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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 different subsets of Treg cells.Activated PBMC (1×106/ml) from Control and RA were incubated for 2 h in the presence or absence (NS) of GXMGal (10 µg/ml) or MTX (10 ng/ml). After incubation, cells were stained for cell surface expression of CD4, CD25 and CD127 and then intracellular stained for FOXP3. During the acquisition step a population of PBMC enriched of CD4+ T cells was obtained. For analysis, the CD4+ lymphocytes were gated on PBMC (based on side light scatter and CD4 staining: R1) and analyzed for CD25 and FOXP3 expression (CD25+FOXP3+: R2 and CD25−FOXP3+: R3). The expression of CD127 was shown as FACS histograms in R2 and R3 cells. The gating strategy was shown (A). The percentage of CD25+FOXP3+ and CD25−FOXP3+ cells are shown as mean ± SEM of ten independent experiments. p = 0.0211 (triplicate samples of 10 different Control and RA; RA GXMGal-treated vs untreated cells); p = 0.0357 (triplicate samples of 10 different Control and RA; RA MTX-treated vs untreated cells (B). The mean of fluorescence intensity (MFI) of FOXP3 in CD25+FOXP3+ and CD25−FOXP3+ cells (C) or magnetically purified Treg (D) from RA after 18 h of GXMGal or MTX treatment was shown as mean ± SEM of five independent experiments. *, p<0.05 (triplicate samples of 5 different RA; RA GXMGal-treated vs untreated cells).
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pone-0111163-g002: GXMGal effect on different subsets of Treg cells.Activated PBMC (1×106/ml) from Control and RA were incubated for 2 h in the presence or absence (NS) of GXMGal (10 µg/ml) or MTX (10 ng/ml). After incubation, cells were stained for cell surface expression of CD4, CD25 and CD127 and then intracellular stained for FOXP3. During the acquisition step a population of PBMC enriched of CD4+ T cells was obtained. For analysis, the CD4+ lymphocytes were gated on PBMC (based on side light scatter and CD4 staining: R1) and analyzed for CD25 and FOXP3 expression (CD25+FOXP3+: R2 and CD25−FOXP3+: R3). The expression of CD127 was shown as FACS histograms in R2 and R3 cells. The gating strategy was shown (A). The percentage of CD25+FOXP3+ and CD25−FOXP3+ cells are shown as mean ± SEM of ten independent experiments. p = 0.0211 (triplicate samples of 10 different Control and RA; RA GXMGal-treated vs untreated cells); p = 0.0357 (triplicate samples of 10 different Control and RA; RA MTX-treated vs untreated cells (B). The mean of fluorescence intensity (MFI) of FOXP3 in CD25+FOXP3+ and CD25−FOXP3+ cells (C) or magnetically purified Treg (D) from RA after 18 h of GXMGal or MTX treatment was shown as mean ± SEM of five independent experiments. *, p<0.05 (triplicate samples of 5 different RA; RA GXMGal-treated vs untreated cells).

Mentions: The proportion of conventional Treg, identified by the expression of CD25 molecule and FOXP3 (CD25+FOXP3+), and non-conventional Treg CD25−FOXP3+ was tested after GXMGal treatment in PBMC from RA patients and Control. CD4+ T cells were gated on PBMC and analyzed for CD25, FOXP3 and CD127 [33] expression (Fig. 2A). It is known that Treg CD25+FOXP3+ are CD127low[34]. As shown in Figure 2B the treatment with GXMGal for 2 h did not modulate the percentage of CD25+FOXP3+ Treg; however, GXMGal increased the percentage of non-conventional CD25−FOXP3+ Treg. The prolonged treatment with GXMGal (18 h) did not modulate the percentage of CD25+FOXP3+ or CD25−FOXP3+ Treg (not shown). Similar effect was observed by using MTX (Fig. 2B). GXMGal-induced FOXP3 expression at 18 h was evidenced in CD25+FOXP3+ but not in CD25−FOXP3+ Treg from RA (Fig. 2C). This was confirmed by using magnetically purified Treg from RA (Fig. 2D). Given that we previously demonstrated that GXMGal is able to induce apoptosis of activated T cells [19], [29], we evaluated whether GXMGal affects apoptosis of Treg. Our results showed that treatment with GXMGal or MTX did not modulate neither apoptosis of naïve and activated RA Treg nor their proliferation (data not shown).


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 different subsets of Treg cells.Activated PBMC (1×106/ml) from Control and RA were incubated for 2 h in the presence or absence (NS) of GXMGal (10 µg/ml) or MTX (10 ng/ml). After incubation, cells were stained for cell surface expression of CD4, CD25 and CD127 and then intracellular stained for FOXP3. During the acquisition step a population of PBMC enriched of CD4+ T cells was obtained. For analysis, the CD4+ lymphocytes were gated on PBMC (based on side light scatter and CD4 staining: R1) and analyzed for CD25 and FOXP3 expression (CD25+FOXP3+: R2 and CD25−FOXP3+: R3). The expression of CD127 was shown as FACS histograms in R2 and R3 cells. The gating strategy was shown (A). The percentage of CD25+FOXP3+ and CD25−FOXP3+ cells are shown as mean ± SEM of ten independent experiments. p = 0.0211 (triplicate samples of 10 different Control and RA; RA GXMGal-treated vs untreated cells); p = 0.0357 (triplicate samples of 10 different Control and RA; RA MTX-treated vs untreated cells (B). The mean of fluorescence intensity (MFI) of FOXP3 in CD25+FOXP3+ and CD25−FOXP3+ cells (C) or magnetically purified Treg (D) from RA after 18 h of GXMGal or MTX treatment was shown as mean ± SEM of five independent experiments. *, p<0.05 (triplicate samples of 5 different RA; RA GXMGal-treated vs untreated cells).
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Related In: Results  -  Collection

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

pone-0111163-g002: GXMGal effect on different subsets of Treg cells.Activated PBMC (1×106/ml) from Control and RA were incubated for 2 h in the presence or absence (NS) of GXMGal (10 µg/ml) or MTX (10 ng/ml). After incubation, cells were stained for cell surface expression of CD4, CD25 and CD127 and then intracellular stained for FOXP3. During the acquisition step a population of PBMC enriched of CD4+ T cells was obtained. For analysis, the CD4+ lymphocytes were gated on PBMC (based on side light scatter and CD4 staining: R1) and analyzed for CD25 and FOXP3 expression (CD25+FOXP3+: R2 and CD25−FOXP3+: R3). The expression of CD127 was shown as FACS histograms in R2 and R3 cells. The gating strategy was shown (A). The percentage of CD25+FOXP3+ and CD25−FOXP3+ cells are shown as mean ± SEM of ten independent experiments. p = 0.0211 (triplicate samples of 10 different Control and RA; RA GXMGal-treated vs untreated cells); p = 0.0357 (triplicate samples of 10 different Control and RA; RA MTX-treated vs untreated cells (B). The mean of fluorescence intensity (MFI) of FOXP3 in CD25+FOXP3+ and CD25−FOXP3+ cells (C) or magnetically purified Treg (D) from RA after 18 h of GXMGal or MTX treatment was shown as mean ± SEM of five independent experiments. *, p<0.05 (triplicate samples of 5 different RA; RA GXMGal-treated vs untreated cells).
Mentions: The proportion of conventional Treg, identified by the expression of CD25 molecule and FOXP3 (CD25+FOXP3+), and non-conventional Treg CD25−FOXP3+ was tested after GXMGal treatment in PBMC from RA patients and Control. CD4+ T cells were gated on PBMC and analyzed for CD25, FOXP3 and CD127 [33] expression (Fig. 2A). It is known that Treg CD25+FOXP3+ are CD127low[34]. As shown in Figure 2B the treatment with GXMGal for 2 h did not modulate the percentage of CD25+FOXP3+ Treg; however, GXMGal increased the percentage of non-conventional CD25−FOXP3+ Treg. The prolonged treatment with GXMGal (18 h) did not modulate the percentage of CD25+FOXP3+ or CD25−FOXP3+ Treg (not shown). Similar effect was observed by using MTX (Fig. 2B). GXMGal-induced FOXP3 expression at 18 h was evidenced in CD25+FOXP3+ but not in CD25−FOXP3+ Treg from RA (Fig. 2C). This was confirmed by using magnetically purified Treg from RA (Fig. 2D). Given that we previously demonstrated that GXMGal is able to induce apoptosis of activated T cells [19], [29], we evaluated whether GXMGal affects apoptosis of Treg. Our results showed that treatment with GXMGal or MTX did not modulate neither apoptosis of naïve and activated RA Treg nor their proliferation (data not shown).

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