Metabolic reprogramming of megakaryocyte subpopulations in ITP: Implications for TSPO in predicting response to TPO-RA Free

Background Single-cell sequencing has revolutionized our understanding of the versatile nature of megakaryocyte subpopulations, among which an immune-skewed subset caught attention. However, the functional heterogeneity of megakaryocytes has not been defined under pathological conditions. Immune thrombocytopenia (ITP) stands as the most common acquired bleeding disorder characterized by dual attack of autoantibodies and cytotoxic T lymphocytes against platelets and megakaryocytes. Impaired megakaryocytes contribute to the pathogenesis of ITP and are associated with treatment outcomes. Although thrombopoietin receptor agonists (TPO-RAs) are fundamental among second-line treatments of ITP, up to 40% of patients turns out unresponsive or refractory to TPO-RAs with uncertain causes. Translocator Protein 18 kDa (TSPO), which locates in the outer mitochondrial membrane, has been shown to inhibit ferroptosis and promote megakaryocyte differentiation. In this study, we aimed to elucidate the mechanisms underlying the differential response to TPO-RAs from a megakaryocytic perspective.

Methods Single-cell RNA sequencing of megakaryocytes from a murine model of active ITP was used to decode megakaryocyte subpopulations and investigate the mechanisms underlying the differential responses to TPO-RAs. Mass cytometry was applied to analyze the bone marrow microenvironments of treated and untreated ITP mice. Electron microscopy and metabolic analysis of the MEG-01 cell line were employed to compare different metabolic spectrums of TPO-RA responders and non-responders. Finally, Cleavage Under Targets and Tagmentation (CUT＆Tag), and Cleavage Under Targets and Release Using Nuclease (CUT＆RUN) were performed to validate the transcriptional regulation of TSPO.

Results Two distinct subpopulations were featured in ITP between TPO-RA responders and non-responders: interactive megakaryocytes (iMK) possessed with an immune nature and platelet-generating megakaryocytes (pMK). ITP patients exhibited alterations in the number and function of these subpopulations, and the imbalance between iMK and pMK was corrected in TPO-RA-responsive ITP mice. Mass cytometry revealed considerable disparities between the bone marrow microenvironments of treated and untreated ITP mice. Furthermore, the gene encoding TSPO, which plays a crucial role in mitochondrial physiology, was differentially expressed between TPO-RA responders and non-responders. We employed TSPO agonists and inhibitors to simulate differential responses to TPO-RAs in the MEG-01 cell line and confirmed that iMK and pMK exhibit distinct metabolic patterns. TSPO also altered the production of reactive oxygen species (ROS) in iMK as well as the level of lipid ROS and ACSL4 in pMK. Additionally, enhanced interaction between these two subpopulations was observed in TPO-RA responders, which favors platelet production.

Conclusion In conclusion, disturbed megakaryocyte subsets participate in the pathogenesis of ITP, which can be rectified in responders to TPO-RAs. TSPO characterizes metabolic reprogramming of megakaryocytes and emerges as a potential biomarker in predicting the efficacy of TPO-RAs.