Cloning Of Zebrafish Complement C5a/C5aR1 And Analysis Of Its Expression And Function In Bacterial Inflammation

The complement system is essential to host defense,but its excessive activation caused by severe pathogen invasion is a driving force in adverse inflammation.The binding of complement component 5a(C5a)and complement component 5a receptor 1(C5a R1)is the key to trigger complement-mediated inflammatory response in mammals.However,the role of C5a-C5a R1 axis in fish immune response remains obscure.Zebrafish(Danio rerio),as an important model,is an ideal material for studying the host immune response to pathogen infection.In this study,we infected zebrafish with Aeromonas hydrophila at different concentrations to evaluate suvival and tissue damage,and to analyze the expression characteristics of complement activator C5a,receptor C5a R1,and inflammation-related cytokines.Furthermore,the role of C5a/C5a R1pathway in the inflammatory response of zebrafish against bacterial infection is discussed.The main results are as follows:(1)The CDS sequences of zebrafish C5a and C5a R1 were obtained by cloning.The length of the sequences was 228bp and 1041bp,encoding 76 and 346 amino acids,respectively.Phylogenetic analysis showed that fish species in Cyprinidae were clustered into one clade,and the grass carp(Ctenopharyngodon idella)was the most closely related to zebrafish,with a similarity of 78.9%.In healthy zebrafish,C5a and C5a R1 were widely expressed in 11 tissues,including liver,intestine,spleen,brain,gills,skin,muscle,heart,eyes,caudal fin and gonad.C5a had the highest expression in the liver and C5a R1 had the highest expression in the spleen.(2)The survival rate and intestinal injury degree of zebrafish were closely related to the concentration of bacterial infection.After zebrafish was infected with A.hydrophila at concentrations of 6.8×10~5,6.3×10~6,1.0×10~7,2.0×10~7,3.4×10~7,5.3×10~7and 1.0×10~8CFU/m L,the survival rates were 100%,80%,63.3%,6.7%,16.7%,0,and 0,respectively.3.0×10~7and 1.0×10~7CFU/m L were selected as the infection concentrations of treatment group 1(TG1)and treatment group 2(TG2)in following experiments,respectively.The survival of control group(CG)was 100%.The survival of TG2 was 59.6%,which was significantly higher than that of TG1(23.3%).The intestinal tissue pathological damage of zebrafish was manifested as separation of mucosal layer and mucosal lamina propria tissue,intestinal villi rupture and a large number of cup cells.The pathological injury was the most serious at 24 h after infection,and decreased at 48 h.In contrast,TG1 was more severely damaged than TG2,and recovered slower.(3)Severe bacterial infection resulted in C5a/C5a R1 pathway over-activation,and the degree of activation was dose-dependent with bacterial infection.The results of q RT-PCR showed that the expression levels of C5a and C5a R1 were significantly up-regulated compared to CG,and the expression pattern of the two genes was the same.Within 72 h after infection,the expression of C5a and C5a R1 in the liver,intestine,spleen and gill increased first and then decreased.In TG1,the relative expression of C5a and C5a R1 reached the highest level at 12 h or 24 h after infection.In TG2,the relative expression of C5a and C5a R1 reached the highest level at 24 h or 48 h after infection.The expression levels of C5a and C5a R1 in TG1 were higher than TG2.(4)Over-activation of the C5a/C5a R1 pathway induced a"cytokine storm".At the m RNA level,the expressions of pro-inflammatory cytokines interleukin-1β(IL-1β)、interleukin-8(IL-8)、interleukin-17(IL-17)and anti-inflammatory cytokine transforming growth factor-β(TGF-β)in TG1 and TG2 were significantly up-regulated after A.hydrophila infection,and the expressions in the liver,intestine,spleen,and gill all increased first and then decreased.In TG1,the relative expression of IL-1β,IL-8,and IL-17 in different tissues reached the highest level within 6-24 h after infection,and the relative expression of TGF-βreached the highest level within 12-48 h after infection.In TG2,the peak expression of IL-1β,IL-8 and IL-17 was 12-48 h after infection,and the peak expression of TGF-βwas 48-72 h after infection.The relative expression levels of the four cytokines in TG1 were higher than TG2,and the changes of the relative expression levels of the pro-inflammatory cytokines were much greater than those of the anti-inflammatory cytokines.At the protein level,the results of ELISA were similar to those at the m RNA level,the content of pro-inflammatory cytokines(IL-1β,IL-8,IL-17)and anti-inflammatory cytokine(TGF-β)in TG1 and TG2 increased significantly after infection,but the changes were delayed compared to m RNA.The levels of the four cytokines in TG1 were higher than TG2,and the changes in the expression of pro-inflammatory cytokines were much greater than those of anti-inflammatory cytokine.(5)C5a R1 antagonist treatment could reduce the excessive activation of C5a/C5a R1pathway after bacterial infection and reduce the inflammatory response in zebrafish.Zebrafish pretreated with C5a R1 antagonist(W-54011)had a significantly higher survival rate(56.7%)than that in the non-antagonist group(43.3%).Through the observation of intestinal pathological sections,it was found that the damage degree of intestinal tissue of zebrafish treated with antagonists after infection was significantly less than that of the untreated group.The results of q RT-PCR showed that the relative expression levels of C5a,C5a R1,pro-inflammatory factor(IL-1β,IL-8,IL-17)and anti-inflammatory factor(TGF-β)in the liver,intestine,spleen and gills of the infected zebrafish treated with antagonists were significantly lower than those of the non-antagonist group.In conclusion,this study described the relationship between the activation of C5a/C5a R1 pathway and the inflammatory response after bacterial infection in zebrafish,analyzed the effect of C5a R1 antagonist(W-54011)on the inflammatory response,and explored the role of C5a/C5a R1 pathway in bacterial inflammation in zebrafish.These results confirmed that C5a/C5a R1 pathway may be an important target for the prevention and control of bacterial inflammation in fish,which lays a theoretical foundation for the research on the regulation mechanism of fish disease resistance traits and provides a new candidate gene for the cultivation of fish disease resistance families,and which is of great significance for the healthy and sustainable development of aquaculture industry.