Study On The Mechanism Of Action Of IL-34 And Leukemia-associated Macrophages Trained Immunity In Leukemi

Objective:Acute myeloid leukemia(AML)is the most common type of acute leukemia in adults.Although the clinical treatment has been greatly improved,most AML patients will eventually relapse and die.As a highly heterogeneous disease,endogenous factors and microenvironment abnormalities are involved in the transformation,progression and recurrence of AML.There are many adverse phenotypes in the development of AML,including excessive proliferation,more leukemia stem cells(LSC),and extensive extramedullary infiltration,etc.Elucidating the mechanism leading to these phenotypes will be helpful for better treatment in AML.Interleukin 34(IL-34)was first identified as another ligand of colony stimulating factor-1(CSF-1,also known as macrophage colony stimulating factor,M-CSF)receptor(CSF-1R,CD115).IL-34 plays physiologic and pathologic roles mainly through the signal axis of CSF-1/IL-34-CSF-1R complex multi-ligand signal system,which shows functional redundancy,tissue limitation and diversity.This axis is critical for the survival,differentiation and function of monocyte lineage cells,and plays pathologic roles in many diseases.Previous studies have shown that overexpression of IL-34 in AML cells can accelerate the progression of disease,shorten the survival time of AML,and cause subcutaneous infiltration of AML cells.By exploring the effect of overexpression of IL-34 on the characteristics of AML cells,we found that overexpression of IL-34 significantly enhanced the proliferation ability and LSC frequency of AML cells,but had no significant effect on the apoptosis of AML cells.However,the mechanism of the effect of IL-34 on the characteristics of AML cells is not clear.This paper aims to reveal the endogenous and microenvironment mechanisms of IL-34 in AML.Methods:Overexpression of IL-34 and control AML mice are referred to as MA9-IL-34 and MA9.Based on c-Kit expression,MA9 cells were divided into two groups,i.e.c-Kit+and c-Kit-groups,while over 95%of MA9-IL-34 cells were c-Kit+.RNA sequencing was performed on MA9-c-Kit+,MA9-c-Kit-,and MA9-IL-34 cells.Candidate genes were identified through analysis of GSEA,GO,and gene related expression patterns.The shRNA technology was used to knockdown select candidate genes in MA9-IL-34 cells to investigate whether the knockdown could reverse the phenotype of MA9-IL-34 cells caused by overexpression of IL-34.The AML model caused by MA9-IL-34 cells knocked down of Sox 13 was established by tail intravenous injection,and the proportion of peripheral blood AML cells and survival time were detected.Ki-67-staining was used to detect the proliferation ability of leukemia cells,colony formation experiments were used to detect their colony formation ability,and the c-Kit expression was detected by FACS analysis.Next,the role of IL-34 in AML microenvironment was explored.In MA9 and MA9-IL-34 mice,flow cytometry was used to detect the proportion of macrophages in bone marrow,spleen,and liver.Bone marrow macrophages were sorted to test their phagocytic ability and qRT-PCR was used to detect the polarization status of macrophages.Results:The RNA sequencing results of MA9-c-Kit+,MA9-c-Kit-and MA9-IL-34 cells were analyzed.The candidate genes,Fgl2,Gfi1,Ikzf2,Prom1 and Sox13,were obtained through GSEA,GO and gene-related expression pattern analysis.The candidate gene Sox13 was identified through qRT-PCR verification and GSE database query.Then,Sox13 was knocked down in AML cells overexpressing IL-34 for functional verification.Knocking down of Sox 13 significantly delayed the progression of AML,prolonged the survival time of AML mice and reduced the subcutaneous infiltration of AML cells.Knocking down of Sox 13 reduced the proliferation and colony forming ability of AML cells and the expression of c-Kit.These results suggested that knockdown of Sox13 reversed the malignant phenotype of AML caused by overexpression of IL-34.In addition,more leukemia-associated macrophages(LAM)were detected in the bone marrow,spleen and liver of MA9-IL-34 mice.These LAMs showed M2-like phenotype and had reduced phagocytotic potential,which indicated that LAM was also related to the malignant phenotype caused by IL-34.Conclusion:On the one hand,overexpression of IL-34 accelerated the progression of AML by upregulating the expression of Sox 13 to enhance the proliferation of leukemia cells,to increase the number of LSCs and to enhance the subcutaneous infiltration of AML cells.On the other hand,overexpression of IL-34 increased the M2 phenotype of macrophages,which also contributed to AML progression.Objective:Macrophages are important members of the innate immune system and play important roles in both physiologic and pathologic states.The traditional view is that only the adaptive immune system has immune memory,but recent studies have found that the innate immune system also has immune memory.The main cells involved in the innate immune response are innate immune cells including monocytes,macrophages,neutrophils and natural killer cells,etc.The definition of "innate immune memory" or"trained immunity" is that:After being first stimulated,these innate immune cells undergo changes in their epigenetic and cellular metabolism,and can exhibit enhanced or weakened responses when encountered with secondary stimulation.The innate immune memory of macrophages plays important roles in infectious diseases,transplant rejection,atherosclerosis and autoimmune diseases,but its role in tumors remains to be explored.In leukemia,leukemia-associated macrophages(LAM)are highly heterogeneous and simultaneously express Ml and M2 related genes,which play a role in the progression of leukemia.Here we aim to explore whether LAM has immune memory and what role it plays in the progression of leukemia.Methods:Firstly,we investigated whether stimulated macrophages could be detected after adoptive transplantation,which is a prerequisite for trained macrophages to function.The AML cells were transplanted into C57 mice through tail intravenous injection.In the early stages of AML,mouse bone marrow and spleens were extracted,and a single cell suspension was prepared.The cells were enriched with F4/80 magnetic beads and labeled antibodies to sort macrophages,which were transplanted into B6 mice through tail intravenous injection.After 20 days,flow cytometry was used to detect whether the stimulated macrophages could be detected in recipient mice.AML cells were intraperitoneally injected into C57 mice.After 2 days,peritoneal cells were extracted and peritoneal macrophages were sorted before transplanted into B6 mice through tail intravenous injection.After 20 days,flow cytometry was used to detect whether the above stimulated macrophages could be detected in recipient mice.Secondly,two models were used to investigate the role of trained macrophages in the progression of AML.In the late stages of AML-RFP,peritoneal macrophages were sorted and transplanted intoB6 mice through tail intravenous injection.After 7 days,AML-GFP cells were transplanted into B6 mice through tail intravenous injection.The progression and survival of AML mice were observed.AML-RFP cells or irradiated AML-RFP cells were intraperitoneally injected into the normal mice.After 2 days,peritoneal macrophages were extracted,sorted and transplanted into B6 mice through tail intravenous injection.After 7 days,AML-GFP cells were transplanted into B6 mice through tail intravenous injection.The progression and survival of AML mice were observed.Third,RNA sequencing was performed on the peritoneal macrophages trained by AML cells,which were divided into the following groups:no stimulation(WT-PEM),2 days after first stimulation(RA-PEM),7 days after first stimulation(D7-PEM),2 days after stimulation without first stimulation(second-WT-PEM),and 2 days after second stimulation after 7 days post first stimulation(second-AML-PEM),to identify the changed genes and related pathways.Finally,in order to investigate the role of LAM trained immunity in leukemia recurrence,we constructed an AML-TK model After transplantation of AML-TK cells,GCV was injected intraperitoneally at a dose of 100 mg/kg.After continuous injection for 20 days,it was observed whether AML cells could be completely cleared.Results:After 20 days of transplantation of LAM,donor-originated macrophages from the bone marrow and abdominal cavity,but not from the spleen,can be detected in the bone marrow,spleen,and abdominal cavity in the recipient mice.AML-RFP cells or irradiated AML-RFP cells were intraperitoneally injected into the normal mice.Peritoneal macrophages were sorted and transplanted into B6 recipient mice,and AML-GFP cells were transplanted after 7 days.The stimulated macrophages accelerated AML progression.After trained by AML cells,the gene expression profile of macrophages changed.Comparison between WT-PEM and RA-PEM as well as second-WT-PEM and second-AML-PEM,the results showed changes in genes related to T cell activation and phagocytic function.Analyzing the differential genes among WT-PEM,RA-PEM,D7-PEM and second-AML-PEM groups,a gene set was identified.The gene expression pattern was that significant changes in gene expression were detected after first stimulation while the gene expression level returned to normal 7 days after first stimulation.Furthermore,the change amplitude after second stimulation was greater than that after first stimulation.These genes are related to processes such as histone modification and cell cycle regulation.We successfully constructed an AML-TK model,which can completely eliminate transplanted AML cells after continuous intraperitoneal administration of GCV for 20 days.Conclusion:After 20 days of transplantation of LAM,macrophages from the donor mouse bone marrow and abdominal cavity could be detected in the recipient mouse.Trained peritoneal macrophages,7 days after transplantation into recipient mice,had the function to promote AML progression.After trained by AML cells,the gene expression profile of macrophages changed.A gene set with significantly greater change amplitude after second stimulation was identified.These genes may be involved in the formation of macrophage trained immunity stimulated by AML cells.