(d) The percentage of Th17 cells for WW, WD, or DD were 1.94%, 3.23%, or 2.76% (= 0.042) in the untreated group and 2.48%, 5.78%, or 3.51% after being stimulated (= 0.405). 3.5. proteins that cleaves and activates caspase-1, followed by the processing of the inactive proinflammatory cytokines IL-1and 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 Edaravone (MCI-186) regulates caspase-1-dependent IL-1expression and nuclear factor- (NF-) (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 Edaravone (MCI-186) 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 healthy controls who had diabetes, pregnancy, obesity, cardiovascular disease, active or chronic infections, or connective tissue diseases were excluded in our research [26]. The response criteria and severity of the disease were defined according to the guideline [27]. The patients’ characteristics were shown in Table 1. After one or two pulses of high-dose dexamethasone (HDD, 40?mg/d for 4 days), on the 14th day after treatment, we accessed the response by platelet counts and bleeding score. All the patients were followed up at least 12 months from diagnosis [27]. The ITP patients and the controls are matched for ethnicity. This study was approved by the ethics committee of Qilu Hospital of Shandong University. Informed consents were obtained from patients and controls. Table 1 Clinical characteristics of ITP patients and controls. = 403)= 336)for 8 minutes. Plasma supernatant was frozen at ?80C for the assay of MAIPA. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque density gradient centrifugation and stored at ?80C for further analysis and functional study. Moreover, Rabbit Polyclonal to ETV6 heparin-anticoagulated blood samples were collected for T helper subset analysis. 2.3. Genotyping of NLRP3 Inflammasome Genes The genotyping of NLRP3 (rs35829419), IL-1(rs16944), IL-18 (rs1946518), or CARD8 (rs2043211) in all subjects was performed using a standard TaqMan? allelic discrimination assay (Applied Biosystems, USA). The NF-value? ?0.05 was considered statistically significant. 3. Results 3.1. The Polymorphism of NF-= 0.672) or gender (= 0.052) between ITP patients and controls. SNP genotypic frequencies, except for the NF- 0.001) or the homozygote deletion (DD) genotype (OR?=?1.591, 95% CI: 1.061C1.368, = 0.024). As for the frequency of the allele, the -94insATTG (W) allele was significantly higher in ITP cases compared to controls (62.53% versus 54.61%, = 0.002). Moreover, the W allele was significantly associated with ITP susceptibility (OR?=?1.387, 95% CI 1.126C1.708). Table 3 Genotype and allele distribution of NLRP3 gene polymorphisms. (%)(%)value(rs16944)Genotype?GG121 (30.02)81 (24.11)?GA183 (45.41)167 (49.7)1.363 (0.960C1.936)0.083?AA99 (24.57)88 (26.19)1.328 (0.888C1.985)0.166Allele?G425 (52.73)329 (48.96)?A381 (47.27)343 (51.04)1.163 (0.947C1.427)0.149NLRP3 (rs35829419)Genotype?AA0 (0)0 (0)?CA0 (0)0 (0)?CC403 (100)336 (100)Allele?A0 (0)0 (0)?C806 (100)672 (100) Edaravone (MCI-186) Open in a separate window 3.2. The Association between NF-= 0.054). Moreover, ITP patients were also divided into sITP and nsITP according to the platelet count before treatment, but no significant difference was found. Table 4 The results of NF-= 0.032, Figure 1(a)). We further analyzed the association between the distribution of NF-= 0.085, Figure 1(b)). Open in a separate window Figure 1 (a) The platelet counts of ITP patients with the WW genotype (7??109/L) or WD genotype (7.5??109/L) were lower than those with the DD genotype (12.5??109/L) (= 0.032). (b) As for megakaryocyte counts in ITP patients, no significant correlation was found among the three genotypes (= 0.085). Our data showed that there was a significant difference (= 0.037) in gender between patients with NF-= 0.038). In addition, no statistical difference was found between genotype distribution and age of onset. A total of 148 ITP patients were included to determine the antiplatelet autoantibodies against GPIIb/IIIa and GPIb/IX, including 69 patients for positive autoantibodies and 79 for negative autoantibodies. However, there was no significant correlation between the frequencies of NF- 0.0001. Figure 2(a)). Moreover, NF-= 0.033), which indicated a gene dose-dependent expression (Figure 2(b)). Open in a separate window Figure 2 (a) Significantly lower NF- 0.0001). (b) NF-= 0.045). (d, e) However, it showed no difference in IL-1or IL-18 mRNA expression among the three genotypes. With a similar trend, the expression of NLRP3 was also found significantly different in ITP patients with the WW genotype (median 0.023), WD genotype (median 0.011), and DD genotype (median 0.013, = 0.045, Figure 2(c)). However, it showed no difference of IL-1or IL-18 mRNA expression among the three genotypes (Figures 2(d) and 2(e)). 3.4. Th17 Was Correlated with NF- 0.05, Figure 3(d)). However, the statistical differences between the three groups disappeared after the.