The Mechanism Study Of Severe Infection Escherichia Coli Causes The Emergency Myelopoiesis In Zebrafish Larval

Inflammation is a very common and important basic pathologic process.Under the stress conditions,such as infection,the body needs to quickly generate a large number of immune cells in response to this challenge,which called emergency hematopoiesis.However,whether and how the immune cell perform the reaction are still unknown.The response of HSPC,which serve as to produce all the blood components required lifelong,under demand-derived emergency conditions are not yet well addressed.When vertebrates are systemically infected by pathogens,particularly bacteria,a bacteremia-likesyndromeusuallyensues.Inthisscenario,theblood components—leukocytes in particular—respond quickly and a large number of cytokines play an important role,such as Irf8 and Pu.1.The myeloid leukocytes,includingmononuclear(macrophages)andpolymorphonuclearphagocytes(granulocytes),usually serve as the first line of defense against the foreigner invaders,protecting the health of an organism via their non-specific phagocytotic capability.Granulocytes,which account for a large portion of the peripheral blood members,usually play the major role during this process via various routes to bactericide.The tremendous involvement of granulocytes results in their immediate exhaustion and subsequent significant expansion,which called emergency granulopoiesis and it is an indispensible immune reaction to eliminate bacteria.Macrophages are another key player in clearing both the pathogens and dead cells throughout the process,taking advantage of their powerful phagocytotic and digestive ability.Thus,macrophages play indispensible roles in resolving the infection-induced inflammation.Zebrafish(Danio rerio)is a popular aquarium fish native in SE of Himalayan region.In the past decades,zebrafish is becoming more and more popular as a model animal in scientific research,due to its large reproducing rate,Easy to raise,optical transparence and small size.In our study,we established an emergency myelopoiesis model by injecting the E.coli into the blood circulation of 2dpf zebrafish embryos.After infection the macrophages and neutrophils all responded quickly to act as phagocytes against the microbes.Macrophages were endowed with enormous phagocytotic ability,whereas neutrophils behaved differently for different infective methods and microbes.As we all know,the blood cells,a family which are made up of Erythroid Cells(including erythrocytes and platelets),Myeloid Cells(including macrophages and neutrophils)and lymphoid cells(including T and B lymphocytes),originally derived from a common progenitor cells that are derived from mesoderm hematopoietic stem cell(HSC).HSCs are a special group of quiescent cells with the self-renewal capability to maintain homeostasis,which in turn serve to produce all the blood components required lifelong.We know that the amplification of myeloid cells can cause the activation of its progenitor cells,but whether HSC will be activated in immune response are not yet well addressed.Based on lineage-specific markers,many studies in the literature have indicated that HSPCs undergo intensive expansion when challenged by bacteria or related particles,including LPS.However,other publications have argued that HSPCs did not perform this reaction or even undergo reduction.It appears that different reactions of HSPCs have been caused by different pathogens.High-throughput sequencing technologies is also called"next generation"sequencing technology,it makes a species of the transcriptome and a detailed analysis of the whole genome come ture,so its also known as deep sequencing.In order to further explore the change of RNA after infection,we choose the deep sequencing.The results suggesting that the immune response was initially repressed then the immune reaction was activated until finish the clearance of bacteria.This result was consistent with the transient depletion of both myeloid phagocytes during the early period,followed by obvious expansion.According to the transcriptome data we selected pu.1as the key object of study,which is important for the blood cells development.In the development of primitive hematopoietic,the level of Pu.1 is important for the differentiation of macrophage or granulocyte.Further research shows that the pu.1has a dose dependent relationship in the regulation of lymphoid cells and myeloid cell differentiation process.When Pu.1 high expression can promote myeloid,especially macrophages,to generation,while lower expression to promote development of B lymphocyte.In the pu.1 mutant,after infection,the myeloid phagocytes and its progenitor cells would still be a lot of amplification.Compared with the wild type,the increase amplitudes of macrophage is higher,but,its weaker about granulocyte and myeloid progenitor cell.However,when pu.1~G242D/G242D242D/G242D embryos were challenged,the cmyb~+HSPCs showed a notable reduction when compared with the PBS-treated embryos.Overall,Pu.1 was dispensable for the required expansion of myeloid phagocytes,particularly macrophages,but it played essential roles for their functional maturation and maintained HSPCs homeostasis after the E.coli challenge.In this study,we established a systemic infection model by injecting the non-pathogenic bacteria,E.coli into the blood circulation of zebrafish embryos.The cellular and molecular reactions of various blood components,particularly the HSPCs,in the transient and opened hematopoietic organ-CHT-were carefully investigated in this case.The results indicated that the myeloid phagocytes responded immediately to engulf and clear the bacteria,leading to their obvious exhaustion as of the first 6 hpi.However,a remarkable expansion of both macrophages and neutrophils was quickly revealed from 1 day post injection(dpi)on,reaching its maximal level at 2-4 dpi.Thereafter,both myeloid phagocytes gradually returned to their physiological level.Meanwhile,the immune response-related genes presented initially repressed expression followed by intensive up-regulation.Consistently with the fluctuation of myeloid phagocytes,their progenitors underwent a similar but earlier expansion and recovery process;however,no initial exhaustion of myeloid progenitors appeared.Surprisingly,the HSPC reaction was dependent on the bacterial burden.A lower dose of bacteria led to a slight expansion of HSPCs,but an excessive volume caused obvious exhaustion of HSPCs.Molecularly,a cluster of hematopoiesis-related factors showed noticeable transcriptional alteration after inoculation.Pu.1 was one such factor,important in the maintenance of HSPCs homeostasis,effective response of myeloid phagocytes and tight control of the pro-inflammatory cytokines in our study.