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X. Zhu et al.
roles in immune modulatory functions with effects on T- and B-cell activation.15,16 Notably, we found that MSC from ITP patients exhibited increased apoptosis and senescence, which was associated with the regulation of T-cell subsets.17-19 However, the underlying mechanisms of the dysfunction of MSC in ITP bone marrow remain unclear. We, therefore, wondered whether complement activation in bone marrow was associated with defective MSC in ITP.
Complement components can enhance pro-inflamma- tory receptor-mediated signaling in phagocytes, leading to increased production of interleukin-1β (IL-1β).20-22 IL-1β is critically involved in several inflammatory diseases and its levels have been reported to be elevated in ITP.23 Interestingly, bone marrow MSC have been demonstrated to be capable of synthesizing and releasing IL-1β.24,25
All-trans retinoic acid (ATRA) has revolutionized the therapy of acute promyelocytic leukemia.26 We previously reported that the combination of ATRA with any one of methylprednisolone, danazol or cyclosporine A produced better responses in patients with corticosteroid-resistant or relapsed ITP (54th American Society of Hematology Annual Meeting and Exposition; Poster ID: 3338). Recently we also reported the findings of a multicenter, randomized, open-label, phase II trial, suggesting that ATRA represents a promising candidate treatment for patients with corticosteroid-resistant or relapsed ITP.27 Panzer and Pabinger positively appraised our findings of a high response rate to ATRA as well as the few, mild adverse events associated with this drug compared with other second-line treatments for ITP.28 However, few stud- ies have focused on the mechanisms underlying the effects of ATRA.29 Furthermore, the role of ATRA in regu- lating MSC function in ITP bone marrow is poorly under- stood. It has not been elucidated whether the complement system and associated pro-inflammatory cytokines are targeted by ATRA.
Here, we present evidence strongly suggesting that the complement-IL-1β loop mediates bone marrow MSC impairment in ITP. More importantly, ATRA protects MSC from dysfunction and apoptosis by upregulating DNA hypermethylation of the IL-1β promoter, which is conducive to restoring the thrombopoietic niche. We believe that these findings will serve to shift the focus of future studies on the complement system in the pathogen- esis of ITP and interventions with ATRA to factors that regulate thrombocytopoiesis.
Methods
Patients and study design
The blood samples utilized in this study were collected between December 2016 and November 2017 from 58 consecu- tive, newly diagnosed ITP patients at the Institute of Hematology, Peking University People’s Hospital, Beijing, China. Approval to take blood and bone marrow samples from healthy volunteers and patients was granted by the Ethics Committee of Peking University People’s Hospital, and written informed con- sent was obtained from all subjects according to the Declaration of Helsinki.
Only untreated patients over 18 years old at diagnosis with platelet counts <30×109/L were enrolled. ITP was diagnosed based on guidelines for ITP.30,31 Bone marrow samples were also taken from transplant donors (n=42), who were considered as
healthy controls. The healthy control cohort comprised 17 males and 25 females, aged 18-59 years (median, 38 years).
Patients in the group given combination therapy received 10 mg of oral ATRA twice daily and concomitant therapy with oral danazol at a relatively low dose of 200 mg twice daily consecu- tively. Initial response was assessed after 4, 8 and 12 weeks of treatment. The primary endpoint of the study was overall response. Secondary endpoints were complete response, response, time to response, peak platelet count, reduction in bleeding symptoms, and safety. A complete response was defined as a platelet count of at least 100×109/L. Response was defined as a platelet count between 30×109/L and 100×109/L and at least doubling of the baseline count. Overall response includ- ed complete responses and responses. No response was defined as a platelet count lower than 30×109/L or less than doubling of the baseline count.31 Additionally, we defined time to response as the time from starting treatment to the time to achieve the response. We defined bleeding in accordance with the World Health Organization’s bleeding scale (0 = no bleeding, 1 = petechiae, 2 = mild blood loss, 3 = gross blood loss, and 4 = debilitating blood loss). Lastly, we graded adverse events accord- ing to the Common Terminology Criteria for Adverse Events (version4.0).
Animal model and treatment
All animal experiments were approved by the Ethics Committee of Peking University People’s Hospital and undertaken in accordance with the Institutional Guidelines for the Care and Use of Laboratory Animals.
The inclusion and exclusion criteria for patients, the methods for MSC isolation, RNA extraction and microarrays, the immuno- fluorescence assays, enzyme-linked immunosorbent assays (ELISA), cell proliferation assays, apoptosis assays, western blot- ting, determination of CXCR4 expression on megakaryocyte sur- faces, analysis of CD34+ cells, analysis of colony-forming unit- megakaryocytes, the modified monoclonal antibody-specific immobilization of platelet antigens assay, analysis of DNA methy- lation, real-time polymerase chain reaction analysis, gene silenc- ing, gene overexpression, immunostaining, in situ hybridization, animal model and treatment, and statistics are explained in the Online Supplement.
Results
Complement activation in the bone marrow of patients with immune thrombocytopenia
To illuminate the role of the complement system in the bone marrow of ITP patients, we used indirect ELISA to detect the deposition of complement proteins C1q, C4d, C3b and C5b-9 on the surface of MSC from ITP patients and healthy volunteers. Reference ranges of complement deposition on MSC from the healthy controls were deter- mined: C1q (1.0 ± 0.1), C4d (0.9 ± 0.2), C3b (0.9 ± 0.1) and C5b-9 (1.0 ± 0.4). The cutoff values defining deposition of C1q, C4d, C3b and C5b-9 were 1.2, 1.3, 1.1 and 1.8, respectively. These cutoffs represent a level of complement deposition that falls approximately two standard devia- tions above the reference mean for the majority of comple- ment components. Patients were categorized as having complement activation if the level of one or more of the measured complement components was equal to or more than the cutoff value. In this study, 26 of the 58 patients were assigned to the group with complement activation (MSC-ITP-C+ group), while the remaining 32 patients were
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haematologica | 2019; 104(8)