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

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.

Figure 1. 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).

Figure 2. 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).

Figure 3. GXMGal effect on Treg cell intracellular cytokines production.

Activated purified CD4⁺ T cells (1×10⁶/ml) from RA were incubated for 18 or 96 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 CD25 then intracellular stained for FOXP3 and TGF-β1 or IL-10. For the analysis of intracellular cytokines, CD25⁺FOXP3⁺ (R1) and CD25⁻FOXP3⁺ (R2) cells were gated on purified CD4⁺ T cells. The gating strategy was shown (A). The percentage of intracellular TGF-β1 and IL-10 on CD25⁺FOXP3⁺ and CD25⁻FOXP3 positive cells were shown after 18 h or 96 h (B) as mean ± SEM of ten independent experiments. *, p<0.05 (triplicate samples of 10 different RA; treated vs untreated cells).

Figure 4. 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).

Figure 5. GXMGal effect on Th1 response.

Activated PBMC (A, B and D) (5×10⁶/ml) from Control and RA were incubated for 20 min, 2, 18 and 72 h in the presence or absence (NS) of GXMGal (10 µg/ml), MTX (10 ng/ml) or DEX (10 nM). After 20 min of incubation, cell lysates were analyzed by western blotting. Membranes were incubated with Ab to T-bet. Actin was used as an internal loading control. Normalization was shown as mean ± SEM of five independent experiments. *, p<0.05 (triplicate samples of 5 different Control and RA; treated vs untreated cells) (A). Culture supernatants were collected after 2, 18 and 72 h to test IFN-γ, IL-12p70 (B) and IL-8 (D) 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); ‡, p<0.05 (triplicate samples of 7 different Control and RA; RA DEX-treated vs untreated cells). Activated purified CD4⁺ T cells (1×10⁶/ml) (C) from RA were stimulated as above described and intracellular stained for T-bet and IFN-γ. The percentage of T-bet⁺/IFN-γ⁺ CD4⁺ T cells 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 five different RA; treated vs untreated cells).

Figure 6. GXMGal effect on Th17 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), MTX (10 ng/ml) or FLLL31 (5 µM). After 18 h of incubation, cell lysates were analyzed by western blotting. Membranes were incubated with Abs to pSTAT3 and STAT3. Actin was used as an internal loading control. Normalization was shown as mean ± SEM of five independent experiments (A) or as one representative experiment of three with similar results (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 IL-21, IL-22, IL-23, IL-6 and IL-17A 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).

Figure 7. Schematic representation of GXMGal effects on Treg (A) and on Treg/Th17 balance (B).