Deciphering the mechanism of Tinospora cordifolia extract on Th17 cells through in-depth transcriptomic profiling and in silico analysis

Abstract

Naive CD4⁺ T cells differentiate into effector (Th1, Th2, Th17) cells and immunosuppressive (Treg) cells upon antigenic stimulation in the presence of a specific cytokine milieu. The T cell <i>in vitro</i> culture system provides a very efficient model to study compounds' therapeutic activity and mechanism of action. <i>Tinospora cordifolia</i> (Willd.) Hook.f. & Thomson (Family. Menispermaceae) is one of the widely used drugs in <i>Ayurveda</i> (ancient Indian system of medicine) for various ailments such as inflammatory conditions, autoimmune disorders, and cancer as well as for promoting general health. <i>In vitro</i> and <i>in vivo</i> studies on immune cells comprising dendritic cells, macrophages, and B cells suggest its immune-modulating abilities. However, to date, the effect of <i>T. cordifolia</i> on individual purified and polarized T cell subsets has not been studied. Studying drug effects on T cell subsets is needed to understand their immunomodulatory mechanism and to develop treatments for diseases linked with T cell abnormalities. In this study, we examined the immunomodulatory activity of <i>T. cordifolia</i> on primary CD4⁺ T cells, i.e., Th1, Th17, and iTreg cells. An aqueous extract of <i>T. cordifolia</i> was non-cytotoxic at concentrations below 1500 µg/ml and moderately inhibited the proliferation of naive CD4⁺ T cells stimulated with anti-CD3ε and anti-CD28 for 96 h. <i>T. cordifolia</i> treatment of naive CD4⁺ T cells differentiated under Th17-polarizing conditions exhibited reduced frequency of IL-17 producing cells with inhibition of differentiation and proliferation. For the first time, in-depth genome-wide expression profiling of <i>T. cordifolia</i> treated naive CD4⁺ T cells, polarized to Th17 cells, suggests the broad-spectrum activity of <i>T. cordifolia</i>. It shows inhibition of the cytokine-receptor signaling pathway, majorly <i>via</i> the JAK-STAT signaling pathway, subsequently causing inhibition of Th17 cell differentiation, proliferation, and effector function. Additionally, the molecular docking studies of the 69 metabolites of <i>T. cordifolia</i> further substantiate the inhibitory activity of <i>T. cordifolia via</i> the cytokine-receptor signaling pathway. Furthermore, <i>in vitro</i> polarized Th1 and iTreg cells treated with <i>T. cordifolia</i> extract also showed reduced IFN-γ production and FoxP3 expression, respectively. This study provides insight into the plausible mechanism/s of anti-inflammatory activity of <i>T. cordifolia</i> involving T cells, mainly effective in Th17-associated autoimmune and inflammatory diseases.