Células T reguladoras na tuberculose pleural: caracterização fenotípica e interação com o microambiente de mediadores inflamatórios

Abstract

Regulatory T cells (Tregs) play an indispensable role in tolerance and immune homeostasis. Data from the literature have shown an increased frequency of Tregs as well as other subtypes of effector T lymphocyte producing inflammatory mediators in pleural space during the pleural tuberculosis (TBPl), the most common extrapulmonary manifestation of Mycobacterium tuberculosis (Mtb) infection. Given the heterogeneity and difficulty of identifying human Tregs, these studies lack markers that indicate well-defined phenotypes. Thus, this study aimed to characterize the Tregs in TBPl and also to understand their relationship with the microenvironment of cytokines where they are immersed. For this, the following investigations were performed: i) paired pleural fluid (PF) and peripheral blood (PB) samples from patients with TBPl and other causes of exudative effusion non-TB (NTB) were stimulated or not with anti-CD3 OKT-3 and evaluated for the frequency of Tregs by flow cytometry; ii) quantification of the cytokines IFN-γ, TNF, IL-2, IL-6 IL-17A by fluorescence bead-based flow cytometry assay, as well as TGF-β and IP-10 by enzyme-linked immunosorbent assay in serum and LP; iii) gene expression analysis for the transcription factors T-bet, GATA3, ROR-c, FOXP3 and CTLA-4 molecule. Our results showed higher frequencies of CD4+, CD25high, CD127low, CD45RO+, FOXP3+, CD39+ Tregs in LP of TBPl patients, when compared to SP in the same group. A higher proportion of CD39+ memory Tregs by normalized conventional TCD4+ cells was found on LP from TBPl patients compared to blood from patients in the same group, as well as when compared to LP from the NTB group. The analysis between the frequencies of Tregs and ultrasound data revealed a higher frequency of Tregs in less complex spills. Quantifications of cytokines showed higher concentrations of IFN-γ, TNF, TGF-β and IP-10 in LP of TBPl patients, compared to LP of NTB patients. Principal components analysis revealed that the cytokines IP-10 (PE), TGF-β (serum), TNF (serum), IL-10 (serum) IL-17A (PE) e IL-6 (serum) showed the largest variations associated with a partial discrimination between TBPl and NTB patients. The IL-17A cytokine showed a positive correlation with the frequencies of Tregs in the PE of TBPl patients. No significant differences were found among gene expression results. From the present study, it was possible to understand a little more about the biology of Tregs in the pleural microenvironment during Mtb infection. We hope that new diagnostic strategies and management of the complications associated with TBP1 can be investigated from the results obtained in this work.