Study On The Mechanism Of TLR-MyD88-NFκB Signaling Pathway In The Emergency Myelopoiesis In Zebrafish

Inflammation is a common pathological process which is characterized by redness,swelling,heating,and pain.Meanwhile,it is a complicated biological process including the reaction of granulocytes and macrophages.As we know,inflammation is caused by many factors,such as chemical radiation,injury,or infection.If the injury occurs locally,the vascular endothelial cells express P-selection,E-selection and other factors,which attract circulating granulocytes and nearby macrophages or dendritic cells to move to the damaged sites.During earlier inflammatory response,the immune cells accumulate in the damaged site,releasing a variety of inflammatory factors which recruit more immune cells in turn.Finally,it causes the signaling transduction and cascade amplification.With the progress of the inflammation,the injured tissues are gradually repaired.Consequently,the inflammatory signals terminate and immune cells gradually recover into the steady state.On the contrary,if the infection caused by microbes are not completely removed,the microbes will expand to the whole body through circulatory system,which leads to systemic inflammation.A large number of myeloid cells are produced at this point,which is called emergency myelopoiesis.The Emergency myelopoiesis consists of granulopoiesis and monopoiesis which caused by bacteria such as intestinal enteritis Yersinia infection.When pathogenic microorganism break the barrier of the body,the organism recognizes components of bacteria,pathogen-associated molecular pattern(PAMP),through pattern-recognize receptor(PRRs)which activate signaling pathway to regulate the response of the immune cells.Different Toll-like receptors(TLRs),one of the most important pattern recognition receptors,recognize different bacterial components.For example,TLR4 specifically recognizes LPS while TLR3 recognizes Poly I: C.Once receptors are activated,the downstream target gene will respond largely through myeloid differentiation factor 88(MyD88).It phosphorylate nuclear factor κB inhibition Kinase complex(IKK complex)that are composed of IKK-α,IKK-β and IKK-γ,and subsequently phosphorylate IκB.The inhibitor of NFκB(P50 and P65 combination)is released,leading to NFκB into the nucleus initiating the expression of downstream genes such as tnfα,il1 b expression.In addition,the G-CSF—G-CSFR signaling pathway is involved in this process,mainly through the CCAAT enhancer binding protein β(C/EBPβ).The CCAAT enhancer binding protein family contains several members,such as C/EBPα,C/EBPβ,C/EBPδ,C/EBPε.The differentiation of myeloid cells in C/ebpα-deficient mutants is blocked in the pluripotent precursor stage,and the number of granulocytes significantly reduce during the steady state.While C/ebpβ-deficience only affect emergency granulopoiesis and the development of granulocytes haven’t been affected in the steady state.As regard to emergency myelopoiesis,there are still many questions required to be explored further.Zebrafish(Danio rerio)is a popular model organism in recent years.They are suitable for large-scale genetic mutation screening to study the development of blood cells.In addition,both innate immunity and acquired immunity are accessible in zebrafish as that in mammals.The endotoxin LPS was injected into the blood of zebrafish at 2dpf(2 days post fertilization),which caused systemic inflammation.TLR4,a specific receptor for LPS,which activates inflammatory factors,such as il1 b,tnfα and so on,through MyD88-NFκB signaling pathway.In order to study whether MyD88-NFκB signaling pathway was involved in the process,myd88 mutant and Tg(NFκB-GFP)fish were applied.The results showed that emergency myelopoiesis was blocked in myd88 mutant.What’s more,Tg(NFκB:GFP)fish was used to verify whether NFκB signaling was activated.The green fluorescence enhanced significantly after injecting LPS when compared with that of PBS.It reached the maximum at approximately 6 hpi(6 hours post injection)and maintained for about 24 hours.The zebrafish was treated with Jsh-23,inhibitor of NFκB,before LPS was injected.The results showed that myeloid cells including progenitors reduced in LPS.Subsequently,the different inflammatory factors was detected by qPCR,which showed that il1 b changed seriously after injection of LPS.In order to confirm the role of il1 b in myelopoiesis,both overexpression of il1 b and il1 b morpholino are applied.The results showed that the emergency myelopoiesis was enhanced with overexpression of il1 b while the emergency myelopoisis was inhibited after il1 b morpholino.In addition,the expression of C/ebpβ increased after injection of LPS in myd88 mutant while its expression was significantly reduced compared with wild type.In order to investigate the role of C/ebpβ in this process,LPS was injected into C/ebpβ mutant.The results showed that the expansion of granulocytes were completely blocked and the macrophages significantly decreased compared with wild type.Further study indicated that the expression of C/ebpβ was down-regulated after NFκB inhibitor treatment.Besisdes,overexpression C/ebpβ could enhanced the emergency myelopoiesis in Wild type.Our preliminary results suggested that emergecy myelopoiesis induced by LPS emerged was regulated through MyD88,which activated NFκB signaling pathway.The inflammatory factors,such as il1 b produced during induction of LPS intensively,which further activated downstream signaling pathways.The expression of C/ebpβ was up-regulated in the process and C/ebpβ was essential for the emergency myelopoiesis induced by LPS.