EVALUATION OF THE IMPACT OF THE INHIBITOR OF Γ-SECRETASE ON THE POLARIZATION OF IMMUN RESPONSE IN PULMONARY TUBERCULOSIS

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Introduction. Immune imbalance with Th2-polarization of immune response is considered to be one of the factors of antigen-specific immune response in pulmonary tuberculosis. The Notch receptor family is an important modulator of differentiation and activation of the effector function of T cells. A key role in the functioning of the signaling cascade belongs to the enzyme γ-secretase, which through proteolytic release of the intracellular domain of the receptor, NICD, affects the activation of transcription of target genes that regulate cell differentiation. One of the known inhibitors of γ-secretase is DAPT - N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester).
Objective of the study. To establish the role of γ-secretase inhibitor (DAPT) in modulation of Th1- and Th2-lymphocyte differentiation in pulmonary tuberculosis (PT).
Materials and Methods. The study included 15 patients (mean age 45.4±6.58 years) with newly diagnosed THB isolating mycobacteria sensitive to major antituberculosis drugs (ML, 8 patients) and resistant to at least isoniazid and rifampicin (IR, 7 patients). The control group consisted of 8 healthy donors. Mononuclear leukocytes were isolated from blood by gradient centrifugation. CFP10-ESAT6 antigens (AG) (Diakintest, Generium, Russia) at a dose of 10 μg/ml or γ-secretase inhibitor (DAPT, Tocris Bioscience, UK) at a dose of 10 μM/L in combination with AG were added to incubation medium. The cells were cultured in complete nutrient medium at 37°C and 5% CO2 for 72 h. Th1- and Th2-lymphocyte content was assessed by determining the expression of CD4 receptor (FITC, BD Biosciences, USA) and intracellular transcription factors T-bet (Alexa Fluor 405, R&D Systems Inc, USA), GATA-3 (PerCP-eFluor 710, BD Biosciences, USA) by flow cytofluorimetry. Statistical processing was performed using IBM SPSS Statistics 20 software package.
Results. The relative numbers of Th1 (CD4+T-bet+) and Th2 (CD4+GATA-3+) lymphocytes were found to be 1.8 and 2.4 times higher (p<0.001) on average in the groups with LF and UL TH than in healthy volunteers, respectively. The number of Th1-lymphocytes was lower (p=0.005) and the number of Th2-cells was higher (p=0.035) in LU TL patients than in LH TL patients. After stimulation of AG cell cultures, an increase in the number of cells expressing CD4+T-bet+ and CD4+GATA-3+ was recorded relative to baseline values in all groups of subjects. Addition of DAPT and AG to the incubation medium in LF and LU TH patients resulted in a statistically significant increase in the number of Th1-lymphocytes and, conversely, a decrease in the number of Th2 cells as compared with the corresponding figures during AG stimulation.
Conclusion. The γ-secretase inhibitor (DAPT) has a modulating effect on T-lymphocyte differentiation in patients with pulmonary tuberculosis. At a concentration of 10 μM/l DAPT promotes differentiation of Th1-cells and reduces the number of Th2-lymphocytes.

About the authors

Alina Evgenievna Sanina

Siberian State Medical University

Email: albe4303@mail.ru
ORCID iD: 0000-0002-9261-635X
Russian Federation, 2, Moscow tract, Tomsk, 634050, Russia

Valentina Aleksandrovna Serebryakova

Siberian State Medical University

Email: serebryakova-val@mail.ru
ORCID iD: 0000-0001-7078-4988
SPIN-code: 5320-3029
Russian Federation, 2, Moscow tract, Tomsk, 634050, Russia

Olga Ivanovna Urazova

Siberian State Medical University

Author for correspondence.
Email: urazova72@yandex.ru
ORCID iD: 0000-0002-9457-8879
Russian Federation, 2, Moscow tract, Tomsk, 634050, Russia