Effects of the β2-GPI DNA vaccine and FK506 treatment on β2-GPI-specific spleen cell proliferation.

<p>Spleen cells were purified from the different mouse groups on day 56 and stimulated with recombinant β2-GPI protein (10 μg/mL). After 96 h, cell proliferation was assessed by [³H]-TdR incorporation. The results are presented as the means ± standard deviation of triplicate assays from six mice/group. ^nsp>0.05, *** p < 0.001 versus the control APS group.</p

Tolerogenic β2-glycoprotein I DNA vaccine and FK506 as an adjuvant attenuates experimental obstetric antiphospholipid syndrome

<div><p>DNA vaccines have recently emerged as a therapeutic agent for treating autoimmune diseases, such as multiple sclerosis. Antiphospholipid antibody syndrome (APS) is an autoimmune disease characterized by β2-glycoprotein I (β2-GPI)-targeting antiphospholipid antibodies (APAs) and vascular thrombosis or obstetrical complications. To examine the therapeutic potential of a β2-GPI DNA vaccine, we administered a vaccine mixed with FK506 as an adjuvant to a mouse model of obstetric APS. First, the pCMV3-β2-GPI DNA vaccine, which encodes the full-length human β2-GPI gene, was constructed. Then, we administered the β2-GPI DNA vaccine in 0.1 ml of saline, mixed with or without 100 μg of FK506, intramuscularly to the mice on days 28, 35 and 42. Blood titers of the anti-β2-GPI antibody, platelet counts, activated partial thromboplastin times (aPTTs), and the percentage of fetal loss were measured. We also stimulated murine splenic T cells ex vivo with β2-GPI and determined the T helper cell proportion and cytokine secretion. The administration of the β2-GPI DNA vaccine mixed with FK506 reduced the blood IgG anti-β2-GPI antibody titers and suppressed APS manifestations in mice. The combination also suppressed interferon-γ and interleukin (IL)-17A secretion but increased the Treg cell proportion and IL-10 secretion in murine splenic T cells following ex vivo stimulation with β2-GPI. Our results demonstrated the therapeutic efficacy of a β2-GPI DNA vaccine and FK506 as an adjuvant in a murine model of obstetric APS. Possible mechanisms include the inhibition of Th1 and Th17 responses and the up-regulation of Treg cells.</p></div

Characterization of the DNA vaccine.

<p>(A) Schematic diagram of the β2-GPI-expressing vectors. The plasmids were named “β2-GPI DNA vaccine” (B) Expression of β2-GPI in vitro. COS-1 cells were transfected with the “β2-GPI DNA vaccine” plasmids, and β2-GPI expression levels were determined by western blotting.</p

Effects of the β2-GPI DNA vaccine and FK506 treatment on β2-GPI-specific Treg cell response.

<p>Spleen cells were purified from the different mouse groups on day 56 and stimulated with recombinant β2-GPI protein (10 μg/mL). After 96 h, (A) the percentage of Foxp3-expressing CD4+ T cells was determined by flow cytometry. The dot plot shows data from one representative mouse from each group. The bar graph represents the mean ± standard deviation (SD) of six mice from three independent experiments. (B) The culture supernatants were collected, and IL-10 and TGF-β production was analyzed in triplicate by ELISA. The data are presented as the means ± SD of triplicate assays from six mice/group. ^nsp>0.05, **p < 0.01 versus the control APS group.</p

Effects of the β2-GPI DNA vaccine and FK506 treatment on IgG anti-β2-GPI antibody levels.

<p>Serum samples were obtained from each mouse group on day 56, and IgG anti-β2-GPI antibody levels were analyzed by ELISA. The data are presented as the means ± standard deviation of triplicate assays from six mice/group. ^nsp>0.05, *p < 0.05, ** p < 0.01 versus the control APS group.</p

Clinical manifestations in mice.

<p>Clinical manifestations in mice.</p

Clinical manifestations in mice.

<p>Clinical manifestations in mice.</p

Effects of the β2-GPI DNA vaccine and FK506 treatment on cytokine production.

<p>Spleen cells were purified from the different mouse groups on day 56 and stimulated with recombinant β2-GPI protein (10 μg/mL). After 96 h, (A) the culture supernatants were collected, and interferon (IFN)- γ, interleukin (IL)-4 and IL-17A production was analyzed in triplicate by ELISA. The data are presented as the means ± standard deviation (SD) of triplicate assays from six mice/group. (B) The percentages of IFN-γ-expressing CD4+ T cells, IL-4-expressing CD4+ T cells and IL-17A-expressing CD4+ T cells were determined by flow cytometry. The dot plot shows data from one representative mouse from each group. The bar graph represents the mean ± SD of six mice from three independent experiments. ^nsp>0.05, *p < 0.05, **p < 0.01 versus the control APS group.</p

Additional file 1 of SARS-CoV-2 primed platelets–derived microRNAs enhance NETs formation by extracellular vesicle transmission and TLR7/8 activation

Additional file 1

Additional file 1: of Association between autophagy and inflammation in patients with rheumatoid arthritis receiving biologic therapy

Figure S1. Representative cytometric histograms of Cyto-ID staining in circulating CD4+ T cells (A1), CD8+ T cells (A2), and CD19+ B cells (A3) from one patient with rheumatoid arthritis (RA) and one healthy control subject (HC). Comparisons of autophagosome levels reflected by Cyto-ID-staining MFI, in CD4+ T cells (B), CD8+ T cells (C) and CD19+ B cells (D) between patients with RA and HC. Data are presented as box plot diagrams, with the box encompassing the 25th percentile (lower bar) to the 75th percentile (upper bar). The horizontal line within the box indicates median value for each group. *p < 0.05 versus HC. Representative cytometric histograms of Cyto-ID staining in peripheral blood (PB)-derived granulocytes (E) and synovial fluid (SF)-derived granulocytes (F). Comparisons of autophagosome levels in PB-derived and SF-derived granulocytes in patients with RA (G). (TIF 1427 kb