Interleukin-17C promotes Th17 cell responses and autoimmune disease via interleukin-17 receptor E.

Although several interleukin-17 (IL-17) family members and their receptors have been recently appreciated as important regulators in inflammatory diseases, the function of other IL-17 cytokines and IL-17 receptor-like molecules is unclear. Here we show that an IL-17 cytokine family member, IL-17C, was induced in a Th17 cell-dependent autoimmune disease and was required for its pathogenesis. IL-17C bound to IL-17RE, a member of IL-17 receptor family whose full-length isoform was selectively expressed in Th17 cells and signaled via an IL-17RA-RE receptor complex and the downstream adaptor Act1. IL-17C-IL-17RE induced the expression of a nuclear IkappaB family member, IκBζ, in Th17 cells to potentiate the Th17 cell response. Thus, our work has identified a cytokine-receptor pair with important function in regulating proinflammatory responses. This pathway may be targeted to treat autoimmune diseases.

Toll-like receptor 2 signaling in CD4(+) T lymphocytes promotes T helper 17 responses and regulates the pathogenesis of autoimmune disease.

Toll-like receptors (TLRs) have previously been shown to play critical roles in the activation of innate immunity. Here, we describe that T cell expression of TLR2 regulates T helper 17 (Th17) cell responses. Stimulation with TLR2 agonists promoted Th17 differentiation in vitro and led to more robust proliferation and Th17 cytokine production. Using the experimental autoimmune encephalomyelitis (EAE) model, we found that TLR2 regulated Th17 cell-mediated autoimmunity in vivo and that loss of TLR2 in CD4(+) T cells dramatically ameliorated EAE. This study thus reveals a critical role of a TLR in the direct regulation of adaptive immune response and pathogenesis of autoimmune diseases.

Molecular antagonism and plasticity of regulatory and inflammatory T cell programs.

Regulatory T (Treg) and T helper 17 (Th17) cells were recently proposed to be reciprocally regulated during differentiation. To understand the underlying mechanisms, we utilized a Th17 reporter mouse with a red fluorescent protein (RFP) sequence inserted into the interleukin-17F (IL-17F) gene. Using IL-17F-RFP together with a Foxp3 reporter, we found that the development of Th17 and Foxp3(+) Treg cells was associated in immune responses. Although TGF-beta receptor I signaling was required for both Foxp3 and IL-17 induction, SMAD4 was only involved in Foxp3 upregulation. Foxp3 inhibited Th17 differentiation by antagonizing the function of the transcription factors RORgammat and ROR*. In contrast, IL-6 overcame this suppressive effect of Foxp3 and, together with IL-1, induced genetic reprogramming in Foxp3(+) Treg cells. STAT3 regulated Foxp3 downregulation, whereas STAT3, RORgamma, and ROR* were required for IL-17 expression in Treg cells. Our data demonstrate molecular antagonism and plasticity of Treg and Th17 cell programs.

TL1A-DR3 interaction regulates Th17 cell function and Th17-mediated autoimmune disease.

T helper type 17 (Th17) cells play an important pathogenic function in autoimmune diseases; their regulation, however, is not well understood. We show that the expression of a tumor necrosis factor receptor family member, death receptor 3 (DR3; also known as TNFRSF25), is selectively elevated in Th17 cells, and that TL1A, its cognate ligand, can promote the proliferation of effector Th17 cells. To further investigate the role of the TL1A-DR3 pathway in Th17 regulation, we generated a TL1A-deficient mouse and found that TL1A(-/-) dendritic cells exhibited a reduced capacity in supporting Th17 differentiation and proliferation. Consistent with these data, TL1A(-/-) animals displayed decreased clinical severity in experimental autoimmune encephalomyelitis (EAE). Finally, we demonstrated that during EAE disease progression, TL1A was required for the optimal differentiation as well as effector function of Th17 cells. These observations thus establish an important role of the TL1A-DR3 pathway in promoting Th17 cell function and Th17-mediated autoimmune disease.

Regulatory mechanisms of helper T cell differentiation: new lessons learned from interleukin 17 family cytokines.

Interleukin 17 (IL-17) family consists of six cytokines in mammals. Among them, IL-17 and IL-17F are expressed by a novel subset of CD4+ helper T (Th) cells and play critical function in inflammation and autoimmunity. On the other hand, IL-17E, also called IL-25, has been associated with allergic responses. Here we summarize recent work by us as well as other investigators in understanding the regulation and function of these three cytokines. From these studies, IL-17 family cytokines may serve as novel targets for pharmaceutical intervention of immune and inflammatory diseases.

T helper 17 lineage differentiation is programmed by orphan nuclear receptors ROR alpha and ROR gamma.

T cell functional differentiation is mediated by lineage-specific transcription factors. T helper 17 (Th17) has been recently identified as a distinct Th lineage mediating tissue inflammation. Retinoic acid receptor-related orphan receptor gamma (ROR gamma) was shown to regulate Th17 differentiation; ROR gamma deficiency, however, did not completely abolish Th17 cytokine expression. Here, we report Th17 cells highly expressed another related nuclear receptor, ROR alpha, induced by transforming growth factor-beta and interleukin-6 (IL-6), which is dependent on signal transducer and activator of transcription 3. Overexpression of ROR alpha promoted Th17 differentiation, possibly through the conserved noncoding sequence 2 in Il17-Il17f locus. ROR alpha deficiency resulted in reduced IL-17 expression in vitro and in vivo. Furthermore, ROR alpha and ROR gamma coexpression synergistically led to greater Th17 differentiation. Double deficiencies in ROR alpha and ROR gamma globally impaired Th17 generation and completely protected mice against experimental autoimmune encephalomyelitis. Therefore, Th17 differentiation is directed by two lineage-specific nuclear receptors, ROR alpha and ROR gamma.

Phosphorylation and ubiquitination of the IkappaB kinase complex by two distinct signaling pathways.

The IkappaB kinase (IKK) complex serves as the master regulator for the activation of NF-kappaB by various stimuli. It contains two catalytic subunits, IKKalpha and IKKbeta, and a regulatory subunit, IKKgamma/NEMO. The activation of IKK complex is dependent on the phosphorylation of IKKalpha/beta at its activation loop and the K63-linked ubiquitination of NEMO. However, the molecular mechanism by which these inducible modifications occur remains undefined. Here, we demonstrate that CARMA1, a key scaffold molecule, is essential to regulate NEMO ubiquitination upon T-cell receptor (TCR) stimulation. However, the phosphorylation of IKKalpha/beta activation loop is independent of CARMA1 or NEMO ubiquitination. Further, we provide evidence that TAK1 is activated and recruited to the synapses in a CARMA1-independent manner and mediate IKKalpha/beta phosphorylation. Thus, our study provides the biochemical and genetic evidence that phosphorylation of IKKalpha/beta and ubiquitination of NEMO are regulated by two distinct pathways upon TCR stimulation.

B cell lymphoma 10 is essential for FcepsilonR-mediated degranulation and IL-6 production in mast cells.

The adaptor protein B cell lymphoma 10 (Bcl10) plays an essential role in the functions of the AgRs in T and B cells. In this study, we report that Bcl10 also plays an important role in mast cells. Bcl10 is expressed in mast cells. Although Bcl10-deficient mast cells undergo normal development, we demonstrate that Bcl10 is essential for specific functions of FcepsilonR. Although Bcl10-deficient mast cells have normal de novo synthesis and release of the lipid mediator arachidonic acid, the mutant cells possess impaired FcepsilonR-mediated degranulation, indicated by decreased serotonin release, and impaired cytokine production, measured by release of IL-6. In addition, Bcl10-deficient mice display impaired IgE-mediated passive cutaneous anaphylaxis. Moreover, although Bcl10-deficient mast cells have normal FcepsilonR-mediated Ca(2+) flux, activation of PI3K, and activation of the three types of MAPKs (ERKs, JNK, and p38), the mutant cells have markedly diminished FcepsilonR-mediated activation of NF-kappaB and decreased activation of AP-1. Thus, Bcl10 is essential for FcepsilonR-induced activation of AP-1, NF-kappaB, degranulation, and cytokine production in mast cells.

The CARMA1-Bcl10 signaling complex selectively regulates JNK2 kinase in the T cell receptor-signaling pathway.

Members of the c-Jun NH(2)-terminal kinase (JNK) family play crucial roles in cell activation, differentiation, and apoptosis. Although many studies have indicated that JNK1 and JNK2 have functional differences and redundancy, the upstream signaling pathway that selectively activates JNK1 or JNK2 remains unknown. In this study, we have revealed a selective mechanism of JNK activation, in which JNK2, but not JNK1, was regulated by CARMA1, a scaffold molecule, after stimulation of the T cell receptor (TCR). This CARMA1-dependent regulation of JNK2 worked through the scaffold molecule Bcl10, which was inducibly associated with JNK2 and served as a JNK-interacting protein (JIP)-like scaffold to assemble the kinases JNK2, MKK7, and TAK1. Finally, we showed that CARMA1- and Bcl10-mediated JNK2 activation had a critical role in regulating the amount of c-Jun protein. Together, our studies provide genetic evidence that JNK1 and JNK2 are differentially regulated in the TCR-signaling pathway and play different functions.

Measurement of interleukin-17.

Upon antigenic stimulation, naive CD4+ T cells undergo proliferation and differentiate into cytokine-producing T helper (T(H)) effector cells. T(H)1 cells secrete effector cytokine IFN-gamma and regulate cell-mediated immunity, whereas T(H)2 cells produce IL-4, IL-5, and IL-13 cytokines, and mediate immunity against extracellular pathogens and allergic reactions. Recent studies have identified a novel T(H) subset, called T(H)17, TH(IL-17), or inflammatory T(H) (THi) cells, characterized by the production of a proinflammatory cytokine, IL-17, and regulating inflammatory responses. In this unit, we describe the protocols for the differentiation of mouse IL-17-expressing T cells in vitro, detection of IL-17-expressing T cells by intracellular cytokine staining, and measurement of IL-17 secretion in culture supernatants by ELISA. Generation of IL-17-expressing T cells in vitro under defined culture conditions allows investigation of their differentiation regulation. Detection of IL-17 in cell culture and tissue samples helps in monitoring inflammatory diseases and determining efficacy of therapeutic interventions.

Potential role of CARMA1 in CD40-induced splenic B cell proliferation and marginal zone B cell maturation.

NF-kappaB activation through B cell receptor (BCR) ligation is critical for B cell development, survival and antigen-mediated activation of B cells. CARD domain and MAGUK-domain containing protein-1 (CARMA1), recently identified adaptor molecule, has been shown to play an essential role in BCR-induced NF-kappaB activation. CARMA1-deficient B cells fail to proliferate upon BCR stimulation, leading to defective humoral responses. Surprisingly, CARMA1-deficient B cells are also defective in CD40-induced proliferation. The mechanisms responsible for CD40-induced proliferation defect have not yet been characterized. In this study, we show that signaling cascades activated by CD40 stimulation are largely unaffected in CARMA1-deficient B cells. Instead, we have found that the defective proliferation of CARMA1-deficient B cells is due to two events. First, CARMA1-deficient B cells show defective cell-cycle progression. Secondly, the numbers of marginal zone (MZ) B cells, which are the main responders upon CD40 stimulation, are greatly diminished in CARMA1-deficient mice. Since B cell maturation requires basal signaling through BCR and NF-kappaB activation, we propose that impaired BCR signaling in CARMA1-deficient mice leads to defective maturation of MZ B cell population, which in turn, contributes to impaired proliferation upon CD40 stimulation.

Alteration of cell surface sialylation regulates antigen-induced naive CD8+ T cell responses.

The strength of interactions with APC instructs naive T cells to undergo programmed expansion and differentiation, which is largely determined by the peptide affinity and dose as well as the duration of TCR ligation. Although, most ligands mediating these interactions are terminally sialylated, the impact of the T cell sialylation status on Ag-dependent response remains poorly understood. In this study, by monitoring TCR transgenic CD8+ T cells, OT-I, we show that biochemical desialylation of naive OT-I T cells increases their sensitivity for agonist as well as partial agonist peptides. Desialylation enhances early activation and shortens the duration of TCR stimulation required for proliferation and differentiation, without increasing apoptosis. Moreover, desialylation of naive OT-I T cells augments their response to tumor-presented Ag. These results provide direct evidence for a regulatory role for sialylation in Ag-dependent CD8+ T cell responses and offer a new approach to sensitize or dampen Ag-specific CD8+ T cell responses.