Additional file 1: of Cytokines in the pathogenesis of rheumatoid arthritis: new players and therapeutic targets

Reviewer reports and AU response to reviewers. (DOCX 17 kb

Schematic representation of GalXM effect in T cells from RA patients.

<p>RA differentiated Th17 cells express high levels of CD45RO and produce IL-17A through pSTAT3 activation, causing cartilage destruction and bone erosion. GalXM interacts with Th17 cells by selectively binding to CD45RO. This interaction leads to the inhibition of pSTAT3 activation, IL-17A production and apoptosis induction.</p

IL-17A production and caspase-3 activation.

<p>CD3⁺ T cells (5×10⁶/ml, A or 1×10⁶/ml, B and C) were activated for 30 min in the presence or absence (NS) of soluble anti-CD3 (3 µg/ml) and anti-CD28 (3 µg/ml) mAbs (A, B and C) or rhTGF-β1 (5 ng/ml) and rhIL-6 (20 ng/ml) in mAb to CD3 (B and C) or mAbs to CD3 plus CD28 (A) pre-coated 48-well culture plates, washed, and subsequently incubated for 72 h (A) or 18 h (B and C) in the presence or absence of GalXM (10 µg/ml). In A, after incubation, IL-17A levels were evaluated in culture supernatants by specific ELISA assay. *, <i>p</i><0.05 (Control and RA, n = 7; GalXM treated <i>vs</i> untreated cells). In B, after incubation, cells were labelled with PE anti-active caspase-3 and Alexa Fluor anti-IL-17A mAbs and analysed using FACScan flow cytometry. The percentage of double positive cells is shown as dot plots (B) or bar graph (C). *, <i>p</i><0.05 (Control and RA, n = 7; GalXM treated <i>vs</i> untreated cells). The results reported in the bar graphs are the mean ± SEM.</p

Percentage of GalXM binding to CD45 isoforms on T cells from seven RA patients.

*<p><i>p</i><0.01 (Student’s T test), CD45RO⁺/GalXM⁺<i>vs</i> CD45RA⁺/GalXM^+.</p

Evaluation of proliferation.

<p>CD3⁺ T cells (1×10⁶/ml) were activated for 30 min in the presence or absence (NS) of anti-CD3 mAb (3 µg/ml) and rhIL-2 (20 ng/ml) or PHA (2 µg/ml), washed, and subsequently incubated for 72 h in the presence or absence of GalXM (10 µg/ml). After incubation, cell proliferation was evaluated by ViaLight Plus Kit. *, <i>p</i><0.05 (Control and RA n = 10; GalXM treated <i>vs</i> untreated cells). The results reported in the bar graphs are the mean ± SEM.</p

Phospho-STAT3 activation and IL-17A production.

<p>PBL (5×10⁶/ml) were activated for 30 min with soluble anti-CD3 (3 µg/ml) and anti-CD28 (3 µg/ml) mAbs, washed, and subsequently incubated for 2 or 18 h in the presence or absence of GalXM (10 µg/ml) or FLLL31 (5 µmol/L). After incubation, cell lysates were analysed by Western blotting. Membranes were incubated with anti-pSTAT3 and anti-STAT3 Abs. Actin was used as an internal loading control (A). Normalization was shown in panel B. Culture supernatants were collected to test IL-17A levels by specific ELISA assay (C). *, <i>p</i><0.05 (Control and RA, n = 7; GalXM treated <i>vs</i> untreated cells). The results reported in the bar graph of panel C are the mean ± SEM.</p

Association between CD45 and GalXM.

<p>CD3⁺ T cells (1×10⁶/ml) were incubated for 30 min in the presence or absence of GalXM-FLUOS or an irrelevant-FLUOS molecule (both 10 µg/ml) (A, B and C). After incubation, cells were labelled with PE anti-CD45, anti-CD45RA or anti-CD45RO mAbs and analysed using FACScan flow cytometry. Dot plots of the percentage of double positive cells, from one representative experiment out of seven with similar results, were reported (Control and RA n = 7) (A). In B the percentage of CD45⁺/GalXM⁺ T cells in RA patients compared to control is reported. *, <i>p</i><0.05 (RA <i>vs</i> Control, n = 7). In C the relative variation in percentage of CD45RA⁺/GalXM⁺ or CD45RO⁺/GalXM⁺ in relation to double positive CD45⁺/GalXM⁺ in RA patients was calculated. *, <i>p</i><0.01 (CD45RO⁺/GalXM⁺<i>vs</i> CD45⁺/GalXM⁺). The results reported in the bar graphs are the mean ± SEM.</p

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

<p>Schematic representation of GXMGal effects on Treg (A) and on Treg/Th17 balance (B).</p

Evaluation of apoptosis.

<p>CD3⁺ T cells (1×10⁶/ml) were incubated for 18 h in the presence or absence (unstimulated, NS) of GalXM (10 µg/ml) (A and B), caspase-3 inhibitor (IC-3) (C) or activated for 30 min in the presence or absence (NS) of anti-CD3 mAb (3 µg/ml) and rhIL-2 (20 ng/ml) or PHA (2 µg/ml), and subsequently incubated for 72 h in the presence or absence of GalXM (10 µg/ml) (D). In A, B and D, after incubation, the percentage of cells undergoing apoptosis was evaluated. Data are expressed as percentage of apoptotic CD3⁺ T cells (A and D) *, <i>p</i><0.05 (GalXM treated <i>vs</i> untreated cells). In B, the fold increase of percentage of GalXM-induced apoptosis was shown for each RA patient. In C, after incubation, cells were labelled with PE anti-active caspase-3 mAb and analysed using FACScan flow cytometry. Mean ± SEM of MFI of labelled cells is shown as bar graphs and representative FACScan histogram. <i>p</i> = 0.0292 (GalXM treated <i>vs</i> untreated cells). Error bars denote SEM in all graphs. Panel A and B: Control (n = 10) or RA (n = 30). Panel C: Control and RA (n = 7). Panel D: Control and RA (n = 10).</p

Expression of CD45 isoforms.

<p>Purified CD3⁺ T cells (1×10⁶/ml), isolated from control and RA patients, were labelled with PE anti-CD45, anti-CD45RA or anti-CD45RO mAbs and analysed using FACScan flow cytometry. The MFI of labelled cells from one representative experiment out of seven with similar results, were reported (Control and RA n = 7). Background fluorescence values of cells were obtained using isotype control.</p