Crassolide Suppresses Dendritic Cell Maturation and Attenuates Experimental Antiphospholipid Syndrome.

Antiphospholipid syndrome (APS) is an autoimmune disease characterized by the production of ß2-glycoprotein I (ß2GPI)-dependent autoantibodies, with vascular thrombosis or obstetrical complications. Around 20% of APS patients are refractory to current treatments. Crassolide, a cembranoid diterpene extracted from soft corals, is a potential therapeutic candidate. Here, to examine the anti-inflammatory properties of crassolide, we first determined its effects on bone marrow-derived and splenic dendritic cells (DC). Specifically, we applied lipopolysaccharide (LPS) or ß2GPI stimulation and measured the expressions of CD80 and CD86, and secretions of cytokines. We also determined in the OT-II mice, if bone marrow-derived DC was able to stimulate antigen-specific T cells. Moreover, we examined the therapeutic potential of crassolide postimmunization in a murine model of APS that depended on active immunization with ß2GPI. The vascular manifestations were evaluated in terms of fluorescein-induced thrombi in mesenteric microvessels, whereas the obstetric manifestations were evaluated based on the proportion of fetal loss after pregnancy. We also measured blood titers of anti-ß2GPI antibody, splenic cell proliferative responses and cytokine secretions after ß2GPI stimulation ex vivo. Finally, we determined in these mice, hematological, hepatic and renal toxicities of crassolide. Crassolide after LPS stimulation suppressed DC maturation and secretion of tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-12 and IL-23, and downstream T cell activation. Crassolide could partially ameliorate both the vascular and obstetric manifestations of APS in BALB/c mice. Both blood titers of anti-ß2GPI antibody and splenic cell proliferation after ß2GPI stimulation were reduced. Splenic Th1 and Th17 responses were also lowered after ß2GPI stimulation. Finally, within therapeutic doses of crassolide, we found no evidence of its toxicity. In conclusion, we showed the ability of crassolide to suppress DC and downstream T cell responses. Crassolide is therefore a potential candidate for adjunctive therapy in APS.

Hydroxychloroquine partially prevents endothelial dysfunction induced by anti-beta-2-GPI antibodies in an in vivo mouse model of antiphospholipid syndrome.

BACKGROUND: Antiphospholipid syndrome is associated with endothelial dysfunction, which leads to thrombosis and early atheroma. Given that hydroxychloroquine has anti-thrombotic properties in lupus, we hypothesized that it could reduce endothelial dysfunction in an animal model of antiphospholipid syndrome. We evaluated the effect of hydroxychloroquine in preventing endothelial dysfunction in a mouse model of antiphospholipid syndrome. METHODS: Antiphospholipid syndrome was induced by an injection of monoclonal anti-beta-2-GPI antibodies. Vascular reactivity was evaluated in mesenteric resistance arteries isolated from mice 3 weeks (APL3W) after receiving a single injection of anti-beta-2-GPI antibodies and after 3 weeks of daily oral hydroxychloroquine treatment (HCQ3W) compared to control mice (CT3W). We evaluated endothelial dysfunction by measuring acetylcholine-mediated vasodilation. A pharmacological approach was used to evaluate NO synthase uncoupling (tetrahydrobiopterin) and the generation of reactive oxygen species (Tempol). RESULTS: Impaired acetylcholine-mediated dilation was evidenced in mice 3 weeks after anti-beta-2-GPI antibodies injection compared to CT3W, by reduced maximal dilation (p<0.0001) and sensitivity (pKd) (p = 0.01) to acetylcholine. Hydroxychloroquine improved acetylcholine-dependent dilation, on pKd (p = 0.02) but not maximal capacity compared to untreated mice. The addition of tetrahydrobiopterin (p = 0.02) and/or Tempol (p = 0.0008) improved acetylcholine-mediated dilation in APL3W but not in HCQ3W. CONCLUSIONS: We demonstrated that endothelial dysfunction in mouse resistance arteries persisted at 3 weeks after a single injection of monoclonal anti-beta-2-GPI antibodies, and that hydroxychloroquine improved endothelium-dependent dilation at 3 weeks, through improvement of NO synthase coupling and oxidative stress reduction.

IgG accumulates in inhibitory hippocampal neurons of experimental antiphospholipid syndrome.

Mice immunized with ß2-glycoprotein I (ß2GPI) are an experimental model of the antiphospholipid syndrome (eAPS) displaying elevated titers of antiphospholipid antibodies (aPL). We presently studied whether the behavioral hyperactivity in eAPS mice is associated with in vivo binding and accumulation of IgG in the brain. At 6 weeks post immunization eAPS mice had significantly higher levels of aPL (1.32 ± 0.28 and 0.02 ± 0.01 AU, p < 0.001 by t-test) compared to adjuvant immunized controls, as measured by ELISA. Significant hyperactivity in a staircase test in the eAPS mice compared to controls was found in stair-climbing (18.4 ± 0.9 and 12.0 ± 1.7, respectively) and rearing measures (23.5 ± 2.1 and 12.5 ± 1.9, p < 0.01 by t-test). Immunofluorescence staining in eAPS mice revealed significant in vivo accumulation of IgG in cortical and hippocampal neurons which was not seen in controls. Staining for IgG was markedly intense in inhibitory interneurons co-stained for GAD67 in the hippocampus of eAPS mice. The integrity of the blood brain barrier (BBB) evaluated by injection of Evans blue (EB) was impaired in eAPS and adjuvant immunized mice compared to naïve mice. Electrophysiological recordings in hippocampal brain slices showed altered response to paired pulse stimulation as well as dysregulation of carbachol-induced γ- oscillations in eAPS mice compared to control. Penetration into the brain and direct interaction of aPL with inhibitory interneurons in the hippocampus may explain the hyperactive behavior of the eAPS mice. A direct role of aPL in causing CNS dysfunction points to these antibodies as an important therapeutic target in APS.