The Role And Mechanism Of Gut Microbiota And Its Metabolites In The Development Of Acute Myeloid Leukemia

PART I Changes of gut microbiota in acute myeloid leukemia and its regulation on disease progressionBackground Acute myeloid leukemia(AML)is a hematological malignancy characterized by excessive proliferation of immature myeloid cells.Although chemotherapy is currently effective in AML,the long-term survival rate of patients is not high.The long-term survival and prognosis of AML are closely related to many influencing factors in the process of AML progression.Therefore,a better understanding of the mechanisms of AML progression has important implications for improving AML prognosis and survival.There are a large variety of bacteria living in the human gut,collectively referred to as gut microbiota.Under normal circumstances,in the gut microbiota,various bacteria are combined according to a certain proportion,restrict each other,depend on each other,and form an ecological balance in terms of quality and quantity.Gut microbiota homeostasis plays an important role in maintaining the normal function of the body.The imbalance of gut microbiota can cause an increase in the proportion of opportunistic pathogens and a decrease in the proportion of symbiotic bacteria,thereby causing various diseases.Therefore,the gut microbiota is considered to be an unrecognized "organ" in the human body,which is of great significance for studying disease mechanisms and developing new therapeutic methods.The progression of acute myeloid leukemia is influenced by multiple factors including genetics and homeostasis.There is increasing evidence that alterations in the diversity of the human gut microbiota affect the homeostasis and are associated with inflammation,autoimmune disease,and cancer.Studies have reported that the effect of CTLA-4 antibody anti-tumor immunotherapy mainly depends on the gut microbiota;in addition,in patients with melanoma,non-small cell lung cancer,renal cancer and urothelial tumors,gut microbiota disturbance can Significantly reduce the anti-tumor effect of PD-1 inhibitors,and fecal microbiota transplantation(FMT)or oral probiotics to improve gut microbiota can significantly enhance its anti-tumor effect.In conclusion,gut microbiota imbalance is involved in the occurrence and development of various tumors,but its role in hematological tumors is still unclear,and its research is of great significance.With the deepening of research,the relationship between gut microbiota and blood system has been gradually reported.According to current evidence,gut microbiota can regulate and maintain normal homeostatic hematopoiesis.Recently,it has been reported that the structure of gut microbiota is altered in patients with acute lymphoblastic leukemia(ALL),and the diversity of the microbial community is significantly reduced.In addition,it has been reported that changes in gut microbiota after allogeneic hematopoietic stem cell transplantation(HCT)are associated with the occurrence of gastrointestinal graft-versus-host disease(GVHD),suggesting that gut microbiota may be an important factor in the pathogenesis of GVHD.However,the role of the gut microbiota in acute myeloid leukemia is not yet clear,how the gut microbiota is causally related to AML,and how the gut microbiota participates in the progression of the disease,these issues need to be further studied and resolved.Objectives 1.To clarify the composition of gut microbiota in the gut of AML patients and its relationship with the clinical characteristics of patients.2.To explore the changes of gut microbiota during the occurrence and development of AML and the impact of disrupted microbiota on the disease,and to clarify the causal relationship between the two.3.Interfering with the gut microbiota of AML through fecal flora transplantation to explore the specific role of the flora on disease progression.Methods 1.Specimen collection:Collect feces,peripheral blood and bone marrow from 31 newly diagnosed acute myeloid leukemia patients and 30 control groups who were treated in Qilu Hospital of Shandong University;part of the patient’s feces were frozen at-80℃,and the excess feces were mixed Double-distilled water was stirred evenly,filtered,added with glycerol and frozen at-80℃;peripheral blood and bone marrow were collected in EDTA anticoagulation tubes,centrifuged to take the supernatant,and then stored in a-80℃ refrigerator.2.Detection of intestinal flora in AML patients:16s rRNA sequencing method was used to detect the bacterial species in the stool of patients and normal controls,and the α andβ diversity analysis was carried out through the Meiji cloud platform database.T-test and rank-sum test were used to determine the magnitude of the difference.3.Correlation analysis between intestinal flora and clinical characteristics of patients:Logistic regression was used to analyze the correlation between the number of bacteria and risk classification,Spearman correlation was used to analyze the correlation between the number of bacteria and white blood cells,and the Shapiro-Wilk test was used for normality test.Normally distributed data were analyzed using t-test or paired t-test.4.Related detection of Th subsets in bone marrow of AML patients:Flow cytometry was used to detect the proportion of Th subsets in bone marrow of AML patients and control groups,including Th1,Th17 and Treg.5.Exploring the causal relationship between intestinal flora and AML progression through animal models:using the bone marrow primary cells of MLL-AF9 and AML 1ETO two AML model mice to inject into wild-type female mice with C57BL/6J background,Two AML mouse models were established respectively,and the C1498 mouse leukemia cell line was injected into the wild-type female mice with C57BL/6J background by tail vein to establish the C1498 mouse AML model.16s rRNA sequencing was used to detect the changes of intestinal flora in the above three model mice.At the same time,the antibiotic mixture of ampicillin,streptomycin,neomycin and vancomycin was taken orally to remove the original intestinal flora of mice,and the stored fecal hydration solution of humans or mice was administered by gavage.The fecal bacterial transplantation was carried out by the method of flow cytometry,and the proportion of leukemia cells in the peripheral blood,spleen and bone marrow of the animal model was determined.Result 1.Changes in gut microbiota in AML patients:Through 16s rRNA sequencing,we found that the diversity of gut microbiota in AML patients was significantly lower than that in normal controls,among which the differences in α and β diversity were the most significant.The differential flora between AML patients and healthy controls has obvious species specificity:cloud platform analysis proved that at the phylum level,Bacteroidetes had significant differences between the two groups,while at the genus level,Faecalibacterium and Roseburia were significantly different in AML.significantly decreased in the patient group.The Spearman correlation analysis demonstrated that the two bacteria(Faecalibacterium and Roseburia)significantly reduced in the feces of AML patients had a strong positive correlation,suggesting that the two bacteria may play a synergistic effect.2.Correlation between gut microbiota and clinical characteristics of patients:The clinical characteristics and differential bacteria were analyzed by Spearman correlation analysis and Logistic regression analysis,and it was found that Faecalibacterium and Roseburia were significantly negatively correlated with the number of peripheral blood leukocytes of patients;Risk classification was significantly positively correlated;Faecalibacterium was significantly negatively correlated with the proportion of Treg cells in the patient’s bone marrow.3.Changes of gut microbiota in AML model mice:As the disease progresses in AML model mice,the gut microbiota changes significantly.On the 14th day after the onset of the disease,the diversity of the gut microbiota of the mice in the AML group was significantly lower than that of the normal group.4.The effect of gut microbiota on leukemia in AML model mice:Compared with ordinary AML model mice,AML model mice that use antibiotics to destroy gut microbiota have more serious leukemia cell infiltration in the spleen,bone marrow and peripheral blood,the survival time was significantly shortened.After the use of bacterial flora transplantation to restore the gut microbiota of the antibiotic-treated mice,the burden of AML in the mice was significantly relieved.Conclusions 1.There is a decrease in the diversity of gut microbiota in AML patients.Faecalibacterium and Roseburia are significantly reduced in the stool of AML patients,and are negatively correlated with bone marrow Treg cells,peripheral WBC and risk classification.2.The progression of AML leads to the imbalance of gut microbiota,and the imbalanced gut microbiota has a significant impact on the progression of AML.3.In a mouse model of AML,fecal bacterial transplantation of human and mouse feces can significantly affect the progression of AML in vivo.PART Ⅱ Study on the regulation of gut microbiota metabolites and intestinal barrier function on the progression of acute myeloid leukemiaBackground Gut microbiota is a complex ecosystem composed of microorganisms that maintain the homeostasis of the gastrointestinal tract and play a role by producing bioactive metabolites.The metabolic capacity of intestinal microbiota greatly exceeds that of human cells,and these bioactive metabolites can directly or indirectly affect the physiological function of the host.More and more studies have shown that most of the effects of gut microbiota on host physiology are related to microbial metabolites,and the metabolites produced by microorganisms are the key media based on diet affecting host physiological processes.Metabolites can not only directly enter the blood to play a role,but also affect the collective indirectly by affecting the intestinal region or intestinal barrier.Some microbial metabolites come from intestinal microflora rather than host origin,and they mediate the biological effects of intestinal microflora.Microbial metabolites such as short-chain fatty acid(SCFAs)are associated with blood diseases.Payen et al.The results showed that the levels of major fatty acids(acetic acid,propionic acid and butyric acid)decreased significantly in patients with severe graft-versus-host disease.In addition,butyric acid,as an important SCFA,has been reported as the main energy source of intestinal epithelial cells.Butyric acid can effectively alleviate GVHD.This finding suggests that intestinal microflora may play an important role in diseases through metabolic pathways,especially in GVHD.However,there is a lack of research on short-chain fatty acids in acute myeloid leukemia.The existence of intestinal microflora and its nutritious SCFA metabolites can make the growth of intestinal epithelial cells more active and regulate their differentiation and repair.17-19.The intestinal barrier is the only door that prevents intestinal microflora or metabolites from entering the bloodstream.Its integrity and its normal function are directly related to the fluctuation of the number of bacteria and their metabolites in the blood.Cui et al.It has been found that the physical,chemical,immune and microbial barriers in the intestinal tract constitute a complete intestinal barrier and play an important role in preventing the invasion of harmful substances from the intestinal tract.Another study confirmed that in patients with hematological diseases,patients with graft-versus-host disease showed a significant loss of intestinal barrier function compared with healthy volunteers.The integrity and permeability of the intestinal barrier usually determine how gut microbiota and their metabolites affect systemic immunity.The intestinal barrier of acute myeloid leukemia is often ignored.Paying attention to the intestinal barrier may provide a new perspective for improving the therapeutic effect of acute myeloid leukemia.Objectives 1.To clarify the difference of metabonomics in feces of patients with AML and normal controls.2.To explore the correlation between intestinal differential metabolites and differential flora.3.To explore the effects of metabolites of gut microbiota on primary and leukemic cell lines.4.To investigate the effect and mechanism of gut microbiota metabolites on intestinal barrier function.5.To explore the effect of metabolites of gut microbiota on the progression of AML in vivo and its specific mechanism.Methods 1.Metabonomic detection:the collected fecal and serum samples were thawed on ice and analyzed by gas chromatography-mass spectrometry(GC-MS)with Agilent7890A/5975C GC-MS system.The quantitative determination curve of single chain fatty acid was established in the concentration range of 0.1～100 μg/mL.IS was used to correct the injection variability between samples and small changes in the instrument response.The quantitative determination curve of single chain fatty acids was established in the concentration range of 0.1～100 μg/mL.2.Functional prediction of gut microbiota:using the picrust database,the OUT of gut microbiota was compared with the data in the database,and the functional abundance corresponding to gut microbiota and the functional differences between multiple groups were inferred by a variety of statistical methods.3.Difference analysis of gut microbiota and metabolites:select the short-chain fatty acids(butyric acid,propionic acid,acetic acid)with the most significant differences in metabonomic,on the other hand,select the bacteria with the most significant differences for Spearman thermograph correlation analysis to observe whether there is a close correlation between them.4.Intestinal microorganism transplantation:use antibiotics to destroy the original gut microbiota of AML mice.AML mice were treated with the prepared mixture of feces and water every day,and the colonization of bacteria was detected by 16sRNA.5.Inhibitory effect of butyric acid on AML primary cells and cell lines in vitro:the primary cells of AML cell lines kasumi and THP-1 and AML mice were selected,and the proliferation was detected by CCK8 at 24 and 48 hours after butyric acid treatment.On the other hand,the apoptosis curve was detected by flow cytometry after butyric acid treatment for 24 hours and 48 hours.6.Intestinal barrier function was impaired:the intestinal epithelial cells of mice were extracted,the expression of secret connexin(zo-1,claudin-1,claudin-2)was detected by PCR,Western and other methods,the tight junction protein was localized by immunofluorescence,and the gap between epithelial cells was further observed by electron microscope.Result 1.Non-targeted metabolomics indicated that the metabolites in feces of AML patients were significantly different from those of the normal control group,among which the short-chain fatty acids butyric acid and propionic acid had the most obvious differences.2.The abundance of functional types of metabolites in the stool of AML patients was significantly decreased,and the metabolism of short-chain fatty acids(butyric acid)was significantly lower than that of normal controls.3.Targeted metabolomics showed that the content of fecal short-chain fatty acids(acetic acid,butyric acid,propionic acid)was significantly decreased in AML patients,and butyric acid was significantly positively correlated with differential gut microbiota(Faecalibacterium and Roseburia).4.Butyric acid co-cultured with leukemia cell lines THP-1,Kasumi and C1498 can significantly inhibit their proliferation and promote apoptosis.In vivo,butyric acid gavage significantly delayed disease progression and prolonged survival in AML mice.5.The intestinal barrier function of AML mice is damaged,and the expression of tight junction proteins ZO-1 and claudin-1 is decreased.Giving butyric acid gavage can repair the damaged intestinal barrier.Conclusions1.The metabolomics in the stool of AML patients was significantly different from that of the normal control group,and the functional diversity was significantly decreased,and the short-chain fatty acid metabolism was significantly different;2.There is a significant positive correlation between differential metabolites and differential flora;3.Short-chain fatty acid butyric acid can significantly inhibit the proliferation of leukemia cells;4.The intestinal barrier function of AML mice is impaired and can be reversed by butyric acid.PART Ⅲ Role and mechanism of LPS mediated NLRP3 inflammasome in acute myeloid leukemiaBackground Acute myeloid leukemia(AML)is a hematological malignant tumor caused by the malignant proliferation and accumulation of myeloid stem and progenitor cells in bone marrow and other tissues.The cure rate for adult acute myeloid leukemia is only 20 per cent and 40 per cent.In the past decade,although some progress has been made in improving prognosis and developing new treatments for specific subtypes,understanding the pathogenesis of the disease is still the key to changing the cure rate of AML.According to the previous part of the study,we found that the introduction of LPS,a metabolite of gut microbiota,aggravated the progress of AML in mice,but its specific mechanism has not been deeply studied.As the main bacterial component of Gramnegative bacteria,LPS can cause inflammation.According to several reports,lipopolysaccharide(LPS)can activate NLRP3 inflammasome in human cells through TLR4 pathway,which has a great effect on the body.However,in the progress of AML,whether LPS can affect the progress of the disease by activating NLRP3 is worthy of our further study.Inflammatory corpuscles are recognized as an important role in innate immunity.Among them,NLRP3 inflammasome is the most thoroughly studied.NLRP3 inflammasome complex belongs to the family of nucleotide binding and oligomerization domain-like receptors(nucleotide-binding oligomerization domainlike receptors,NLRs),also known as "protein 3 containing pyrin domain" and It mainly includes NLRP3,ASC and caspase-1.NLRP3 inflammasome are closely related to many diseases,including multiple sclerosis,inflammatory bowel disease and other autoimmune and autoinflammatory diseases.Although the role of NLRP3 inflammasome in tumorigenesis has been controversial,more and more evidence emphasize that NLRP3 inflammasome promote chronic inflammatory response,thus promoting the occurrence,development and progression of tumors.NLRP3 inflammasome-effector cytokines IL-1β or IL-18 have also been shown to be involved in cell proliferation and differentiation,and are considered to play a key role in tumorigenesis.NLRP3 has been widely involved in the occurrence and development of various blood diseases.However,how LPS promotes the occurrence and development of AML through NLRP3 inflammasome is still unknown.Therefore,we explored the gene expression of NLRP3 inflammasome in primary mouse bone marrow cells stimulated by LPS in order to clarify its role in the occurrence and development of AML.Objectives 1.To clarify the route of intestinal LPS into the blood and its concentration in the blood;2.To clarify the difference in the expression of NLRP3 in mouse leukemia cells stimulated by LPS;3.To explore the effect of NLRP3 on AML progression in vivo;4.To clarify the effect and mechanism of IL-1β,a downstream effector molecule of NLRP3,on the proliferation and apoptosis of leukemia cells.Methods 1.Determination of LPS concentration in AML patients and model mice:Enzymelinked immunosorbent assay(ELISA)was used to detect the LPS concentration in peripheral blood of AML patients and mice;LPS in the butyric acid-treated group and the antibiotic-destroyed bacteria group was also detected in the AML mice.content.2.The effect of LPS stimulation on the expression of NLRP3 inflammasome-related genes in AML cells was detected by PCR.3.The NLRP3 lentiviral vector was used to transfect the mouse leukemia cell line C1498.After successful transfection,PCR and Western blot were used to detect the expression of the target gene.After successful transfection,normal C57BL/6J mice were injected into the tail vein to observe the incidence of mice.4.Extract the bone marrow of NLRP3 knockout mice,magnetically sort C-kit+ HSC cells,transfect them with MLL-AF9 lentivirus,and inject normal C57BL/6J mice into the tail vein to establish the NLRP3 knockout AML mouse model.After the establishment of the model,the mice were sacrificed and their bone marrow was taken as NLRP3 knockout mouse leukemia cells.C57BL/6J was injected into the tail vein to establish NLRP3 knockout AML mice,and the difference in the onset and progression between them and ordinary AML mice was observed.5.The expression of IL-1β in leukemia cell lines was interfered with small interference in vitro,the proliferation of cells was detected by CCK8,and the apoptosis and sensitivity to chemotherapeutic drugs(ADR,DNR)were detected by flow cytometry.Western blot was used to detect the expression of apoptosis proteins such as BCL-2 and caspase-3.Result 1.AML mice have obvious impaired intestinal barrier and function,and the impaired intestinal barrier function increases the amount of LPS released into the blood,and the elevated LPS accelerates the progression of AML in mice.2.LPS up-regulated the expression of NLRP3 in mouse leukemia cells,and all its downstream genes CASPASE-1 and IL-1β were also up-regulated.3.Up-regulation of NLRP3 in mouse leukemia cells by lentivirus accelerates the progression of AML in mice,manifesting as hepatosplenomegaly,severe bone marrow infiltration,and significantly shortened survival.4.In AML mice with NLRP3 knockout in leukemia cells,the spleen and bone marrow infiltration were significantly reduced,the spleen enlargement was relieved,the number of bone marrow GFP+leukemia cells was significantly reduced,and the survival period was prolonged.5.We used siRNA to down-regulate the expression of IL-1β in THP-1 cell line,inhibited the proliferation of THP-1 cells,promoted their apoptosis,and promoted the sensitivity to cytarabine and daunorubicin.Conclusion 1.Damage to the intestinal barrier can accelerate the entry of enterogenic LPS into the blood and accelerate the progression of AML.2.Under the stimulation of exogenous LPS,the expression of NLRP3 gene in mouse leukemia cells was significantly increased.3.Up-regulation of NLRP3 by lentivirus can promote the progression of AML in mice,and knockout of NLRP3 in mouse leukemia cells can delay disease progression.4.After down-regulation of IL-1β,the downstream effector molecule of NLRP3 in leukemia cell line THP-1,the proliferation rate was significantly decreased,the apoptosis was increased,and the drug sensitivity to Ara-c and DNR was also significantly increased.It is proved that IL-1β can promote the proliferation of leukemia cells,inhibit their apoptosis,and reduce their sensitivity to chemotherapeutic drugs.