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

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

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

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

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

Image5_Suppression of Dendritic Cell Maturation by Kefir Peptides Alleviates Collagen-Induced Arthritis in Mice.TIF

Arthritis is a disorder that is characterized by joint inflammation and other symptoms. Rheumatoid arthritis (RA), an autoimmune disease, is one of the most common arthritis in worldwide. Inflammation of the synovium is the main factor that triggers bone erosion in the joints in RA, but the pathogenesis of RA is not clearly understood. Kefir grain-fermented products have been demonstrated to enhance immune function and exhibit immune-modulating bioactivities. This study aims to explore the role of kefir peptides (KPs) on the regulation of dendritic cell, which are found in RA synovial fluid, and the protection effects of KPs on mice with collagen-induced arthritis (CIA). Immature mouse bone marrow-derived dendritic cells (BMDCs) were treated with KPs (2.2 and 4.4 mg/ml) and then exposed to lipopolysaccharide (LPS) to study the immune regulation function of KPs in dendritic cells. Mice with CIA (n = 5 per group) were orally administrated KPs (3.75 and 7.5 mg/day/kg) for 21 days and therapeutic effect of KPs on mice with arthritis were assessed. In this study, we found that KPs could inhibit surface molecule expression, reduce inflammatory cytokine release, and repress NF-κB and MAPK signaling in LPS-stimulated mouse BMDCs. In addition, a high dose of KPs (7.5 mg/kg) significantly alleviated arthritis symptoms, decreased inflammatory cytokine expression, suppressed splenic DC maturation and decrease the percentage of Th1 and Th17 in the spleens on mice with CIA. Our findings demonstrated that KPs ameliorate CIA in mice through the mechanism of suppressing DC maturation and inflammatory cytokine releases.</p

Image1_Suppression of Dendritic Cell Maturation by Kefir Peptides Alleviates Collagen-Induced Arthritis in Mice.TIF

Arthritis is a disorder that is characterized by joint inflammation and other symptoms. Rheumatoid arthritis (RA), an autoimmune disease, is one of the most common arthritis in worldwide. Inflammation of the synovium is the main factor that triggers bone erosion in the joints in RA, but the pathogenesis of RA is not clearly understood. Kefir grain-fermented products have been demonstrated to enhance immune function and exhibit immune-modulating bioactivities. This study aims to explore the role of kefir peptides (KPs) on the regulation of dendritic cell, which are found in RA synovial fluid, and the protection effects of KPs on mice with collagen-induced arthritis (CIA). Immature mouse bone marrow-derived dendritic cells (BMDCs) were treated with KPs (2.2 and 4.4 mg/ml) and then exposed to lipopolysaccharide (LPS) to study the immune regulation function of KPs in dendritic cells. Mice with CIA (n = 5 per group) were orally administrated KPs (3.75 and 7.5 mg/day/kg) for 21 days and therapeutic effect of KPs on mice with arthritis were assessed. In this study, we found that KPs could inhibit surface molecule expression, reduce inflammatory cytokine release, and repress NF-κB and MAPK signaling in LPS-stimulated mouse BMDCs. In addition, a high dose of KPs (7.5 mg/kg) significantly alleviated arthritis symptoms, decreased inflammatory cytokine expression, suppressed splenic DC maturation and decrease the percentage of Th1 and Th17 in the spleens on mice with CIA. Our findings demonstrated that KPs ameliorate CIA in mice through the mechanism of suppressing DC maturation and inflammatory cytokine releases.</p