Effects of lidocaine on regulatory T cells in atopic dermatitis
Authors: Huaguo Li, Chunxiao Li, Hui Zhang, Lingyun Zhang, Ruhong Cheng, Ming Li, Yifeng Guo, Zhen Zhang, Zhiyong Lu, Yin Zhuang, Ming Yan, Yan Gu, Xiaobo Feng, Jianying Liang, Xia Yu, Honglin Wang, Zhirong Yao
Journal: Journal of Allergy and Clinical Immunology (2015)

Atopic dermatitis (AD) is a chronic and relapsing skin disease characterized by inflammation and pruritus. In our previous hospital-based study of 1008 patients with AD, we found that the proportion of patients with severe AD was 10.7%.1Shi M. Zhang H. Chen X. Guo Y. Tao J. Qi H. et al.Clinical features of atopic dermatitis in a hospital-based setting in China.J Eur Acad Dermatol Venereol. 2011; 25: 1206-1212Crossref PubMed Scopus (22) Google Scholar Moreover, AD is a huge economic burden for families and society in general due to the long course of relapsed disease. Regulatory T (Treg) cells control immune homeostasis and balance immune responses during inflammation. Treg cells suppress immune responses by interacting with effector T cells or antigen-presenting cells.2Pellerin L. Jenks J.A. Bégin P. Bacchetta R. Nadeau K.C. Regulatory T cells and their roles in immune dysregulation and allergy.Immunol Res. 2014; 58: 358-368Crossref PubMed Scopus (76) Google Scholar Recent clinical research has found that parents, particularly mothers, who have a history of atopy could have babies with less stable FOXP3+ Treg cells in cord blood. These babies have the onset of AD less than 1 year after birth.3Hinz D. Bauer M. Roder S. Olek S. Huehn J. Sack U. et al.Cord blood Tregs with stable FOXP3 expression are influenced by prenatal environment and associated with atopic dermatitis at the age of one year.Allergy. 2012; 67: 380-389Crossref PubMed Scopus (157) Google Scholar Therefore, it is likely that the abnormal numbers of Treg cells weaken the inhibition of TH2 lymphocytes, thus resulting in AD inflammation. Lidocaine is a widely used short-acting local anesthetic and antiarrhythmic agent. Previous studies demonstrated that lidocaine attenuated bronchoconstriction in patients with severe asthma, which enabled the dosage of oral corticosteroids to be reduced or eliminated in long-term treatment.4Hunt L.W. Frigas E. Butterfield J.H. Kita H. Blomgren J. Dunnette S.L. et al.Treatment of asthma with nebulized lidocaine: a randomized, placebo-controlled study.J Allergy Clin Immunol. 2004; 113: 853-859Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar Because of the similarity of allergic diseases, lidocaine was used as a treatment for AD in China. Previous studies have shown that lidocaine dose-dependently inhibits the proliferative response and release of inflammatory factors from Staphylococcal enterotoxins A- and Staphylococcal enterotoxins B–stimulated PBMCs in patients with AD, contributing to clinical remission.5Jiao Q. Wang H. Hu Z. Zhuang Y. Yang W. Li M. et al.Lidocaine inhibits staphylococcal enterotoxin-stimulated activation of peripheral blood mononuclear cells from patients with atopic dermatitis.Arch Dermatol Res. 2013; 305: 629-636Crossref PubMed Scopus (7) Google Scholar In vivo, we sought to explore the effect of lidocaine on Treg cells and other key cytokines in patients with AD and murine AD models. Twenty patients were administered lidocaine (3 mg/kg per day) via a slow intravenous drip for 14 days. During the treatment period, no adverse effects were observed in these patients. The clinical manifestations of AD markedly improved (Fig 1, A), and the SCORing Atopic Dermatitis index significantly declined (Fig 1, B). We found that FOXP3 mRNA expression and the proportion of Treg cells in PBMCs from patients with AD were significantly decreased compared with that in healthy individuals. In addition, FOXP3 mRNA expression level and the proportion of Treg cells in patients with AD after lidocaine treatment significantly increased compared with before treatment (Fig 1, C and D). Four patients permitted skin biopsies and their skin sections were used to evaluate FOXP3 mRNA and protein expression levels. Consistent with FOXP3 mRNA expression levels in PBMCs, the expression of FOXP3 mRNA and protein levels in lesioned skin was also upregulated (see Fig E1, B, in this article's Online Repository at www.jacionline.org; Fig 1, E). Immunochemistry analysis showed that FOXP3+ cell numbers in the skin increased in the 3 patients with AD after lidocaine therapy (Fig 1, F and G). Furthermore, we established AD murine models with topical ovalbumin (OVA) as previously described,6Spergel J.M. Mizoguchi E. Brewer J.P. Martin T.R. Bhan A.K. Geha R.S. Epicutaneous sensitization with protein antigen induces localized allergic dermatitis and hyperresponsiveness to methacholine after single exposure to aerosolized antigen in mice.J Clin Invest. 1998; 101: 1614-1622Crossref PubMed Scopus (536) Google Scholar of which each group included 5 mice. Then, we treated OVA-sensitized mice (via tail vein injection) with different doses of lidocaine: 1.5 mg/kg per day (OVA Lido1.5) and 3.0 mg/kg per day (OVA Lido3.0) for 7 days. A recent report has shown that cyclosporine elevates the proportion and absolute number of Treg cells in patients with AD; however, the function was not significantly different from that in healthy controls.7Brandt C. Pavlovic V. Radbruch A. Worm M. Baumgrass R. Low-dose cyclosporine A therapy increases the regulatory T cell population in patients with atopic dermatitis.Allergy. 2009; 64: 1588-1596Crossref PubMed Scopus (81) Google Scholar Therefore, cyclosporine (2.5 mg/kg per day) was intragastrically administered for 7 days as a positive control (OVA Cys). The phenotypes of mice treated with lidocaine and cyclosporine improved compared with mice treated with saline (see Fig E2, A, in this article's Online Repository at www.jacionline.org). In addition, the index of lesion severity and histopathological findings were consistent with the improved phenotypes (Fig E2, B and C). We subsequently measured FoxP3 mRNA and protein expression in whole lesioned skin using RT-PCR and Western blot. We also used flow cytometric analysis to determine the proportion of Treg cells in the dermis of lesioned skin, spleen, and lymph nodes in the 4 treatment groups. Compared with the OVA saline group, the proportion of Treg cells of murine lesions and FoxP3 mRNA and protein expression levels in the lidocaine treatment groups and the cyclosporine treatment group were significantly upregulated (see Fig E3, A-C, in this article's Online Repository at www.jacionline.org). However, no significant difference was detected in the proportion of Treg cells in murine spleen and lymph nodes from the OVA Lido1.5 and OVA Lido3.0 treatment groups and the OVA Cys treatment group (Fig E3, E). Furthermore, lidocaine therapy ameliorated the TH1/TH2 cell and IL-17A/IL-17E cytokine imbalance in patients with AD and murine AD models. In patients with AD, IFN-γ, IL4, and IL17A mRNA expression levels were significantly upregulated in PBMCs after lidocaine treatment in contrast to those before treatment while IL17E mRNA level was downregulated significantly (Fig E1, A). Furthermore, IL17E mRNA level of whole lesioned skin decreased significantly in patients with AD after lidocaine treatment (Fig E1, B). IL-17E is involved in the TH2-dominant immune response and its downregulation might contribute to the amelioration of symptoms. Moreover, the relationship between IFN-γ and IL-4 as well as that between IL-17A and IL-17E is interacting. Consequently, we presumed that IL-4 mRNA expression was upregulated to inhibit the elevation in IFN-γ mRNA expression while IL-17A mRNA expression increased in response to the decrease in IL-17E mRNA expression level. Although lidocaine therapy did not totally block the upregulation of cytokines mentioned above, it still disrupted the amplification cycle of skin inflammation and ameliorated symptoms. Owning to the less complicated neuro-endocrine-immune networks and habitation in pathogen-free environment, the regulation of inflammatory factors in murine models was more explicit. Compared with the OVA saline group, the expression of IFN-γ mRNA in the OVA Lido3.0 group was significantly upregulated, whereas IL-4 and IL-17E mRNA levels in the OVA Lido1.5, OVA Lido3.0, and OVA Cys groups were significantly downregulated (Fig E3, D). In vitro, the splenocytes of FoxP3-EGFP mice were purified to prepare CD4+CD25− cells as naive CD4+ T cells. Cells were stimulated with antibodies against CD3 (10 μg/mL), CD28 (2 μg/mL), TGF-β (5 ng/mL), and IL-2 (40 ng/mL). The cells were collected for flow cytometry and Western blot analysis 4 days later. First, we used CCK8 analyses to identify safe levels of lidocaine and determined that 0.8 mM was appropriate (see Fig E4, A, in this article's Online Repository at www.jacionline.org). At this concentration, lidocaine upregulated the proportion of Treg cells and FoxP3 protein expression level (Fig 2, A and B). Western blot analyses were used to elucidate potential target signaling pathways. We found that 0.8 mM lidocaine significantly promoted Treg-cell differentiation, activating the Smad3/TGF-β signaling pathway (Fig 2, C), but not through the Smad2/TGF-β or nuclear factor kappa B signaling pathways (Fig 2, C; Fig E4, B). SIS3 (5 μM), an inhibitor of Smad3, was used to define the authenticity of lidocaine-induced regulation of Treg cells. We found that SIS3 significantly downregulated the proportion of Treg cells in the presence of 0.8 mM lidocaine, which established categorically that Treg cells regulated by lidocaine were at least in part, if not wholly, dependent on Smad3 activity (Fig 2, D). To our knowledge, the present study is the first report to demonstrate that lidocaine therapy can ameliorate the severity of lesioned skin in patients with AD and murine AD models. Our study has also uncovered the critical role and molecular mechanism of lidocaine in Treg-cell regulation. In addition, we found that lidocaine promoted FoxP3 transcription by activating TGF-β–induced Smad3 phosphorylation. Lidocaine therapy also ameliorated the TH1/TH2 cell and IL-17A/IL-17E cytokine imbalance in patients with AD and murine AD models. These novel findings indicate that lidocaine might exert regulatory effects on immune cells and thus reduce AD inflammation. Before the study, patient consent and ethical approval were obtained. Subjects included in this study were 20 patients with AD with lidocaine therapy and 20 healthy age- and sex-matched volunteers. Patients with AD younger than 65 years were severely affected and fulfilled the criteria of Hanifin and Rajka. The clinical severity during therapy was monitored by using the SCORing Atopic Dermatitis index (0-24, mild AD; 25-50, moderate AD; 51-103, severe AD). None of the patients had other skin diseases besides AD, and they did not receive any systemic corticosteroids in the recent 3 months. Balb/C mice (6- to 8-week-old female mice) were purchased from Slack Laboratory (Shanghai, China). The mice were anesthetized and their backs were shaved. Skin injury was induced by repeated tape-stripping, after which 100 mg of OVA in 100 mL of saline, or saline only, was applied on a specific patch and attached to the injured area. Treatment was repeated with the 1-week sensitization period after which a 2-week resting period was kept before the second and similar sensitization week. The lesional severity was assessed every week. The standards used are shown as follows: 0 point for none, 1 point for mild, 2 points for moderate, and 3 points for severe lesioned skin. The symptoms included erythema, papule/edema, excoriation, oozing, lichenification, and dry skins. At the end of the seventh week, the mice in the saline group and the OVA group were sacrificed and samples were collected 24 hours after the last treatment. The other 4 groups were treated with saline, lidocaine 1.5 mg/kg per day and lidocaine 3.0 mg/kg per day with tail vein injection, and cyclosporin 2.5 mg/kg per day by intragastric administration for 7 days, which were accompanied by sustained OVA sensitization (each group n = 5). Mice in the above 4 groups were sacrificed at the eighth week. FoxP3-EGFP mice were offered by the research group of Professor Honglin Wang in the Institute of Medical Sciences, Shanghai Jiao Tong University School of Medicine. Naive CD4+ T cells were prepared with FoxP3-EGFP mice. PBMCs were isolated from heparinized venous blood from patients with AD with Ficoll-Paque plus (Pharmacia, Uppsala, Sweden) within 6 hours after bleeding by means of density gradient centrifugation and cells were resuspended in RPMI 1640 medium supplemented with 5% pooled type AB normal human serum (Sigma-Aldrich, St Louis, Mo) for further total RNA extraction and flow cytometry. Murine CD4+ T cells were purified from murine splenocytes by negative selection, which used an MACS system with biotin-antibody cocktail and antibiotin microbeads. Negatively selected CD4+ cells were further positively selected into CD4+CD25− cells as naive CD4+ T cells by using CD25− PE antibody and anti-PE MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany). Single-cell suspensions from the murine inflamed skins were prepared by cutting the skin samples into small pieces, then passing them through a 70-μm cell strainer, washing with PBS, and filtrating with a 40-μm cell strainer again (Becton-Dickinson & Company, Franklin Lakes, NJ). For the preparation of dermal single-cell suspension, lesioned skins were cut into 3 mm × 3 mm sections and immersed in DispaseII at 4°C overnight. The sections were then washed thrice by Hank's fluid without Ca2+ and Mg2+. The dermis was gently isolated with ophthalmic forceps and vibrated with collagenase at 37°C for 20 minutes and with DNAase for the next 25 minutes. (All the products mentioned above were from Sigma-Aldrich.) CD4+ T cells (1 × 105 for cells/well) were seeded onto a 96-well plate in medium with FBS. Lidocaine of different concentrations was added into the plate (0, 0.1, 0.2, 0.4, 0.8, 1.2, 1.6, and 2.0 mM). Water-soluble tetrazolium salts were added to each well (CCK-8, Dojindo, Mashiki-machi, Kamimashiki-gun, Kumamoto, Japan). Cells were incubated for 4 hours at 37°C. Microplate reader was used to detect absorbance, which was measured at 450 nm with a reference filter at 650 nm. Naive CD4+ T Cells were cultured in complete medium. Purified CD4+CD25− cells were stimulated with antibodies against CD3 (10 μg/mL) and CD28 (2 μg/mL) (Becton-Dickinson & Company) and TGF-β (5 ng/mL) and IL-2 (40 ng/mL) (R&D Systems, Minneapolis, Minn). SIS3 (5 μM) was added to the culture at 18 hours after iTreg-cell differentiation to define the specificity of lidocaine-mediated Smad3 induction. Anti-mouse CD4 APC and anti-mouse CD25 PerCP-Cyanine5.5 were used for surface staining. Intracellular staining for FoxP3 and Helios was done with anti-mouse/rat FoxP3 PE and anti-Helios FITC according to the manufacturer's instruction (eBioscience, San Diego, Calif). Isolated 1 × 106/mL PBMCs were stained with human Treg-cell staining kit. Briefly, cells were suspended in a staining buffer and incubated with a surface marker (CD4, CD25, CD127) for 30 minutes at 4°C in the dark. After 6 minutes of washing with the staining buffer at 300 g, cells were incubated overnight with fixation/permeabilization buffer at 4°C in the dark. Subsequently, the cells were stained with PE anti-human FOXP3 for 30 minutes at room temperature in the dark and washed twice (eBioscience). Total RNA was extracted from PBMCs and murine skins with TRIzol Reagent (Invitrogen, Carlsbad, Calif) according to the manufacturer's instructions. It was used as a template for cDNA synthesis by using SuperScript III reverse transcriptase (Invitrogen). Real-time quantitative PCR analysis was performed with 7500 Fast Real Time PCR system (Applied Biosystems, Foster City, Calif) using primers and probes. β-Actin was used as an endogenous control. All primers and probes were purchased from Sangon Biotech (Shanghai, China). Western blot analysis was performed with anti-FoxP3 (eBioscience), Smad3, Phospho-Smad3, Smad2, Phospho-Smad2, nuclear factor kappa b p65, Phospho-nuclear factor kappa b p65, IκBα, Phospho-IκBα rabbit mAb, and anti–β-actin mouse mAbs (Cell Signaling Technology, Beverly, Mass) followed by horseradish peroxidase–conjugated goat anti-rabbit IgG and goat anti-mouse IgG (H+L) (Beyotime Biological Technology, Jiangsu, China) and ECL detection (Thermo Fisher Scientific, Inc, Waltham, Mass). Skins were fixed in 10% formalin and embedded in paraffin. Multiple 4-μm-thick sections were stained with hematoxylin-eosin. Samples obtained from exposed skin were immersed in 4% formamint-fixed, and paraffin-embedded blocks were archived. Sections (4 μm) were used for immunoperoxidase staining to detect FOXP3 (eBiosciences). Horseradish peroxidase-conjugated secondary antibody anti-rat IgG (H+L) was purchased from Sangon Biotech. Data were analyzed by using the GraphPad Prism 5 package (GraphPad Inc, La Jolla, Calif). Two-tailed student t tests and nonparametric Mann-Whitney test were used to compare values. P values are designated as P < .05 (*), P < .01 (**), and P < .001 (***). All participants or their legal guardians provided informed consent. All procedures, forms, and protocols were approved by the Ethics Committee of Xinhua Hospital, affiliated with Shanghai Jiao Tong University School of Medicine. The Animal Care and Use Committee of Xinhua Hospital approved all animal experiments.Fig E2Effects 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E3Effects 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E4Effects 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT)