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.