Background and Objectives We previously demonstrated that the purified microbial polysaccharide Galactoxylomannan (GalXM) from the opportunistic fungus Cryptococcus neoformans is able to impair T-cell proliferation and to induce T lymphocyte apoptosis following the interaction with CD45 in normal subjects. Since T lymphocytes are crucially involved in the pathogenesis of rheumatoid arthritis (RA), aim of the present study was to investigate the effects of GalXM on circulatingT-cell subsets in RA and provide the rationale for potential therapeutic application of this compound.
Materials and Methods Sixty RA patients and 40 healthy donors (HD) were included in the study. RA and HD magnetic sorted-CD3+, CD4+ or CD4+CD25high T cells were cultured in presence or absence of GalXM (10 ng/ml), Dex (10nM), MTX (10ng/ml) or FLLL31 (5µM). Lymphocyte apoptosis was performed at different time-points evaluating propidium iodide staining and in selected experiments cleaved caspase 3 staining by flow cytometry. Flow cytometry was also employed for phenotypic analysis of T cell subsets (CD4, CD25, CD127, FoxP3, IL-17, IL-10, IL-17, TGF-β1) and for the analysis of cell proliferation evaluating CFSE dilution. Western blot was employed to assess phospho-STAT3, STAT3, FoxP3 and T-bet expression in cell lysates. Cytokine concentration in culture supernatants (IL-21, IL-22, IL-23, IL-6, IL-17A, IFN-γ, IL-12p70, IL-8, TGF-β1 and IL-10) was assessed with commercial ELISA kits.
Results GalXM selectively reduced proliferation and increased apoptosis rate in RA Th1 and Th17 cells. GalXM was also able to reduce STAT3 phosphorilation eventually leading to decreased IL-17 production in surviving Th17 cells. To note, GalXM strongly up-regulated FoxP3 expression in CD4+T cells, increased the percentage of non-conventional CD4+CD25-FOXP3+ Treg cells and enhanced suppressive activity of both CD4+CD25highFOXP3+and CD4+CD25-FOXP3+Treg cells. All these findings were further supported by a rebalance of effector/regulatory cytokines in culture supernatants.
Conclusion Our results suggest that GalXM represents a powerful compound able to rebalance Treg/T-effector ratio in RA and therefore it is worth to be employed for therapeutic purposes in this disease.