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Recent findings have shown that the level of interleukin‐35 (IL‐35) is abnormal in several autoimmune diseases. Nonetheless, whether IL‐35 participates in the pathogenesis of immune thrombocytopenia (ITP) remains unclear. The current study investigates whether IL‐35 modulates megakaryopoiesis. The results show that IL‐35 receptors are progressively expressed on bone marrow megakaryocytes during the in vitro differentiation of CD34+ progenitors. IL‐35 increases the number of megakaryocyte colony‐forming units through the Akt pathway. The level of bone marrow IL‐35 is reduced in ITP patients, and the decreased level of IL‐35 may inhibit megakaryopoiesis. Then, the potential causes of decreased IL‐35 in ITP patients are explored. The primary type of cell that secretes IL‐35, known as IL‐35‐producing regulatory T cells (iTr35), is reduced in ITP patients. Bone marrow mesenchymal stem cells (MSCs) from ITP patients exhibit an impaired capability of inducing iTr35 due to enhanced apoptosis, which may contribute to the reduced level of bone marrow IL‐35 in ITP patients. Iguratimod promotes megakaryocyte development and differentiation by elevating the expression of IL‐35 receptors on megakaryocytes. Iguratimod improves response rates and reduces bleeding symptoms in corticosteroid‐resistant ITP patients. [ABSTRACT FROM AUTHOR]

Summary: The abnormal immunomodulatory functions of mesenchymal stem cells (MSCs) have been implicated in the development of immune thrombocytopenia (ITP). Recent studies have suggested important effects of complement on immune cell function. However, whether complement modulates bone marrow MSCs function in ITP is poorly defined. Tacrolimus has recently been applied to the treatment of autoimmune diseases. Here, we explored whether impaired ITP‐MSCs could be targeted by tacrolimus. Our results showed that the Nod‐like receptor family pyrin domain containing 3 (NLRP3) inflammasome was activated in ITP MSCs with complement deposition (MSCs‐C+) and initiated caspase‐1‐dependent pyroptosis. Transcriptome sequencing results showed abnormal fatty acid metabolism in MSCs‐C+. Enhanced fatty acid β‐oxidation and reactive oxygen species production activated the NLRP3 inflammasome. Adipocytes derived from MSCs‐C+ secreted less adiponectin. Adiponectin promoted the differentiation of megakaryocytes and inhibited the destruction of platelets. Tacrolimus inhibited NLRP3 inflammasome activation and MSCs‐C+ pyroptosis in vitro and in vivo. Tacrolimus plus danazol elicited a higher sustained response than danazol monotherapy in corticosteroid‐resistant patients with ITP. Our findings demonstrate that the activation of the NLRP3 inflammasome in ITP MSCs mediated by complement could be inhibited by tacrolimus, which might be a potential new therapy for ITP. [ABSTRACT FROM AUTHOR]

Summary: Immune thrombocytopenia (ITP) is an autoimmune disease. Mesenchymal stem cells (MSCs) play important roles in the physiology and homeostasis of the haematopoietic system, including supporting megakaryocytic differentiation from CD34+ haematopoietic progenitor cells. Tumour necrosis factor alpha‐induced protein 3 (TNFAIP3, also termed A20) plays a key role in terminating NF‐κB signalling. Human genetic studies showed that the polymorphisms of the TNFAIP3 gene may contribute to ITP susceptibility. In this study, we showed a significant decrease in TNFAIP3 and increase in NF‐κB/SMAD7 in ITP‐MSCs. In co‐cultures with CD34+ cells, NF‐κB was overexpressed in MSCs from healthy controls (HC‐MSCs) after transfection with NFKBIA (IκB)‐specific short hairpin (sh)RNAs, resulting in MSC deficiency and a reduction in megakaryocytic differentiation and thrombopoiesis. Knockdown of TNFAIP3 expression using TNFAIP3‐specific shRNAs in HC‐MSCs affected megakaryocytopoiesis. However, IKBKB knockdown corrected megakaryocytopoiesis inhibition in the ITP‐MSCs by decreasing NF‐κB expression. Amplified TNFAIP3 expression in ITP‐MSCs by TNFAIP3 cDNA can facilitate megakaryocyte differentiation. shRNA‐mediated knockdown of SMAD7 expression rescued the impaired MSC function in ITP patients. Therefore, we demonstrate that a pathological reduction in TNFAIP3 levels induced NF‐κB/SMAD7 pathway activation, causing a deficiency in MSCs in ITP patients. The ability of ITP‐MSCs to support megakaryocytic differentiation and thrombopoiesis of CD34+ cells was impaired. [ABSTRACT FROM AUTHOR]