Decreased CD19+CD24hiCD38hi Regulatory B Cells in Alopecia Areata

Regulatory B cells (Bregs) produce anti-inflammatory factors, such as IL-10, IL-35, and TGF-β, and exert immunosuppressive effects in autoimmune diseases by inducing regulatory T cells.(Rosser and Mauri, 2015Rosser E.C. Mauri C. Regulatory B cells: origin, phenotype, and function.Immunity. 2015; 42: 607-612Abstract Full Text Full Text PDF PubMed Google Scholar) Deficiency of Bregs, particularly the IL-10-producing subset, has been implicated in several autoimmune diseases involving cutaneous manifestations, such as lupus erythematosus, psoriasis and atopic dermatitis.(Wang et al., 2020Wang W.M. Guo L. Jin H.Z. Role of B cells in immune-mediated dermatoses.Mol Immunol. 2020; 126: 95-100Crossref PubMed Scopus (4) Google Scholar) However, their role in alopecia areata (AA), an autoimmune disease involving hair follicles, is unclear. We used flow cytometry to characterize immunosuppressive B-cell subsets in peripheral blood mononuclear cells of 22 patients with AA (8 females and 14 males, 34.7 ± 13.6 years; 15 patchy AA and 7 alopecia totalis/universalis) and 13 healthy volunteers (HVs; 4 females and 9 males, 28.2 ± 5.1 years). The participant characteristics and methods are detailed in Supplementary Materials (Methods, Supplementary Table S1). The frequency of CD19+CD24hiCD38hi (immature B cell), CD19+CD38intCD24int (mature B cell), and CD19+CD24-CD38+ (plasmablast), and IL-10-producing immature Bregs were examined after the stimulation of CPG using flow cytometry. The association between frequency of B-cell subsets and clinical characteristics was also analyzed. The effect of IL-10-producing Bregs on the key immune mediators of AA development was also evaluated using a co-culture system. Statistical analysis was performed by conducting two-way Analysis of variance and the Holm–Sidak's multiple comparisons test using Prism Software (GraphPad 9.5.1). p < 0.05 were considered statistically significant. This study was performed in accordance with the Declaration of Helsinki. Collection of human samples for this study was approved as part of the study protocol. This human study was approved by the Review Board of Jeonbuk National University Hospital (JNUH-2022-05-002). All the participants provided written informed consent to participate in this study. The frequency of CD19+CD24hiCD38hi cells (immature B cells) was significantly lower in patients with AA than in HVs, regardless of the clinical subtype of AA; however, there were no significant differences in the frequencies of the CD19+CD24+CD38- (memory B cell), CD19+CD38intCD24int (mature B cell), or CD19+CD24-CD38+ (Plasmablast B cell) subsets (Figure 1a). The number of IL-10-producing cells by CD19+CD24hiCD38hi B cells was significantly lower in AA patients than in HVs, while IL-10 production from the remaining B-cell subsets was not different between the two groups (Figure 1b and Supplementary Figure S1). There were generally fewer CD19+CD24hiCD38hi cells and considerably more CD19+CD38intCD24int B cells in alopecia totalis/universalis patients than in HVs, but no differences were observed in patients with patch AA. CD19+CD24hiCD38hi B cells from patients with both alopecia totalis/universalis and patchy AA produced lower levels of IL-10 and higher TNF-α expansion than those from HVs, regardless of the severity (Supplementary Figure S2). The frequency of Breg-producing IL-10 did not differ significantly according to the duration of the current AA episode (Supplementary Figure S3). In further experiments to determine whether Breg/IL-10 regulates the production of the key inflammatory mediators of AA, CD19+CD24hiCD38hi B cells from AA patients significantly reduced the frequency of CD8+T , CD8+NKG2D+T, and NKG2D+ NK cells and the production of IFN-γ, which was reversed by anti-IL-10 (Figure 2a and Figure 2b). The frequency of CD4+T cells and Foxp3+Treg was not changed by CD19+CD24hiCD38hi B cells and anti-IL-10. The IFN-γ level produced by the Breg subsets was consistently low and did not differ between AA and HVs (Supplementary Figure S4). Finally, we compared the phosphorylated (p)-STAT levels in IL-10-producing B cells to investigate the regulatory signaling mechanism of Bregs in AA. CD19+CD24hiCD38hi B cells from AA patients produced significantly higher p-STAT1 and lower p-STAT3 levels than those from HVs (Supplementary Figure S5).Figure 2CD19+CD24hiCD38hi B cells from AA patients negatively regulate the production of key effect cells (NKG2D+CD8+ T cell) and mediators of alopecia areata development and IFN-γ through the IL-10 dependent manner (a) Representative flow cytometry plots (left panel) and graphs (right panel) showing the frequencies of CD8+, CD4+, CD8+ NKG2D+ T cells, Foxp3+ Tregs, and NKG2D+CD56+ NK cells within PBMCs after co-culture with CD19+CD24hiCD38hi B cells and anti-IL-10 antibodies. (b) Representative flow cytometry plots (left panel) and graphs (right panel) showing the frequencies of IFN-γ production by CD8+, CD4+, and NKG2D+CD56+ NK cells within PBMCs after co-culture with CD19+CD24hiCD38hi B cells and anti-IL-10 antibodies. All analyses were performed using flow cytometry. Values are expressed as means ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001, One-way ANOVA followed by the Holm–Sidak post hoc test. ANOVA, analysis of variance; AA, alopecia areata; HV, healthy volunteers, PBMCs, peripheral blood mononuclear cells.View Large Image Figure ViewerDownload Hi-res image Download (PPT) AA is an autoimmune disease resulting from the collapse of hair follicle-immune privilege and the subsequent Th1-cell mediated immune-inflammatory response. IL-10 is the guardian of hair follicle immune privileges, and it negatively IFN-γ or granzyme-producing cytotoxic CD8+ T cells in AA lesions (Matsumura et al., 2022Matsumura Y. Watanabe R. Koguchi-Yoshioka H. Nakamura Y. Saito A. Kume M. et al.IL-10‒Producing Potency from Blood B Cells Correlates with the Prognosis of Alopecia Areata.J Invest Dermatol. 2022; Google Scholar, Simakou et al., 2019Simakou T. Butcher J.P. Reid S. Henriquez F.L. Alopecia areata: A multifactorial autoimmune condition.J Autoimmun. 2019; 98: 74-85Crossref PubMed Scopus (130) Google Scholar). In this study, we identified a significant decrease in IL-10-producing Bregs in the peripheral blood mononuclear cells of patients with AA. CD19+CD24hiCD38hi B cells negatively regulated the production of NKG2D+CD8+ T cells and IFN-γ that primarily contribute to the pathogenesis of AA in an IL-10 dependent manner, suggesting a protective role of IL-10-producing Bregs in AA development. Concurrent with our observations, serum IL-10 levels significantly decreased with higher IFN-γ levels in the active phase of AA (Ma et al., 2017Ma X. Chen S. Jin W. Gao Y. Th1/Th2 PB balance and CD200 expression of patients with active severe alopecia areata.Exp Ther Med. 2017; 13: 2883-2887Crossref PubMed Scopus (10) Google Scholar). Moreover, B cells from patients with AA with better prognoses produced significantly higher levels of IL-10, implying that IL-10 is a positive prognostic indicator of AA. However, contrary to our findings, IL-10 levels in B cells were higher in patients with AA than in controls, probably due to the negative feedback response (Matsumura et al., 2022Matsumura Y. Watanabe R. Koguchi-Yoshioka H. Nakamura Y. Saito A. Kume M. et al.IL-10‒Producing Potency from Blood B Cells Correlates with the Prognosis of Alopecia Areata.J Invest Dermatol. 2022; Google Scholar). Systemic treatment including glucocorticoids has been associated with increased Breg populations with higher serum IL-10 concentrations (Garcia et al., 2021Garcia S.G. Sandoval-Hellín N. Franquesa M. Regulatory B Cell Therapy in Kidney Transplantation.Front Pharmacol. 2021; 12791450Crossref Scopus (3) Google Scholar, Wang et al., 2014Wang L. Zhao P. Ma L. Shan Y. Jiang Z. Wang J. et al.Increased interleukin 21 and follicular helper T-like cells and reduced interleukin 10+ B cells in patients with new-onset systemic lupus erythematosus.J Rheumatol. 2014; 41: 1781-1792Crossref PubMed Scopus (39) Google Scholar). The discrepancy in IL-10 levels may also be due to differing participant characteristics between previous and the present study, such as the severity and activity of AA (Torkestani et al., 2021Torkestani S. Moghimi H. Farsiabi R. Khazaei S. Eftekharian M.M. Dalvand E. Evaluation of serum levels of IL-6, IL-10, and TNF-alpha in alopecia areata patients: A systematic review and meta-analysis.Biomedical Research and Therapy. 2021; 8: 4668-4678Crossref Google Scholar). Our cohort included patients with a duration of current AA episodes ≤ 12 months without a history of systemic treatment. Mechanistically, the STAT signaling pathway is implicated in IL-10 production by B cells as well as AA development (Blair et al., 2010Blair P.A. Norena L.Y. Flores-Borja F. Rawlings D.J. Isenberg D.A. Ehrenstein M.R. et al.CD19(+)CD24(hi)CD38(hi) B cells exhibit regulatory capacity in healthy individuals but are functionally impaired in systemic Lupus Erythematosus patients.Immunity. 2010; 32: 129-140Abstract Full Text Full Text PDF PubMed Scopus (1252) Google Scholar). We found altered p-STAT1 and p-STAT3 activation in GPG-activated Bregs in AA patients, suggesting that the regulatory role of Bregs in AA pathogenesis are mediated by the STAT signaling pathway. Since T cell populations inducing STATs have been implicated in driving autoimmunity in AA (Deenick et al., 2018Deenick E.K. Pelham S.J. Kane A. Ma C.S. Signal Transducer and Activator of Transcription 3 Control of Human T and B Cell Responses.Front Immunol. 2018; 9: 168Crossref PubMed Scopus (45) Google Scholar), whether these are B-cell-intrinsic effects of STAT1/STAT3 overactivation or secondary to defects in T cells remains unclear. The small sample size and lack of information on the lesional B-cell in hair follicles are limitations of this study. Nevertheless, these results support a possible connection between IL-10-producing Bregs and AA, opening up novel diagnostic and therapeutic targets for AA. Further studies with larger blood and skin samples are needed to confirm this association and elucidate the exact mechanism by which Bregs affect AA development. Our publication contains no large datasets such as RNA sequencing or proteomics. The data that support the findings of this study are available from the corresponding author upon reasonable request. The authors declare no conflicts of interest. Conceptualization: J-P, JK-C; data curation: HJ-L, GJ-L; formal analysis: JY-L, HJ-L; funding acquisition: J-P, JK-C; investigation: SH-K, KH-N; methodology: HJ-L, SH-K; project administration: JK-C; resources: JK-C; software: HJ-L; supervision: KH-N; validation: SH-K, KH-N; visualization: JY-L, GJ-L; writing – original draft preparation: JY-L, J-P, JK-C; writing – review and editing: J-P, JK-C. JK-C is the guarantor for this work. This study was supported by the Fund of the National Research Foundation of the Korean Government (Grant number: 2022R1A2C4001257, 2021R1C1C1006089, 2022M3A9H1015892) and BK21FOUR 21st Century of Medical Science Creative Human Resource Development Center. Download .pdf (.04 MB) Help with pdf files Download .pdf (.08 MB) Help with pdf files Download .pdf (.06 MB) Help with pdf files Download .pdf (.17 MB) Help with pdf files Download .pdf (.13 MB) Help with pdf files Decreased CD19+CD24hiCD38hi Regulatory B cells in Alopecia Areata Jong Yeong Lee, Hyo Jung Lim, Sang-Hyun Kim, Kyung-Hwa Nam, Jin Park, Jin Kyeong Choi Corresponding author: Jin Park and Jin Kyeong Choi E-mail: [email protected]; [email protected] Methods Figure S1-S5 Table S1. Clinical characteristics of the patients with alopecia areata Patients and healthy controls Blood samples were obtained from 22 patients with alopecia areata (AA) (age range: 16–63 years, 14 males and 8 females) and 13 healthy volunteers (HVs) (age range: 22–39 years, 4 males and 9 females). The duration of the current AA episode at the baseline was 2–12 months, (mean: 6.9 ± 3.6 months). Those with other illnesses, such as chronic systemic or cutaneous diseases or infectious conditions at the time of blood sampling, and those receiving systemic treatments, such as immunosuppressive agents, were excluded. This study was performed in accordance with the Declaration of Helsinki. Collection of human samples for this study was approved as part of the study protocol. This human study was approved by the Review Board of Jeonbuk National University Hospital (JNUH 2022-05-002). All the participants provided written informed consent to participate in this study. PBMCs were isolated from whole blood using Ficoll-Paque Plus density gradient centrifugation (Amersham Biosciences). The cells were cultured in complete RPMI 1640 supplemented (Gibco) with 10% fetal bovine serum (Hyclone), 1% penicillin/streptomycin (Invitrogen), 1% L-glutamine (Gibco), and 0.2% 2-mercaptoethanol (Gibco). For activation of B cells, PBMCs were stimulated with CPG (ODN2006 3 μg/ml, Invivogen) for 96 h. PBMCs from AA patients and HVs were used to conduct surface and intracellular FACS analyses, respectively. For intracellular cytokine detection, cells were stimulated for 5 h with PMA (50 ng/ml, Sigma-Aldrich), ionomycin (250 ng/ml; Sigma-Aldrich), and GolgiPlug (BD Biosciences), and intracellular cytokine staining was performed using the BD Biosciences Fixation/Permeabilization Kit, as recommended. Dead cells were stained using the LIVE/DEAD Fixable Stain Kit (Invitrogen). Human regulatory B cell subsets were characterized by analyzing the expansion of BV510 anti-CD19 (Clone SJ25C1; BD Biosciences), BV421 anti-CD24 (Clone ML5; BD Biosciences), PE-cy7 anti-CD27 (Clone M-T271; BD Biosciences), and PE-Cy5 anti-CD38 (Clone HIT2; BD Biosciences). To detect intracellular cytokines, the cells were washed, fixed, permeabilized, and stained with PE anti-IL-10 (Clone JES3-19F1; BD Bioscience) or FITC anti-TNF-α (Clone MAb11; BioLegend). Samples were analyzed using an Attune NxT acoustic focusing cytometer (Thermo Fisher Scientific) and the data were processed using FlowJo (v10.7.1., BD Biosciences). CD19+CD24hiCD38hi B cells and B cell-depleted PBMCs from AA patients were sorted by flow cytometry. The CD19+CD24hiCD38hi cells, treated with CPG (3 μg/ml), were co-cultured 1:1 for 72 h in 96-well transwell plates with PBMCs and plate-bound anti-CD3 monoclonal antibodies (1 μg/ml), with or without the addition of neutralizing IL-10 antibodies (5 μg/ml). PMA/Ionomycin and Golgi plug were added in the final 5 h of culture. The results represent data from five patients in each AA group. For the T-cell subset analysis, cells were stained with BV421 anti-CD4 (Clone SK3; BD Biosciences), PE-Cy7 anti-CD8 (Clone HIT8a; BD Biosciences), BV510 anti-CD56 (Clone B159; BD Biosciences), PE anti-NKG2D (Clone JM7A4; BioLegend), and APC anti-NKG2D (Clone 1D11; BioLegend). Intracellular cytokines and Tregs were identified using PerCP-Cy5.5 anti-IFN-γ (Clone 4SB3; BD Biosciences) and FITC anti-Foxp3 (Clone PCH101; Invitrogen). PBMCs were surface-stained with anti-CD19, anti-CD24, anti-CD27, and anti-CD38 antibodies in stain buffer (BD Biosciences) for 20 min and then rested for 1 h in complete RPMI 1640 medium. Cells were stimulated by incubation with 3 μg/ml CPG for 30 min and then fixed with fixation buffer for 30 min at 4 °C, washed with stain buffer, and permeabilized with phospho-STAT1 (Thermo) and phospho-STAT3 (Thermo) antibodies for 1 h. Statistical analysis was performed by conducting two-way Analysis of variance and the Holm–Sidak's multiple comparisons test using Prism Software (GraphPad 9.5.1). *p < 0.05, **p< 0.01, ***p < 0.001 and ****p < 0.0001 were considered statistically significant. Figure S1. Comparison of IL-10 production in B cell subsets between alopecia areata (AA) patients and AA and healthy volunteers (HV). The dot graph represents the percentage of IL-10 produced by CD19+ (Total Pan B cell), CD19+CD20 + (Naïve B cell), and CD20+CD27+ (Follicular B cell) B cells in PBMCs from AA patients (n = 8) and HV (n = 6) after 72 h of stimulation with CPG (3 μg/ml). AA, alopecia areata; HV, healthy volunteers, PBMCs, peripheral blood mononuclear cells. Figure S2. Decreased IL-10-producing Bregs compartment in patients with a patch and/or alopecia totalis /universalis (AT/AU). (a) Bar chart showing mean percentages of B cells within each subset in the PBMCs of healthy donors (n = 8) and patients with a patch (n = 12) or AT/AU (n = 5). (b) Bar graph show the percentage of IL-10+ B cells within CD19+CD24hiCD38hi, CD19+CD24+CD38-, CD19+CD24-CD38+, and CD19+CD24intCD38int B cells. (c) Frequencies of TNF-α+ and IL-10: TNF-α+ B cells within the CD19 + CD24hiCD38hi B cell subset. *p < 0.05; **p < 0.01; ***p < 0.001, One- or Two-way ANOVA followed by the Holm-Sidak post hoc test. ANOVA, analysis of variance; AT, Alopecia totalis; AU, alopecia unversalis. Figure S3. Frequency of IL-10 produced by Regulatory B cell subsets according to the duration of alopecia areata. The bar graphs represent the percentage of IL-10 produced by CD19+CD24hiCD38hi, CD19+CD24+CD38-, CD19+CD24-CD38+, and CD19+CD24intCD38int B cells in PBMCs from AA patients and HV after 72 h of stimulation with CPG (3 μg/ml). The duration of the current episode at the sampling time was classified into two groups, < 6 months and ≥ 6 months. All analyses were performed using flow cytometry. Values are expressed as means ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001, Two-way ANOVA followed by the Holm-Sidak post hoc test. ANOVA, analysis of variance; AA, alopecia areata; HV, healthy volunteers, PBMCs, peripheral blood mononuclear cells. Figure S4. The frequency of IFN-γ produced by regulatory B cell subsets in alopecia areata (AA) patients and healthy volunteers (HV). The dot plot represents the percentage of IFN-γ produced by CD19+CD24hiCD38hi, CD19+CD24+CD38-, CD19+CD24-CD38+, and CD19+CD24intCD38int B cells in PBMCs from AA patients and HV after 72 h of stimulation with CPG (3 μg/ml). Values are expressed as means ± SEM. Two-way ANOVA followed by the Holm-Sidak post hoc test. ANOVA, analysis of variance; AA, alopecia areata; HV, healthy volunteers, PBMCs, peripheral blood mononuclear cells. Figure S5. Alteration of STAT1 and STAT3 signaling in CD19+CD24hiCD38hi B cells from alopecia areata (AA) patients. IL-10 signaling response upon CPG stimulation was altered in AA CD19+CD24hiCD38hi B cells. B cell subset activity in response to CPG stimulation for 30 min was assessed using flow cytometry to measure STAT1 and STAT3 phosphorylation after activating PBMC from HVs (n = 5) and patients with AA (n = 5) in vitro with CPG (3 μg/ml). (a) Representative histograms show STAT1 and STAT3 phosphorylation on CD19+CD24hiCD38hi, CD19+CD24+CD38-, CD19+CD24-CD38+, and CD19+CD24intCD38int B cells. (b) Bar charts show percentage phospo-STAT1 and phospho-STAT3 expressions on CD19+CD24hiCD38hi, CD19+CD24+CD38-, CD19+CD24-CD38+, and CD19+CD24intCD38int B cells. *p < 0.05; ****p < 0.0001, Two-way ANOVA followed by the Holm-Sidak post hoc test. ANOVA, analysis of variance; AA, alopecia areata; HV, healthy volunteers, PBMCs, peripheral blood mononuclear cells. Tabled 1VariablesTotal (%)Healthy volunteers13Age (years)28.2 ± 5.1 (22–39)SexMale9Female4AA patients22Age (years)34.7 ± 13.6 (16–63)SexMale14Female8TypePatchy15AT/AU7Duration (current episode at baseline)≤3 month73-6 months56-12 months10Extent (SALT)S1 (<25% loss)8S2 (25–49% loss)5S3 (50–74% loss)3S4/S5 (76–100% loss)6Body hair lossNo14Yes8 Open table in a new tab AA, alopecia areata; AT, alopecia totalis; AU, alopecia universalis; SALT, Severity of alopecia tool.