Chaperone-mediated autophagy reprograms heterogeneous subsets of activated macrophages in immune thrombocytopenia.

IFNG (interferon gamma)-activated macrophages contribute to accelerated clearance of platelets and excessive secretion of inflammatory cytokines in immune thrombocytopenia (ITP). In this study, we identified two distinct subpopulations of activated macrophages: phagocytic macrophages (M[IFNG]p), which predominantly exhibit phagocytic activity, and inflammatory macrophages (M[IFNG]i), characterized by their pro-inflammatory capacity. The M(IFNG)p and M(IFNG)i subsets were functionally disturbed in patients with ITP. Chaperone-mediated autophagy (CMA) deficiency in macrophages was discovered in ITP and has been reported to induce sustained inflammation. Furthermore, CMA interference enhanced pro-inflammatory function rather than phagocytic activity of macrophages in vitro. In parallel, macrophage-specific conditional lamp2a (lysosome associated membrane protein 2A) knockout mice exhibited expansion and excessive cytokine release of M(IFNG)i. Restoring CMA restrains M(IFNG)i subset and reprograms M(IFNG)p subset via ALDH2 (aldehyde dehydrogenase 2 family member) in ITP. Here, anti-ITGB3/CD61 immune-sensitized splenocytes were transferred into severe combined immunodeficient mice to establish an active murine model of ITP. CMA activation diminished the pro-inflammatory and phagocytic activity of activated macrophages and ameliorated thrombocytopenia in ITP mice. MeRIP-sequencing identified PTEN (phosphatase and tensin homolog) as a crucial activator for LAMP2A, exhibiting decreased m6A methylation and subsequent downregulation, which indicated a potential mechanism underlying CMA deficiency in activated macrophages. In conclusion, restoring CMA restrains the M(IFNG)i subset and reprograms the M(IFNG)p subset via ALDH2 to raise platelet counts in ITP. Targeting CMA in activated primary human macrophages presents a promising therapeutic strategy to rapidly and sustainably increase platelet counts and restore immune balance in ITP.Abbreviation ALDH2: aldehyde dehydrogenase 2 family member; ALKBH5: alkB homolog 5, RNA demethylase; CCL: C-C motif chemokine ligand; CMA: chaperone-mediated autophagy; CSF1/M-CSF: colony stimulating factor 1; CUT&Tag: Cleavage Under Targets and Tagmentation; CXCL: C-X-C motif chemokine ligand; FBS: fetal bovine serum; FCGR/FcγR: Fc gamma receptor; HLA-DR: major histocompatibility complex, class II, DR; IFNG/IFN-γ: interferon gamma; IGF2R: insulin like growth factor 2 receptor; IL: interleukin; ITP: immune thrombocytopenia; LAMP2A: lysosome associated membrane protein 2A; m6A: N6-methyladenosine; MeRIP-seq: methylated RNA immunoprecipitation sequencing; METTL3: methyltransferase 3, N6-adenosine-methyltransferase complex catalytic subunit; MFI: mean fluorescence intensity; mRNA: messenger RNA; NFE2L2/NRF2: NFE2 like bZIP transcription factor 2; NLRP3: NLR family pyrin domain containing 3; NOS2/iNOS: nitric oxide synthase 2; PBMCs: peripheral blood mononuclear cells; PTEN: phosphatase and tensin homolog; PYCARD: PYD and CARD domain containing; STAT: signal transducer and activator of transcription; Th1: T-helper 1; TNF: tumor necrosis factor; WTAP: WT1 associated protein.