Study On The Mechanism Of Macrophagederived IL-1β Regulating Emergency Myelopoiesis In Zebrafish

As the major components of the innate immunity,myeloid phagocytes(neutrophils and macrophages)serve as the first line of defense against pathogenic microorganisms.These myeloid phagocytes,especially the neutrophils,are endowed with short life span and need to be constantly generated from their progenitor cells to maintain the homeostasis of the immune system.Upon encountering the foreigner invaders,in particularly the pathogenic microorganisms like bacteria,these cells immediately respond to phagocyte and clear the invaders.Resultantly,they are consumed in large quantities and require constant replenishment,which causes the dramatic proliferation and differentiation of myeloid cells,then produces a large number of myeloid precursors,granulocytes and macrophages,even hematopoietic stem and progenitor cells(HSPCs).This process termed emergency myelopoiesis or demand-adapted myelopoiesis.Sensing the invading pathogens,directly or indirectly,is the crucial first step in the emergency myelopoiesis cascade.While pathogen recognition TLRs are widely expressed in both non-hematopoietic and hematopoietic derived cells,including hematopoietic progenitor cells and mature myeloid cells,so the definition of the pathogen’s primary monitoring cells has been controversial.Macrophages are probably an excellent candidate,according to their wide distribution and equipment with the molecular machineries,including PRRs and cytokines regulating myelopoiesis,which are required for the pathogen recognition and signals translation in the emergency myelopoiesis.However,this assumed roles and mechanisms for macrophages in emergency myelopoiesis have not been directly proven by rigorous in vivo.In addition,inflammatory factors reported to promote the hematopoietic process are unusually numerous.Interleukin-1(IL-1),the first interleukin identified,consists of two related genes,IL-1αand IL-1β.Acute IL-1 signaling is associated with increased myeloid cell production,loss of lymphoid cell generation and chronic anemia,However,the mechanism by which IL-1 contributes to emergency myelopoiesis is largely unknown.Considering the gradation of hematopoietic system,it is not clear which type of hematopoietic cells dose emergency myelopoiesis mainly originate from and what is the molecular mechanism?In addition,although it has previously been shown that interaction between NF-κB and C/EBPβregulated inflammatory cytokines in inflammatory conditions and both them contribute to emergency hematopoiesis,further study is needed to determine whether this interaction between them contributes to emergency myelopoiesis.The zebrafish(Danio rerio)is a vertebrate with conserved hematopoietic function and unique experimental advantages,which is suitable for the study of human emergency myelopoiesis.The optical transparency of zebrafish embryos is an excellent indicator of the details of how inflammation occurs and develops when cells are tracked with the genetically encoded fluorescent reporter and imaging techniques.Therefore,zebrafish is an ideal model of systemic infection.Because of the obscure mechanism of emergency myelopoiesis at present,in this study,we setup a systemic infection in the embryonic zebrafish by circulative administration of LPS(lipopolysaccharides,cell wall component of gram-negative bacteria)and concurrently focus on the demand-adapt myeloid development in the CHT.We find that systemic infection induced by LPS can fortify the myeloid progenitor proliferation,and generate a large number of myeloid cells,including myeloid progenitors,macrophages,especially granulocytes,but no alteration is observed in the erythroid,lymphoid,hematopoietic stem cells and the proliferation and apoptosis of hematopoietic stem cell when compared to the PBS control group.The emergency myelopoiesis induced by LPS was significantly inhibited when we deplete macrophages through the pharmaceutical application of metronidazole(MTZ)or with genetic irf8 mutants and irf8morpholino.To further explore the molecular mechanism of macrophages regulating emergency myelopoiesis,we found that macrophages mainly secreted il1βfrom the TLR4-MyD88 signaling pathway to regulate emergency myelopoiesis.After specificity improving the transcription level of il1βin macrophages through the mpeg1 promoters,whether in the PBS or LPS group can obviously augment the number of myeloid cells.On the contrary,knocking down il1βby using morpholino significantly blocks inflammatory emergency myelopoiesis.In addition,the expression of il1βand emergency myelopoiesis are obviously blocked in myd88 mutants.However,the specific remediation of myd88 expression in macrophages can completely rescue the phenotype of il1βdefect in myd88 mutants.In order to further explore how the myeloid precursor responds to the macrophage-derived il1βsignaling to achieve mass amplification.IL-1βhas been reported to activate NF-κB,and NF-κB signaling is widely involved in inflammatory response and critical for emergency hematopoiesis.We found that NF-κB activity is enhanced after LPS treatment.and when NF-κB is inhibited with inhibitor Jsh-23 or activated by ikbαa morpholino,which could effectively inhibit or promote emergency myelopoiesis respectively.In order to explore how the NF-κB signal promotes the emergency myelopoiesis,because of the attention to the previous in vitro study found the NF-κB and C/EBPβcould form a new transcription factor by protein interactions and C/ebpβplays a critical role in emergency granulopoiesis,and our immunohistochemical staining shows that most of the p65(a subunit of NF-κB)and C/EBPβsignals co-locate in lyz~+myeloid cells,so we hypothesize that C/ebpβmay be involved in the regulation of emergency myelopoiesis.To further explore the relationship between C/EBPβand NF-κB during emergency myelopoiesis,firstly,we find that the emergency myelopoiesis is obviously failed in C/ebpβmutants.Secondly,we find that the activation of NF-κB in C/ebpβmutants can`t rescue the defect phenotype of emergency myelopoiesis.In return,the overexpression of C/ebpβin the myd88 mutants with impaired NF-κB activity also fail to rescue the defect phenotype of emergency myelopoiesis.This implies that both NF-κB and C/EBPβare indispensable in emergency myelopoiesis.Therefore,the interaction between these two proteins is detected through co-precipitation assay and the result shows interaction indeed happens between them,consistent with previous report,and simultaneously activated both proteins can significantly promote myelopoiesis in both homeostasis and inflammation.In summary,we firstly demonstrate in vivo that during the LPS induced emergency myelopoiesis,the effective reaction and communication of macrophages with the myeloid progenitors are quite essential.The macrophages quickly detect LPS through the TLR4signals to start the emergency warn mainly via IL-1β.Upon the response,the progenitors turn on the NF-κB signaling and function together with C/EBPβto synergistically regulate the emergency myelopoiesis.This study explored the molecular mechanism of emergency myelopoiesis and monitoring pathogen cell type,providing a theoretical basis for the clinical treatment of various human infectious and malignant blood diseases.