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Effects of lidocaine on regulatory T cells in atopic dermatitis

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1 Effects of lidocaine on regulatory T cells in atopic dermatitis
Huaguo Li, MD, Chunxiao Li, MS, Hui Zhang, PhD, Lingyun Zhang, PhD, Ruhong Cheng, MD, Ming Li, PhD, Yifeng Guo, MD, Zhen Zhang, MS, Zhiyong Lu, PhD, Yin Zhuang, MS, Ming Yan, MS, Yan Gu, MD, Xiaobo Feng, MS, Jianying Liang, MS, Xia Yu, MS, Honglin Wang, PhD, Zhirong Yao, PhD  Journal of Allergy and Clinical Immunology  Volume 137, Issue 2, Pages e5 (February 2016) DOI: /j.jaci Copyright © 2015 The Authors Terms and Conditions

2 Fig 1 Effects of lidocaine therapy on lesion severity and Treg cells in patients with AD. A, Photographs of 4 patients with AD before and after lidocaine therapy (14 days). B, The SCORAD index of patients with AD declined significantly after lidocaine treatment (n = 20). C and D, FOXP3 mRNA expression in PBMCs (via RT-PCR) and the proportion of Treg cells in PBMCs (via flow cytometric analysis) were upregulated in patients with AD after lidocaine treatment compared with before treatment (n = 20). E, FOXP3 protein expression in whole lesioned skin from patients with AD after lidocaine treatment was significantly increased compared with before treatment (n = 4) (via Western blot analysis). F and G, FOXP3+ cell numbers in the lesioned skin increased in patients with AD after lidocaine therapy compared with before treatment (n = 3) (via immunochemistry analysis). Original magnification: a and c, ×100; b and d, ×200. *P < .05, **P < .01, and ***P < .001. Bars indicate mean ± SEM of 3 independent experiments. Statistical analyses were performed with paired t test. HC, Healthy control; SCORAD, SCORing Atopic Dermatitis. Journal of Allergy and Clinical Immunology  , e5DOI: ( /j.jaci ) Copyright © 2015 The Authors Terms and Conditions

3 Fig 2 Effects of lidocaine on murine Treg-cell differentiation and its specific signaling pathway. A-C, In vitro Treg-cell differentiation: Lidocaine could upregulate the proportion of Treg cells (via flow cytometry), FoxP3 protein expression level, and P-Smad3/Smad3 level (via Western blot) at a concentration of 0.8 mmol/L. D, SIS3 (5 μM) significantly blocked the promotion of lidocaine to Treg-cell differentiation at a concentration of 0.8 mmol/L (via Western blot). *P < .05, **P < .01, and ***P < .001. Bars indicate mean ± SEM of 3 independent experiments. Statistical analyses were performed with the paired t test. Journal of Allergy and Clinical Immunology  , e5DOI: ( /j.jaci ) Copyright © 2015 The Authors Terms and Conditions

4 Fig E1 Lidocaine regulated other cytokines in PBMCs and lesioned skins of patients with AD. A, IFN-γ, IL-4, and IL-17A mRNA expression levels were significantly upregulated in PBMCs of patients with AD after lidocaine treatment in contrast to those before treatment, whereas IL-17E mRNA level was downregulated significantly (n = 20). B, FOXP3 mRNA level significantly increased, whereas IL-17E mRNA level decreased in whole lesioned skins of patients with AD after lidocaine treatment in contrast to those before treatment (n = 4). *P < .05 and **P < .01. Bars indicate mean ± SEM of 3 independent experiments. Statistical analyses were performed with nonparametric Mann-Whitney test. HC, Healthy control. Journal of Allergy and Clinical Immunology  , e5DOI: ( /j.jaci ) Copyright © 2015 The Authors Terms and Conditions

5 Fig E2 Effects of lidocaine therapy on lesional severity in OVA-sensitized murine models. A, Lidocaine significantly improved the phenotypes in mice of lidocaine (OVA Lido1.5 and OVA Lido3.0) and cyclosporin (OVA Cys) treatment groups compared with the OVA saline group (n = 5). B, Lidocaine significantly improved the histopathologic findings with significant decline in the number of infiltrating cells in mice of lidocaine (OVA Lido1.5 and OVA Lido3.0) and cyclosporin (OVA Cys) treatment groups compared with the OVA saline group (n = 5). Original magnification: ×50. C, Except the OVA saline group, the murine scores of severity in the other 3 groups (OVA Lido1.5, OVA Lido3.0, ad OVA Cys) after treatment were significantly decreased in contrast to those before treatment (n = 5). Bars indicate mean ± SEM (n = 5) of 2 independent experiments. Statistical analyses were performed with the paired t test. ∗∗∗P < .001. Journal of Allergy and Clinical Immunology  , e5DOI: ( /j.jaci ) Copyright © 2015 The Authors Terms and Conditions

6 Fig E3 Effects of lidocaine therapy on OVA-sensitized murine models. A-C, Compared with those of the OVA saline group, the proportion of Treg cells (via flow cytometry) in murine dermis of lesioned skin and FoxP3 mRNA (via RT-PCR) and protein expression level (via Western blot) of whole lesioned skins in the lidocaine treatment group and the cyclosporin treatment group (OVA Cys) were significantly upregulated. D, Compared with those of the OVA saline group, murine IFN-γ mRNA expression level of the OVA Lido3.0 group was significantly upregulated while IL-4 and IL-17E mRNA expression levels of lidocaine treatment groups (OVA Lido1.5 and OVA Lido3.0) and the cyclosporin treatment group (OVA Cys) were significantly downregulated. No significant difference in IL-17A mRNA expression level was detected in any groups. E, No significant difference was detected in the proportion of Treg cells in murine spleen and lymph nodes in lidocaine treatment groups (OVA Lido1.5 and OVA Lido3.0) and the cyclosporin treatment group (OVA Cys). *P < .05 and **P < .01. Bars indicate mean ± SEM (n = 5) of 2 independent experiments. Statistical analyses were performed with nonparametric Mann-Whitney test. Journal of Allergy and Clinical Immunology  , e5DOI: ( /j.jaci ) Copyright © 2015 The Authors Terms and Conditions

7 Fig E4 Effects of lidocaine on murine Treg-cell differentiation and its specific signaling pathway. A, The safety level of lidocaine was verified as 0 to 0.8 mmol/L by CCK8 analysis. B, At the concentration of 0.8 mmol/L, the promotion of lidocaine to Treg-cell differentiation was not significantly relevant with nuclear factor kappa B signaling pathway (via Western blot). *P < .05, **P < .01, and ***P < .001. Bars indicate mean ± SEM of 3 independent experiments. Statistical analyses were performed with paired t test. Journal of Allergy and Clinical Immunology  , e5DOI: ( /j.jaci ) Copyright © 2015 The Authors Terms and Conditions

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