NF-κB-94ins/del ATTG Genotype Contributes to the Susceptibility and Imbalanced Th17 Cells in Patients with Immune Thrombocytopenia

Hindawi Journal of Immunology Research Volume 2018, Article ID 8170436, 12 pages https://doi.org/10.1155/2018/8170436 Research Article NF-κB-94ins/del ATTG Genotype Contributes to the Susceptibility and Imbalanced Th17 Cells in Patients with Immune Thrombocytopenia Jie Yu,1,2 Mingqiang Hua,1 Xueyun Zhao,1 Rui Wang,1 Chaoqing Zhong,1 Chen Zhang,1 Ruiqing Wang,1 Guosheng Li,1 Na He,1 Ming Hou,1 and Daoxin Ma 1 1 2 Department of Hematology, Qilu Hospital, Shandong University, Jinan, China Department of Hematology, Weihai Municipal Hospital, Weihai 264200, China Correspondence should be addressed to Daoxin Ma; Received 8 March 2018; Accepted 19 June 2018; Published 22 July 2018 Academic Editor: Francesca Santilli Copyright © 2018 Jie Yu et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background. The NLRP3 inflammasome plays important roles in the pathogenesis of autoimmune diseases. However, the role of the NLRP3 inflammasome in the pathophysiology of immune thrombocytopenia (ITP) remains unclear. Methods. RT-PCR was used to examine the polymorphism and expression of genes involved in the NLRP3 inflammasome in ITP patients. T helper cells and apoptosis of PBMC from ITP patients were analyzed by flow cytometry. The antiplatelet autoantibodies in plasma were determined by modified monoclonal antibody-specific immobilization of platelet antigens (MAIPA). Results. We found that the NF-κB-94ins/del ATTG genotype contributed to the susceptibility of ITP. Furthermore, the platelet counts of ITP patients with the WW genotype or WD genotype were lower than those with the DD genotype of NF-κB-94ins/del ATTG. Compared with controls, NF-κB gene expression was significantly decreased and WW or WD genotype ITP patients displayed higher mRNA expression than DD individuals. Similarly, the mRNA expression of NLRP3 was also increased in the WW genotype. There was a significant gene dose effect of the percentage of Th17 cells for the WW, WD, and DD genotypes (WW < WD < DD) in the unstimulated group and no significant difference was found after being stimulated. The activation of the NLRP3 inflammasome could upregulate Th17 in ITP patients. Conclusion. The NF-κB-94ins/del ATTG genotype might serve as a novel biomarker and potential target for ITP. 1. Introduction Primary immune thrombocytopenia (ITP) is an acquired autoimmune disease characterized by a transient or persistent decrease of the platelet count and increased risk of bleeding [1]. Although autoreactive B lymphocytes, secreting antiplatelet antibodies, are considered as the primary immunological defect in ITP, T cellular immunity abnormalities are considered important in the pathogenesis of ITP [2, 3]. ITP has also been documented to be connected with cytokine response and dysregulation in the cytokine network [4]. Furthermore, there have been several studies reporting that the genetic variants contribute to ITP pathogenesis [5, 6]. NLRP3 (NOD-like receptor pyrin domain-containing protein 3), a well-characterized inflammasome, belongs to the innate immune system that responds to cellular stress by producing the proinflammatory cytokines IL-1β and IL-18. The NLRP3 inflammasome is composed of NLRP3, the adaptor protein ASC, and caspase-1. Activation of the inflammasome leads to the homotypic interaction of the PYD (pyrin domain) of NLRP3 and ASC proteins that cleaves and activates caspase-1, followed by the processing of the inactive proinflammatory cytokines IL-1β and IL-18 to their active forms that trigger downstream inflammatory response [7]. Caspase recruitment domain-containing protein (CARD) 8, also known as TUCAN (tumor upregulated CARD-containing antagonist of caspase nine), interacts physically with caspase-1 and negatively regulates caspase1-dependent IL-1β expression and nuclear factor- (NF-) κB activation. The NLRP3 inflammasome plays a role in the 2 pathogenesis of numerous inflammatory and autoimmune diseases such as diabetes, obesity, and atherosclerosis [8, 9]. Single nucleotide polymorphisms (SNPs) are the most abundant types of DNA sequence variation in the human genome. SNPs provide a well-established method for analyzing complex gene-associated diseases and individual susceptibility for some disorders. Polymorphisms in NLRP3-associated genes are linked to common immune inflammatory diseases [10–12]. The SNP of CARD8 rs2043211 changing cysteine at codon 10 to a premature termination codon (c.30T>A; p.C10X) in this gene was found to be associated with the inflammatory activity in early rheumatoid arthritis (RA) and inflammatory bowel disease (IBD) [13, 14]. A common insertion (ins)/deletion (del) promoter polymorphism (-94ins/del ATTG polymorphism) of NF-κB seems to be related to several inflammatory diseases such as ulcerative colitis (UC), Graves’ disease, and psoriasis vulgaris [15–17]. Polymorphisms of the IL-18 gene have been shown to influence some chronic inflammatory diseases including diabetes, RA, systemic lupus erythematosus (SLE), and IBD [18–21]. The SNP of IL-1β-511T>C has been associated with promoter activity and the risk of diabetes nephropathy, RA, and UC [22–24]. There are several studies about the gene polymorphisms closely associated with the risk for ITP [5, 6]. Nevertheless, the involvement of SNPs with the NLRP3 inflammasome in ITP has not yet been demonstrated. Meanwhile, the NLRP3 inflammasome has effects on CD4+ T cell differentiation via the production of the caspase-1-dependent cytokines, IL-18 and IL-1β, directing Th17 responses. Th17 was an importantly proinflammatory Th cell subset, characterized by the expression of the key transcription factor RORC and secretion of IL-17 which may induce IL-17R expressed cells to produce inflammatory cytokines. Moreover, the release of IL-18 may promote the activity of IL-22, the effect factor of Th22 cells expressing the key transcription factor AHR. Our previous research reported that Th17 and Th22 cells were significantly elevated in ITP patients [25]. However, no data has been reported about the association of the NLRP3 inflammasome and T helper cells in ITP until now. To determine the susceptibility and clinical significance of the NLRP3 inflammasome in ITP, we examined the SNPs of three NLRP3 inflammasome components and two inflammatory cytokines, including NLRP3 (rs35829419), CARD8 (rs2043211), NF-κB-94ins/del ATTG, IL-18 (rs1946518), and IL-1β (rs16944). Then, we further carried out the functional study to explore the role of NLRP3 in Th cell development in ITP patients. 2. Materials and Methods 2.1. Subjects. A total of 403 ITP patients and 336 sex- and age-matched healthy controls were recruited prospectively in Qilu Hospital of Shandong University from July 2011 to March 2016. The diagnosis of the enrolled ITP patients was based on the American Society of Hematology guideline. Patients and health (...truncated)