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The complement component 5a/complement component 5 receptor 1 (C5a/C5aR1) pathway plays a crucial role in the onset and development of inflammation, but relevant studies in fish are lacking. In this study, we successfully characterized the relationship between half-smooth tongue sole (Cynoglossus semilaevis) C5aR1 (CsC5aR1) and bacterial inflammation. First, we showed that the overexpression of CsC5aR1 significantly increased bacterial pathological damage in the liver and intestine, whereas inhibition attenuated the damage. The in vitro experiments suggested that CsC5aR1 was able to positively regulate the phagocytic activity and respiratory burst of tongue sole macrophages. In terms of both transcriptional and translational levels, overexpression/inhibition of CsC5aR1 was followed by a highly consistent up-regulation/decrease of its downstream canonical inflammatory factor interleukin-6 (CsIL-6). Furthermore, we stimulated macrophages by lipopolysaccharide (LPS) and lipoteichoic acid (LTA) and found a broad-spectrum response to bacterial infections by the C5a/C5aR1 complement pathway together with the downstream inflammatory factor CsIL-6. Subsequently, we directly elucidated that CsIL-6 is an indicator of C5a/C5aR1-mediated inflammation at different infection concentrations, different infectious bacteria (Vibrio anguillarum and Mycobacterium marinum), and different detection levels. These results might provide a new inflammation bio-marker for early warning of bacteria-induced hyperinflammation leading to fish mortality and a promising target for the treatment of bacterial inflammation in teleost. • By directly manipulating CsC5aR1, it was demonstrated that its overactivation could lead to bacterial inflammatory damage. • The IL-6 gene in C. semilaevis was functionally characterized for the first time. • CsC5aR1 and CsIL-6 were widely involved in bacterial inflammation triggered by Gram-positive/negative bacteria. • CsIL-6 could serve as an indicator of bacterial inflammation. [ABSTRACT FROM AUTHOR]

MicroRNAs play crucial roles in various biological processes, including but not limited to differentiation, development, disease, and immunity. However, their immunoregulatory roles in half-smooth tongue sole are lacking. Our previous studies indicated that miR-722 could target C5aR1 to modulate the complement pathway to alleviate inflammatory response and even affect the mortality after the bacterial infection with Vibrio anguillarum. Driven by the purpose of revealing the underlying mechanisms, in this study, we investigated the effects of miR-722 on the gene expression and alternative splicing (AS) in the liver of half-smooth tongue sole after Vibrio anguillarum infection, with the approach of miR-722 overexpression/silencing and subsequent RNA-seq. Among the different comparisons, the I group (miR-722 inhibitor and V. anguillarum) versus blank control (PBS) exhibited the highest number of differentially expressed genes (DEGs), suggesting that the immune response was overactivated after inhibiting the miR-722. In addition, enrichment analyses were performed to reveal the functions of DEGs and differential AS (DAS) genes, reflecting the enrichment of RNA splicing and immune-related pathways including NF-κB and T cell receptor signaling pathway. Comparing the M group (miR-722 mimic and V. anguillarum) with the negative control (random sequence and V. anguillarum), two immune-related genes, cd48 and mapk8 , were differentially expressed, of which mapk8 was also differentially spliced, indicating their importance in the immune response. Furthermore, representative gene analysis was performed, suggesting their corresponding functional changes due to AS. To verify the RNA-seq data, quantitative real-time PCR was employed with twenty pairs of primers for DEGs and DAS events. Overall, our results demonstrated that miR-722 could mediate the transcriptome-wide changes of gene expression and AS in half-smooth tongue sole, and provided insights into the regulatory role of miR-722 in immune responses, laying the foundation for further functional analyses and practical applications in aquaculture. • miR-722 mediated the transcriptome-wide changes of gene expression and AS in tongue sole. • DAS contributed significantly to the immune response after the bacterial infection. • mapk8 was the only gene that underwent both differential expression and DAS in M vs N. [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]