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MicroRNAs (miRNAs) are small non-coding RNAs involved in various biological processes, including immunity. Previously, we investigated the miRNAs of half-smooth tongue sole (Cynoglossus semilaevis) and found that miR-722 (designated Cse -miR-722) was significantly differentially expressed after infection with Vibrio anguillarum , reflecting its importance in immune response. Our preliminary bioinformatic analysis suggested that Cse -miR-722 could target C5aR1 (designated CsC5aR1), which was known to play crucial roles in complement activation and inflammatory response, as a receptor of C5a. However, the underlying mechanisms of their interactions and specific functions in inflammatory and immune response are still enigmas. In this study, we successfully cloned the precursor sequence of Cse -miR-722 (94 bp) and the full length of CsC5aR1 (1541 bp, protein molecular weight 39 kDa). The target gene of Cse -miR-722 was verified as CsC5aR1 by a dual luciferase reporter assay, and Cse -miR-722 was confirmed to regulate CsC5aR1 at the protein level using quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting. The expression of CsC5aR1 and Cse -miR-722 in liver cells and four immune tissues of half-smooth tongue sole changed significantly after LPS stimulation and infection with V. anguillarum. To explore the functional role of Cse -miR-722 in half-smooth tongue sole, we performed both in vitro and in vivo experiments. Cse -miR-722 was observed to affect phagocytosis and respiratory burst activity of macrophages by regulating CsC5aR1 in half-smooth tongue sole. Furthermore, we found that Cse -miR-722 regulated the expression of CsC5aR1 , CsC5a , and the inflammatory factors CsIL1-β , CsIL6 , CsIL8 , and CsTNF-α both in vitro and in vivo. In addition, Cse -miR-722 reduced mortality and pathological damage. This study clarified the regulatory mechanism of Cse- miR-722 on CsC5aR1 and provided insight into the regulatory roles of Cse- miR-722 in immune responses, laying a theoretical foundation for the feasibility of using miR-722 to prevent and control bacterial diseases in teleost. • Cse -miR-722 targeted CsC5aR1 and silenced CsC5aR1 at the translation level. • Cse -miR-722 affected the phagocytic activity and respiratory burst of macrophages. • Cse -miR-722 regulated immune responses through the C5a/C5aR1 complement pathway. • Cse -miR-722 reduced mortality and body damage after Vibrio anguillarum infection. [ABSTRACT FROM AUTHOR]

Frequently occurred bacterial diseases have seriously affected the aquaculture industry of half-smooth tongue sole (Cynoglossus semilaevis). Notably, vibriosis, with Vibrio anguillarum as one of the causative pathogens, is the most severe bacterial disease with severe inflammatory response of the host, leading to high mortality rates. In the present study, we explored the relationship between bacterial concentrations and host mortality, inflammatory reaction, and immune response in half-smooth tongue sole after infection with V. anguillarum at different concentrations (Treatment 1, 6.4 × 105 CFU/mL; Treatment 2, 6.4 × 106 CFU/mL). The mortality of Treatment 2 (77.5%) was significantly higher than that of Treatment 1 (10%), corresponding with bacterial concentrations. Although the number of deaths varies, intensive deaths were observed within 24 h post infection (hpi) in both bacterial concentration groups. Histopathological analyses revealed that fish tissues were most severely damaged at 24 or 48 hpi, and Treatment 2 was more severe than Treatment 1. A qRT-PCR-based detection method with virulence factor gene empA was established to quantify the bacterial loads in various tissues, and the bacterial loads were the highest at 24 hpi in Treatment 2, and at 48 hpi in Treatment 1. Additionally, the expression levels of complement genes (C5a , C3 , C5 , and C6), inflammatory factors (IL-1β , TNF-α , and IL-1 0), and other immune-related genes (jak2 , NF-κB1 , stat3 , and tlr3) were increased in various tissues after infection in both treatment groups, with most genes being most expressed at 24 or 48 hpi, and expression levels of inflammatory factors in Treatment 2 were higher than those in Treatment 1. Moreover, the expression of C5a was positively correlated with that of proinflammatory cytokines in both bacterial concentration groups. According to the results of this study, 24–48 hpi was a key node for early vibriosis detection and intervention. Compared with the low mortality of Treatment 1, the mass death of fish in Treatment 2 was suggested to be caused by uncontrolled excessive inflammatory reaction induced by the overactivation of complement system, especially C5a. We believe these results could provide theoretical basis for prevention, evaluation, and treatment of vibrio disease in tongue sole aquaculture, and lay a solid foundation for future functional analyses. • The mortality, inflammation response, host histopathological damage and bacterial loads of tongue sole after Vibrio anguillarum infection peaked at 24–48 hpi. • Inflammatory factors (IL-1β , TNF-α , and IL-10), complement genes (C5a , C3 , C5 , and C6), and other immune genes (jak2 , NF-κB1 , stat3 , and tlr3) were up-regulated after infection, with a bacterial dose effect. • The observed mass death under high bacterial concentration infection was suggested to be caused by the excessive inflammatory response mainly induced by C5a overactivation. [ABSTRACT FROM AUTHOR]

The complement system is essential to host defense, but its excessive activation caused by severe pathogen invasion is a driving force in adverse inflammatory. The binding of complement component 5a (C5a) and complement component 5a receptor 1 (C5aR1) is the key to trigger complement-mediated inflammatory response in mammals. However, the role of C5a-C5aR1 axis in fish immune response remains obscure. In this study, the role of C5a-C5aR1 axis of zebrafish (Danio rerio) after serious infection with Aeromonas hydrophila was investigated. C5a and C5aR1 of zebrafish were cloned, with CDS sequences of 228 and 1041 bp, respectively, and they were widely expressed in various tissues with the highest expression in the liver and spleen, respectively. The survival of zebrafish was closely correlated to the dose of A. hydrophila. The cytokine storm occurred at high concentrations of A. hydrophila infection. At 24 h post infection (hpi), the expression of C5a and C5aR1 in the spleen increased 26.8-fold and 9.9-fold in treatment group 1 (TG1, 3.0 × 107 CFU/mL) (P < 0.01), and 4.7-fold and 3.4-fold in treatment group 2 (TG2, 1.0 × 107 CFU/mL) (P < 0.05), respectively. Correspondingly, proinflammatory cytokines interleukin-1β (IL-1β), interleukin-8 (IL-8), and interleukin-17 (IL-17) were positively correlated to C5a and C5aR1 at mRNA and protein expression levels. The expression of IL-1β was significantly increased in the spleen at 6 hpi, with a 599.2-fold and 203.2-fold upregulation in TG1 and TG2 (P < 0.001), respectively. Moreover, after inhibition of C5a-C5aR1 binding treated with C5aR1 antagonist (W-54011), zebrafish showed lower expression of C5a , C5aR1 , and cytokines, less intestinal damage, and significantly enhancement of survival (P < 0.05) after A. hydrophila challenge. This study revealed that the inflammatory effect of C5a was achieved by binding to C5aR1 in zebrafish, providing novel insights into using C5a-C5aR1 axis as an effective target to reduce bacterial inflammation and disease in fish. • C5a-C5aR1 axis was activated by bacterial infection with dose effect. • Hyperactivation of C5a-C5aR1 axis triggered a "cytokine storm" at both mRNA and protein levels in zebrafish. • Inhibition of C5a-C5aR1 axis significantly reduced the inflammatory response after bacterial infection in zebrafish. • The inflammatory effect of C5a was achieved by binding to C5aR1. [ABSTRACT FROM AUTHOR]