Effects of selected natural products on human immunocompetent cells

The identification of new lead compounds, and the development of novel drugs for the treatment of autoimmune diseases, are of great importance, since today’s available pharmaceuticals often have substantial limitations. Glucocorticoids and drugs that inhibit the deoxyribonucleic acid (DNA) synthesis (e.g. cyclophosphamide) often cause severe side effects, while state-of-the-art biologicals usually impose a heavy financial burden. Plant extracts are a good starting point for the development of immunosuppressive leads, since they are evolutionarily optimized to serve numerous biological functions. The track record of natural product drug discovery for immunosuppressive leads has been distinguished by blockbuster drugs, such as cyclosporin A or tacrolimus; however, a well-planned, multidisciplinary research approach is required for screening plant extracts, characterizing their effects, clarifying targets, and isolating bioactive compounds.
Enhanced T cell proliferation is a feature of autoimmune diseases such as rheumatoid arthritis or multiple sclerosis; therefore, as a starting point, this study investigated the T cell proliferation inhibitory potential of a library of 435 extracts, prepared from plants used in traditional Chinese medicine (TCM). The immunosuppressive activity of the extracts was assessed by a proliferation-based assay utilizing physiologically-relevant anti-CD3 and anti-CD28 stimulated primary human T lymphocytes. It showed that an Artemisia argyi (Asteraceae, A. argyi) ethyl acetate extract and a Boswellia carteri (Burseraceae, B. carteri) dichloromethane (DCM) extract were active, reflected by a half maximal inhibitory concentration (IC50) of 16.2 µg/mL for the A. argyi extract and 27.0 µg/mL for B. carteri extract. The observed inhibitory effect on T cell proliferation was based on a specific intervention of T cell signaling via an interleukin-2 (IL-2)-dependent mechanism, rather than induced apoptosis or necrosis. Further characterizations revealed a reduced expression of the T cell activation markers CD25 and CD69, as well as a decreased production of IL-2 and interferon-γ (IFN-γ), by the A. argyi extract; the B. carteri extract also suppressed the IL-2 and IFN-γ secretion. Moreover, treatment with B. carteri extract resulted in a reduced degranulation capacity of stimulated T cells.
Both extracts were subjected to high-performance liquid chromatography (HPLC)-mass spectrometry (MS)-based activity profiling. A T cell proliferation assay identified 8-acetyl-artanomaloide, arteglasin A, jaceosidin, 1R-canin, and (4S,5S,6S,7S)- and (4R,5R,6S,7S)-seco-tanapartholides as active constituents of A. argyi. The proliferation assay showed that for B. carteri, 3-O-acetyl-8,24-dienetirucallic acid, 3-O-acetyl-7,24-dienetirucallic acid, 3-oxo-8,24-dienetirucallic acid and 3-O-acetyl-α-boswellic acid suppressed the proliferation of stimulated T lymphocytes.
To validate the target of the active A. argyi and B. carteri compounds, monitoring of the T cell signaling cascade was performed, starting with the IL-2 transcription factor activator protein 1 (AP-1), the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), and the nuclear factor of activated T cells (NFAT). Suppression of the NF-κB and NFAT activity, with IC50 values between 2.0 and 9.3 µM for NF-κB and 1.6 and 9.3 µM for NFAT, was detected for the A. argyi sesquiterpene lactones. 3-O-acetyl-alpha-boswellic acid was found to be the most promising candidate among the B. carteri compounds, as reflected by an IC50 value of 5.6 µM for NFAT activity suppression. For A. argyi the T cell signaling cascade monitoring was extended to the calcium flux in anti-CD3 stimulated Jurkat T cells. The results indicated a suppression of the calcium flux by 30 µg/mL A. argyi extract; however, no influence on the calcium flux of stimulated Jurkat T cells could be shown for the A. argyi compounds, suggesting that the crude plant extract may affect the signaling on a more upstream level than the single compounds, isolated thus far.
This study also evaluated the potential wound healing and immune modulating capacities of extracts from nine plants that are traditionally used in Nepal to improve wound healing. An ethyl acetate extract of Gmelina arborea (Lamiaceae, G. arborea) positively influenced the wound-healing capacity of human keratinocytes and fibroblasts.
For satisfactory wound healing, a balance between pathogen clearance by inflammatory feedback loops, and regulatory mechanisms to prevent fatal inflammatory responses, is essential; thus, the influence of the extracts on inflammation parameters was addressed. The G. arborea ethyl acetate extract, and an ethyl acetate extract from Bassia longifolia (Sapotaceae, B. longifolia), concentration-dependently inhibited the proliferation of stimulated T cells. This proliferation inhibition was not related to induced apoptosis or necrosis. The observed suppression of T cell proliferation could be linked to a decreased secretion of IL-2, which is essential for the proliferation and differentiation of T lymphocytes. Furthermore, the degranulation capacity of stimulated T cells was shown to decrease in response to treatment with either B. longifolia or G. arborea extract, emphasizing the anti-inflammatory potential of both extracts. Dendritic cells (DCs) play an important role in wound closure, since they increase the cell migration rate of keratinocytes by secreting interleukin-8 (IL-8). A slightly enhanced IL-8 secretion by DCs was detected after treatment with ethyl acetate extracts of either G. arborea or B. longifolia.