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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 response.Activated PBMC (A and C) or purified CD4+ T cells (B) (both 5×106/ml) from Control and RA were incubated for 2, 18 and 72 h in the presence or absence (NS) of GXMGal (10 µg/ml) or MTX (10 ng/ml). After 2 and 18 h (A) or 18 h (B) of incubation, cell lysates were analyzed by western blotting. Membranes were incubated with Ab to FOXP3. Actin was used as an internal loading control. Normalization was shown as mean ± SEM of five independent experiments (A and B). *, p<0.05 (triplicate samples of 5 different Control and RA; RA treated vs untreated cells). Culture supernatants were collected after 2, 18 and 72 h to test TGF-β1 and IL-10 levels by specific ELISA assays. *, p<0.05 (triplicate samples of 7 different Control and RA; RA GXMGal-treated vs untreated cells); †, p<0.05 (triplicate samples of 7 different Control and RA; RA MTX-treated vs untreated cells) (C).
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pone-0111163-g001: GXMGal effect on Treg cell response.Activated PBMC (A and C) or purified CD4+ T cells (B) (both 5×106/ml) from Control and RA were incubated for 2, 18 and 72 h in the presence or absence (NS) of GXMGal (10 µg/ml) or MTX (10 ng/ml). After 2 and 18 h (A) or 18 h (B) of incubation, cell lysates were analyzed by western blotting. Membranes were incubated with Ab to FOXP3. Actin was used as an internal loading control. Normalization was shown as mean ± SEM of five independent experiments (A and B). *, p<0.05 (triplicate samples of 5 different Control and RA; RA treated vs untreated cells). Culture supernatants were collected after 2, 18 and 72 h to test TGF-β1 and IL-10 levels by specific ELISA assays. *, p<0.05 (triplicate samples of 7 different Control and RA; RA GXMGal-treated vs untreated cells); †, p<0.05 (triplicate samples of 7 different Control and RA; RA MTX-treated vs untreated cells) (C).

Mentions: 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 [33]. 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.


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 response.Activated PBMC (A and C) or purified CD4+ T cells (B) (both 5×106/ml) from Control and RA were incubated for 2, 18 and 72 h in the presence or absence (NS) of GXMGal (10 µg/ml) or MTX (10 ng/ml). After 2 and 18 h (A) or 18 h (B) of incubation, cell lysates were analyzed by western blotting. Membranes were incubated with Ab to FOXP3. Actin was used as an internal loading control. Normalization was shown as mean ± SEM of five independent experiments (A and B). *, p<0.05 (triplicate samples of 5 different Control and RA; RA treated vs untreated cells). Culture supernatants were collected after 2, 18 and 72 h to test TGF-β1 and IL-10 levels by specific ELISA assays. *, p<0.05 (triplicate samples of 7 different Control and RA; RA GXMGal-treated vs untreated cells); †, p<0.05 (triplicate samples of 7 different Control and RA; RA MTX-treated vs untreated cells) (C).
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pone-0111163-g001: GXMGal effect on Treg cell response.Activated PBMC (A and C) or purified CD4+ T cells (B) (both 5×106/ml) from Control and RA were incubated for 2, 18 and 72 h in the presence or absence (NS) of GXMGal (10 µg/ml) or MTX (10 ng/ml). After 2 and 18 h (A) or 18 h (B) of incubation, cell lysates were analyzed by western blotting. Membranes were incubated with Ab to FOXP3. Actin was used as an internal loading control. Normalization was shown as mean ± SEM of five independent experiments (A and B). *, p<0.05 (triplicate samples of 5 different Control and RA; RA treated vs untreated cells). Culture supernatants were collected after 2, 18 and 72 h to test TGF-β1 and IL-10 levels by specific ELISA assays. *, p<0.05 (triplicate samples of 7 different Control and RA; RA GXMGal-treated vs untreated cells); †, p<0.05 (triplicate samples of 7 different Control and RA; RA MTX-treated vs untreated cells) (C).
Mentions: 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 [33]. 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.

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