Alternation of circadian clock modulates forkhead box protein-3 gene transcription in CD4+ T cells in the intestine Gui Yang, MD, Hong Zhang, PhD, Yi.

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Alternation of circadian clock modulates forkhead box protein-3 gene transcription in CD4+ T cells in the intestine Gui Yang, MD, Hong Zhang, PhD, Yi.

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Presentation transcript:

Alternation of circadian clock modulates forkhead box protein-3 gene transcription in CD4+ T cells in the intestine Gui Yang, MD, Hong Zhang, PhD, Yi Liu, PhD, Ying Feng, PhD, Xiang-Qian Luo, MD, PhD, Zhi-Qiang Liu, MD, PhD, Xiao-Rui Geng, MD, Shuai Wang, PhD, Peng-Yuan Zheng, MD, PhD, Bai-Sui Feng, MD, PhD, Zhi-Gang Liu, MD, PhD, Ping-Chang Yang, MD, PhD, Hua-Bin Li, MD, PhD, Shan-Dong Wu, PhD Journal of Allergy and Clinical Immunology Volume 138, Issue 5, Pages 1446-1449.e10 (November 2016) DOI: 10.1016/j.jaci.2016.04.035 Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 A-F, AC facilitates intestinal allergy. BALB/c mice (n = 10) were treated with regular circadian clock (RC) or altered circadian clock (AC) and fed with OVA (mixed in the mouse food). The data of bars are presented as mean ± SD. ∗P < .01, compared with naive group. #P < .01, compared with the saline group in AC group. Journal of Allergy and Clinical Immunology 2016 138, 1446-1449.e10DOI: (10.1016/j.jaci.2016.04.035) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E1 Assessment of OVA-specific CD4+ T cells. CD4+ T cells were isolated from the intestine, labeled with CFSE, and stimulated with OVA or BSA for 72 hours in the presence of dendritic cells. The gated flow cytometry histograms indicate the frequency of CD4+ T-cell proliferation. Each group consists of 10 mice. Samples from individual mice were analyzed separately. CFSE, Carboxyfluorescein diacetatesuccinimidyl ester. Journal of Allergy and Clinical Immunology 2016 138, 1446-1449.e10DOI: (10.1016/j.jaci.2016.04.035) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E2 ACC suppresses Treg cells in the intestine. A, The gated dot plots are CD4+ T cells of LPMCs. B and C, The gated dot plots are Treg cells. D, The bars (mean ± SD; *P < .01, compared with the RCC group) are the summarized data of Fig E2, B and C. Each group consists of 10 mice. Samples from individual mice were processed separately. LPMC, Lamina propria mononuclear cell; SSC, side-scattered light. Journal of Allergy and Clinical Immunology 2016 138, 1446-1449.e10DOI: (10.1016/j.jaci.2016.04.035) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E3 Adoptive transfer with Treg cells suppresses ACC (altered circadian clock)-induced food allergy. The ACC mice were treated with Treg cells, or naive CD4+ T cells, at 106 cells/mouse via tail vein injection. The bars indicate the serum TH2 cytokine levels (A), serum specific IgE levels (B), intestinal epithelial barrier permeability (C), infiltration of mast cell/eosinophil in the intestinal mucosa (D), the frequency of antigen-specific CD4+ T cell in the intestinal mucosa (E) and the number of diarrhea mice after antigen challenge (F). The data of bars are presented as mean ± SD. *P < .01, compared with the saline group. Samples from individual mice were processed separately. Each group consists of 10 mice. Journal of Allergy and Clinical Immunology 2016 138, 1446-1449.e10DOI: (10.1016/j.jaci.2016.04.035) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E4 ACC upregulates NFIL-3 expression in CD4+ T cells. A, The bars indicate the NFIL-3 promoter binding rate by ACC molecules in mice treated with ACC. B and C, The bars indicate the NFIL-3 mRNA (Fig E4, B) and NFIL-3 binding to Foxp3 promoter in peripheral B cells of subjects engaging day-/night-shift rotation. *P < .01, compared with the RCC group (Fig E4, A), or the “Before” group (Fig E4, B and C). Samples from the individual subjects were analyzed separately. Journal of Allergy and Clinical Immunology 2016 138, 1446-1449.e10DOI: (10.1016/j.jaci.2016.04.035) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E5 NFIL-3 represses Foxp3 expression in CD4+ T cells. A, Medium: CD4+ T cells were cultured with medium alone. NFIL-3 plasmid or control plasmid: CD4+ T cells were transfected with NFIL-3-expression plasmids or control plasmids. B, Sketch of Foxp3 promoter reporter construct. C, The bars indicate the Foxp3 gene activity. D, The gated cells indicate the frequency of Foxp3+ T cells. E, The bars show the summarized data of panel D. F, The immune blots indicate an immune complex of Foxp3 and NFIL3. The data of bars are presented as mean ± SD. *P < .001, compared with the medium group. The data represent 3 independent experiments. Ab, Antibody; SSC, side scattered light. Journal of Allergy and Clinical Immunology 2016 138, 1446-1449.e10DOI: (10.1016/j.jaci.2016.04.035) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E6 ACC-induced NFIL-3 increases IL-4 expression in CD4+ T cells. #NFIL-3–deficient mice. The bars of (A-D) indicate the levels of p300, acetylated H3K4, Pol II, and GATA3 at the IL-4 promoter locus. E, The bars indicate the IL-4 mRNA levels in CD4+ T cells. F, The immune blots indicate the protein levels of IL-4 in CD4+ T cells. The data of bars are presented as mean ± SD. *P < .001, compared with the medium group RCC (regular circadian clock). The data represent 3 independent experiments. Journal of Allergy and Clinical Immunology 2016 138, 1446-1449.e10DOI: (10.1016/j.jaci.2016.04.035) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E7 Deficiency of NFIL-3 abolishes ACC-induced food allergy in mice. Wild and NFIL-3-deficient mice were sensitized and sacrificed next day after the last antigen challenge. A and B, The bars indicate the serum levels of TH2 cytokines (A) and antigen-specific IgE (B). C, The bars indicate the frequency of mast cell/eosinophil in the intestinal mucosa. D-G, The flow cytometry histograms indicate the frequency of antigen-specific CD4+ T cells in the intestinal mucosa. H, The bars indicate the summarized data of D-G. I-J, The bars indicate the intestinal epithelial barrier function. The data of bars are presented as mean ± SD. *P < .01, compared with the RCC group. Samples from individual mice were processed separately. Each group consists of 10 mice. NFIL-3d, NFIL-3–deficient. Journal of Allergy and Clinical Immunology 2016 138, 1446-1449.e10DOI: (10.1016/j.jaci.2016.04.035) Copyright © 2016 American Academy of Allergy, Asthma & Immunology Terms and Conditions