Molecular characteristics and functional differentiation of two IL-10 genes in barbel chub Squaliobarbus curriculus

Interleukin-10 (IL-10), an anti-inflammatory cytokine, plays crucial roles in balancing excessive inflammation in fish during infectious diseases. In this study, we identified two IL-10 gene copies in barbel chub (Squaliobarbus curriculus), designated as ScIL-10A and ScIL-10B. Gene collinearity analysis revealed that ScIL-10A and ScIL-10B were located on different chromosomes, and only ScIL-10A shared a conserved gene block with IL-10s from other vertebrates. Both ScIL-10A and ScIL-10B possessed a typical IL-10 protein domain and showed substantial sequence homology with their counterparts in other cyprinid species. Subcellular localization analysis and CHO-S cell expression experiments indicated that ScIL-10A was secreted, whereas ScIL-10B was membrane-localized. Tissue-specific expression profiling manifested that ScIL-10A was predominantly expressed in immune-related tissues, including the spleen, kidney, and head kidney, while ScIL-10B exhibited ubiquitous expression across all examined tissues. Following LPS challenge, both ScIL-10A and ScIL-10B showed the significantly upregulated mRNA expression levels and the similar expression patterns in S. curriculus kidney (ScK) cells. Transcriptomic data revealed that overexpression of ScIL-10A and ScIL-10B induced two distinct lists of differentially expressed genes in ScK cells. Specifically, ScIL-10B overexpression significantly downregulated the expression of multiple pro-inflammatory factors, including CXCL8, CXCL11, IL-8, and IL-34, while ScIL-10A overexpression showed minimal impact on these pro-inflammatory factors expression. In LPS-stimulated ScK cells, however, Western blot analysis demonstrated that both ScIL-10A and ScIL-10B overexpression significantly reduced the protein levels of key pro-inflammatory cytokines TNF-α and IL-1β, suggesting their potential synergistic role in inflammation regulation. This study first discovers the molecular characteristics and functional differentiation of two copied IL-10s in barbel chub, providing a novel insight into the evolutionary biology of IL-10 and the diversity of anti-inflammatory mechanisms in fish.