The selective JAK1 inhibitor upadacitinib restores impaired neutrophil chemotaxis in the proplatelet formation of immune thrombocytopenia Free

Introduction Neutrophils are innate immune phagocytes that have a central role in immune defense. Neutrophils have been shown to play critical roles in the initiation and perpetuation of autoimmune disorders. Recent studies revealed direct cellular interactions between neutrophils and megakaryocytes (MKs) in the bone marrow (BM). Neutrophils are recruited to the proplatelet budding site of MKs and control thrombopoiesis by plucking on forming proplatelets, in turn accelerating their growth and release into the circulation (Immunity 2022, Blood 2025). The pathogenic mechanisms of immune thrombocytopenia (ITP) are not fully understood. Our previous data indicated that impaired proplatelet formation (PPF) contributed to the development of thrombocytopenia in ITP. To further explore the underlying mechanism of impaired PPF in ITP, we found that activation of the type I interferon (IFN) pathway caused dysfunction of neutrophil chemotaxis via CXCL12/CXCR4. The oral selective Janus kinase 1 (JAK1) inhibitor upadacitinib, which has been approved for the treatment of multiple autoimmune diseases, including ulcerative colitis and rheumatoid arthritis, could restore neutrophil chemotaxis and promote PPF.

Methods Thirty ITP patients and healthy controls from March 2023 to December 2023 were included in the study. Single-cell RNA sequencing, 4D label-free proteomics and Olink proteomics were used for the first time to characterize the changes in bone marrow neutrophil development and heterogeneity. Bone marrow neutrophils were isolated from ITP patients and controls. The chemotactic motility of neutrophils was observed through TAXIScan cell migration visualization and transwell migration assays. A coculture system of neutrophils and MKs was established. Pf4-cre(+)/confetti/Lyz2-eGFP dual reporter mice and multi-photon microscopy were used to visualize the interaction between MKs and neutrophils. An ITP mouse model was established to observe the therapeutic effects of upadacitinib on PPF.

Results ITP neutrophils displayed impaired neutrophil chemotaxis, with reduced directional motility and velocity. Based on single cell sequencing data, we identified 6 neutrophil subsets including Neu_cycling, MPO_Neu, CAMP_Neu, MMP9_Neu, CXCR2_Neu, and ISG15_Neu. In patients with ITP, the number of neutrophils significantly changed during differentiation and maturation, the number of ISG15_Neu significantly increased. And pathway enrichment analysis of differential expression genes revealed that type I interferon pathway was the most enriched. After IFN-α stimulation in vitro, the chemotactic function and orientation of neutrophils were decreased. Together, the activation of type I interferon pathway may contribute to the impaired neutrophil chemotaxis in ITP.

Neutrophils promote PPF through the direct cellular interactions with MKs. The neutrophil and MKs co-localization are reduced in the ITP BM microenvironment. The expression of neutrophil chemokine CXCL12 in BM demonstrated no significant differences between ITP and controls. The neutrophil chemokine receptor CXCR4 in the BM of ITP patients was significantly decrease. Taken together, the activation of type I interferon pathway accounted for impaired neutrophil chemotaxis through CXCL12/CXCR4 signaling axis, which resulted in the defective PPF in ITP.

After binding to receptors, type I interferon activate the JAK/STAT pathway and exert their effects on different cytokine receptors. Upadacitinib is an oral JAK1 inhibitor that has high selectivity for JAK1 inhibition. We then investigated the effect of upadacitinib on PPF in ITP patients. Our data showed that in vitro treatment with upadacitinib inhibited the activation of the type I interferon pathway, upregulated the expression of CXCR4, and restored neutrophil chemotaxis. In vivo studies showed that upadacitinib could rescue the impaired PPF in both patients and mice model with ITP.

Conclusions The BM of ITP patients displayed impaired neutrophil chemotaxis regulated by type I interferon pathway. Upadacitinib restores neutrophil chemotaxis and corrected impaired PPF by inhibiting type I interferon pathway and upregulating CXCR4. It sheds light on a novel mechanism of ITP pathogenesis and provides a basis for the therapeutic potential of upadacitinib in ITP patients.