Date Published: October 22, 2014
Publisher: Public Library of Science
Author(s): Eva Pericolini, Elena Gabrielli, Alessia Alunno, Elena Bartoloni Bocci, Stefano Perito, Siu-Kei Chow, Elio Cenci, Arturo Casadevall, Roberto Gerli, Anna Vecchiarelli, Maurizio Del Poeta.
Regulatory T cells (Treg) play a critical role in the prevention of autoimmunity, and the suppressive activity of these cells is impaired in rheumatoid arthritis (RA). The aim of the present study was to investigate function and properties of Treg of RA patients in response to purified polysaccharide glucuronoxylomannogalactan (GXMGal).
Flow cytometry and western blot analysis were used to investigate the frequency, function and properties of Treg cells.
GXMGal was able to: i) induce strong increase of FOXP3 on CD4+ T cells without affecting the number of CD4+CD25+FOXP3+ Treg cells with parallel increase in the percentage of non-conventional CD4+CD25−FOXP3+ Treg cells; ii) increase intracellular levels of TGF-β1 in CD4+CD25−FOXP3+ Treg cells and of IL-10 in both CD4+CD25+FOXP3+ and CD4+CD25−FOXP3+ Treg cells; iii) enhance the suppressive activity of CD4+CD25+FOXP3+ and CD4+CD25−FOXP3+ Treg cells in terms of inhibition of effector T cell activity and increased secretion of IL-10; iv) decrease Th1 response as demonstrated by inhibition of T-bet activation and down-regulation of IFN-γ and IL-12p70 production; v) decrease Th17 differentiation by down-regulating pSTAT3 activation and IL-17A, IL-23, IL-21, IL-22 and IL-6 production.
These data show that GXMGal improves Treg functions and increases the number and function of CD4+CD25−FOXP3+ Treg cells of RA patients. It is suggested that GXMGal may be potentially useful for restoring impaired Treg functions in autoimmune disorders and for developing Treg cell-based strategies for the treatment of these diseases.
Rheumatoid arthritis (RA) is an autoimmune disease characterized by destructive joint inflammation. The synovial inflammation is characterized by non-specific infiltration of both lymphocytes and innate immune cells, such as synoviocytes, macrophages and neutrophils . RA is generally considered to be an autoimmune disease in which pathogenic T cells such as T helper (Th)1 and Th17 cells play an important role . An exciting aspect of Th17 cell homeostasis is the reciprocal relationship with regulatory T cells (Treg), whose imbalance is believed to play a major role in the development of autoimmune disease –. Treg were originally identified by high surface expression of CD25 ,  and subsequently by the forkhead box protein P3 (FOXP3) transcriptional factor, which controls their development and suppressive function , . Interestingly, it was suggested that activated FOXP3+ non-Treg cells may be a reservoir of silent Treg that regain their function following activation . The data on the effective number of Treg cells in RA patients are inconsistent. Some studies report a decreased number of Treg cells in the blood of RA , while others report increased numbers of Treg cells , however it is clear that Treg cells appear unable to suppress inflammation in the rheumatoid joints . Thus, paradoxically, CD4+CD25+ T cells, including CD4+CD25+FOXP3+ Treg cells, circulate in RA patients despite the still ongoing inflammation , . The capacity of Treg cells to suppress several arthritic responses both in humans and animal models makes them potential therapeutic targets in arthritic conditions such as RA . A prior study suggested that the therapeutic efficacy of methotrexate (MTX), the current “gold standard” treatment in experimental RA, was partly attributed to the increased development of CD4+CD25+ Treg cells . Furthermore, anti-rheumatic biotechnological therapies may offer a means of restoring the Th17/Treg cell balance in favor of Treg, thereby re-establishing immune tolerance . It has been demonstrated that some fungal products, such as those from Cryptococcus neoformans (C. neoformans), directly induce lymphocytes apoptosis . Accordingly, we demonstrated that glucuronoxylomannogalactan (GXMGal), a purified capsular polysaccharide from the opportunistic fungus C. neoformans, directly induce apoptosis of activated lymphocytes . This was recently evidenced also using activated RA T cells producing IL-17A suggesting the potential role of GXMGal in fighting deleterious Th17 cells . In light of this finding, the aim of this study was to understand whether GXMGal could restore the Th17/Treg balance, both by dampening Th17 response  and modulating Treg cell suppressive capacity.
Firstly, we analyzed the effect of GXMGal on FOXP3 expression in PBMC and CD4+ T cells from RA patients. PBMC from RA patients and from healthy donors (Control) were treated with GXMGal or MTX for 2 and 18 h and the expression of FOXP3 was evaluated. The results showed that GXMGal markedly induced FOXP3 activation after 2 and 18 h of incubation, while MTX only after 2 h (Fig. 1A). GXMGal and MTX treatment did not produce any modulation on PBMC from Control. GXMGal-induced FOXP3 activation at 18 h was also confirmed by using purified CD4+ T cells from RA (Fig. 1B). Furthermore, we analyzed key cytokines such as TGF-β1 and IL-10 involved in Treg activation . To this purpose PBMC were treated with GXMGal for 2, 18 and 72 h and the possible modulation of TGF-β1 and IL-10 production was tested. Both cytokines were produced at higher levels in culture supernatants of PBMC from RA patients with respect to Control. The production of TGF-β1 and IL-10 was further enhanced by GXMGal treatment after 18 h or 18 and 72 h of incubation respectively (Fig. 1C). The lack of the increased production of TGF-β1 after 72 h of incubation could be due to the reutilization of this cytokine by the cells during the incubation period. MTX produced similar effects.
FOXP3 is thought to be the main marker of Treg, since it plays a critical role in their development and maturation . Compelling evidence show that FOXP3-deficient mice develop autoimmune disease , . The role of Treg in RA has been partially elucidated and different results have been reported. In particular, a decreased number of Treg in the blood of RA patients has been observed ; however, other reports show high levels of circulating conventional CD4+CD25+FOXP3+ Treg cells in RA , , while additional studies reported an unaltered number . Because of these controversial results about the number of circulating Treg cells, the interpretation of the data is problematic. Nevertheless, compelling evidence has emerged about the impaired function of these cells in RA patients . Recent investigations revealed that a potentiation of Treg cells is beneficial in RA , . In this study we report that the treatment with GXMGal induces strong and long-lasting increase of FOXP3 expression in RA Treg cells still evident 18 h after stimulation. This was accompanied by early and transient enhancement of TGF-β1 production and long-lasting production of IL-10. The major source of early production of TGF-β1 appeared to be the CD4+CD25−FOXP3+ T cell subset. On the contrary, the early production of IL-10 seems to be due to both CD4+CD25+FOXP3+ and CD4+CD25−FOXP3+ cells, although only CD4+CD25+FOXP3+ cells seemed to be responsible of its long-lasting production. We previously demonstrated that GXMGal inhibits pro-inflammatory cytokine secretion. Since the suppressive activity of Treg cells could be counteracted by inflammatory mediators , , particularly by TNF-α , it is possible that the increased activity of Treg cells induced by GXMGal could also include the previously described inhibition of TNF-α . Accordingly, the treatment of RA subjects with an anti-TNF-α specific antibody was shown to restore Treg cell function via increased expression of FOXP3 phosphorylation . Similarly, GXMGal could retain beneficial effects similar to those of anti-TNF-α treatment by influencing Treg cell activity. GXMGal did not modulate the percentage of CD4+CD25+FOXP3+ cells, while atypical CD4+CD25−FOXP3+ cells appeared to be numerically increased after GXMGal addition. The percentage of both cells was calculated on CD4+ T cells gated on PBMC, suggesting that the small and not statistically significant decline of CD4+CD25+FOXP3+, observed after GXMGal treatment, could at least in part account for the increased number of CD4+CD25−FOXP3+; however it is likely that besides the shift of CD4+CD25+FOXP3+ into CD4+CD25−FOXP3+, there is also an increase of the CD4+CD25−FOXP3+ cell number per se. Moreover, the significant increased expression of FOXP3 observed in conventional RA Treg cells after 18 h of GXMGal treatment clearly demonstrated that the associated increase of suppressive activity of all Treg is ascribed to CD4+CD25+FOXP3+ cells. GXMGal effects on different Treg subsets have been summarized in Figure 7.