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Th22 Cells as Well as Th17 Cells Expand Differentially in Patients with Early-Stage and Late-Stage Myelodysplastic Syndrome

Background Immunological mechanisms are increasingly recognized in the progression of myelodysplastic syndrome (MDS). Early-stage MDS (E-MDS) is characterized by autoimmune-mediated myelosuppression whereas late-stage MDS (L-MDS) involves immune evasion, giving dysplastic cells growth potential to progress into acute myeloid leukemia. T-helper (Th) 22 is involved in the pathogenesis of inflammatory autoimmunity and tumorigenesis. The roles of Th22 cells in the pathophysiology of E-MDS and L-MDS remain unsettled. Design and Methods We studied 37 MDS patients (E-MDS, n = 17; L-MDS, n = 20) and 20 healthy controls to characterize their peripheral blood (PB), as well as 25 MDS patients and 10 healthy controls to characterize their bone marrow(BM). The expression of Interleukin-22 (IL-22), IL-17 or interferon gamma (IFN-γ) was examined in E-MDS, L-MDS patients and controls by flow cytometry. The mRNA expression levels of RAR-related orphan receptor C (RORC), IL-6, tumor necrosis factor alpha (TNF-α) and IL-23 in peripheral blood mononuclear cells (PBMCs) were determined by real-time quantitative polymerase chain reaction. The levels of IL-22 and IL-17 both in PB and BM plasma were examined by enzyme-linked immunosorbent assay. Results In E-MDS, peripheral Th17 cells were significantly elevated and correlated with peripheral Th22 cells compared with healthy controls and L-MDS. Significantly higher levels of peripheral Th22 expansion, mRNA expression of IL-6, TNF-α and lower level of RORC mRNA expression were observed in L-MDS compared with E-MDS. No statistical difference was found in IL-23 mRNA expression or plasma IL-22, IL-17 levels among E-MDS, L-MDS and controls. Conclusions Our data demonstrated that L-MDS cohort had increased frequencies of peripheral Th22 cells and higher mRNA expression levels of IL-6 and TNF-α, indicating that Th22 cells along with Th17 cells or not are involved in the dynamic immune responses of MDS.

Th17 and Treg Cells in Bone Related Diseases

Bone-related diseases share the process of immune response that targets bone tissue and bone marrow and then induce adverse effects on structure and function. In recent years, reciprocal relationship between immune cells and bone systems has been uncovered gradually. Regulatory T (Treg) and T helper 17 (Th17) cells are newly identified subsets of CD4+ T cells, and the balance between them is particularly essential for maintaining immune homeostasis. Accumulated data have demonstrated quantitative or functional imbalance between Th17 and Treg in bone related diseases, suggesting that Th17 and Treg cells are involved in these bone diseases. Understanding the molecular mechanisms regulating Th17 and Treg cells will create opportunities for the development of therapeutic approaches. This review will present the role of Th17 and Treg cells in the inflammatory bone diseases and bone marrow malignancies and find the potential therapeutic target for immunotherapy.

Gene expression profiling of the DNMT3A R882 mutation in acute leukemia

DNA methyltransferase 3A (DNMT3A) is one of two human de novo DNA methyltransferases essential for the regulation of gene expression. DNMT3A mutations and deletions have been previously observed in acute myeloid leukemia (AML), myelodysplastic sydromes and myeloproliferative neoplasms. However, the involvement of DNMT3A in acute lymphoblastic leukemia (ALL) has rarely been reported. In the present study, PCR and direct sequencing was performed to analyze mutations of DNMT3A amino acid residue 882 in 99 acute leukemia patients, including 57 AML patients, 41 ALL patients and a single biphenotypic acute leukemia (BAL) patient. DNMT3A expression was detected in mono-nuclear cells of the bone marrow in these patients and in normal individuals using real-time quantitative polymerase chain reaction, and 17.5% (10/57) of AML patients were found to exhibit DNMT3A mutations. Four missense mutations were observed in the DNMT3A-mutated AML patients, including R882 mutations and a novel single nucleotide polymorphism resulting in the M880V amino acid substitution. However, the ALL and BAL patients were not found to exhibit DNMT3A mutations. The DNMT3A expression levels in the AML patients were significantly higher compared with those of the ALL patients or normal controls. The reduced expression levels of DNMT3A were associated with a significantly lower complete remission rate in the AML patients. However, in the ALL patients, no statistical significance was identified. The results of the present study indicate that DNMT3A may play varying roles in the regulation of DNA methylation in AML and ALL.

Increased Th22 cells as well as Th17 cells in patients with adult T-cell acute lymphoblastic leukemia

BACKGROUND Immune regulation is important for the pathogenesis of T-cell acute lymphoblastic leukemia (T-ALL). Th22 cells are recently-identified CD4(+) T cells that implicated in the pathogenesis of many hematological diseases, such as AML. However, the role of Th22 cells in the pathophysiology of T-ALL remains unclear. We examined the Th22, Th17 and Th1 cell frequencies in peripheral blood of T-ALL patients. METHODS We studied 24 newly-diagnosed (ND), 17 morphologic complete remission (CR) T-ALL patients and 30 healthy controls. Th subsets were examined by flow cytometry. Plasma IL-22 and IL-17 concentrations were measured by ELISA. Transcription factor RORC, T-bet and AHR mRNA expressions were examined by RT-PCR. RESULTS Th22, Th17 frequencies, plasma IL-22 concentration and AHR expression were significantly increased in ND or CR T-ALL patients compared with controls. Moreover, Th22 showed positive correlation with Th17 or Th1 cells in T-ALL patients. However, a significant decrease of IL-17 concentration, Th1 frequency and T-bet expression was found in ND or CR ALL patients compared with controls. Furthermore, Th17 cells showed positive correlation but Th1 cells showed negative correlation with white blood cell counts. CONCLUSION The profile of Th subsets was distinct for T-ALL patients and showed some correlations with clinical index, which suggest that these Th subsets may be implicated in the pathogenesis of T-ALL and be reasonable targets for therapeutic intervention.

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.

The Alteration and Clinical Significance of Th22/Th17/Th1 Cells in Patients with Chronic Myeloid Leukemia

T helper- (Th-) cell immunodeficiency plays important roles in tumor development and their effects in chronic myeloid leukemia (CML) remain unclear. In the present study, we mainly investigated the role of Th22, Th17, and Th1 cell and their related cytokines (IL-22, IL-17, and IFN-r) in the pathophysiology of CML. Bone marrow (BM) and peripheral blood (PB) were extracted from newly diagnosed (ND), chronic phase- (CP-) CML patients, and controls. Th subsets were examined by flow cytometry. Plasma IL-22, IL-17, and IFN-r concentrations were measured by ELISA. AHR and RORC mRNA expressions were examined by RT-PCR. The frequencies of Th22, Th17, and Th1 cells, along with the expression of specific transcription factors RORC and AHR, were significantly decreased in ND patients compared with healthy controls, while all these abnormality recovered in CP patients. In addition, there existed a significantly positive relationship between Th22 and Th17 cells in PB or BM. A significantly negative relationship was found between Th cells (Th22, Th17, or Th1) and BCR-ABL (%) IS or the number of PB white blood cells. All these results demonstrated that Th22, Th17, and Th1 cells might be important therapeutic targets in CML and could facilitate a better outcome for tumor immunotherapy.

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.

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.

The Mutation Profile of Calreticulin in Patients with Myeloproliferative Neoplasms and Acute Leukemia.

Objective: Calreticulin (CALR) plays important roles in cell proliferation, apoptosis, and immune responses. CALR mutations were described recently in Janus kinase 2 gene (JAK2)-negative or MPL-negative primary myelofibrosis (PMF) and essential thrombocythemia (ET) patients. CALR trails JAK2 as the second most mutated gene in myeloproliferative neoplasms (MPNs). However, little is known about CALR mutation in Chinese patients with leukemia. In the present study, a cohort of 305 Chinese patients with hematopoietic neoplasms was screened for CALR mutations, with the aim of uncovering the frequency of CALR mutations in leukemia and MPNs. Materials and Methods: Polymerase chain reaction and direct sequencing were performed to analyze mutations of CALR in 305 patients with hematopoietic malignancies, including 135 acute myeloid leukemia patients, 57 acute lymphoblastic leukemia patients, and 113 MPN patients. Results: CALR mutations were found in 10.6% (12 of 113) of samples from patients with MPNs. CALR mutations were determined in 11.3% (6 of 53), 21.7% (5 of 23), and 9.1% (1/11) of patients with ET, PMF, and unclassifiable MPN, respectively. Conclusion: We showed that MPN patients carrying CALR mutations presented with higher platelet counts and lower hemoglobin levels compared to those with mutated JAK2. However, all of the leukemia patients had negative results for CALR mutations.

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.