Mechanism Of Proinflammatory Factor Interleukin 6 Signaling Regulates Embryonic Hematopoietic Stem Cell Production And Development

Hematopoietic stem cells(Hematopoietic stem cells,HSCs)are self-renewing multipotent stem cells capable of replenishing all mature blood lineages and maintaining hematopoietic ability during the lifetime of an individual.HSCs emergence and development is a stepwise regulation process by signalling network composed of various microenvironment,transcription factors,cytokines,epigenetic factors and so on.Imbalance or abnormalities in any node of this signalling network may lead to hematopoietic diseases and even evolve to bone marrow failure or leukemia.The more complete mechanisms that regulate HSCs emergence is,the more beneficial it will be to the in vitro use of pluripotent progenitor cells to be induced anddifferentiatedinto HSCs which are required for hematopoietic stem cell transplantation(HSCT).But until now,this goal has not been achieved,partily because of our poor and incomplete understanding of the inherent regulatory network molecules and their mechanisms in the regulation of HSCs emergence.The role of proinflammatory factor in the normal embryonic hematopoiesis has become a key point of HSC biological research in recent three years,among which TNFa?IFNr and TLR4 all have been revealed that they play an essential role in the regulation of HSCs emergence,maintenance and differentiation in embryonic mouse and zebrafish.Besides,these three proinflammatory signallings are associated with Notchla signaling.Under the infection pressure for adult human and mouse,these factors are involved in the regulation of hematopoietic stem and progenitor cells(HSPCs)proliferation and differentiation in an effort to ultimately resist the infection and repair damage tissues.IL6 plays an important role in numerous aspects such as the mobilization of adult HSPCs,the maturation of T,B lymphocytes and megakaryocyte,and the proliferation of tumor plasma cells as well.However,the role of this signaling in the development,proliferation and differentiation of embryonic HSCs and its possible mechanisms are still less clear and absent of comprehensive experimental evidences.Due to the similarity and conservation of zebrafish with human and mouse for embryonic hematopoiesis in terms of hematopoietic microenvironment and important signaling pathways,zebrafish has become a canonical model in the study of hematopoietic development.In this study,we used zebrafish as a model to explore whether or not IL6 and IL6R affect the emergence,proliferation and differentiation of HSCs;whether or not they affect the development of primitive erythropoiesis and primitive myeloid cells;whether or not IL6 signaling regulates the development of HSCs in a direct or a indirect way;whether the IL6 signaling is related to those classical HSCs regulation signals such as Notchl,NF-kB and Stat3;All the problems will be systematically explored in our study.This paper consists of two parts:1)IL6 and IL6R signaling regulates the emergence,proliferation and differentiation of embryonic HSCs;2)the mechanism of IL6 and IL6R signaling in the regulatation of emergence and development of HSCs.Chapter I:IL6 and IL6R signaling regulates the production,proliferation and differentiation of HSCs.Objective:to explore the role of IL6 and IL6R signaling in the regulation of the emergence and proliferation of embryonic HSCs and their differentiation to lymphoid and myeloid cells.Methods:Firstly,fregments of hematopoietic specific genes were constructed into the pEASY-T3 plasmid,and then utilized ApaI or SalI restriction endonuclease to linearize the circuiar plasmid,purified as a template in vitro transcription synthesis of the corresponding antisense probes.The expression of normal transcripts of target gene IL6 and IL6R was knocked down by injecting the targeted morpholino(MO),and the negative control group was offered by Genetools company as reference standard Standard MO(Std MO).We utilized cd41:eGFP transgenic embryos to explore the optimal concentration of IL6 and IL6R MOs,the optimal dose of Std MO derived from references.We performed the experiments including whole embryo in situ hybridization(WISH),the common fluorescence microscope and the confocal array scanning arrays to observe the changes in the number of runx1,cmyb positive cells,or cmyb and kdrl double positive cells in the floor of dornal aorta(DA)compared with the control group;and the number of cd41 positive cells in the tailor hematopoietic tissue(CHT);and the number of rag1 and lck positive cells in the thymus;and the number of lyz positive cells in the trunk and CHT regions at the different time points of postfertilization(hpf).In addition,we utilized the published sequencing data to analyze the expression levels of IL6,IL6R and GP130 from hematopoietic endothelial cells(HEs)to adult HSCs at the different developmental time points.Results:1)In this study,we successfully constructed the target gene vector and synthesized their antisense probes(including runx1,cmyb,gata1a,pu.1,mpx,1-plastin,ragl,rag2,lmo2,scl,..kdrl,efnb2a,dlc,foxnl;2)we defined that the optimal working concentration as follows:0.6 mM for IL6 MO,0.4 mM for IL6R MO,0.2 mM for Std MO,1 nl for microinjection per embryos,and the time range of injection was at 1-2 cell stage;3)Compared with the Std MO group at 36hpf,the cmyb positive cells in or near the ventral wall of the DA were significantly decreased in IL6 and IL6R MO groups,and the number of embryos with this phenotype in the two groups was more than 75%.At the same time,the number of HSCs(cmyb+kdrl+)cells in the ventral wall of the DA area was reduced by about 50%(P<0.001)compared with the control group.The WISH results showed that the numbers of runxl positive cells in IL6 and IL6R MO groups were significantly decreased in the ventral wall of DA at 24hpf and 28hpf,and the number of embryos with this phenotype increased by more than 70%.The number of cd41:eGFP positive cells in CHT region at 72hpf was significantly decreased,and the number of embryos with this phenotype increased significantly by about 50%.4)The WISH results embryos at 96hpf showed that the thymus ragl positive cells in the IL6 and IL6R MO groups were almost absent and the number of positive embryos was more than 95%.At the same time point,the number of lck:GFP cells in the experimental groups was significantly decreased than that in the control group,the number of positive phenotypic embryos was more than 80%.Transgenic embryos at 72hpf showed that the number of lyz:dsRed positive cells in the trunk and CHT regions in IL6 and IL6R MO groups was significantly decreased(P<0.0001).5)Single-cell RNA sequencing results showed that the expression levels of IL6R and its co-receptor GP130 were increased from HEs to HSCs,and reached the highest levels in adult HSCs,while IL6 expression was too low to detect.Conclusions:1)Once the signaling of IL6 and IL6R was blocked,and the number of cmyb positive cells or HSCs at 36 and 48hpf was significantly decreased,suggesting that the signaling was necessary for the maintenance of HSCs in DA area.HSCs were transferred by HEs(endothelial-to-hematopoietic transition,EHT).WISH was used to confirm that the early process(24 to 28 hpf)in the DA region of the early HSCs through blocking the IL6 and IL6R signaling.This indicates that the signaling is important for regulation of the EHT process.The CHT region(similar to fetal liver of mouse)is an important microenvironment of HSCs for abtaining proliferation and self-renewal abilities.Blocking IL6 and IL6R signaling,the proliferation of HSCs in this region significantly decreased;2)Blocking the signal,thymus ragl,lck positive cells at 96hpf significantly reduced,trunk lyz positive cells at 72hpf were significantly reduced,suggesting that HSCs differenting into lymphoid and myeloid cell also relys on IL6 and IL6R signaling;3)Both HEs and HSCs express membrane IL6R/GP130 co-receptors,implying that the formation and development of HSCs are directly regulated by this signaling,but the expression of 116 is very low,suggesting that it may play a role in a paracrine fashion.Chapter II:The mechanism of IL6 and IL6R signaling in the regulation of HSCs production and development.Objective:To explore the possible source of IL6,and reveal the relationship with the canonical HSCs regulation signalings such as Notchl signaling;whether the IL6 and IL6R signaling indirectly affects HSCs by regulating vascular development;whether HSCs apoptosis would be induced once the signaling is blocked.Methods:zebrafish-derived il6r gene coding sequence(CDS)was constructed into the PCS2 +plasmid by the homologous recombination method.We used the Not I restriction endonuclease to linearize the plasmid,which was used as template for synthesizing I16r mRNA in vitro.Notch la targeted inhibitor DAPT was added to the zebrafish embryo incubator holt buffer from 14 to 48 hpf,the final concentration is 100?M according to literature report;IL6R mRNA was injected into the embryonic yolk sac at a single cell stage with the concentration gradient of 50pg,100pg,150pg,200pg and 250pg per embryos,respectively,a total of five groups to investigate its optimal concentration:Using a labeled ata1a,mpx,l-plastin probe and mpeg1:eGFP transgenic embryos to figure out whether the development of primitive erythrocyte,primitive neutrophils and macrophages would be affected when Il6-IL6R signaling was blocked;we calculated the number of double positive cells in the DA region of tpl:eGFP(Notch1a marker)and kdrl:mCherry(arteriovenous maker)double transgenic embryos after block the signaling;we assessed the vascular development using kdrl,efnb2a,dlc probes;usingthe TUNEL immunofluorescence kit to detect the apoptosis of vascular endothelial cells and HSCs,we sorted mpx and mpegl positive cells using flow cytometry,and detected the expression levels of il6,il6r and gp130 using the one-step PCR.Results:1)Complete il6r CDS sequence was constructed into the plasmid PCS2 and IL6R mRNA was successfully synthesized;2)Compared with the Std MO control group,the number of primitive erythrocytes in the ICM(intermediate cell mass)of the 24 hpf embryo were not significantly changed in the IL6 and IL6R MO groups,while the primitive neutrophils and the primitive myeloid cells were significantly reduced in the trunk and PBI(posterior blood island)of 28 hpf embryos;However,the distribution and number of mpegl:eGFP cells of 32-36 hpf embryos did not change significantly(P>0.05);3)Through blocking the IL6 and IL6R signaling,we observed that the tpl + kdrl + double positive cells in the ventral floor of the dorsal aorta(DA)of 32-36hpf embryos had no significant change(P>0.05),but when DAPT blocked the Notchla signaling,the number of cmyb positive cells was significantly reduced in the DA of 48hpf embryos,the positive phenotypic embryos accounts for 75%of total embryos,under the condition,we injected IL6R mRNA 200pg into embryos simultaneously,the cmyb cell deficient phenotype could be rescued,the efficiency was up to 30%;Similarly,DAPT can inhibit the proliferation of cd41:Egfp positive cells in CHT of 72hpf embryos,and this phenotype can be partially reversed in the presence of 200pg IL6R mRNA injection,the rescued efficiency reached up to 30%;4)There was no significant difference between the Standard MO group and I16/IL6R MO groups in terms of the vascular markers as kdrl,efnb2a and dlc at 28hpf,as did about the number of TUNEL and flila double positive cells in the DA of 30hpf embryos;5)Both primitive neutrophils and macrophages had significantly higher expression of il6 and il6r compared with the average expression level of whole embryos,but the gp130 expression levels were not different among groups for the macrophage.Conclusions:1)I16/IL6R signaling regulates the development of primitive neutrophils,but has no significant effect on the development of primitive macrophages,so it is suggested that neutrophils may promote autogenesis by auto secreting 116 and may also promote the production and development of HSCs by acting on local microenvironment in a paracrine fashion;2)IL6R acts downstream of Notchla and both of them regulate the production of HSCs;3)Vascular development is not affected,suggesting that IL6R receptor signaling regulates HSCs independent of vascular development;4)Blocking 116 and IL6R signaling did not cause apoptosis of vascular endothelial cells and HSCs,suggesting the regulation of HSCs is indeed achieved through the EHT process;5)Both primitive neutrophils and macrophages can secrete IL6 to and may promote the emergence and development of HSCs.