Efficacy Of Camptothecins And IFN-? Blocking In Treating Hemophagocytic Lymphohistiocytosis

Part one Camptothecins effectivelytreat hemophagocytic lymphohistiocytosis in a murine modelObjective We aim to develop new therapies for HLH with superior efficacy and safety compared to etoposide and thus improve long term outcome of HLH.Methods1.In vitro drug screening:Activated and resting T cells of mouse spleen or human peripheral blood were treated with different dilutions of chemotherapeutics including etoposide,topotecan,cyclophosphamide,mitoxantrone,cis-platinum,methotrexatein and 5-fluorouracil in vitro,and drug-induced apotosis was tested using flow cytometry in order to screen potentially effective agents.2.In vivo experiments:(1)Murine model:A classic murine model of HLH was established by infecting perforin-deficient(Prf^-/-)mice with lymphocytic choriomeningitis virus(LCMV)intraperitoneally and wide type mice were infected as a control group.(2)Five groups:Negtive control group was defined as wide type mice infected with LCMV and treated with drug carrieronly,model control group was defined as Prf^-/-mice infected with LCMV and treated with drug carrier only,while three test groups were Prf^-/-mice infected with LCMV and subsequently treated withetoposide,topotecan or irinotecan.(3)Serum IFN-?level,disease-related biomarkers such as serum soluble CD25 and ferritin levels,and alanine aminotransferase(ALT)were detected by enzyme linked immunosorbent assay(ELISA).Complete blood cell counts,as well as spleen histology in hematoxylin and eosin(HE)stainwere examined.LCMV-specific activated T cells were quantitified using tetramer staining by flow cytometry.Overall survival by 100 days and clinical disease severity scores were assessed.3.Statistics analysis was performed by using SPSS 16.0 software.One-way ANOVA or non-parametric tests were used to compare continuous variables among groups and two-tailed P<0.05 was considered significantly different.Results1.In vitro,apoptosis rate of activated mouse or human CD8⁺T lymphocyte in 1?Metoposide was about 50%,while apoptosis rate of na?ve or resting CD8⁺T lymphocyte in 1?Metoposidewas about 6%.In contrast,apoptosis rate of activated CD8⁺T lymphocyte in0.03?Mtopotecan was over 50%,while apoptosis rate of resting CD8⁺T lymphocyte in0.03?Mtopotecan wasabout 7%;apoptosis rate of activated CD8⁺T lymphocyte in0.1?Mtopotecan was over 60%,while apoptosis rate of resting CD8⁺T lymphocyte in0.1?Mtopotecan wasonly 8%.These results showed that topotecan,a representative camptothecin,exibited similar efficacy and selectivity as etoposide for killing activated mouse or human T lymphocyte in vitro.2.In vivo experiments:(1)Results showed that topotecan,irinotecan and etoposide all effectively suppressed IFN-?production,largely reduced serum soluble CD25 and ferritin levels,selectively deleted LCMV-specific T cells in mouse spleens,and rescued mice from develping acute anemia(all P<0.05).(2)ALT level of topotecan treated mice on day 12 was significantly lower than that of irinotecan treated mice(P=0.003),but there was no significant difference between topotecan and etoposide treated mice in terms of ALT levels(P=0.70).Ferritin level of topotecan treated mice on day 12 was significantly lower than that of irinotecan treated mice(P=0.01),but there was no significant difference between topotecan and etoposide treated mice in terms of ferritin levels(P=0.99).Topotecan treated mice had better neutrophils recovery upon day 15 after infection than etoposide treated mice(P=0.046).Morover,topotecan treated mice had better histologic structure of spleen with less inflammatory infiltration on day15 than other agents treated mice.(3)There was no significant difference among etoposide,topotecan and irinotecan treated groupsin terms of100-day overall survival(75%,90%and 90%,respectively;P=0.37).However,topotecan treated mice had significantly lower peak of clinical disease severity score than etoposide treated mice(P=0.02),while there was no significant difference between irinotecan and etoposide treated mice in terms of peaks of clinical disease severity score(P=0.61).Conclusion1.Topotecan,a representative camptothecin,can effectively and selectivelyinduce apoptosis of activated T lymphocyteas etoposidein vitroand in vivo.2.Topotecan has superior efficacy than etoposide in treating murine HLH and irinotecan has similar efficacy with etoposide in treating murine HLH.3.Camptothecinsexhibit obvious efficacy of treating murine HLH,and their use is not associated with the risk of secondary malignancies,which is a concerning long-term adverse effect of etoposide.Thus,camptothecins,especially topotecan are promising candidates for treating HLH in clinical use.Part two The effect of IFN-? receptor and macrophages deactivation on murine hemophagocytic lymphohistiocytosisObjective The objective of the present study is to explore potential targets in the IFN-? signalling pathway for treating HLH,including IFN-? receptor and macrophages.Methods Mice were divided into four groups with 6-8 mice in each group according to the genotype of mice as follows: wild type(WT)mice as negtive control group,perforin gene knock out(Prf-/-)mice as positive control group,perforin gene and IFN-? receptor gene double knock out(Ifngr.Prf DKO)mice as one test group,and macrophages insensitive to IFN-? transgenic mice with perforin gene knock out(MIIG.Prf-/-)as another test group.All these mice were infected with LCMV and without any subsequent treatment.Then these mice were scored for HLH associated disease severity 2-3 times per week and the survival was also recorded.Complete blood cell counts of all mice were carried out at day 15 post LCMV infection.Statistics analysis was performed by using SPSS 16.0 software.One-way ANOVA or non-parametric testswere used to compare continuous variables and two-tailed P<0.05 was considered significantly different.Results The results of complete blood cells counts showed that hemoglobin level(P=0.001)and platelets count(P<0.001)were significantly increased in Ifngr.Prf DKO mice as compared to Prf-/-mice,though the increase of absolute neutrophils was not yet signifcant(P=0.07).Hemoglobin level(P=0.008),Platelets(P<0.001)and neutrophils(P<0.001)were all significantly increased in MIIG.Prf-/-mice as compared to Prf-/-mice.All Prf-/-mice developed typical HLH-like disease after LCMV infection and deceased within 12-40 days.Ifngr.Prf DKO mice developed very mild coure of HLH and the 100-day survial rate was 87.5%,which was the same as the survival rate of WT mice.However,MIIG.Prf-/-mice developed severe course of HLH without the trend to recover,and the 100-day survival rate was only 16.7%.Conclusion The deletion of IFN-? receptor is able to treat HLH in a murine model.The abrogation of macrophage function induced by IFN-? prevents the development of pancytopenia,but cannot rescue the mice from adverse outcome.