Study On The Role Of Bone Marrow Sympathetic Nerve And Imbalance In The Development Of Acute Leukemia

PART I Study on the Correlation between Neuropathy and Th Subsets Imbalance in the Bone Marrow Microenvironment of Acute Myeloid LeukemiaBackgroundAcute myeloid leukemia(AML)is a heterogeneous group of malignant diseases characterized by excessive proliferation of leukemia stem cells and/or progenitor cells.Even with intensive chemotherapy and stem cell transplantation,overall survival of AML remains poor.The microenvironment of malignant leukemia cells will be reshaped into an environment benefits leukemia cells' survival.Such a bone marrow microenvironment can protect AML cells from chemotherapy-induced cell death.Explore the factors in the bone marrow microenvironment that can protect leukemia cells from chemotherapeutic drugs,and in order to find effective targets for AML treatment.The sympathetic nervous system in the localized region of the bone marrow can regulate the normal hematopoietic function of the bone marrow by directly contacting hematopoietic stem cells(HSC)or in the form of secreted cytokines to maintain the survival of hematopoietic stem cells in the bone marrow.It is reported that with the mouse's sympathetic nerve destroyed,loss of innervation of the bone marrow will cause a sharp decrease in the number of hematopoietic stem cells in the bone marrow and inhibit hematopoietic function.Bone marrow sympathetic nerve tissue plays a important role in regulating hematopoietic and immune functions of the bone marrow microenvironment.However,bone marrow sympathetic nerve injury is closely related to the occurrence and development of hematological diseases.Studies have found that the bone marrow microenvironment of patients with bone marrow proliferative tumors(Myeloproliferative Neoplasms,MPN)and model mice have significant damage to sympathetic nerve fibers,and these injuries can cause normal HSC apoptosis.CD4+T cells play an important role in the immune system,and their differentiated Th(T helper)subgroups also tae a part in the development of many tumors.The research team found that expression levels of sympathetic nerve-associated moleculars tyrosine hydroxylase(TH)and Nestin in the bone marrow of AML patients were positively correlated with the expression of Th cell-related molecule IL-17,and negatively correlated with the ratio of Foxp3/IL-17.Therefore,we speculate that during the occurrence and development of AML,pathological changes of bone marrow sympathetic nerves cause abnormalities in the Th cell immune network of the bone marrow microenvironment,resulting in the suppression of immune balance in the bone marrow microenvironment and the development of leukemia.However,the mechanisms that how sympathetic nerve injury in AML changes the bone marrow Th cell immune network,and how the abnormality of Th cell immunity promotes the development of leukemia cells need further study.Objectives1.To clarify the local nerve damage in the bone marrow of patients with AML and its association with the clinical characteristics.2.Detect the changes of Th subpopulations in bone marrow microenvironment,confirm that imbalance of Th subsets in bone marrow microenvironment,and infer the key points by system biology analysis.3.To investigate the changes of Th subsets after the sympathetic nerve has been destroyed in the AML animal model.4.Applying the method of intramedullary cavity injection to destroy the sympathetic nerves in the bone marrow cavity of AML animal models,in order to clarify the role of neuropathology in the development of leukemia.Methods1.In this study,bone marrow fluids from 63 newly diagnosed patients of acute myeloid leukemia and 16 control groups from Qilu Hospital of Shandong University were collected;BMMCs were isolated and CD4+T cells were sorted by MACS,and the bone marrow supernatants were collected..2.Neuropathology in bone marrow microenvironment of patients with AML:RT-PCR was used to detect the mRNA levels of neuron-specific molecules Nestin,GFAP,TUB3,MAP2 and S-100 in bone marrow of patients and controls.3.Correlation analysis between adrenergic receptor expression levels in bone marrow of patients with AML and clinical characteristics:RT-PCR was used to detect three different subtypes of ?-adrenergic receptor ?1-ADR,?2-ADR and ?3-ADR,and analyze their correlations with clinical characteristics4.Immunological imbalance of Th subsets in the bone marrow of AML patients:Flow cytometry was used to detect the proportion of Th subsets in the bone marrow of AML patients and control groups,including Th1,Th2,Th17,Th9,Th22 and Treg;RT-PCR was applied to detect mRNA levels of Th subset-specific transcription factors T-bet,RORc,GATA3,AHR and Foxp3;ELISA was used to detect cytokines IFN-?,IL-17A,TGF-?,IL-22,IL-10 and IL-4 concentrations in the bone marrow supernatant.5.System biology method analysis:We use Bayesian network in systems biology method to analyze Th subsets in bone marrow microenvironments of leukemia patients and control group6.Effect of sympathetic nerve destruction on leukemia and Th subsets in AML animal models:AML mouse models were constructed by injecting MLL-AF9 cells through tail vein into wild-type female C57BL/6J mice;Intraperitoneal injection of 6-Hydroxydopamine hydrobromide(6-OHDA)and 4-Methylcatechol(4-MY)in mice for neurological destruction and neuroprotective intervention respectively;Flow cytometry was used to measure the proportion of Th subpopulations in the bone marrow of mice.7.Impact of bone marrow sympathetic nerve destruction on leukemia:6-OHDA was injected into the bone marrow to destroy nerves in mouse bone marrow.Results1.Neuropathology in bone marrow of patients with AML:By comparing gene expression levels in bone marrow between patients and control groups,it was found that the mRNA levels of neuron-specific molecules Nestin,GFAP,TUB3,MAP2,and S-100 were much lower in leukemia patients.Suggesting that there has the neuropathology in the bone marrow microenvironment of AML patients2.Correlation analysis between local adrenergic receptor expression and clinical characteristics in bone marrow of AML patients:By detecting three different subtypes of P-adrenergic receptor ?1-ADR,?2-ADR and ?3-ADR,we found that ?1 subtype has low expression in bone marrow of leukemia patients.By analyzing its relationship with clinical characteristics,it was found that AML-M5 highly expressed ?1-ADR,AML-M3 highly expressed ?2-ADR,and patients with high expression of ?2-ADR were more likely to have extramedullary infiltration.3.There were immune imbalances of Th subsets in the bone marrow of AML patients(1).Compared with the control group,the percentages of Th1 and Th 17 in bone marrow of AML patients were significantly reduced,and the percentages of Treg and Th22 were significantly increased;(2).The mRNA expression levels of T-bet,RORc,and GATA3 in the bone marrow of AML patients were significantly lower than those in the control group,while Treg specific transcription factors Foxp3 and Th22 specific transcription factor AHR increased;(3).About the cytokines secreted by Th subsets of AML patients,IFN-?,IL-17A,TGF-? and IL-22 were much lower than those in the control group,while the secretions of IL-10 and IL-4 were higher than those in the control group.(4).Analysis of the above results by Bayesian network found that the end point of the Bayesian network in the control group points were IFN-? and IL-4,and the end points of the network in patients with AML changed to IL-17A and IL-104.The effects of sympathetic nerve destruction on leukemia and Th subgroups in AML animal models:(1).The survival time of AML mice in the nerve injury group was shorter than that of other groups;the leukemia load in the nerve injury group was significantly higher than other groups;(3).In the local microenvironment of bone marrow,Th1 and Th17 were lower in the nerve injury group than in the other groups,and percentages of Treg and Th2 were much higher in the nerve injury group than the control groups.The ratio of Th17/Treg was lower in the nerve injury group than in the other groups.5.Impact of bone marrow sympathetic nerve destruction on leukemia and Th subgroup:After the sympathetic nerve of a single femur of a leukemia model mouse was destroyed by intramedullary injection,the leukemia load was observed.The leukemia load in the medullary cavity on one side was significantly higher than the load on the side without nerve destruction.Conclusions1.The bone marrow sympathetic nerve of AML patients was damaged,and?-adrenergic receptors were highly expressed in the bone marrow of AML patients.2.There was an immune imbalance of Th subsets in the bone marrow of AML patients.The proportions of Th1 and Th17 were significantly reduced,and the proportions of immunosuppressive subgroups such as Treg were significantly increased.3.In the AML mouse model group,the survival time of the nerve injury group was significantly shorter and the leukemia load increased,while the neuroprotectant group had a longer survival time and less leukemia load;there was an immune imbalance in the bone marrow of the nerve injury group.4.Partial destruction of unilateral bone marrow sympathetic nerves in AML mice revealed that the leukemia load in the damaged medullary cavity was significantly higher than that in the intact myeloid cavity,which proved that neuropahology could promote the development of leukemia.PART ? The Studies in Immune Regulation of Sympathetic Neurotransmitters on Th Subsets and Acute Myeloid Leukemia CellsBackgroundAcute myeloid leukemia(AML)is a biological and clinical heterogeneous disease.Although advances in supportive care and prognostic risk stratification have optimized established therapies,overall long-term survival is still low,so identifying factors that can influence the development of the disease is necessary.It is well known that acute stress response is a positive response of the human body to external stimuli,while chronic stress is usually harmful and may cause serious health complications.Stress triggers neuroendocrine chain reactions under the control of specific parts of the central nervous system,which mainly include catecholamines and glucocorticoids secreted by the adrenal and sympathetic nervous systems.Catecholamines are the earliest signal molecules that respond to stress,and they are also the main molecules that take part in stress responses.Although the main function of catecholamines(mainly adrenaline and norepinephrine)is to affect cardiac function,recent studies have shown that these stress hormones also show important roles in cancer.T helper(Th)subsets are CD4+T cells that differentiate into different subsets after activation and play a key role in various immune responses.Early discovered Thl and Th2 subsets or the recently discovered Th17 and Treg subsets are considered to be involved in the pathogenesis of malignancies such as ovarian cancer and melanoma.The immune imbalance of the Th subsets is considered to be a key factor in the development of diseases.Moreover,accumulating evidences showed that the receptors of multiple neurotransmitters are expressed on the surface of lymphocytes and monocytes,while sympathetic neurotransmitters not only suppress the immune system,but also cause Thl/Th2 cytokines to balance towards the main Th2 cytokines response shifts and promotes asthma and allergic diseases.However,it is unclear how sympathetic neurotransmitters participate in the development of AML.Based on previous detection of a certain degree of neuropathology and immune imbalance in the Th subsets in the bone marrow microenvironments of AML patients,we hypothesized that in the AML bone marrow microenvironment,sympathetic neurotransmitters will cause changes of Th17,Treg,Thl,Th2,etc.The imbalance of multiple Th cell immunity eventually promotes the development of AML leukemia.Objectives1.To investigate the effect of local sympathetic neurotransmitters in the bone marrow on the immune balance of the Th subsets,and to clarify its mechanisms.2.To investigate the effects of sympathetic neurotransmitters on primary leukemia cells and leukemia cell lines at different concentrations,and to clarify how they promote the development of leukemia by changing the immune balance of the Th subsets.3.To confirm that the sympathetic neurotransmitters could inhibit the differentiation of CD4+T to the immunoprotective subgroups Thl and Th17 through ?-adrenergic receptors.4.Further explore the neurotransmitter-inhibited Thl and Th17 subpopulations could weaken the killing effect on leukemia cells,and then promote the occurrence and development of leukemia.Methods1.Specimen collection:This study collected bone marrow fluids from newly diagnosed patients with acute myeloid leukemia from Qilu Hospital of Shandong University;bone marrow mononuclear cells were isolated and CD4+T cells were sorted by MACS,and the bone marrow supernatant were retained.2.Sympathetic neurotransmitters can promote the proliferation of primary and leukemia cell lines:acute myeloid leukemia cell lines MV4-11 and THP-1 and primary cells from newly diagnosed patients with acute myeloid leukemia;CCK8 was used to detect the proliferations of AML cells at 24 and 48 hours.3.Expression of ?-adrenoceptor on different cells:RT-PCR was used to detect the expression levels of ?1/(32/?3-adrenoceptor receptors mRNA on different cells.4.The effect of sympathetic neurotransmitters at different concentrations on the Th subsets:set the concentration gradients of 0,10-8M,10-7M,10-6M,10-5M,and 10-4M;MACS immunomagnetic beads were used to sort CD4+T cells;flow cytometry was used to detect the proportion of Th subsets;ELISA was used to detect the concentrations of IFN-y and IL-17A cytokines in the culture supernatants.5.Sympathetic neurotransmitters affect the immune balance of Th subsets through P-adrenergic receptors:MACS immunomagnetic beads were used to sort mouse CD4+T cells;agarose gel DNA electrophoresis detects genotypes of ?-adrenergic receptor knockout mice;Flow cytometry was used to detect changes in Th subpopulations of mice;ELISA was used to detect the concentrations of IFN-y and IL-17A in cell culture supernatants.6.The effect of neuro transmitters on the differentiation of CD4+T and the killing effect on leukemia cells:MACS immunomagnetic bead sorting of CD4+T cells;flow cytometry for the proportion of Th subpopulations;CCK8 method for the detection of cell proliferation.Results1.Sympathetic neurotransmitters can promote the proliferations of primary and leukemia cell lines:different concentrations of epinephrine and norepinephrine were applied to MV4-11 and THP-1 and primary cells of newly diagnosed leukemia patients,respectively,and tested proliferation rate at 24 and 48 hours using the CCK8 method.As the concentration increased,the effect of sympathetic neurotransmitters on the proliferation of leukemia cells became more obvious.Both epinephrine and norepinephrine have the most obvious proliferation-promoting effect at aconcentration of 10-4M,and adrenaline-promoting effect on leukemia cells is better than noradrenaline.2.P-adrenergic receptor expression levels on different cells:(1)RT-PCR was used to detectp-adrenergic receptors expression levels of four different leukemia cell lines,HL60,THP-1,U937,Kasumi,and CD4+T cells.Among them,(32-adrenoceptor expression level was much higher than the other two subtypes.(2)The expression levels of P-adrenergic receptors in the spleen,bone marrow,peripheral blood,sorted CD4+T cells,and skeletal muscle cells were also detected in mice.?2-adrenoceptor expression level in every tissues was still much higher than the other two subtypes.3.Low concentrations of sympathetic neurotransmitters have no significant effect on the immune balance of Th subsets:(1)Add concentrations from 10-8M to10-5M of epinephrine and norepinephrine to the CD4+T cell culture system.After 3 days,the percentages of Thl,Th2,Th17,and Treg subsets were detected by flow cytometry.The study found that in the concentration range from 10-8M to 10-5M,there was no statistical difference among Th1,Th2,Thl7 and Treg compared with the blank control group.(2)The supernatants of the cultured cells were taken,and the concentrations of secreted IFN-y and IL-17A cytokines were detected by enzyme-linked immunolabeling(ELISA).There was no statistical difference between each concentration gradient group and the blank control group.4.High concentrations of sympathetic neurotransmitters can affact the immune balance of Th subsets:(1)10-4M concentration of epinephrine and norepinephrine were added to the CD4+T cell culture system,and flow cytometry was applied to detect the percentages of Thl,Th2,Th17,and Treg subsets.Our study had found that both epinephrine and norepinephrine can significantly reduce the percentages of Thl and Th17,and can increase the percentage of Treg.(2)Enzyme-linked immunosorbent assay(ELISA)was used to detect the cell supernatants after culture.The concentrations of IFN-y and IL-17A cytokines in the supernatants were reduced after the culture of adrenaline and norepinephrine.5.Sympathetic neurotransmitters also change the immune balance of Th subsets in mice:After sorting mouse CD4+T cells,apply the method described above to add sympathetic neurotransmitters for cell culture.The percentages of Thl and Thl7 decreased significantly,and the percentage of Treg increased significantly.The mRNA levels of IFN-y and IL-17A detected by RT-PCR were significantly lower than those of the control group.6.Sympathetic neurotransmitters affect the immune balance of the Th subsets through?-adrenergic receptors:(1)Detect the genotype of ?1/?2-adrenergic receptor knockout mice.(2)After sorting CD4+T cells of adrenergic receptor knock-out mice,the above method was used to add sympathetic neurotransmitters for culture.Flow cytometry was used to detect the percentages of Thl,Th17,Th2 and Treg.Th1 and Th17 subsets were not inhibited by sympathetic neurotransmitters.It is proved that the sympathetic neurotransmitters epinephrine and norepinephrine could change the Th subsets through the ?-adrenergic receptor.7.The effect of neurotransmitters on the differentiation of CD4+T and then the killing effect on leukemia cells:10-4M epinephrine and norepinephrine were added to the culture system of CD4+T cells,and cultured for 3 days.The cells were co-cultured with leukemia cells,and the proliferation of leukemia cells was detected by the CCK8 method at 24 hours and 48 hours.Both epinephrine and norepinephrine can promote the proliferation of leukemia cells by changing the balance of Th subsets,and the effect of adrenaline is more obvious.Conclusions1.The sympathetic neurotransmitters epinephrine and norepinephrine can promote the proliferation of primary leukemia cells and leukemia cell lines at 24 hours and 48 hours,and the proliferation effect is more obvious with increasing concentration.2.Sympathetic neurotransmitters can inhibit the differentiation of human and mouse CD4+T cells toward Thl and Th17.3.After the ?-adrenergic receptor was knocked out,sympathetic neurotransmitters could not inhibit the differentiation of mouse CD4+T cells to Thl and Th17.4.Sympathetic neurotransmitters inhibit the killing effect on leukemia cells by inhibiting the differentiation of CD4+T cells into Thl and Th17 directions.PART ? Single Nucleotide Polymorphisms of NLRP3 Inflammasome-related Genes in Acute Lymphoblastic LeukemiaBackgroundAcute lymphoblastic leukemia(ALL)is a hematological malignancy that is caused by the malignant proliferation and accumulation of lymphoid stem progenitor cells in the bone marrow.Although the most common ALL is childhood acute lymphoblastic leukemia(80%of ALL cases),the cure rate for adult acute lyrnphoblastic leukemia is only 20%-40%.In the past decade,despite improving prognosis and developing new therapies targeted at specific subtypes,the overal survival remains low,understanding the pathogenesis of the disease remains a key point in incresing the cure rate of ALL.Inflammatory bodies are recognized as important roles in innate immunity.Among them,the most thoroughly studied are NLRP3 inflammatory bodies,and the NLRP3 inflammatory body complex belongs to a family of nucleotide-binding oligomerization domain-like receptors(NLRs),also known as "Pyrin domain-containing protein 3".NLRP3 inflammatory bodies are involved in the occurrence and progression of various diseases,including metabolic disorders,multiple sclerosis,inflammatory bowel disease,and other autoimmune diseases.Single nucleotide polymorphism(SNP)mainly refers to DNA sequence polymorphism caused by mutation of a single nucleotide at the genome level.SNPs differ in at least 1%of the population and account for the majority of normal human genetic variation.Although most SNPs do not have a clear phenotypic effect,many SNPs are believed to promote disease development and affect the efficacy of drug treatment.SNPs of NLRP3-related molecules play a significant role in the onset and progression of many tumors and chronic diseases.For example,researches show that CARD8(rs2043211)is related to the occurrence of cardiovascular disease,and NF-KB-94ins/del ATTG polymorphism is closely related to the incidence of cancers such as gastric cancer,hepatocellular carcinoma and lung cancer.A single nucleotide polymorphism rs 1946518 of IL-18 has been shown to be involved in the pathogenesis of periodontitis and hepatocellular carcinoma,while IL-1? rs 16944 is thought to cause cervical cancer and gastritis.Therefore,the polymorphism of NLRP3 inflammasome-related genes has a very strong correlation with malignant tumors.NLRP3 inflammatory bodies have been widely involved in the occurrence and development of various blood diseases.However,how NLRP3 inflammatory bodies promote the pathogenesis and clinical characteristics of acute lymphocytic leukemia remains unknown.Therefore,we studied the single nucleotide polymorphisms of NLRP3 inflammasomes and the expression of NLRP3 inflammasome-related genes in the bone marrow of ALL patients in order to clarify their roles in the development of acute lymphocytic leukemia.ObjectivesTo explore the relationship between single nucleotide polymorphisms of NLRP3 inflammatory body components and susceptibility of ALL;analyze the relationship between single nucleotide polymorphisms of NLRP3 inflammatory body components and clinical characteristics under different modes.In order to clarify the specific mechanism of NLRP3 single nucleotide polymorphism,explore its effect on the expression levels of NLRP3 components,downstream factor expressions and cytokine secretions.Methods1.Detection of single nucleotide polymorphisms of CARD8(rs2043211),IL-?(rs16944),IL-18(rs1946518)and NF-KB-94ins/del ATTG genes in ALL patients and healthy controls by PCR.2.Analysis of CARD8(rs2043211),IL-1?(rs16944),IL18(rs1946518)and NF-?B-94ins/del ATTG in leukemia patients and healthy controls by row x column chi-square test and single factor logistic regression analysis.3.Analysis of CARD8(rs2043211),IL-1?(rs16944),IL-18(rs1946518),and NF-?B-94ins/del ATTG genes in patients with leukemia by row x column chi-square test and single factor logistic regression the relationship between polymorphism and clinical characteristics in patients with prognostic relevance.4.RT-PCR was used to detect the mRNA expression of IL-1? and IL-18 in ALL patients,and analyzed the correlation between the expression levels and the NPRP3 inflammatory body related gene SNPs.5.ELISA was used to detect the levels of IL-1? and IL-18 in the serum of ALL patients,and the correlation between their concentrations and the NPRP3 inflammatory body-associated gene SNP in patients was analyzed.Results1.The NF-?B-94ins/del ATTG and CARD8(rs2043211)gene polymorphisms are related to the susceptibility of ALL:detecting single nucleotide polymorphisms of NLRP3 related components in the ALL patients and the control group,we found that CARD8(rs2043211)AT/TT genotype under dominant mode and TT genotype under codominant model were significantly related to ALL susceptibility.In addition,the frequency of the D allele of NF-?B-94ins/del ATTG and the DD genotype under the dominant mode were significantly related to ALL susceptibility.We used single factor logistic regression analysis to analyze NF-?B and CARD8.The study found that the single nucleotide polymorphism of NF-?B showed a protective effect,while the AT/TT genotype of CARD8(rs2043211)increased the susceptibility to ALL significantly.2.Patients with ALL expressing NF-KB-94ins/del ATTG DD genotype have lower WBC counts.CARD8(rs2043211)AT/TT genotype is associated with T cell immune phenotype:according to the "NCCN Guideline for Acute Lymphocytic Leukemia" several clinical indicators can provide the clinical prognostic information.Our study analyzed the associations between these prognostic indicators and the four SNPs of the NLRP3 inflammasome-associated genes in order to clarify the correlation between NLRP3 genetic polymorphisms and the prognosis of ALL.According to the guidelines,WBC counts exceeding 30×109/L are considered to be a poor prognostic indicator,and our study found that ALL patients expressing the DD genotype of NF-KB-94ins/del ATTG had lower WBC counts.The immune phenotype of ALL is mainly divided into T cell immune phenotype and B cell immune phenotype.T cell immune phenotype is also an important poor prognostic factor,and the AT/TT genotype of CARD8(rs2043211)showed more relevant to T cell immune phenotype.Therefore,we confirmed that the DD genotype of NF-KB-94ins/del ATTG showed as a protective factor,while the AT/TT genotype of CARD8(rs2043211)is associated with poor prognosis.3.Correlation analysis between NF-KB-94ins/del ATTG,IL-18(rs 1946518),IL-1p(rs 16944)and clinical characteristics of ALL patients:compared with WWAVD genotype of NF-KB-94ins/del ATTG under recessive mode,DD genotype is associated with lower hemoglobin content.Under the codominant model,splenomegaly was more likely to occur in the DD genotype and D allele.The TT genotype of IL-18(rs1946518)under recessive mode was statistically correlated with splenomegaly and high AST concentrations in the serum.In addition,the TT genotype and T allele under the codominant model were associated with higher levels of fasting blood glucose.The polymorphism in IL-1?(rs16944)is related to creatinine concentrations.Under recessive mode,compared with the GG/GT genotype,the TT genotype was significantly associated with high creatinine concentrations.Regarding the frequency of alleles,the G allele was also significantly associated with high creatinine concentrations.4.The relationship between NLRP3 inflammatory body single nucleotide polymorphisms and their related gene expression:The AT genotype of CARD8(rs2043211)expresses higher levels of NLRP3 and ASC.Compared with AA and AT genotypes,TT genotypes have higher caspase-1 mRNA levels.In addition,compared with,the AA genotype,the TT genotype was also related to the IL-18 concentration in the bone marrow microenvironment of ALL.Regarding the IL-1?(rs16944)polymorphism,compared with the GG and TT genotypes,the GT genotype was significantly related to the mRNA expression of IL-18.And compared with the TT genotype,the GT genotype expressed higher ASC levels and secreted more IL-1?.Compared with the GG genotype,the TT genotype of the IL-18(rs 1946518)polymorphism was also correlated with IL-1? and IL-18 concentrations.However,the GT genotype of the IL-18 polymorphism is associated with higher expression levels of NLRP3 and ASC.In addition,we also found a significant correlation between the concentration of IL-1? in the bone marrow microenvironment and the mRNA expression levels of IL-1 and ASC.Therefore,the AT and TT genotypes of CARD8(rs2043211)?the GT and TT genotypes of IL-1?(rsl6944),and the TT genotype of IL-18(rs1946518)are related to high mRNA expression and secreted cytokines of NLRP3-related genes.Conclusions1.NF-KB-94ins/del ATTG and CARD8(rs2043211)gene polymorphisms are associated to susceptibility of ALL.2.Patients with ALL expressing the DD genotype of NF-?B-94ins/del ATTG have lower WBC counts.The AT/TT genotype of CARD8(rs2043211)is associated with the T cell immune phenotype.3.NF-KB-94ins/del ATTG is associated with lower hemoglobin content and splenomegaly;ALL patients expressing the IL-18(rs1946518)genotype are more likely to have splenomegaly,high AST concentrations and high fasting blood glucose levels;The IL-1?(rs16944)genotype ALL patients more likely to have high creatinine concentrations.4.The AT and TT genotypes of CARD8(rs2043211)?the GT and TT genotypes of IL-1?(rs 16944),and the TT genotype of IL-18(rs1946518)are related to the high mRNA expression levels and secreted cytokines of NLRP3-related genes.