Nocardia seriolae mediates granulomatous chronic inflammation of spleen in Micropterus salmoides through necroptosis

Granulomatous chronic inflammation is a typical pathological characteristic of largemouth bass (Micropterus salmoides) infected by Nocardia seriolae, and it is also an important breakthrough to solve the problem of pathogen prevention and treatment. Spleen, the target organ of N. seriolae infection, is significant for studying pathogenic mechanism. However, histopathological characteristics and immunological molecular mechanisms of the development of splenic granulomas in fish have not been extensively studied. In the initial process of exploring the molecular mechanism of N. seriolae infectious splenic granuloma formation, we accidentally found positive signals of necroptosisrelated gene by immunohistochemical staining, suggesting that necroptosis may be involved in the formation of granulomas. Nevertheless, the role of necroptosis in the formation of splenic granuloma is still unclear. In this study, we established a granuloma model in the spleen of largemouth bass infected by N. seriolae. Through multiple tissue staining, we compared splenic granulomas with hepatic and renal granulomas, and preliminarily clarified the histopathological characteristics of splenic granulomas and the formation pattern of fibrosis, calcium salt deposition and other lesions. Notably, we found that the expression of inflammatory factors and necroptosisrelated molecules in spleen were significantly upregulated during N. seriolae infection. Further immunohistochemical staining showed that the splenic granulomas were accompanied by extensive and intense p-MLKLpositive signals in the peripheral tissue. In addition, western blot and immunofluorescence results showed that N. seriolae could induce necroptosis in primary spleen cells of largemouth bass in vitro. Our data indicated that necroptosis is closely related to the formation of splenic granulomas in largemouth bass, and provide some ideas for the study of the formation mechanism of fish granuloma.