Nuclear IκB family proteins form a complex with NF-κB and control NF-κB target gene regulation. IκB-ζ, a member of this protein family, can be induced in T cells in response to TGF-β+IL-6 and positively regulates Th17 differentiation. Thus, IκB-ζ-deficient mice are able to resist some Th17-dependent autoimmune diseases, such as experimental autoimmune encephalomyelitis (EAE). However, these mice develop different types of autoimmune disorders such as Sjogren syndrome with advanced age, limiting the utility of IκB-ζ as a therapeutic target. In this study, we focus on another nuclear IκB family protein, IκB NS (IκB NS is a most similar homolog of IκB-ζ) and asked whether IκB NS have a potential therapeutic target of EAE without any immune dysregulations.Ankyrin-repeatsAnkyrin-repeats Transactivation domain NuclearLocalizeSignal I  B-  I  B NS 43% match ・ Less Th17 differentiation Nature ;464: ・ Sjogren syndrome like diseases Immunity ;38: ・ Not observe immune dysregulation with ages Deficient mice Plasmids: Expression vectors encoding FLAG-tagged mouse I  B-  were constructed as described previously (JBC : ). Mouse I  B NS was inserted into a pcDNA3-FLAG vector at the EcoRI and BamHI sites. Mouse IL-17A promoter (-6647 to +1) was inserted into a pGL4.12 vector at the NheI & HindIII sites. EAE induction: Mice were injected subcutaneously (on the lower back) on day 0 with emulsions containing complete Freund’s adjuvant (CFA), 100 μg MOG peptide, and 0.5 mg Mycobacterium tuberculosis H37RA. In addition, these mice received 500 ng pertussis toxin by i.p. injection to boost immunological responses on day 0 and 2. In vitro T cell culture: Purified CD4 + CD25 - T cells were cultured in RPMI 1640 medium containing 10% heat-inactivated fetal calf serum at 37°C in 5% CO 2. Th0 condition: anti-CD3 (1 μg/mL) + anti-CD28 (1 μg/mL) stimulation. Th17 condition: anti-CD3 (1 μg/mL) + anti-CD28 (1 μg/mL) with TGF-  1 (2 ng/mL) and IL-6 (50 ng/mL) stimulation. Data Analysis: Data (Fig.1-3) shown represent mean ± S.E. Paired data were evaluated using the Student’s t test. *p < 0.05, **p < Fig.2 Less severity in I  B NS deficient T cells transfer model (A) Diagram of methods of Passive EAE models. (B) Disease progression of EAE (n = 3-4) EAE model in I  B NS deficient (Nfkbid -/- ) mice Fig.1 I  B NS deficient mice have resistant to EAE (A) Disease progression of EAE in Nfkbid +/+ (n = 11–13) and Nfkbid -/- mice (n = 9­­–11). (B-C) Analysis of mice 12 days after immunization. (B) Measurement of IL-17A supernatant concentrations by ELISAs (Nfkbid +/+ : n = 5; Nfkbid -/- : n = 6), using cultured draining LNs incubated in the presence or absence of MOG peptide (10 ng/ml) for 72h. (C) Histology of spinal cord specimens in EAE models. Sections were stained with hematoxylin and eosin (HE) or Klüver- Barrera staining (KB) A B C Passive EAE model using CD4 + T cells MOG+CFA 12 days after immunization Inguinal draining LNs Cultured in the presence of MOG Purified CD4+ T cells & transfer to WT mice A B Th17 generation In vitro Spleen Naïve CD4 + TCultured With/without TGF-  +IL-6 ELISA (A) Supernatant RT-PCR (B) Cells A B Fig.3 I  B NS deficient T cells fail to generate Th17 (A, B) Expression of IL-17A protein (A) or Il-17a mRNA (B) in CD4 + T cells from Nfkbid +/+ and Nfkbid -/- mice, cultured for 48 h under Th0 or Th17 conditions. Transcriptional activity of I  B NS to IL-17A gene TCR ROR  t I  B-  TGF-  IL-17 A IL-6 I  B NS ? A B Fig.4 I  B NS did not have transcriptional activity to IL-17A (A) Schematic of Th17 development controlled by nuclear I  B family proteins. (B) Il-17a reporter activity presented as the fold-increase over that observed in HEK293 cells transfected with the empty vector. Data shown are the mean ± S.D. of duplicate samples and are representative of 3 independent experiments. Status of Acetyl Histone & Th17 master regulator ‘ROR  t’ expression Fig.5 I  B NS deficient T cells were less acetylation of CNS1 & ROR  t expression (A) Scheme of IL-17A gene conservation. (B) ChIP analysis of H3K27Ac. Cells were cultured under Th17 conditions for 48 h. (C) RORγt expression in CD4 + T cells from Nfkbid +/+ and Nfkbid -/- mice, cultured for 72 h under Th17 conditions. Data are representative of three independent experiments. Spleen Naïve CD4 + TCultured (48h) With TGF-  +IL-6 ChIP (B) Cells FACS (C) ROR  t & I  B-  binding element A B C Our results indicate that Nfkbid -/- T cells showed impaired Th17 cells differentiation because of a reduction in RORγt expression and histone H3 acetylation in the CNS 1 region. In conclusion, we show that I  B NS deficiency causes resistance to Th17-dependent autoimmune disease through the control of Th17 differentiation. PLoS One 2014 Oct 27;9(10):e I am grateful to Dr. Muta, Tatsushi (Tohoku University) for supervising this research and for helpful discussions. We are also grateful to Mr. Goto, Yasuyuki (Tohoku University) and Ms. Takahashi, Kyoko (Gifu University) for providing technical assistance.