Iguratimod Could Correct the Defective IL-35-Mediated Megakaryopoiesis in Immune Thrombocytopenia

Abstract

Introduction: IL-35 is an inhibitory cytokine of the IL-12 family, and comprises an EBI3 subunit and a p35 subunit. Previous data indicated that serum levels of IL-35 were significantly lower in immune thrombocytopenia (ITP) patients than in healthy controls. Moreover, the concentration of plasma IL-35 in ITP patients achieving complete response returned to normal, but was not corrected in those who failed to achieve response. However, the underlying pathological function of IL-35 in impaired megakaryopoiesis in ITP remains unexplored. Our study aimed to evaluate the immunoregulatory role of IL-35 in megakaryocytes (MKs) differentiation. Iguratimod, a traditional therapy for inflammatory disorders, has been demonstrated to promote megakaryopoiesis. The effect of iguratimod on the treatment of ITP was retrospectively reviewed. Methods: Bone marrow samples were obtained from newly-diagnosed primary ITP patients and healthy donors. CD34+ cells from ITP patients and healthy donors were differentiated into MKs with and without IL-35 to determine the mechanism of IL-35 in megakaryopoiesis. Iguratimod was added to the culture medium of MKs to determine its function in MKs differentiation. Additionally, ITP mouse model was established to further validate the function of IL-35. Results: In comparison with healthy controls, ITP patients showed lower bone marrow levels of IL-35. IL-35 receptor expression was progressively induced on bone marrow MKs from healthy controls during the differentiation of CD34+ progenitors. In contrast, MKs from ITP patients exhibited defective expression of the IL-35 receptor. We then determined the effect of IL-35 on the megakaryocytic potential of CD34+ progenitor cells in culture medium containing TPO. IL-35 increased the number of medium-sized and large-sized colonies, and increased the total number of CD41+ cells. In IL-35-treated MKs, higher levels of Akt phosphorylation were observed. Pretreatment with LY294002, an inhibitor of Akt phosphorylation, blocked the differentiation of MKs induced by IL-35. LY294002 inhibited proplatelet formation and aggregation of β1-tubulin in MKs stimulated by IL-35. Flow cytometry analysis revealed that platelet production decreased in the LY294002 pretreatment group. These results demonstrated that IL-35 can enhance the proliferation and differentiation of MKs through Akt pathways. The decreased level of bone marrow IL-35 may partially explain the defective megakaryopoiesis in ITP. We then explored the potential causes of the decreased IL-35 levels in ITP. Flow cytometry analysis showed that the percentage of bone marrow iTr35 cells, the primary sources of IL-35, was significantly lower in ITP patients. Furthermore, we cocultured CD4+ T cells with MSCs from healthy controls and ITP patients, respectively. The percentage of iTr35 cells was lower when cultured with ITP-MSCs. These results showed that ITP-MSCs exhibited an impaired capability of inducing iTr35 cells, which might contribute to the reduced level of bone marrow IL-35 in ITP patients. We further investigated the effects of iguratimod on the differentiation of CD34+ progenitors into MKs. When CD34+ cells were cultured with TPO alone, the addition of iguratimod upregulated the expression of IL-35 receptors on MKs from ITP patients and a murine model of this disease, and consequently promoted the formation of MK colonies. In the retrospective study of iguratimod, a total of 68 adult patients with corticosteroid-resistant primary ITP were included. Initial response was achieved in 42/68 (62%) patients, and complete response was achieved in 25/68 (37%) patients. In patients who achieved initial response, the median time to response was 28 days. Sustained response at 6 months was seen in 35/68 (51%). Bleeding symptoms were reduced from 37/68 (54%) patients at baseline to 14/68 (21%) patients at 6 months. Most adverse events were mild (grade 1-2), and there were no grade 4 or worse adverse events in our patients. The most common adverse events were elevated ALT, gastrointestinal disorders, and nausea. Conclusions: ITP-MSCs exhibited an impaired capability of inducing iTr35 cells, which contributed to the reduced level of bone marrow IL-35 in ITP. IL-35 promoted MKs differentiation through the Akt pathway. Iguratimod promoted megakaryopoiesis by inducing the expression of IL-35 receptors on MKs. Iguratimod had good efficacy and safety in the treatment of ITP.