Imbalanced NLRP3 Inflammasome Associated With T Helper Cells And Their Clinical Relevance In Patients With Acute Myeloid Leukemia

BackgroundAcute myeloid leukemia (AML) is a common malignant tumor originated from bone marrow. A large number of myeloid and its precursor cells accumulated in bone marrow even in peripheral blood of patients with acute myeloid leukemia. Abnormalities in bone marrow and accumulation of leukemia cells,destroy or inhibit the normal bone marrow hematopoietic function, so the normal blood cells in AML patients is reduced. AML is a serious threat to human health.AML can occur in all ages, but the incidence in adult is far higher than that in the young people. In recent years,despite the improvement of chemotherapy and the development of novel drugs, clinical outcomes for most AML subtypes remain poor. At the present,chemotherapy is first choice for AML patients. For most patients with AML chemotherapy has certain effect, but long-term survival rate has not improved. A large number of studies have shown that the imbalance of immune system plays an important role in the development of AML. Previous studies have demonstrated that aberrant Th cells participated in the progress of AML. It is well-known that the innate immune closely associated with the adaptive immune, but the research for the innate immune in AML is limited. NLRP3 inflammation as a typical example of innate immunity has been demonstrated associated with a variety of autoimmune diseases, metabolic diseases and tumor. In the study of autoimmune diseases,metabolic disease and tumor NLRP3 inflammation is found related to Th subgroup cells and promote the development of the diseases. Aromatic hydrocarbon receptor (AHR) has been shown that playing an important role in the regulation of immune response, especially in the regulation of Th cells. The role of NLRP3 inflammation in AML and its connection with AHR is not clear. Our previous studies had demonstrated that Th cells imbalanced in AML, but there is no reported about the NLRP3 inflammation in AML patients.In this research we investigated the NLRP3 inflammasome and its related cytokines IL-1β and IL-18 in the development of AML as well as the correlations between NLRP3 inflammasome and Th subsets in PB and BM microenvironment, and explored their clinical relevance.ObjectedThe purpose of this research:1. To explore the role of NLRP3 inflammation,AHR in the patients with AML and to find the connection between them.2. To detected the relationship among the NLRP3 inflammation,AHR and Th cells in AML patients and further explore their roles in the development of AML.3. Targeting NLRP3 inflammasome may be considered as a novel potential treatment for AMLMethodsNinety newly-diagnosed (ND) AML patients (42 females and 48 males; age range, 15-75 years, median age,49 years) and seventy-nine complete remission (CR) AML patients (31 females and 48 males; age range,15-75 years; median age,37 years) were involved in this research. AML was diagnosed according to French-American-British (FAB) classification system and CR was consistent with International Working Group Criteria. AML patients who suffered from hypertension, cardiovascular diseases, infection, connective tissue diseases as well as autoimmune diseases were excluded. Twenty-eight healthy controls took part in this research. Bone marrow mononuclear cells (BMMCs) and peripheral blood mononuclear cells (PBMCs) were isolated from AML patients and controls. Enrollment happened between September 2014 and September 2015 in Qilu Hospital of Shandong University (Jinan, China). This study achieved approves from the Medical Ethical Committee of Qilu Hospital of Shandong University. All patients were informed before enrollment in the study. The expressions of NLRP3 inflammasome associated molecules(NLRP3,ASC,Caspase-1,IL-1β and IL-18) and AHR were detected by Quantitative Real-time PCR. The concentrations of IL-18 in plasma were measured by enzyme-linked immunosorbent assay (ELISA).The frequencies of Thlsubset cells and Th22 subset cells were detected by flow cytometry.Results1. NLRP3 inflammasome molecules were aberrantly expressed in AML patients1.1. In BM microenvironment, the expression of NLRP3 was significantly higher in ND AML group than in CR AML group (p<0.001).Our data showed that ASC expression was elevated in ND groups compared with CR group (p<0.05). No statistical significance was found between the expressions of Caspase-1 in ND patients and in CR group.1.2. In peripheral blood, we also found the elevated expression of ASC in ND group compared with CR group (p<0.05) or normal control group (p<0.05). Moreover, there was no significant difference of NLRP3 expression among ND patients, CR patients and controls. However, Caspase-1 in ND patients was decreased relative to CR group and control group (p<0.001).2. NLRP3 effector cytokines (IL-1 β and IL-18) were abnormal in AML patients2.1. In BM microenvironment of AML patients, IL-18 expression was significantly increased in ND group relative to CR group (p<0.001). Moreover, IL-1β was also marginally elevated in the ND patients compared with CR patients but no statistical significance was found.2.2. In peripheral blood, IL-18 in ND group was significantly higher than that in CR group and controls (p<0.001). However, IL-1β was obviously increased in ND and CR group compared with controls,but no statistical significance was found. And no obvious difference of IL-1β was found between ND and CR group.2.3. ELISA was used to detect the level of IL-18 in PB plasma. IL-18 was found significantly increased in ND patients compared to CR patients (p<0.05) or controls (p<0.001). IL-18 in CR patients was also decreased relative to normal controls (p<0.05).3. The relationships between NLRP3 inflammasome molecules and effector cytokines3.1. In PBMCs the results suggested that NLRP3 inflammasome molecules NLRP3, ASC and Caspase-1 were positively correlated with each other in both ND and CR patients, but in controls we only found the positive correlation between NLRP3 and ASC. Moreover the effective cytokines IL-1β and IL-18 were found positively correlated with NLRP3 inflammasome molecules in ND and CR AML patients. But in controls no correlation between NLRP3 inflammasome molecules and the effective cytokines was found.3.2. In BMMCs, we found the three NLRP3 molecules were positively correlated with each other and the NLRP3 inflammasome molecules were also found positively correlated with the effective cytokines. In CR patients, we only found the positive correlation between ASC and Caspase-1.4. Abnormal Th cells in AML patientsThe frequency of Th22 was increased in ND patients relative to CR patients (p<0.001) or controls (p<0.001). Moreover, though Thl cells have the decreased tend in ND patients compared with CR group and controls, no statistical significance was found.5. mRNA expressions of Th-related molecule AHR5.1. Our present study showed that no significant difference of AHR expression was found in PBMCs of ND AML, CR patients and controls. But in the bone marrow we found that AHR was obviously increased in ND patients compared to CR patients (p<0.05).6. The relationship between NLRP3-related molecules and Th-related molecules6.1. Our data in BMMCs showed the positive correlations between NLRP3 inflammasome associated molecules and AHR in AML patients.6.2. In PBMCs, we found that in ND patients the expression of NLRP3 (ASC, Caspase-1,IL-18 or IL-1β) had positive correlations with AHR. In CR patients we also found that the levels of inflammasome molecules and effector cytokines were positively correlated with AHR. In controls we found that AHR was negatively correlated with Caspase-1.7. The correlations of NLRP3 with clinicopathological characteristics of AML patients7.1. IL-18 was found negatively correlated with the level of serum albumin (p<0.001) or a positively correlated with LDH (p<0.001) in all patients including ND and CR patients.7.2. In ND AML patients, we found significantly positive correlations between NLRP3 inflammasome molecules (NLRP3,ASC,IL-1β and IL-18) and WBC count.We also found AHR had positive correlation with WBC count in ND AML patients.8. Chemotherapy recovered the aberrant expression of IL-18 and Caspase-1 in PBMCs of AML patientsTo further understand the influence of chemotherapy on AMLwe observed the whole treatment process of 28 AML patients. CR was obtained after the standard induction chemotherapy. We found that in PBMCs the expression of IL-18 became normalized after the ND AML patients achieved complete remission as well as Caspase-1.However, no statistical significance was found for other NLRP3 inflammasome molecules.Conclusions1. Taken those findings together, we could conclude that NLRP3 inflammasome and Th subsets cells were implicated in the development of AML.2. NLRP3 inflammasome associated with AHR were implicated in the development of AML and might influence the differentiation of Th subsets. Targeting NLRP3 inflammasome may be considered as a new potential treatment for AML.