Mingqiang Hua

The aberrant expression of microRNAs and correlations with T cell subsets in patients with immune thrombocytopenia

Both microRNAs and T helper (Th) cells involve in autoimmune diseases and their effects and interactions in immune thrombocytopenia (ITP) remain unclear. In the present study, we investigated the expression profiles of seven immune-related microRNAs (miR-155, 146a, 326, 142-3p, 17-5p, 21 and 181a) and the frequencies of four Th cells (Th1, Th2, Th17 and Treg) in peripheral blood mononuclear cell (PBMCs) of ITP patients and healthy controls. Platelet autoantibodies specific for GPIIb/IIIa or GPIb/IX were measured using MAIPA method. The regulating effect of miR-146a on Th differentiation was evaluated after using agomir. Our results showed that the expression of miR-146a, miR-326 or miR-142-3p in ITP patients was lower than that of controls. The frequencies of Treg cells were decreased, whereas the frequencies of Th17 and Th22 cells were increased significantly in ITP patients compared to those in controls. The expression levels of miR-142-3p and miR-146a were negatively correlated with Th17 cells, respectively. The expression of miR-146a was positively correlated with the frequencies of Treg cells and platelet counts. No significant correlation was found between the miRNAs expression and different autoantibody groups. The up-regulated miR-146a expression with agomir contributed to the differentiation of Th17 and Treg in ITP patients. Moreover, miR-146a was increased in the presence of DEX in PBMCs of ITP patients in vitro. Our study represents the abnormal expression profile of immune-related miRNAs in ITP patients, and miR-146a may be involved in Tregs differentiation and function.

Genetic polymorphisms of IL-18 rs1946518 and IL-1β rs16944 are associated with prognosis and survival of acute myeloid leukemia

Background Though the pathogenesis of AML is still unknown, accumulating evidence revealed that immune response plays a vital part in it. NLRP3 inflammasome as a component of immune system has been found related to several cancers. The single nucleotide polymorphisms (SNPs) of NLRP3 inflammasome genes may be related to pathogenesis and prognosis of AML.Methods and resultsWe determined polymorphisms of NLRP3 (rs35829419), CARD8 (rs2043211), IL-1β (rs16944), IL-18 (rs1946518) and NF-κB −94 ins/del ATTG in de novo AML patients to find out whether they play roles in the susceptibility and severity of AML. In our study, 383 AML cases and 300 randomly selected healthy individuals were examined for the polymorphisms and expression of NLRP3 genes. IL-1β (rs16944) polymorphism in different risk AML subgroups was found statistically different, with more GA genotype in favorable-risk cytogenetics group. We also demonstrated that the bone marrow blasts of patients carrying IL-18 (rs1946518) GG or GT genotype were higher than patients of TT genotype. IL-18 plasma level of patients with IL-18 (rs1946518) GT or TT genotype was higher than GG genotype. Moreover, the GT genotype of IL-18 (rs1946518) led to statistically poorer AML-specific survival.Conclusion IL-1β (rs16944) and IL-18 (rs1946518) may be served as potential predictors for AML.

Neuropathy correlated with imbalanced Foxp3/IL-17 in bone marrow microenvironment of patients with acute myeloid leukemia

Bone marrow (BM) neural tissues are important components of bone marrow microenvironment and play important roles in normal hematopoiesis. Neuropathy of BM can cause immunological alteration in hematopoietic microenvironment. It also can induce the impairment of normal hematopoiesis and promote the development of hematologic diseases. In the present study, we determined the expression levels and clinical significances of nerve-related molecules [nestin, tyrosine hydroxylase (TH), Glial Fibrillary Acidic protein (GFAP) and S100B] and T helper-related molecules (IL-17, Foxp3) in BM of AML patients and controls by immunohistochemical analysis and RT-PCR. Our results showed that the positive rates and expression levels of nestin, TH, GFAP and IL-17 were significantly decreased while Foxp3 and the ratio of Foxp3/IL-17 were statistically elevated in BM of AML patients. We found that there were significantly positive correlations between nestin with TH and IL-17 in BM of AML patients. We also observed significantly negative correlations between nestin with TH and Foxp3/IL-17 ratio. Moreover, the expression of nestin was positively correlated with the overall survival of AML patients. Our study suggests that neuropathy together with imbalanced T helper immunology in bone marrow might play important roles in AML.

The genetic polymorphism and expression profiles of NLRP3 inflammasome in patients with chronic myeloid leukemia.

NLRP3 inflammasome has been recently reported as an important risk factor in the development of cancer. But the relationship between polymorphisms of NLRP3 inflammasome related genes and chronic myeloid leukemia (CML) is rarely reported. Therefore, the aim of the present study was to investigate the association of five genetic polymorphisms (NLRP3, IL-1β, IL-18, CARD8 and NF-κB) in 267 CML patients and 344 healthy controls. We found that the AT genotype of CARD8 (rs2043211) was significantly higher compared to TT genotype in high and intermediate risk CML patients. IL-1β (rs16944) polymorphism in early molecular response at 6 months was marginally different, with more GG and less AA genotype in BCR-ABLIS >1% group. IL-18 (rs1946518) polymorphism was significantly different with more GG genotype in BCR-ABLIS >1% group at 6 months. We also demonstrated that WBC count of newly diagnosed patients carrying AG genotype was significantly higher than that of GG or AA genotype of IL-1β (rs16944). The onset age of patients carrying ins/ins genotype of NF-κB (rs28362491) was significantly older than that of ins/del and del/del genotype. Moreover, IL-1β or NLRP3 mRNA expression was decreased and IL-18 mRNA expression was increased significantly in CML patients compared with controls. In conclusion, the genetic polymorphisms of NLRP3 inflammasome may be served as potential predictors for CML.

Increased Regulatory T Cells in Peripheral Blood of Acute Myeloid Leukemia Patients Rely on Tumor Necrosis Factor (TNF)-α–TNF Receptor-2 Pathway

Acute myeloid leukemia (AML) harbors an immune suppression environment, featured by increased regulatory T cells (Tregs). The expression of tumor necrosis factor receptor-2 (TNFR2) on Tregs could be used to identify the maximally suppressive Treg population, and TNF-α furtherly promoted the expansion and function of Tregs via TNFR2 in mice. However, the role of TNF-α has not been determined in AML patients. In view of high levels of TNF-α and Tregs in AML patients, we hypothesized that the increased frequency of Tregs may rely on TNF-α–TNFR2 pathway. We investigated the levels of TNFR2+ Tregs and TNF-α secreted by T cells in peripheral blood (PB) of AML by flow cytometry and enzyme-linked immunosorbent assay, respectively. Our results showed the elevated plasma TNF-α in PB of newly diagnosed (ND) AML patients. The production of TNF-α by CD4+ T cells, especially by T helper (Th)17 cells was remarkably higher in ND AML patients than in complete remission (CR) patients and healthy controls. Then, we found that the circulating frequencies of CD4+CD25+ Tregs and CD4+CD25high Tregs in AML patients were elevated compared with those in healthy controls and CR patients. TNFR2 expression was much higher on Tregs in AML patients and was preferentially expressed on CD4+CD25high T cells. Furthermore, we confirmed that, in vitro, the additional TNF-α can increase the frequency of Tregs through TNFR2 in both AML patients and healthy controls. Summarily, in AML patients, the abnormally elevated level of TNF-α secreted by CD4+ T especially Th17 cells promoted the higher Tregs frequency via the TNF-α–TNFR2 pathway.

Polymorphisms of Interlukin-1β rs16944 confer susceptibility to myelodysplastic syndromes

Genetic factors have been shown to be associated with Myelodysplastic syndromes (MDS) susceptibility. In recent years, the role of inflammation in the promotion of tumor growth is supported by a broad range of experimental and clinical evidence. But the relationship between polymorphisms in NOD-like receptor protein 3 (NLRP3) inflammasome and MDS is rarely reported. Thus, we conducted a case-control study, and genotyped five single nucleotide polymorphisms (SNPs) (NLRP3, IL-1β, IL-18, CARD8, and NF-κB) in MDS patients and healthy controls. The association of different genotypes with patient characteristics was analyzed. Comparing MDS patients with controls, GG genotype of IL-1β (rs16944) was observed to be associated with a significantly increased risk of MDS 78/166 (48.8%) vs 26/96 (27.0%), OR=2.1, CI (1.0-4.4). No significant association was identified regarding the rest of investigated polymorphisms and MDS susceptibility. Complex karyotypes were more frequent in patients with GG genotype of IL-1β (rs16944). Patients with IL-1β polymorphisms (rs16944) GG and GA had lower hemoglobin than those without. Patients with IL-1β polymorphisms (rs16944) GG had higher IPSS scores than those without IL-1β polymorphisms. In conclusion, our present data shows that the IL-1β polymorphisms (rs16944) GG were frequently occurred in MDS. IL-1β (rs16944) GG genotype might serve as a novel biomarker and potential targets for MDS.

The Genetic Polymorphisms of NLRP3 Inflammasome Associated with T Helper Cells in Patients with Multiple Myeloma

The pathogenesis of multiple myeloma (MM) remains unclear and the NLRP3 inflammasome has been more and more recognized in the progression of many diseases. To investigate the role of the NLRP3 inflammasome in MM, we determined the genetic polymorphisms and expression of NLRP3 inflammasome-related genes (IL-1β, IL-18, CARD8, and NF-κB) in MM patients, and explored their clinical relevance. Furthermore, we investigated the relationship of the NLRP3 inflammasome with Th cells in MM. Our study showed that the CARD8-C10X (rs2043211) AT genotype contributed to the susceptibility of MM. CARD8-C10X TT patients had earlier clinical stage. The WBC count in the three CARD8 genotypes showed an increasing trend (AA<AT<TT). Compared with patients with NF-κB-94 ins/del ATTG ins/ins and ins/del, patients with del/del had the highest myeloma cell ratio. Patients with IL-18 (rs16944) TT had the highest hemoglobin concentration (GG<GT<TT). Furthermore, we found that the genotype of CARD8-C10X (rs2043211) or NF-κB-94 ins/del ATTG was closely related to the frequency of Th1. Therefore, the genetic polymorphisms of the NLRP3 inflammasome associated with Th cells might be involved in the pathogenesis of multiple myeloma.

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

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.

NLRP3 inflammasome activation plays a carcinogenic role through effector cytokine IL-18 in lymphoma

Inflammasomes play important roles in the pathogenesis of tumors, but the roles of NLRP3 inflammasome in the lymphoma remain unclear. Activated NLRP3 inflammasome induces the maturation of its effector cytokine IL-18 which functions in the development of cancer. Here, we investigated the polymorphism and expression of NLRP3 inflammasome related genes and explored their function in lymphoma. We found that IL-18 (rs1946518) and NFκB94 ins/del (rs28362491) contributed to lymphoma susceptibility and allele G in IL-18 was significantly associated with the risk of lymphoma. The mRNA and plasma expression levels of IL-18 were significantly elevated in primary lymphoma patients and decreased after remission. NLRP3 inflammasome could be activated by ATP plus LPS in lymphoma cells accompanied with the increasing expression of NLRP3-related genes. NLRP3 inflammasome activation reduced the dexamethasone-induced proliferation-inhibiting effect by promoting cells into S phase. NLRP3 inflammasome activation promoted lymphoma cells proliferation and inhibited apoptosis through up-regulation of c-myc and bcl-2, and down-regulation of TP53 and bax, and then reduced the anti-tumor effect of dexamethasone. Similar with the activation of NLRP3, the effector cytokine IL-18 also had the proliferation-promoting, apoptosis-inhibiting and resistance-reducing effects on lymphoma cells via shifting the balance of c-myc/TP53 and bcl-2/bax. Moreover, neutralizing IL-18 has the opposite effects. In conclusion, NLRP3 inflammasome contributes to the susceptibility and plays a carcinogenic role through its effector cytokine IL-18 in lymphoma.

Aberrant NLRP3 inflammasome associated with aryl hydrocarbon receptor potentially contributes to the imbalance of T‑helper cells in patients with acute myeloid leukemia

Acute myeloid leukemia (AML) is a hematological malignancy in which the immune response serves a pivotal role in progression. Aryl hydrocarbon receptor (AHR) is involved in the modulation of the immune system, particularly in the differentiation of T-helper cell (Th) subsets. Although the NLR family pyrin domain-containing 3 (NLRP3) inflammasome has been implicated as essential in the pathogenesis of autoimmune and inflammatory diseases, the role it serves in the development of AML remains unknown. Therefore, in order to identify and describe the possible roles of AHR, as well as NLRP3 inflammasome, in the pathogenesis of AML and their relationship with Th subsets (Th1 Th22), the present study investigated the mRNA expression levels of AHR and NLRP3 inflammasome molecules in the peripheral blood and bone marrow. Concentrations of plasma IL-18 were also investigated in peripheral blood by ELISA, as well as the proportions of Th22 and Th1. In the present study, there were three groups: Newly diagnosed (ND) patients; complete remission (CR); and normal controls. A markedly increased expression of NLRP3 inflammasome molecules in bone marrow mononuclear cells (BMMCs) from newly diagnosed (ND) patients compared with patients in complete remission (CR) was identified. NLRP3 inflammasome molecules were also observed to be aberrantly expressed in peripheral blood (PB) mononuclear cells (PBMCs), accompanied with aberrant interleukin (IL)-18 levels in PB plasma. The relative level of IL-18 mRNA became normal after the ND patients with AML achieved CR. In bone marrow, the expression of AHR was significantly higher in ND patients than in CR patients. Furthermore, the expression level of NLRP3 inflammasome molecules was significantly correlated with AHR expression in patients with AML. In the Th subsets, a significantly increased proportion of Th22 in PB from ND patients compared with CR patients or controls was identified, accompanied with decreased Th1. It was concluded that the NLRP3 inflammasome, associated with AHR, was involved in the development of AML and may have influenced the differentiation of Th subsets.