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Additional file 2: Supplementary Figure S2. Mean cumulative scores of EAE after active or passive induction. (a) Steap4+/-and Steap4-/- mice were induced to develop EAE by active immunization with MOG35-55; Mean cumulative scores are shown. Related to Fig. 1 b; (b) Steap4+/-and Steap4-/- mice were induced to develop EAE by MOG35-55-specific Th17 cell adoptive transfer, Mean cumulative scores are shown. Related to Fig. 3 b; (c) Nestin-Cre Steap4fl/+ and Nestin-Cre Steap4fl/fl mice were induced to develop EAE by active immunization with MOG35-55; Mean cumulative scores are shown. Related to Fig. 4 c; (d) Nestin-Cre Steap4fl/+ and Nestin-Cre Steap4fl/fl mice were induced to develop EAE by MOG35-55-specific Th17 cell adoptive transfer, Mean cumulative scores are shown. Related to Fig. 5 a. Data are representative of three independent experiments. n=5/group in each experiment. Error bars, SEM. p values were determined by Mann-Whitney test and shown in each panel.

Additional file 1: Supplementary Figure S1. EAE incidence after active or passive EAE induction. (a) Steap4+/-and Steap4-/- mice were induced to develop EAE by active immunization with MOG35-55; Disease incidence are shown. Related to Fig.1 b; related to Fig. 1 b; (b). MOG35-55-specific Th17 cells from Steap4+/-and Steap4-/- mice were used as donor cells and transferred to naïve wild-type recipient mice. Graph represents EAE incidence after MOG35-55-specific Th17-cell transfer, related to Fig. 2 c; (c) MOG35-55-specific Th17 cells from wild-type mice were used as donor cells and transferred to naïve Steap4-/- mice and the Steap4+/- littermate controls. Graph represents the EAE incidence after MOG35-55-specific Th17-cell transfer, related to Fig. 3 b; (d) MOG35-55-specific Th1 cells from wild-type mice were used as donor cells and transferred to naïve Steap4+/-and Steap4-/- mice. Graph represents the EAE incidence after Th1-cell transfer, related to Fig. 3 f; (e) EAE incidence in Nestin-Cre Steap4fl/+ and Nestin-Cre Steap4fl/fl mice are shown after MOG35-55-specific Th17 cell adoptive transfer, related to Fig. 5 a; (f) EAE incidence in Nestin-Cre Steap4fl/+ and Nestin-Cre Steap4fl/fl mice are shown after MOG35-55-specific Th1 cell adoptive transfer, related to Fig. 5 e. Data are representative of three independent experiments. n=5/group in each experiment. p values were determined by Log-rank test and shown in each panel.

Additional file 3: Supplementary Figure S3. Expression pattern and deletion specificity of Steap4. (a) Spinal cords were harvested from MOG immunized mice at pre-symptomatic stage (clinical score =0), disease onset (clinical score 2~3), peak of the disease (clinical score 4~5) and remission (clinical score 4~3). Harvested spinal cords were analyzed for Steap4 expression by RT-PCR. The average 2-ΔCt values of Steap4 in pre-symptomatic spinal cords were set as 1. The fold changes in spinal cords from disease onset, peak of disease and remission were calculated by divide the 2-ΔCt value of individual biological sample (a spinal cord) by the average 2-ΔCt values of Steap4 in pre-symptomatic spinal cords, which is set as 1. The P value for one-way ANOVA analysis is

The cellular and molecular mechanisms driven by IL-25 and its cognate receptor IL-17RB necessary for the promotion of T helper type 2 (Th2) mediating pathogenic pulmonary inflammation remain to be defined. We have previously reported the critical role of the U-box-type E3 ubiquitin ligase Act1 (1), for the downstream signaling of the IL-17 cytokine family including the Th2 promoting cytokine IL-25 (IL-17E) (2). In this study, we report that IL-25 driven but not conventional IL-4 driven Th2 polarization and cytokine production is impaired in Act1-deficient T cells. Also, Act1 deficiency in the T cell compartment results in the abrogation of eosinophilic airway infiltration as well as airway hyperresponsiveness (AHR) in a mouse model of antigen induced airway inflammation. The in vivo generation of antigen specific Th2 cytokine producing cells is defective in the absence of Act1 expression in T cells following ovalbumin/Alum immunization. Interestingly, the production of ovalbumin specific IgG1 but not IgG2a or IgE is also impaired. At the molecular level, we report that IL-25-mediated induction of Th2 master regulator GATA-3, and the transcription factor GFI-1 is attenuated in Act1-deficient T cells. Taken together, our findings indicated that Act1 expression in T cells is required for cellular and humoral Th2-mediated allergic responses and the development of AHR, in part, through its function in IL-25 induced development of Th2 T cells.

Bone marrow-derived plasmacytoid dendritic cells (pDCs) from IL-1R-associated kinase (IRAK)2-deficient mice produced more IFNs than did wild-type pDCs upon stimulation with the TLR9 ligand CpG. Furthermore, in CpG-stimulated IRAK2-deficient pDCs there was increased nuclear translocation of IFN regulatory factor 7, the key transcription factor for IFN gene transcription in these cells. In IRAK2-deficient macrophages, enhanced NF-κB activation and increased expression of CpG-induced genes were detected within 2 h after treatment. However, at later times, NF-κB activation was decreased and, in contrast to the results with IFN, there was less secretion of other proinflammatory cytokines (such as TNF-α) and chemokines in CpG-stimulated IRAK2-deficient pDCs and macrophages. Therefore, although IRAK2 is a negative regulator of TLR9-mediated IFN production through its modulation of the transcriptional activity of IFN regulatory factor 7, it is also a positive regulator of TLR9-mediated proinflammatory cytokine and chemokine production at some level subsequent to transcription.

As the pathophysiology of acute myelogenous leukemia (AML) involves a block of myeloid maturation, a desirable therapeutic strategy is to induce leukemic cell maturation to increase the efficacy and to avoid the side effects of traditional chemotherapeutics. Through a compound library screen, 6-benzylthioinosine (6BT) was identified as a promising differentiation-inducing agent. 6BT induces monocytic differentiation of myeloid leukemia cell lines such as HL-60 and OCI-AML3, as well as primary patient samples as evidenced by morphology, immunophenotyping, and nitroblue tetrazolium reduction. Not only can 6BT induce differentiation but a subset of AML cell lines such as MV4-11 and HNT34 instead undergo 6BT-mediated cell death. Despite inducing cell death in some leukemic cells, 6BT exhibits extremely low toxicity on several nonmalignant cells such as fibroblasts, normal bone marrow, and endothelial cells. This toxicity profile may relate to the function of 6BT as an inhibitor of the nucleoside transporter, ent1, which is thought to prevent it from entering many cell types. In contrast, 6BT likely enters at least some leukemic cell lines as shown by its requirement for phosphorylation for its differentiation activity. 6BT is also able to synergize with currently used myeloid differentiation agents such as ATRA and decitabine. Early studies indicate that the mechanism of action of this compound may involve ATP depletion that leads to growth inhibition and subsequent differentiation. Besides in vitro activity, 6BT also shows the ability to impair HL-60 and MV4-11 tumor growth in nude mice. 6BT is a promising new monocytic differentiation agent with apparent leukemic cell–specific activity.