Elevated expression of immune checkpoints and pro-inflammatory cytokines as potential biomarkers in pediatric Vulvar...

Vulvar Lichen Sclerosus (VLS) is a chronic inflammatory dermatosis of unknown etiology affecting the external genitalia. In pediatric patients, it can lead to significant discomfort and progressive structural changes in tissues. In recent years, there has been increasing interest in the participation of immune checkpoints and inflammatory mediators in the pathogenesis of chronic diseases, including VLS. Immune checkpoints, such as PD-1, PD-L1, CTLA-4, CD200, and CD200R, play a crucial role in modulating the immune response and may serve as potential diagnostic markers and therapeutic targets. This study aimed to evaluate the expression of PD-1, PD-L1, CTLA-4, CD200, and CD200R molecules on CD4+ T cells, CD8+ T cells, and CD19+ B lymphocytes in prepubertal girls diagnosed with VLS. Additionally, the concentrations of their soluble forms and the levels of proinflammatory cytokines, including IL-2, IL-6, and TNF-α, in serum were determined to assess their potential as diagnostic biomarkers. The study included patients with VLS (study group) and healthy children (control group). The expression of checkpoints was analyzed using flow cytometry, while the concentrations of soluble forms and cytokines were determined using the enzyme-linked immunosorbent assay (ELISA) technique. Statistical significance tests and Spearman's rank correlation analysis were used. Patients with VLS showed markedly higher serum C-reactive protein (CRP) levels compared with healthy controls (49.99 ± 6.09 mg/L vs. 2.70 ± 0.96 mg/L, p < 0.001). A significant increase in checkpoint expression was observed on lymphocyte subsets, including CD4+ T cells expressing PD-1 (4.90 ± 1.67% vs. 0.85 ± 0.56%, p < 0.001) and CTLA-4 (11.58 ± 4.70% vs. 0.96 ± 0.48%, p < 0.001), as well as CD8+ T cells expressing PD-1 (17.31 ± 4.81% vs. 0.76 ± 0.67%, p < 0.001) and CD19+ B cells expressing PD-L1 (16.10 ± 9.50% vs. 1.41 ± 0.45%, p < 0.001). Soluble checkpoint molecules were consistently elevated, for example, sPD-1 (26.69 ± 3.88 pg/mL vs. 4.40 ± 0.75 pg/mL, p < 0.001) and sCTLA-4 (43.96 ± 3.77 pg/mL vs. 5.09 ± 1.09 pg/mL, p < 0.001). Similarly, cytokine levels were significantly increased in VLS patients, including interleukin-2 (28.80 ± 6.36 pg/mL vs. 4.32 ± 1.33 pg/mL, p < 0.001), interleukin-6 (25.35 ± 7.52 pg/mL vs. 2.35 ± 0.78 pg/mL, p < 0.001), and tumor necrosis factor alpha (31.26 ± 3.10 pg/mL vs. 12.80 ± 1.30 pg/mL, p < 0.001). Correlation analyses confirmed significant positive associations between cytokine concentrations and checkpoint expression, highlighting their interdependence in VLS immunopathogenesis. The obtained results confirm an increased immunoactivation profile in children with VLS, characterized by elevated checkpoint expression and increased levels of proinflammatory cytokines. The studied parameters show potential as diagnostic and prognostic biomarkers, which may constitute the basis for the development of new diagnostic tools and targeted therapeutic strategies in VLS in pediatric patients.