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The eye of vertebrates is constantly faced with numerous challenges from aquatic or airborne pathogens. As a crucial first line of defense, the ocular mucosa (OM) protects the visual organ from external threats in vertebrates such as birds and mammals. However, the understanding of ocular mucosal immunity in early vertebrates, such as teleost fish, remains limited, particularly concerning their resistance to bacterial infections. To gain insights into the pivotal role of the OM in antibacterial immunity among teleost fish, we developed a bacterial infection model using Flavobacterium columnare in rainbow trout (Oncorhynchus mykiss). Here the qPCR and immunofluorescence results showed that F. columnare could invade trout OM, suggesting that the OM could be a primary target and barrier for the bacteria. Moreover, immune-related genes (il-6, il-8, il-11, cxcl10, nod1, il1-b, igm, igt, etc.) were upregulated in the OM of trout following F. columnare infection, as confirmed by qPCR, which was further proved through RNA-seq. The results of transcriptome analyses showed that bacterial infection critically triggers a robust immune response, including innate, and adaptive immune-related signaling pathways such as Toll-like, NOD-like, and C-type lectin receptor signaling pathway and immune network for IgA production, which underscores the immune role of the OM in bacterial infection. Interestingly, a substantial reduction in the expression of genes associated with visual function was observed after infection, indicating that bacterial infection could impact ocular function. Overall, our findings have unveiled a robust mucosal immune response to bacterial infection in the teleost OM for the first time, providing valuable insights for future research into the mechanisms and functions of ocular mucosal immunity in early vertebrate species.

To further study the biological function of interferon-gamma (IFN-γ) in the Chinese sturgeon (Acipenser sinensis), we conducted a transcriptome analysis of primary macrophages induced by IFN-γ using Illumina sequencing technology. We obtained 88,879 unigenes, with a total length of 93,919,393 bp, and an average length of 1,057bp. We identified 8,490 differentially expressed genes (DEGs) between the untreated and IFN-γ-treated macrophages, with 4,599 upregulated and 3,891 downregulated. Gene ontology (GO) analysis showed that 4,044 DEGs were enriched in the biological, cellular components, and molecular function categories. Kyoto Encyclopedia of Genes and Genomes (KEGG) identified 278 immunity-related pathways enriched for the DEGs. According to the GO enrichment results, eight key immunity-related genes were screened for verification using qPCR. Results indicate that IFN-γ can activate macrophage Interferon Regulatory Factors (IRFs) and type I interferon (IFN-I), activate RIG-I-like and Toll-like receptor-related pathways, and improve the antiviral ability of macrophages in Chinese sturgeon.

Double Ig IL-1R related molecule (DIGIRR), myeloid differentiation factor 88 (MyD88), interleukin-1 receptor-associated kinase-1 (IRAK-1), tumor necrosis factor receptor-associated factor 6 (TRAF6), and Interferon-c (IFNc), are all signaling molecules involved in Toll-like receptor (TLR)-dependent inflammation. In this study, Chinese sturgeon DIGIRR, MyD88, IRAK1, TRAF6, and IFNc expression levels in immune tissues (blood, spleen, liver, head kidney and middle kidney) were upregulated in response to Citrobacter freundii infection. DIGIRR were highest in the head kidney, MyD88, IRAK1 and TRAF6 were highest in the liver, and IFNc were highest in the blood, respectively. The expression levels of DIGIRR and other genes sturgeons challenged with Plesiomonas shigelloides were similar to those challenged with C. freundii, except that DIGIRR expression was highest in the liver. Surprisingly, the expression levels of the genes after Mycobacterium marinum challenge decreased significantly, DIGIRR, MyD88, IRAK1 were lowest in the liver, TRAF6 were lowest in the blood and IFNc were lowest in the head kidney, respectively. Taken together, the expression levels of DIGIRR and other genes in Chinese sturgeon were upregulated significantly after challenge with C. freundii and P. shigelloides, but decreased significantly by M. marinum. Although their reactions varied, these findings indicate that the DIGIRR signaling pathway in Chinese sturgeon is involved the inflammatory response to diverse bacterial infection.