Effects Of Hypoxia-inducible Factor-1 On The Function Of Tissue-resident Macrophages And Its Mechanism Of Action In Acute Graft-versus-host Diseas

Objectives:Acute graft-versus-host disease(aGVHD)is one of the common complications after hematopoietic stem cell transplantation,which is the multi-system immune damage caused by the the attack of donor lymphocytes on recipient organs.aGVHD seriously affects the survival time and quality of life of patients after transplantation.Macrophages are a classic type of innate immune cells that can be divided into two categories:infiltrating macrophages and tissue resident macrophages.Current research has shown that infiltrating macrophages can cause the proliferation of donor derived alloreactive T cells.Although inhibiting the proliferation of T cells by infiltrating macrophages can reduce the severity of aGVHD,this undoubtedly increases the risk of recurrence in patients after transplantation.However,the tissue resident macrophages are a group of cells that have distinct origin and functions from infiltrating macrophages.If the proliferation of T cells can be suppressed by interfering with the function of these cells,the severity of aGVHD can be reduced without affecting the graft versus leukemia(GVL)effect.In addition,research has found that hypoxia inducible factor 1-alpha(Hif1-α)regulates many important physiological functions such as macrophage polarization and glycolysis.Based on the above research background,the main objectives are:(1)To explore whether Hifl-α has impact on the function of tissue resident macrophages(2)To analyze the effect of Hifl-α on the function of tissue resident macrophages and the mechanisms in aGVHD by single-cell transcriptome sequencing.Methods:(1)We develop the macrophage-specific Hifl-α-deficient conditional knockout mice(LysMCre/Cre Hif1-αF/F,designated Mye-KO)and LysMCre/Cre(designated Mye-WT)as control.(2)Kupffer cells of CD11bintF4/80high were obtained through flow cytometry sorting,and then the functional experiment were conducted.(3)Using Mye-KO and MyeWT as recipients and BALB/c as donors,the allogeneic animal model of aGVHD was established to observe the survival differences between Mye-KO and Mye-WT,and to monitor the changes in body weight,blood routine,histopathology,and aGVHD scoring.(4)CD45+immune cells in liver were isolated by flow cytometry at the most significant time points in the aGVHD model.Besides,single-cell transcriptome sequencing was performed to analyze the transcriptome characteristics of kupffer cells and T cells in the liver during the onset of aGVHD.thereby exploring new underlying mechanism.Results:(1)Hif1-αis an essential transcription factor to maintain the phagocytic ability of kupffer cells,and the knockout Hifl-α decreased significantly the phagocytosis of kupffer cells.(2)The tissue resident macrophages of recipients are involved in the pathogenesis of aGVHD.Compared with Mye-WT,Mye-KO showed higher aGVHD scores,shorter survival,and more severe liver damage.(3)Single-cell transcriptome sequencing showed that apoptosis and lysosomal related signaling pathways in kupffer cells of Mye-KO were significantly upregulated;When aGVHD occurs,the number of effector T cells in MyeKO significantly increases,while the number of regulatory T cells(Treg)significantly decreases.The differentially upregulated expression genes of effector T cells in Mye-KO are enriched in signal pathways such as mitochondria synthesis and oxidative phosphorylation,indicating that the effector T cells of Mye-KO continue to proliferate and activate during aGVHD.(4)Kupffer cells in Mye-KO have the decreased phagocytosis in the key process of damage associated molecular patterns(DAMP)released by damaged cells in aGVHD modeling,while Dnase 1(DNS1)can clear DAMP,reduce the severity of aGVHD,and prolong survival.(3)Single cell transcriptome sequencing showed that when aGVHD occurred,the number of CD8+effector T cells in Mye KO mice significantly increased,while the number of regulatory T cells(Treg)significantly decreased.The interaction between kupffer cells and CD8+effector T cells in Mye-KO mice is strong.In addition,the differentially upregulated genes of effector T cells in Mye KO mice were significantly enriched in ATP synthesis,oxidative phosphorylation and other signaling pathways,indicating that CD8+effector T cells in Mye KO mice were constantly proliferating and activating when aGVHD occurred.(4)The phagocytosis of kupffer cells in Mye-KO mice to the damage related molecular patterns(DAMPs)released by damaged cells during aGVHD decreased significantly,leading to T cell proliferation and activation.For the first time,we found that deoxyribonuclease 1(DNS1)can clear DAMP,reduce the severity of aGVHD,and prolong survival.Conclusion:We innovatively developed macrophage-specific knockout Hifl-α mice for the study of the pathogenesis of aGVHD,and found that the knock out Hifl-α on kupffer cells caused the decreased phagocytosis and the accumulation of extracellular DAMP,which promoted the proliferation and activation of effector T cells,and led to the severe damage of target organs such as the liver.DNS1 can help kupffer cells clear DAMP and reduce cellular immune response,thereby reducing the severity of aGVHD and prolonging the survival of transplanted mice.Therefore,DNS1 is expected to become a new target in the treatment of aGVHD.Purpose:To evaluate the efficacy and safety of allogeneic hematopoietic stem cell transplantation(allo HSCT)in the treatment of adult chronic myelomonocytic leukemia(aCMML).Methods:We retrospectively analyzed 43 aCMML patients who underwent myeloablative conditioning regimen prior to allo-HSCT between January 2009 to December 2021.The 3-year overall survival(OS)rate,disease-free survival(DFS)rate,cumulative incidence of relapse(CIR),non-relapse mortality(NRM)rate and the relevant factors affecting efficacy were analyzed.Results:Among 43 aCMML patients,there were 26 males and 17 females,with a median age of 47(20-59)years.5 cases were CMML-0,12 cases were CMML-1,16 cases were CMML-2,and 10 cases were transformed into acute myeloid leukemia(AML).Sixteen patients had palpable splenomegaly at first diagnosis,17 with grade 2 or higher bone marrow fibrosis,and 20 patients were detected with leukemia related gene mutations.According to the CPSS mol score,1 case is at low risk,10 cases are at immediate-1,23 cases are at immediate-2,and 9 cases are at high risk.There were 27 patients receiving demethylated agents(HMAs)before allo-HSCT,9 patients receiving other chemotherapy without HMAs,and 7 patients who only received supportive treatment such as blood transfusion.Among the 7 patients who received targeted supportive treatment,3 were CMML-0,3 were CMML-1,and 1 was CMML-2.31 patients achieved complete remission(CR)before conditioning,while 12 patients obtained partial or no remission.One patient received matched unrelated donor transplant,1 7 received HLA-haploidentical transplant.and the remaining 25 patients received an HLA-matched sibling transplant.Twenty-nine patients underwent decitabine-based conditioning regimens,while the remaining 14 patients received the myeloablative regimen of Bu/Cy/Flu/Ara-c.After a median followup of 37.5(3-161)months,30 patients survived,13 patients died,and 13 patients experienced relapse after allo-HSCT.Among them,6 patients died of relapse and 7 patients survived after relapse.The overall 3-year OS and DFS were 69.8%(95%CI 79.1%129.6%)and 53.5%(95%CI 53.8%-104.2%),respectively.The 3-year OS and DFS in patients who achieved CR before allo-HSCT were significantly higher than those who did not(3-year OS:80.6%vs.41.7%,P=0.008;3-year DFS:64.5%vs.25.0%,P=0.007).In COX multivariate analysis,the status of complete remission CR before allo-HSCT was the only significant predictor of survival.In addition,we conducted a subgroup analysis of different treatment strategies before transplantation,and the results showed that the 3-year OS and DFS of seven patients receiving support treatment were significantly higher than those who achieved CR after chemotherapy(n=26)and non-CR after chemotherapy(n=10)(3-year OS:85.7%vs.76.9%vs.40.0%,P=0.0153;3-year DFS:71.4%vs.61.5%vs.20.0%,P=0.007).Notably,among 23 patients with HMAs followed by dec-containing conditioning,the 3-year OS between first complete remission(CR1),second complete remission(CR2)or partial remission(PR),and non-remission(NR)were 91.7%,80.0%,and 50.0%(P=0.072).While the 3-year DFS between CR1,CR2 or PR,and NR were 83.3%,60.0%and 16.7%(P=0.006).Conclusion:In conclusion,allo-HSCT remained the curative treatment strategy for patients with CMML,and the achievement of CR prior allo-HSCT was the only independent predictor of prognosis.Patients at early disease stage showed significant therapeutic effects,so once diagnosed,CMML patients should undergo allo HSCT as soon as possible.Besides,patients receiving CR1 during HMAs therapy before allo-HSCT were more suitable to receive decitabine-based conditioning.