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 (CD4(Stat3)(-/-)) do not develop experimental autoimmune uveoretinitis (EAU) or experimental autoimmune encephalomyelitis. Defective Th17 differentiation noted in CD4(Stat3)(-/-) mice is compensated by exaggerated increases in Foxp3-, IL-10-, IL-4-, and IFN-gamma-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-gamma while these double-expressors are absent in CD4(Stat3)(-/-) 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 CD4(Stat3)(-/-) mouse because of the reduction in the expression of activated alpha4/beta1 integrins on CD4(Stat3)(-/-) T cells. Adoptive transfer of activated interphotoreceptor retinoid-binding protein-specific uveitogenic T cells induced in CD4(Stat3)(-/-) 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 CD4(Stat3)(-/-) resistant to CNS inflammatory diseases. STAT3 requirement for IL-17 production by Th17, generation of double positive T cells expressing IL-17 and IFN-gamma, and for T cell trafficking into CNS tissues suggests that STAT3 may be a therapeutic target for modulating uveitis, sceritis, or multiple sclerosis.     

Cutting Edge: Suppression of GM-CSF Expression in Murine and Human T Cells by IL-27

GM-CSF is a potent proinflammatory cytokine that plays a pathogenic role in the CNS inflammatory disease experimental autoimmune encephalomyelitis. As IL-27 alleviates experimental autoimmune encephalomyelitis, we hypothesized that IL-27 suppresses GM-CSF expression by T cells. We found that IL-27 suppressed GM-CSF expression in CD4(+) and CD8(+) T cells in splenocyte and purified T cell cultures. IL-27 suppressed GM-CSF in Th1, but not Th17, cells. IL-27 also suppressed GM-CSF expression by human T cells in nonpolarized and Th1- but not Th17-polarized PBMC cultures. In vivo, IL-27p28 deficiency resulted in increased GM-CSF expression by CNS-infiltrating T cells during Toxoplasma gondii infection. Although in vitro suppression of GM-CSF by IL-27 was independent of IL-2 suppression, IL-10 upregulation, or SOCS3 signaling, we observed that IL-27-driven suppression of GM-CSF was STAT1 dependent. Our findings demonstrate that IL-27 is a robust negative regulator of GM-CSF expression in T cells, which likely inhibits T cell pathogenicity in CNS inflammation.     

Both Th1 and Th17 are immunopathogenic but differ in other key biological activities

The role of Th17 lymphocytes in immunopathogenic processes has been well established, but little is known about their basic cell features. In this study, we compared polarized Th1 and Th17 for key biological activities related to pathogenicity and trafficking. Th1 and Th17 lineages were derived from TCR-transgenic CD4 murine cells specific against hen egg lysozyme. When adoptively transferred into mice expressing hen egg lysozyme in their eyes, both Th1 and Th17 induced ocular inflammation but with slight differences in histological pathology. PCR analysis revealed selective expression of IFN-gamma or IL-17 in eyes of Th1 or Th17 recipients, respectively. Additionally, Th1 and Th17 were found to differ in three other key activities: 1) Th17 cells were inferior to Th1 cells in their capacity to trigger massive lymphoid expansion and splenomegaly; 2) the proportion of Th1 cells among infiltrating cells in inflamed recipient eyes declined rapidly, becoming a minority by day 7, whereas Th17 cells remained in the majority throughout this period; and 3) remarkable differences were noted between Th1 and Th17 cells in their expression of certain surface markers. In particular, reactivated Th1 expressed higher levels of CD49d and alpha(4)beta(7) (mucosal homing) in vitro and higher levels of CXCR3 (Th1 trafficking) in vivo. Reactivated Th17, however, expressed higher levels of alpha(E)beta(7) (epithelial tissue homing) and CD38 (activation, maturation and trafficking) in vitro, but in vivo Th17 expressed higher levels of alpha(4)beta(7) and CCR6 (lymphocyte trafficking). These data reveal that Th1 and Th17 cells differ in several key biological activities influencing migration and pathogenic behavior during inflammatory disease.     

Chronically inflamed human tissues are infiltrated by highly differentiated Th17 lymphocytes

Chronic inflammatory diseases are characterized by local tissue injury caused by immunocompetent cells, in particular CD4(+) T lymphocytes, that are involved in the pathogenesis of these disorders via the production of distinctive sets of cytokines. Here, we have characterized single CD4(+) T cells that infiltrate inflamed tissue taken from patients with psoriasis, Crohn's disease, rheumatoid arthritis, or allergic asthma. Results from a cytokine production and gene profile analysis identified a population of in vivo differentiatedretinoid-related orphan receptor gamma-expressing T cells, producing high levels of IL-17, that can represent up to 30% of infiltrating T lymphocytes. Activated Th17 cells produced IL-26, TNF-alpha, lymphotoxin-beta, and IL-22. IL-17 and IL-22 concentrations secreted by tissue infiltrating Th17 cells could reach up to 100 nM and were inversely correlated with the production of Th1- and Th2-associated cytokines. In addition, tissue-infiltrating Th17 cells are also characterized by high cell surface expression of CCR6, a chemokine receptor that was not expressed by Th1 and Th2 cells, isolated from the same lesions, and by the production of CCL20/MIP3alpha, a CCR6 ligand, associated with tissue infiltration. Culture supernatants of activated Th17 cells, isolated from psoriatic lesions, induced the expression of gene products associated with inflammation and abnormal keratinocyte differentiation in an IL-17 and IL-22-dependent manner. These results show that tissue-infiltrating Th17 cells contribute to human chronic inflammatory disease via the production of several inflammatory cytokines and the creation of an environment contributing to their migration and sequestration at sites of inflammation.     

IFN-alpha is not sufficient to drive Th1 development due to lack of stable T-bet expression

During inflammatory immune responses, the innate cytokine IL-12 promotes CD4+ Th-1 development through the activation of the second messenger STAT4 and the subsequent expression of T-bet. In addition, type I IFN (IFN-alphabeta), secreted primarily during viral and intracellular bacterial infections, can promote STAT4 activation in human CD4+ T cells. However, the role of IFN-alphabeta in regulating Th1 development is controversial, and previous studies have suggested a species-specific pathway leading to Th1 development in human but not mouse CD4+ T cells. In this study, we found that although both IFN-alpha and IL-12 can promote STAT4 activation, IFN-alpha failed to promote Th1 commitment in human CD4+ T cells. The difference between these innate signaling pathways lies with the ability of IL-12 to promote sustained STAT4 tyrosine phosphorylation, which correlated with stable T-bet expression in committed Th1 cells. IFN-alpha did not promote Th1 development in human CD4+ T cells because of attenuated STAT4 phosphorylation, which was insufficient to induce stable expression of T-bet. Further, the defect in IFN-alpha-driven Th1 development was corrected by ectopic expression of T-bet within primary naive human CD4+ T cells. These results indicate that IL-12 remains unique in its ability to drive Th1 development in human CD4+ T cells and that IFN-alpha lacks this activity due to its inability to promote sustained T-bet expression.     

The homeostasis but not the differentiation of T cells is regulated by p27(Kip1)

The cyclin-dependent kinase inhibitor p27(Kip1) is a critical regulator of T cell proliferation. To further examine the relationship of T cell proliferation and differentiation, we examined the ability of T cells deficient in p27(Kip1) to differentiate into Th subsets. We observed increased Th2 differentiation in p27(Kip1)-deficient cultures. In addition to increases in CD4(+) and CD8(+) T cells, there is a similar increase in gamma delta T cells in p27(Kip1)-deficient mice compared with wild-type mice. The increase in Th2 differentiation is correlated to an increase of IL-4 secretion by CD4(+)DX5(+)TCR alpha beta(+)CD62L(low) T cells but not to increased expansion of differentiating Th2 cells. While STAT4- and STAT6-deficient T cells have diminished proliferative responses to IL-12 and IL-4, respectively, proliferative responses are increased in T cells doubly deficient in p27(Kip1) and STAT4 or STAT6. In contrast, the increased proliferation and differentiative capacity of p27(Kip1)-deficient T cells has no effect on the ability of STAT4/p27(Kip1)- or STAT6/p27(Kip1)-deficient CD4(+) cells to differentiate into Th1 or Th2 cells, respectively. Thus, while p27(Kip1) regulates the expansion and homeostasis of several T cell subsets, it does not affect the differentiation of Th subsets.     

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.     

IL-17+ regulatory T cells in the microenvironments of chronic inflammation and cancer

Foxp3(+)CD4(+) regulatory T (Treg) cells inhibit immune responses and temper inflammation. IL-17(+)CD4(+) T (Th17) cells mediate inflammation of autoimmune diseases. A small population of IL-17(+)Foxp3(+)CD4(+) T cells has been observed in peripheral blood in healthy human beings. However, the biology of IL-17(+)Foxp3(+)CD4(+) T cells remains poorly understood in humans. We investigated their phenotype, cytokine profile, generation, and pathological relevance in patients with ulcerative colitis. We observed that high levels of IL-17(+)Foxp3(+)CD4(+) T cells were selectively accumulated in the colitic microenvironment and associated colon carcinoma. The phenotype and cytokine profile of IL-17(+)Foxp3(+)CD4(+) T cells was overlapping with Th17 and Treg cells. Myeloid APCs, IL-2, and TGF-β are essential for their induction from memory CCR6(+) T cells or Treg cells. IL-17(+)Foxp3(+)CD4(+) T cells functionally suppressed T cell activation and stimulated inflammatory cytokine production in the colitic tissues. Our data indicate that IL-17(+)Foxp3(+) cells may be "inflammatory" Treg cells in the pathological microenvironments. These cells may contribute to the pathogenesis of ulcerative colitis through inducing inflammatory cytokines and inhibiting local T cell immunity, and in turn may mechanistically link human chronic inflammation to tumor development. Our data therefore challenge commonly held beliefs of the anti-inflammatory role of Treg cells and suggest a more complex Treg cell biology, at least in the context of human chronic inflammation and associated carcinoma.     

Glycogen synthase kinase-3 is an early determinant in the differentiation of pathogenic Th17 cells

CD4(+) T cells are critical for host defense but are also major drivers of immune-mediated diseases. The classical view of Th1 and Th2 subtypes of CD4(+) T cells was recently revised by the identification of the Th17 lineage of CD4(+) T cells that produce IL-17, which have been found to be critical in the pathogenesis of autoimmune and other diseases. Mechanisms controlling the differentiation of Th17 cells have been well described, but few feasible targets for therapeutically reducing Th17 cells are known. The generation of Th17 cells requires IL-6 and activation of STAT3. During polarization of CD4(+) T cells to Th17 cells, we found that inhibition of glycogen synthase kinase-3 (GSK3) blocked IL-6 production, STAT3 activation, and polarization to Th17 cells. Polarization of CD4(+) T cells to Th17 cells increased by 10-fold the expression of GSK3β protein levels in Th17 cells, whereas GSK3β was unaltered in regulatory T cells. Diminishing GSK3 activity either pharmacologically or molecularly blocked Th17 cell production, and increasing GSK3 activity promoted polarization to Th17 cells. In vivo inhibition of GSK3 in mice depleted constitutive Th17 cells in intestinal mucosa, blocked Th17 cell generation in the lung after Francisella tularensis infection, and inhibited the increase in spinal cord Th17 cells and disease symptoms in the experimental autoimmune encephalomyelitis mouse model of multiple sclerosis. These findings identify GSK3 as a critical mediator of Th17 cell production and indicate that GSK3 inhibitors provide a potential therapeutic intervention to control Th17-mediated diseases.     

Depressed IL-12-mediated signal transduction in T cells from patients with Sézary syndrome is associated with the absence of IL-12 receptor beta 2 mRNA and highly reduced levels of STAT4.

Sézary syndrome (SS) is the leukemic phase of cutaneous T cell lymphoma characterized by the proliferation of clonally derived CD4+ T cells that release cytokines of the Th2 T cell phenotype (IL-4, IL-5, IL-10), whereas Th1 T cell cytokines (IL-2, IFN-gamma) are markedly depressed as is expression of IL-12, a pivotal cytokine for Th1 cell differentiation. Normal Th1 cells express both the beta 1 and beta 2 chains of the IL-12 receptor (IL-12R) and tyrosine phosphorylate STAT4 in response to IL-12. Th2 T cells express only the IL-12R beta 1 and thus do not tyrosine phosphorylate STAT4 in response to IL-12. To determine whether SS cells are Th2-like at the level of IL-12 signal transduction, we analyzed RNA from seven patients for the presence of message for the IL-12R beta 1 and beta 2 genes using RNase protection assays and assessed whether IL-12 induced tyrosine-phosphorylation of STAT4 by immunoblotting. In PBL from six of seven SS patients tested, beta 2 message was expressed at low to undetectable levels and its expression could not be stimulated by either IFN-alpha or IFN- gamma, which stimulated beta 2 expression in control PBL. The absence of beta 2 expression is further supportive evidence for the Th2 lineage of SS cells. However, unlike normal Th2 cells, SS cells also showed severely reduced levels of STAT4, suggesting that they have a depressed response to any inducer of the STAT4 signal transduction pathway, including IFN-alpha. This is the first observation linking STAT4 gene expression with a human disease and suggests that dysregulation of STAT4 expression may be significant to the development and/or progression of SS.     

Tissue specificity of E- and P-selectin ligands in Th1-mediated chronic inflammation.

The demonstrated role of E- and P-selectin ligands in the recruitment of Th1 cells raises the question of tissue specificity determination by pathogenic T cells. We took advantage of the fact that chronic Th1-mediated inflammation in the scid/scid CD4+CD45RBhigh T cell transfer model can occur at multiple tissue sites, resembling inflammatory bowel disease in the colon and psoriasis in the skin. We show that the majority of infiltrating effector T cells from psoriatic skin expresses high levels of functional P-selectin ligand (87 +/- 3%), detected by P-selectin-Ig (PIg), while a significantly smaller subset of T cells from colitic lesions expresses this ligand (24 +/- 2%). Similarly, E-selectin ligand is preferentially expressed on CD4+ T cells infiltrating the skin (24 +/- 2%), but only on very few CD4+ T cells infiltrating the colon (CIT; 1.3 +/- 0.8%). In contrast, CD4+ T cells infiltrating the skin express alpha4beta7 at a significantly lower level than CIT (mean fluorescence intensity, 28 vs 61, respectively), although, interestingly, alphaEbeta7 was expressed at high levels on both populations. Analysis of the disease-inducing potential of PIg+ and PIg- CD4+ CIT cells revealed that both populations not only express similar levels of the gut-homing molecule alpha4beta7 (mean fluorescence intensity, 50 vs 56, respectively), but do not differ in their capacity to express IFN-gamma. Furthermore, CIT depleted of cells expressing functional P-selectin ligand were able to induce colitis upon transfer, suggesting that induction of colitis in this model may be independent of E- and P-selectin. These results indicate that adhesion molecule expression and the homing pattern of inflammatory T cells are regulated by the local environment independently of their inflammatory capacity.     

Lactate Regulates Metabolic and Pro-inflammatory Circuits in Control of T Cell Migration and Effector Functions

Lactate has long been considered a “waste” by-product of cell metabolism, and it accumulates at sites of inflammation. Recent findings have identified lactate as an active metabolite in cell signalling, although its effects on immune cells during inflammation are largely unexplored. Here we ask whether lactate is responsible for T cells remaining entrapped in inflammatory sites, where they perpetuate the chronic inflammatory process. We show that lactate accumulates in the synovia of rheumatoid arthritis patients. Extracellular sodium lactate and lactic acid inhibit the motility of CD4⁺ and CD8⁺ T cells, respectively. This selective control of T cell motility is mediated via subtype-specific transporters (Slc5a12 and Slc16a1) that we find selectively expressed by CD4⁺ and CD8⁺ subsets, respectively. We further show both in vitro and in vivo that the sodium lactate-mediated inhibition of CD4⁺ T cell motility is due to an interference with glycolysis activated upon engagement of the chemokine receptor CXCR3 with the chemokine CXCL10. In contrast, we find the lactic acid effect on CD8⁺ T cell motility to be independent of glycolysis control. In CD4⁺ T helper cells, sodium lactate also induces a switch towards the Th17 subset that produces large amounts of the proinflammatory cytokine IL-17, whereas in CD8⁺ T cells, lactic acid causes the loss of their cytolytic function. We further show that the expression of lactate transporters correlates with the clinical T cell score in the synovia of rheumatoid arthritis patients. Finally, pharmacological or antibody-mediated blockade of subtype-specific lactate transporters on T cells results in their release from the inflammatory site in an in vivo model of peritonitis. By establishing a novel role of lactate in control of proinflammatory T cell motility and effector functions, our findings provide a potential molecular mechanism for T cell entrapment and functional changes in inflammatory sites that drive chronic inflammation and offer targeted therapeutic interventions for the treatment of chronic inflammatory disorders.     

Microenvironment-dependent requirement of STAT4 for the induction of P-selectin ligands and effector cytokines on CD4+ T cells in healthy and parasite-infected mice

T effector cells require selectin ligands to migrate into inflamed regions. In vitro, IL-12 promotes induction of these ligands as well as differentiation of CD4+ T cells into IFN-gamma-producing Th1 but not Th2 cells. STAT4 is strongly involved in these processes. However, the presence of selectin ligands on various T effector cell subsets in vivo points to more complex regulatory pathways. To clarify the role of the IL-12/STAT4 signaling pathway, we analyzed the impact of STAT4 deficiency on the expression of P-selectin ligands (P-lig) on CD4+ T cells in vitro and in vivo, including conditions of infection. In vitro, we found significant expression of P-lig upon activation not only in the presence, but also in the absence, of IL-12, which was independent of STAT4. TGF-beta, an alternative inducer of selectin ligands in human T cells, was not effective in murine CD4+ T cells, suggesting a role of additional signaling pathways. In vivo, a significant impact of STAT4 for the generation of P-lig+CD4+ T cells was observed for cells from peripheral lymph nodes, but not for those from spleen or lung. However, upon infection with the Th2-inducing parasite Nippostrongylus brasiliensis, P-lig expression became dependent on STAT4 signaling. Interestingly, also the frequency of IL-4-producing cells was greatly diminished in absence of STAT4. These data reveal a hitherto unknown contribution of STAT4 to the generation of Th2 cells in parasite infection and suggest that signals inducing inflammation-seeking properties in vivo vary depending on environmental conditions, such as type of organ and infection.