Itgb1b⁺ and Itgb2⁺ leukocytes regulate immune responses to bacterial infection in grass carp

Integrins are important transmembrane receptors involved in cell adhesion, migration, and immune regulation. However, their functional roles remain poorly understood in fish. In this study, the itgb1b and itgb2 genes were cloned from grass carp (Ctenopharyngodon idella). Phylogenetic and synteny analyses showed that teleost itgb1 underwent gene duplication, and diverged into itgb1a and itgb1b, whereas itgb2 remained as a single-copy gene. Polyclonal antibodies against Itgb1b and Itgb2 were generated and characterized. Immunofluorescence staining demonstrated abundant populations of Itgb1b⁺ and Itgb2⁺ cells in the head kidney and spleen of healthy fish. Among head kidney leukocytes, Itgb1b⁺ and Itgb2⁺ cells accounted for 6.75% and 7.68%, respectively, whereas their proportions in the spleen were 9.25% and 3.99%. Notably, Itgb1b⁺/Mcsfr⁺ cells accounted for 4.64% and 5.21% of total live leukocytes in the head kidney and spleen, respectively, whereas Itgb2⁺/Mcsfr⁺ cells accounted for 5.05% and 3.05%, respectively, indicating that Itgb1b and Itgb2 are expressed by subsets of Mcsfr⁺ monocytes/macrophages. Following Aeromonas hydrophila infection, both the mRNA expression of itgb1b and itgb2 and the abundance of Itgb1b⁺ and Itgb2⁺ cells were significantly increased in the head kidney and spleen. Moreover, bacterial challenge promoted the proliferation of Itgb1b⁺ and Itgb2⁺ cells in these immune tissues. These findings indicate that Itgb1b⁺ and Itgb2⁺ leukocytes, including a macrophage subpopulation, participate in the immune response to bacterial infection in fish.