SOCS) in the retina during experimental autoimmune uveitis (EAU): potential neuroprotective role of SOCS proteins.
J. Neuroimmunol.
Egwuagu, C. E., C. R. Yu, M. Zhang, R. M. Mahdi, S. J. Kim, I. Gery.
. Suppressors of cytokine signaling proteins are differentially expressed in Th1 and Th2 cells: implications for Th cell lineage commitment and maintenance.
J. Immunol.
Yu, C. R., R. M. Mahdi, S. Ebong, B. P. Vistica, J. Chen, Y. Guo, I. Gery, C. E. Egwuagu.
. Cell proliferation and STAT6 pathways are negatively regulated in T cells by STAT1 and suppressors of cytokine signaling.
J. Immunol.
Nurieva, R., X. O. Yang, G. Martinez, Y. Zhang, A. D. Panopoulos, L. Ma, K. Schluns, Q. Tian, S. S. Watowich, A. M. Jetten, C. Dong.
. Essential autocrine regulation by IL-21 in the generation of inflammatory T cells.
Korn, T., E. Bettelli, W. Gao, A. Awasthi, A. Jager, T. B. Strom, M. Oukka, V. K. Kuchroo.
. IL-21 initiates an alternative pathway to induce proinflammatory T(H)17 cells.
Kroenke, M. A., B. M. Segal.
. Th17 and Th1 responses directed against the immunizing epitope, as opposed to secondary epitopes, dominate the autoimmune repertoire during relapses of experimental autoimmune encephalomyelitis.
J. Neurosci. Res.
Martin, A. P., L. V. de Moraes, C. E. Tadokoro, A. G. Commodaro, E. Urrets-Zavalia, G. A. Rabinovich, J. Urrets-Zavalia, L. V. Rizzo, H. M. Serra.
. Administration of a peptide inhibitor of α4-integrin inhibits the development of experimental autoimmune uveitis.
Invest. Ophthalmol. Visual Sci.
Greenwood, J., Y. Wang, V. L. Calder.
. Lymphocyte adhesion and transendothelial migration in the central nervous system: the role of LFA-1, ICAM-1, VLA-4, and VCAM-1.
El Kasmi, K. C., J. Holst, M. Coffre, L. Mielke, A. de Pauw, N. Lhocine, A. M. Smith, R. Rutschman, D. Kaushal, Y. Shen, et al
. General nature of the STAT3-activated anti-inflammatory response.
J. Immunol.
Murray, P. J..
. STAT3-mediated anti-inflammatory signalling.
Biochem. Soc. Trans.
Xu, H., A. Manivannan, J. Liversidge, P. F. Sharp, J. V. Forrester, I. J. Crane.
. Requirements for passage of T lymphocytes across non-inflamed retinal microvessels.
J. Neuroimmunol.
Wooten, D. K., X. Xie, D. Bartos, R. A. Busche, G. D. Longmore, S. S. Watowich.
. Cytokine signaling through Stat3 activates integrins, promotes adhesion, and induces growth arrest in the myeloid cell line 32D.
J. Biol. Chem.
Rizzo, L. V., P. Silver, B. Wiggert, F. Hakim, R. T. Gazzinelli, C. C. Chan, R. R. Caspi.
. Establishment and characterization of a murine CD4+ T cell line and clone that induce experimental autoimmune uveoretinitis in B10.A mice.
J. Immunol.
Harris, T. J., J. F. Grosso, H. R. Yen, H. Xin, M. Kortylewski, E. Albesiano, E. L. Hipkiss, D. Getnet, M. V. Goldberg, C. H. Maris, et al
. Cutting edge: an in vivo requirement for STAT3 signaling in TH17 development and TH17-dependent autoimmunity.
J. Immunol.
Acosta-Rodriguez, E. V., L. Rivino, J. Geginat, D. Jarrossay, M. Gattorno, A. Lanzavecchia, F. Sallusto, G. Napolitani.
. Surface phenotype and antigenic specificity of human interleukin 17-producing T helper memory cells.
Nat. Immunol.
Luger, D., P. B. Silver, J. Tang, D. Cua, Z. Chen, Y. Iwakura, E. P. Bowman, N. M. Sgambellone, C.-C. Chan, and R. R. Caspi. 2008. Either a Th17 or a Th1 effector response can drive autoimmunity: conditions of disease induction affect dominant effector category. J. of Exp. Med. In press.