Introduction Immune thrombocytopenia (ITP) is marked by autoimmune platelet destruction and impaired thrombopoiesis, with neuraminidase 1 (NEU1)-mediated desialylation emerging as a key pathogenic factor. Our prior work showed abnormal IL-1β signaling (Haematologica, 2019) and altered megakaryocyte (MK) positioning in ITP marrow and noted that viral infection-induced interferon-β can upregulate NEU1 by reshaping the MHC locus. Thus, IL-1β-driven interferon circuits may regulate NEU1-dependent desialylation and subsequent MK dysfunction. All-trans retinoic acid (ATRA) protects mesenchymal stem cells in ITP by modulating the complement-IL-1β loop (Haematologica, 2019). NEU1-mediated desialylation of platelets and MKs disrupts MK positioning and thrombopoiesis in the marrow. While surface desialylation reduces MKs' proximity to the vascular niche, the upstream regulators of NEU1 are still unknown. We investigated the NEU1-mediated desialylation of key MK membrane proteins, particularly GPIbα, in response to IL-1β stimulation.

Methods Bone marrow aspirates were obtained from patients with corticosteroid-resistant or relapsed chronic immune thrombocytopenia (ITP) and from matched controls [Clinical Trials Register, registration number NCT03304288]. Megakaryocyte desialylation was evaluated by flow cytometric quantification of lectin binding. Western blotting was employed to identify the proteins exhibiting the strongest affinity for RCA-1, thereby pinpointing the principal desialylated moieties on ITP MKs. Megakaryocytic differentiation of the human megakaryoblastic cell line MEG-01 was induced with phorbol 12-myristate 13-acetate (PMA, 10 nM) for 72 h. PMA-induced MEG-01 cells were stimulated with IL-1β (100 ng/mL) for 24 hours in the presence or absence of ATRA (200 ng/mL). GPIbα and NEU1 expression were then measured by qPCR and Western blotting and compared across groups to assess the ability of ATRA to rescue IL-1β-induced desialylation. Platelets isolated from five steroid-resistant/relapsed ITP patients and five healthy donors were analyzed for NEU1 protein expression. Megakaryocyte migration was measured in PMA-induced MEG-01 cells by Boyden chamber assay and staining with 0.5% gentian violet solution.

Results Compared with those from controls, megakaryocytes from patients with resistant/relapsed chronic ITP displayed markedly reduced binding of Sambucus nigra agglutinin (70.59 ± 6.93% vs. 85.22 ± 5.43%, P < 0.0001) and Maackia amurensis lectin (33.83 ± 5.09% vs. 52.98 ± 8.81%, P < 0.0001), indicating significant loss of α2,6- and α2,3-linked sialic acids. Conversely, Ricinus communis agglutinin I (RCA-1) binding was strikingly elevated (62.70 ± 2.07% vs. 23.81 ± 2.23%, P < 0.0001), reflecting increased surface β-galactose residues consistent with increased desialylation. Western blot analysis of RCA-1-bound proteins revealed a prominent ~143 kDa band corresponding to GPIbα. Subsequent immunoblotting with anti-GPIbα antibodies confirmed GPIbα as the primary glycoprotein undergoing desialylation on the megakaryocyte membrane in ITP. Transcriptional analysis by qPCR revealed significant upregulation of NEU1 (1.28-fold, p < 0.0248) and GPIbα (1.51-fold, p < 0.0016) mRNAs in PMA-induced MEG-01 cells exposed to IL-1β compared with the negative controls. Flow cytometry showed a marked increase in membrane NEU1 expression, whereas GPIbα and GPIIb membrane levels remained unchanged. Western blotting confirmed elevated total NEU1 protein (2.8-fold, p < 0.001), with no significant change in total GPIbα expression (1.1-fold, p = 0.42). Transwell migration assays revealed that the number of megakaryocytes traversing the membrane in the IL-1β-stimulated group was significantly lower than that in the control group, indicating marked impairment of MK migration by IL-1β. Cotreatment with ATRA restored the transwell count to a level indistinguishable from that in the control group, demonstrating that ATRA effectively rescues IL-1β-induced migratory dysfunction in megakaryocytes. Further, ATRA reversed the effects of IL-1β, normalizing membrane NEU1 and total NEU1 expression (1.3-fold, p = 0.08) while restoring GPIbα sialylation (85% ± 7% of the control, p < 0.01).

Conclusions IL-1β triggers NEU1-mediated desialylation of GPIbα, disrupting MK signaling and thrombopoiesis. All-trans retinoic acid effectively mitigates this pathway, highlighting its therapeutic potential for IL-1β-driven megakaryocyte dysfunction in ITP.

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2025
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