Abstract
Immune thrombocytopenia (ITP) is an acquired autoimmune bleeding disorder, accounting for about 1/3 of clinical hemorrhagic diseases. Loss of peripheral immune tolerance through simultaneous decrease of CD4+CD25+Foxp3+ regulatory T cells (Tregs) as well as unrestricted proliferation and activation of peripheral CD4+ effector T cells underpin the pathophysiology of ITP.
Atorvastatin (AT), an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, could competitively combine with HMG-CoA reductase and inhibit the production of cholesterol, accompanying with the decrease of some intermediate metabolites, such as small GTPase. Recent studies have found that statins could regulate the homeostasis of effector T cells and Tregs in some autoimmune diseases and enhance bone marrow endothelial cell function in corticosteroid-resistant ITP. However, whether AT could target the Tregs/effector T cell-axis to restore the peripheral immune tolerance in ITP is unknown.
To assess the effect of AT in ITP, CD4+ T cells were isolated magnetically from peripheral blood mononuclear cells of ITP patients and cultured with different doses of AT (0μM, 5μM, 10μM, 20μM) for 3 days. The activation of CD4+ T cells were analyzed by flow cytometry. It was shown that AT could significantly inhibit the expression of CD25 on CD4+ T cells, CD4+CD45RA+ naïve T cells and CD4+CD45RO+ memory T cells and impede the switching from CD45RA to RO dose-dependently. Moreover, AT was also effective in reducing the early activation of CD4+ T cells by decreasing the expression of CD69. The dampened activation of CD4+ T cells could be reversed after blocking AT by L-mevalonate (L-MA). These results suggested that AT can inhibit the activation of CD4+ T cells and naïve T cells in vitro.
We further analyzed the influence of AT on the proliferation, apoptosis and cell cycle progression of CD4+ T cells. The isolated CD4+ T cells were labeled with CFSE and cultured with AT for 7 days. AT was observed to significantly inhibit the proliferation of CD4+ T cells in a dose-dependent manner and found to induce the apoptosis of CD4+ T cells with the cell arrest in G1 phase.
In line with the previous studies about the promotion of Tregs after AT treatment, our in vitro study showed that the ratio of CD4+CD25+Foxp3+ Tregs among CD4+CD25+ T cells were elevated after AT treatment, suggesting that AT could increase the proportion of Treg in activated CD4+ T cells.
Furthermore, as it was reported that AT could target some small GTPase to exert its regulation on T cells, we tested the regulation role of AT on the activation of Rho, Rac and Ras by western blot. It was shown that the expression of Ras and Rho of CD4+ T cells was decreased after AT administration in the culture system, and further influence on activation of small GTPase will be confirmed by pull-down assays.
Finally, in the in vivo study, we established the murine passive ITP models by injecting anti-CD41 antibody and divided them randomly into AT group (AT 40mg/kg/d) and control group (same dose of PBS). The platelet count were detected every other day and the expression of CD25 and Foxp3 on CD4+T in thymus, lymph nodes, spleen and peripheral blood of mice were determined after 7 days. There was no difference on the expression of CD25+ on CD4+ T cells in peripheral blood, lymph nodes, thymus and spleen between the two groups. But increased number of Tregs in the lymph nodes, peripheral blood and spleen of the AT group and decreased number of Tregs in thymus were observed compared to the control group, suggesting that AT could induce the development of peripheral Tregs and facilitate the migration of Tregs from thymus to peripheral organs in the ITP murine models. Due to the short period of the mice model, we didn't observe a significant increase in the platelet number after AT treatment.
Our current results showed that AT played an important role in regulating peripheral immune tolerance by inhibiting the activation, proliferation, expansion and survival of CD4+ T cells, whereas increasing the number of Tregs with dampened GTPase activation. The regulatory role of AT was recapitulated in the ITP murine models. This novel mechanism of AT underlies the potential therapeutic strategy for ITP.
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Disclosures
No relevant conflicts of interest to declare.
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