Threshold cycle numbers (Ct) were determined with Sequence Detector Software (version 2.1.1; PE Applied Biosystems) and transformed using a standard curve generated from serially diluted cDNA samples from WT cells. IL-23, and Th17 cytokine expression in differentiated Th17 cells. Importantly, blockade of IL-1 signaling by IL-1RA inhibited Th17 differentiation and IL-23-induced cytokine expression in differentiated Th17 cells. The results of these studies demonstrate that IL-1-mediated IRAK4 kinase activity in T cells is essential for induction of IL-23 receptor expression, Th17 differentiation, and autoimmune disease. Both, IL-6 and IL-21 bind to their respective receptors and activate STAT3, which is necessary for the induction of IL-17. In addition to STAT3, two additional related transcription factors, ROR and RORt, have been recently shown to be selectively expressed in Th17 cells. The expression of both transcription factors is necessary and sufficient for IL-17 expression. Overexpression of ROR and/or RORt in CD4+ T cells is sufficient to induce IL-17 production in the Pamabrom absence of exogenous cytokines, whereas the deficiency of either factor alone leads to partial inhibition of IL-17 production. Loss of both factors completely blocks Th17 cell differentiation and cytokine production (18). Consistent with their unique expression, the expression of ROR and RORt is increased by TGF- and IL-6 during Th17 differentiation and both are now considered Th17 lineage specific transcription factors (18). In addition to TGF- and IL-6, IL-23 is another critical cytokine involved in Th17 differentiation, cell expansion/survival and stabilization (19). The importance of IL-23 in Th17-mediated inflammation has been revealed mainly through studies in mice. Mice lacking IL-23p19 were shown to be resistant to disease induction in Th17 cell-dependent CIA, EAE, and IBD diseases models (20C22). Administration of anti-IL-23p19 monoclonal antibody (mAb) or anti-IL-12p40 mAb inhibited the production of multiple Pamabrom inflammatory cytokines including IL-17, IL-6, IFN-, IL-1, and TNF-, and consequently, reduced EAE development (20C22). Furthermore, adoptive transfer of myelin oligodendrocyte (MOG33-55)-specific IL-17-producing Pamabrom T cells but not IFN–producing T cells can efficiently induce EAE in recipient mice, indicating that Th17 cells, not Th1 cells, are the pathological effector T cells in this autoimmune disease model (20). Although IL-23 seems to be more directly involved in maintaining or stabilizing Th17 cells, it has been recently shown that IL-23 along with TGF- and inflammatory cytokines such as IL-6 and IL-1 is essential for human Th17 differentiation and cytokine modulation (23, 24). Sutton et al. recently demonstrated that IL-1 also plays a vital role in promoting antigen-specific Th17 cells in response to immunization with foreign or self-antigen and a TLR ligand (25). Upon binding to IL-1R, IL-1 triggers the binding of intracellular adaptor protein, MyD88, to the receptor and consequently leads to the recruitment of IL-1R associated kinases (IRAKs), IRAK4 and IRAK1, to the receptor complex (26). IRAK1 is then phosphorylated, ubiquitinylated and degraded resulting in activation of multiple downstream signaling pathways including JNK, p38, and NF-B (26). IRAK4 is pivotal for IL-1R and TLR-induced signaling and deficiency of IRAK4 in mice abolishes the innate immune response (27). However, the role of IRAK4 in the adaptive immune response is still controversial. IRAK4 has been shown to be recruited to T cell lipid rafts upon T cell receptor activation and IRAK4 deficiency led to impaired T cell responses H37 Ra (Difco) on days 0 and 7, supplemented with intraperotoneal (i.p.) injections of 500 ng pertussis toxin (Calbiochem, San Diego, CA) on days 0 and 2. Clinical symptoms of EAE were scored daily by two separate observers using the following scale; 0 = no symptoms, 0.5 = distal weak or spastic tail, 1 = completely limp tail, 1.5 = limp tail and hind limb weakness (feet slip through cage grill), 2.0 = unilateral partial hind limb paralysis, 2.5 = bilateral partial hind limb paralysis, 3.0 = complete bilateral hind limb paralysis, 3.5 = complete hind limb and unilateral partial forelimb paralysis, 4.0 = moribund or death. The data was recorded as the mean daily clinical score. For Adoptive transfer of EAE, mice were immunized with MOG35-55 plus CFA via conditions that induce active EAE. Lymph nodes were collected 10 days later and single cell suspensions were prepared. Cells (6 106 cells/ml) were cultured in RPMI 1640 medium (supplemented with 10% FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, 100 IU/ml penicillin/streptomycin and 2 10?5 M 2-ME) with MOG35-55 (20 g/ml) and IL-23 (20 ng/ml). Four days after initiation of culture, cells were harvested, washed in PBS and then injected into recipient mice (2 107 cells per mouse) sublethally irradiated (600 rad) 4h before injection. Histology, immunohistochemistry, and flow cytometry MOG35-55-immunized IRAK4 KI and WT mice were euthanized at days 21C25. Spinal cords were removed, fixed overnight in IHC Zinc CDC25A fixative (BD Biosciences PharMingen, San.