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Functional improvement of regulatory T cells from rheumatoid arthritis subjects induced by capsular polysaccharide glucuronoxylomannogalactan.

Pericolini E, Gabrielli E, Alunno A, Bartoloni Bocci E, Perito S, Chow SK, Cenci E, Casadevall A, Gerli R, Vecchiarelli A - PLoS ONE (2014)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Microbiology Section, Department of Experimental Medicine, University of Perugia, Perugia, Italy.

ABSTRACT

Objective: 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).

Methods: Flow cytometry and western blot analysis were used to investigate the frequency, function and properties of Treg cells.

Results: 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.

Conclusion: 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.

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GXMGal effect on Treg cell suppressive activity.Activated RA purified CD4+ T cells (1×106/ml) were stained with CFSE (1 µM) and then co-cultured for 96 h in the presence or absence of magnetically purified autologous RA Treg cells (Tresp/Treg: 16/1) that have been pre-treated for 18 h in the presence or absence of GXMGal (10 µg/ml) or MTX (10 ng/ml). After 96 h the suppressive activity of Treg cells was evaluated by measuring the percentage of inhibition of proliferation of CD4+ responder T cells (Tresp). Representative CFSE histograms show the distribution of proliferating CFSE-labelled Tresp according to the intensity of the CFSE label from the start of the experiment (Time 0) until 96 h. Given that the initial cell labelling is fairly homogeneous, each CFSE peak represents a cohort of cells that proceed synchronously through the division rounds. The areas within each histograms delimitated by the marker represent the percentage of divided CFSE-labeled cells (A). Mean ± SEM of percentage of proliferation inhibition is shown as bar graph (B). *, p<0.05 (triplicate samples of 10 different RA; treated vs untreated cells).
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pone-0111163-g004: GXMGal effect on Treg cell suppressive activity.Activated RA purified CD4+ T cells (1×106/ml) were stained with CFSE (1 µM) and then co-cultured for 96 h in the presence or absence of magnetically purified autologous RA Treg cells (Tresp/Treg: 16/1) that have been pre-treated for 18 h in the presence or absence of GXMGal (10 µg/ml) or MTX (10 ng/ml). After 96 h the suppressive activity of Treg cells was evaluated by measuring the percentage of inhibition of proliferation of CD4+ responder T cells (Tresp). Representative CFSE histograms show the distribution of proliferating CFSE-labelled Tresp according to the intensity of the CFSE label from the start of the experiment (Time 0) until 96 h. Given that the initial cell labelling is fairly homogeneous, each CFSE peak represents a cohort of cells that proceed synchronously through the division rounds. The areas within each histograms delimitated by the marker represent the percentage of divided CFSE-labeled cells (A). Mean ± SEM of percentage of proliferation inhibition is shown as bar graph (B). *, p<0.05 (triplicate samples of 10 different RA; treated vs untreated cells).

Mentions: Given that TGF-β1 and IL-10 production was determined in PBMC supernatants (Fig. 1C), we analyzed the level of these cytokines in both CD25+FOXP3+ and CD25−FOXP3+ cells. For the analysis of intracellular cytokines, CD25+FOXP3+ (R1) and CD25−FOXP3+ (R2) were gated on magnetically purified CD4+ T cells (Fig. 3A). After 18 h of incubation, both GXMGal and MTX induced a significant increase of intracellular TGF-β1 production in purified CD25−FOXP3+ cells, but they did not modulate this cytokine in purified CD25+FOXP3+ cells (Fig 3B). Moreover, intracellular IL-10 was significantly increased by GXMGal stimulation in both CD25+FOXP3+ and CD25−FOXP3+ cells. MTX induced a significant increase of intracellular IL-10 only in CD25+FOXP3+ cells (Fig. 3B). Moreover, when the treatment with GXMGal or MTX was prolonged till 96 h, a persistence of intracellular increase of IL-10 was detected only in CD25+FOXP3+ cells. At this time, no modulation of TGF-β1 was observed in both CD25+FOXP3+ and CD25−FOXP3+ cells (Fig. 3B). Moreover, we verified the in vitro functional activity of Treg towards autologous responder T cells (Tresp). RA activated CFSE-labelled Tresp were cultured for 96 h in the presence or absence of autologous Treg pre-treated for 18 h with or without GXMGal or MTX. Our data demonstrated that proliferation of RA Tresp cells was suppressed by Treg and this effect was significantly potentiated when Treg cells were pre-treated with GXMGal or MTX (Fig. 4A and B). Since it is recognized that Th1 cells play an important pro-inflammatory role in the pathogenesis of RA, the effect of GXMGal on Th1 differentiation was also evaluated. PBMC from RA patients and Control were treated with GXMGal, MTX and DEX for 20 min. It is well established that DEX can inhibit Th1 activation [27] and therefore DEX was used as inhibitor of T-box-containing protein expressed in T cells (T-bet) activation, exclusively expressed in Th1 cells. The results showed that GXMGal treatment was able to significantly suppress T-bet activation in PBMC from RA and Control. A similar effect was observed with DEX, whereas MTX produced inhibitory effects only in PBMC from RA patients (Fig. 5A). To further investigate the role of GXMGal in Th1 response, we analyzed the production of IFN-γ and IL-12p70, that were produced at higher levels in PBMC culture supernatants from RA compared to Control, after 18 and 72 h or 2, 18 and 72 h, respectively (Fig. 5B). A significant inhibition of IFN-γ production was observed after treatment with GXMGal or MTX after 18 and 72 h of incubation. As expected, DEX significantly reduced IFN-γ production (Fig. 5B). IL-12p70 was significantly inhibited by GXMGal at all times tested and by MTX after 18 and 72 h of incubation (Fig. 5B). GXMGal and MTX were also able to significantly reduce the percentage of T-bet+/IFN-γ+ CD4+ T cells from RA after 18 h of incubation (Fig. 5C). We also tested IL-8 production by PBMC from RA patients and Control after GXMGal stimulation. Although IL-8 production was higher in RA than Control PBMC, the levels of this cytokine were not modulated neither by GXMGal nor MTX treatment at all times tested (Fig. 5D).


Functional improvement of regulatory T cells from rheumatoid arthritis subjects induced by capsular polysaccharide glucuronoxylomannogalactan.

Pericolini E, Gabrielli E, Alunno A, Bartoloni Bocci E, Perito S, Chow SK, Cenci E, Casadevall A, Gerli R, Vecchiarelli A - PLoS ONE (2014)

GXMGal effect on Treg cell suppressive activity.Activated RA purified CD4+ T cells (1×106/ml) were stained with CFSE (1 µM) and then co-cultured for 96 h in the presence or absence of magnetically purified autologous RA Treg cells (Tresp/Treg: 16/1) that have been pre-treated for 18 h in the presence or absence of GXMGal (10 µg/ml) or MTX (10 ng/ml). After 96 h the suppressive activity of Treg cells was evaluated by measuring the percentage of inhibition of proliferation of CD4+ responder T cells (Tresp). Representative CFSE histograms show the distribution of proliferating CFSE-labelled Tresp according to the intensity of the CFSE label from the start of the experiment (Time 0) until 96 h. Given that the initial cell labelling is fairly homogeneous, each CFSE peak represents a cohort of cells that proceed synchronously through the division rounds. The areas within each histograms delimitated by the marker represent the percentage of divided CFSE-labeled cells (A). Mean ± SEM of percentage of proliferation inhibition is shown as bar graph (B). *, p<0.05 (triplicate samples of 10 different RA; treated vs untreated cells).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4206502&req=5

pone-0111163-g004: GXMGal effect on Treg cell suppressive activity.Activated RA purified CD4+ T cells (1×106/ml) were stained with CFSE (1 µM) and then co-cultured for 96 h in the presence or absence of magnetically purified autologous RA Treg cells (Tresp/Treg: 16/1) that have been pre-treated for 18 h in the presence or absence of GXMGal (10 µg/ml) or MTX (10 ng/ml). After 96 h the suppressive activity of Treg cells was evaluated by measuring the percentage of inhibition of proliferation of CD4+ responder T cells (Tresp). Representative CFSE histograms show the distribution of proliferating CFSE-labelled Tresp according to the intensity of the CFSE label from the start of the experiment (Time 0) until 96 h. Given that the initial cell labelling is fairly homogeneous, each CFSE peak represents a cohort of cells that proceed synchronously through the division rounds. The areas within each histograms delimitated by the marker represent the percentage of divided CFSE-labeled cells (A). Mean ± SEM of percentage of proliferation inhibition is shown as bar graph (B). *, p<0.05 (triplicate samples of 10 different RA; treated vs untreated cells).
Mentions: Given that TGF-β1 and IL-10 production was determined in PBMC supernatants (Fig. 1C), we analyzed the level of these cytokines in both CD25+FOXP3+ and CD25−FOXP3+ cells. For the analysis of intracellular cytokines, CD25+FOXP3+ (R1) and CD25−FOXP3+ (R2) were gated on magnetically purified CD4+ T cells (Fig. 3A). After 18 h of incubation, both GXMGal and MTX induced a significant increase of intracellular TGF-β1 production in purified CD25−FOXP3+ cells, but they did not modulate this cytokine in purified CD25+FOXP3+ cells (Fig 3B). Moreover, intracellular IL-10 was significantly increased by GXMGal stimulation in both CD25+FOXP3+ and CD25−FOXP3+ cells. MTX induced a significant increase of intracellular IL-10 only in CD25+FOXP3+ cells (Fig. 3B). Moreover, when the treatment with GXMGal or MTX was prolonged till 96 h, a persistence of intracellular increase of IL-10 was detected only in CD25+FOXP3+ cells. At this time, no modulation of TGF-β1 was observed in both CD25+FOXP3+ and CD25−FOXP3+ cells (Fig. 3B). Moreover, we verified the in vitro functional activity of Treg towards autologous responder T cells (Tresp). RA activated CFSE-labelled Tresp were cultured for 96 h in the presence or absence of autologous Treg pre-treated for 18 h with or without GXMGal or MTX. Our data demonstrated that proliferation of RA Tresp cells was suppressed by Treg and this effect was significantly potentiated when Treg cells were pre-treated with GXMGal or MTX (Fig. 4A and B). Since it is recognized that Th1 cells play an important pro-inflammatory role in the pathogenesis of RA, the effect of GXMGal on Th1 differentiation was also evaluated. PBMC from RA patients and Control were treated with GXMGal, MTX and DEX for 20 min. It is well established that DEX can inhibit Th1 activation [27] and therefore DEX was used as inhibitor of T-box-containing protein expressed in T cells (T-bet) activation, exclusively expressed in Th1 cells. The results showed that GXMGal treatment was able to significantly suppress T-bet activation in PBMC from RA and Control. A similar effect was observed with DEX, whereas MTX produced inhibitory effects only in PBMC from RA patients (Fig. 5A). To further investigate the role of GXMGal in Th1 response, we analyzed the production of IFN-γ and IL-12p70, that were produced at higher levels in PBMC culture supernatants from RA compared to Control, after 18 and 72 h or 2, 18 and 72 h, respectively (Fig. 5B). A significant inhibition of IFN-γ production was observed after treatment with GXMGal or MTX after 18 and 72 h of incubation. As expected, DEX significantly reduced IFN-γ production (Fig. 5B). IL-12p70 was significantly inhibited by GXMGal at all times tested and by MTX after 18 and 72 h of incubation (Fig. 5B). GXMGal and MTX were also able to significantly reduce the percentage of T-bet+/IFN-γ+ CD4+ T cells from RA after 18 h of incubation (Fig. 5C). We also tested IL-8 production by PBMC from RA patients and Control after GXMGal stimulation. Although IL-8 production was higher in RA than Control PBMC, the levels of this cytokine were not modulated neither by GXMGal nor MTX treatment at all times tested (Fig. 5D).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Microbiology Section, Department of Experimental Medicine, University of Perugia, Perugia, Italy.

ABSTRACT

Objective: 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).

Methods: Flow cytometry and western blot analysis were used to investigate the frequency, function and properties of Treg cells.

Results: 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.

Conclusion: 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.

Show MeSH
Related in: MedlinePlus