P1595: activation of chaperone-mediated autophagy reduces nlrp3 and platelet clearance in immune thrombocytopenia

Topic: 32. Platelet disorders Background: Chaperone-mediated autophagy (CMA) is an autophagy which selectively degrades chaperone labelled cargos in lysosomes to regulate critical functions of immune cells. The key protein of CMA is lysosome-associated membrane protein 2A (LAMP2A). It is reported that in the mouse model of atherosclerosis, LAMP2A could reduce inflammation levels by transporting NLRP3 into lysosomes and impaired CMA function could lead to an increased production of NLRP3 and IL-1β in macrophages. Immune thrombocytopenia (ITP) is an autoimmune hemorrhagic disorder. The levels of inflammatory factors, such as NLRP3 and IL-1β, are higher in the peripheral blood mononuclear cells (PBMCs) of ITP patients than those of healthy controls. However, the role of CMA in the pathogenesis of ITP remains to be elucidated. Aims: To investigate whether CMA contributes to the pathogenesis and serves as a therapeutic target of ITP. Methods: CD14+ cells were separated from PBMCs of newly diagnosed ITP patients and healthy doners. The expression of LAMP2A in the CD14+ cells was examined via western blotting. Macrophages derived from PBMCs of ITP patients were treated with QX77, an agonist of CMA. Flow cytometry was used to evaluate the phagocytic activity and surface markers of monocyte-derived macrophages. Quantitative PCR (qPCR) and immunoblotting were also performed to determine the expression of LAMP2A and inflammatory factors such as NLRP3 and IL-1β. Furthermore, the treatment effect of QX77 was evaluated in an active murine model of ITP. Briefly, anti-CD61 immune-sensitized splenocytes were transferred into severe combined immunodeficient mice to induce a murine model of severe ITP. The platelet counts were examined weekly after administration, and the polarization level of macrophages from liver, spleen, and bone marrow were detected by flow cytometry. Results: Immunoblotting showed that the expression of LAMP2A was significantly lower in CD14+ cells from ITP patients than from healthy donors. After treatment of QX77, the phagocytic activity of monocyte-derived macrophages was inhibited (Figure 1) and their expression level of CD80 but not CD86 was significantly decreased. qPCR showed that the mRNA level of LAMP2A was significantly increased, while NLRP3 and IL-1β were significantly decreased in macrophages after treatment of QX77 at the concentration of 15μM. Meanwhile, thrombocytopenia was alleviated in mice treated with QX77, showing significantly higher platelet counts than those in the control group at Day 21. The level of CD80 and CD86 were significantly decreased in QX77-modulated macrophages from the liver. Moreover, the levels of CD80 in macrophages from the spleen and bone marrow were significantly lower than those from the control group, which is consistent to the results of in vitro experiments. After QX77 treatment, F4/80+iNOS+ M1 macrophages were significantly decreased in the spleen, while F4/80+CD163+ M2 macrophages were significantly increased in the bone marrow.Summary/Conclusion: This study establishes a pathogenic role of impaired CMA in ITP and provides a novel therapeutic strategy by restoring CMA for ITP patients. Keywords: Macrophage, Autoimmune disease, Immune thrombocytopenia (ITP), Phagocytosis