In vitro modulation of cytokine production during co-culture of human lymphocytes with a multicomponent herbal formulation and Azathioprine

Research and development of novel plant-derived immunomodulators showing in vitro and in vivo pharmacological activity remains a promising area of contemporary phytotherapy. Of particular interest are multicomponent herbal formulations (MHF), which are widely represented on pharmaceutical market. These substances may have several advantages as compared with single-herb preparations, including their complex chemical composition, potential ability to target multiple pathogenetic pathways, mild therapeutic effects without tolerance development, absence of severe adverse effects, and higher efficacy. In previous studies, the authors demonstrated that the proposed multicomponent herbal preparation restored cellular and humoral immune parameters in mice with azathioprine-induced immunosuppression, probably, due to presence of a complex of biologically active compounds, including phenolic constituents such as flavonoids, tannins, phenylpropanoids, and saponins, as well as ascorbic acid and other bioactive substances. According to current concepts, the effects of immunocorrective agents are largely mediated through the cytokine network, which ensures systemic intercellular communication via receptors expressed on the membranes of virtually all animal cells. In this context, the investigation of in vitro production of pro- and anti-inflammatory cytokines during co-culture with the multicomponent herbal preparation and azathioprine is of particular interest. The present study proceeded with evaluation of immunomodulatory activity of the herbal preparation developed as an author’s invention, being focused on the production of TNFα, IL-1ra, and IFNγ by human peripheral blood mononuclear cells in culture supernatants following co-culture with the multicomponent herbal preparation and azathioprine, using Immunal as a reference preparation. Our objective was to assess the production of TNFα, IFNγ, and IL-1ra by human peripheral blood mononuclear cells in vitro during co-culture with a MHF and azathioprine. The study investigated a MHF (the author’s product), containing rose hips, hawthorn fruits, calendula flowers, licorice roots, and Echinacea herb. Baseline contents of the pro- and anti-inflammatory cytokines TNFα, IFNγ, and IL-1ra were measured by enzyme-linked immunosorbent assay in cultures of human peripheral blood mononuclear cells co-incubated with an aqueous extract of the multicomponent herbal preparation (100 mg/mL), with or without azathioprine (50 mg/mL). Immunal (40 mg/mL) was used as the reference bioactive substance. Both Immunal and the MHFn significantly increased TNFα and IFNγ production, whereas IL-1ra levels did not change significantly. Azathioprine reduced TNFα and IFNγ production and markedly increased IL-1ra levels against the intact control group. Likewise, the co-culture of lymphocytes with MFH in the presence of azathioprine resulted in increased TNFα and IFNγ levels and, conversely, a marked reduction in IL-1ra concentration compared with samples treated with azathioprine alone. These effects were comparable to those observed with the reference drug (Immunal). Supplementation of lymphocyte cultures with either Immunal or the MHF resulted in a significant increase in TNFα and IFNγ levels, with no significant effect on IL-1ra production. Azathioprine significantly decreased TNFα and IFNγ levels and markedly increased IL- 1ra production compared with control values. Co-culture of lymphocytes with azathioprine in the presence of either Immunal or the MHF increased TNFα and IFNγ levels and, conversely, markedly decreased IL-1ra levels. Therefore, the proposed herbal formulation may be considered an effective immunocorrective agent with biological activity comparable to that of Immunal.