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

Clinical manifestations in mice.

<p>Clinical manifestations in mice.</p

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

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

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

Inhibitory effects of propofol on Th17 cell differentiation

<p>Propofol (2,6-diisopropylphenol) is probably the most widely used intravenous anesthetic agent in daily practice. It has been reported to show immunomodulatory activity. However, the effect of propofol on the differention of T cells remains unclear. In this study, we demonstrated for the first time that propofol inhibited both interleukin (IL)-6 plus transforming growth factor-β (TGF-β)-induced Th17 cell differentiation <i>in vitro</i> and in LPS-challenged mice. Propofol also suppressed the IL-6-induced phosphorylation of Janus kinase-2 (JAK2)/signal transducer and activator of transcription (STAT3) pathway, a cytokine-activated essential transcription factor in Th17 cell development, which occurred concomitantly with the enhancement of suppressor of cytokine signaling-3 (SOCS3) expression involved in the downregulation of STAT3 phosphorylation. These data extend our knowledge of the immunosuppressive effects of propofol and their underlying mechanism.</p

Plasma levels of high-mobility group box 1 and soluble receptor for advanced glycation end products in primary antiphospholipid antibody syndrome patients

<div><p>Introduction</p><p>Many studies have demonstrated elevated circulating levels of high-mobility group box 1 (HMGB1) and decreased circulating levels of soluble receptor for advanced glycation end products (sRAGE) in patients with autoimmune diseases. In the present study, we investigated plasma levels of both HMGB1 and sRAGE in primary antiphospholipid syndrome (pAPS) patients.</p><p>Methods</p><p>We prospectively recruited 11 pAPS patients, 17 antiphospholipid antibody (APA)-positive SLE patients without APS manifestations (APA⁺SLE) and 12 SLE patients with secondary APS (APS⁺SLE). We also recruited 10 healthy controls (HCs). Plasma levels of HMGB1 and sRAGE were determined using sandwich ELISA kits. In addition, plasma levels of HMGB1 were also determined using Western blot in 6 pAPS patients and 6 HCs.</p><p>Results</p><p>There was no significant difference in plasma levels of HMGB1 measured by ELISA among subgroups of the enrolled subjects. In addition, there was no significant difference in plasma levels of HMGB1 measured by Western blot between pAPS patients and HCs. On the other hand, we observed a trend toward lower plasma levels of sRAGE in APA⁺SLE or APS⁺SLE patients when compared with HCs. However, there was no significant difference in plasma levels of sRAGE between pAPS patients and HCs, or between APA⁺SLE patients and APS⁺SLE patients.</p><p>Conclusion</p><p>There was no significant difference in plasma levels of sRAGE or HMGB1 between pAPS patients and HCs. Plasma levels of sRAGE/HMGB1 could not be utilized to differentiate between APA⁺SLE and APS⁺SLE patients.</p></div