Single-cell transcriptome analysis reveals a cellular immune response in common carp (Cyprinus carpio) infected with...

This study aimed to employ single-cell RNA sequencing technology to comprehensively investigate the cellular immune response mechanisms in the key immune organ, the head kidney, of common carp (Cyprinus carpio) following infection with Aeromonas hydrophila, with a particular focus on the heterogeneity, differentiation, and molecular regulatory networks of macrophages at the cellular level.

An infection model in common carp was established via intraperitoneal injection of A.hydrophila and validated using conventional methods, including pathological examination, serum immune enzyme assays, and immunohistochemistry. The core approach applied single-cell RNA sequencing to the head kidney tissues of infected and control fish. Bioinformatic analyses included cell clustering, high-dimensional weighted gene co-expression network analysis, pseudotime analysis, and cell-to-cell communication analysis to track immune cell dynamics. Proteomics was used to corroborate these key findings.

Infection induced characteristic pathological signs in common carp, altered serum immune-enzyme activities, and caused inflammatory tissue damage. Single-cell transcriptomics of the head kidney revealed ten distinct macrophage subpopulations, with a significant post-infection shift in the proportion of CXCL19-expressing cells. CXCL19⁺ macrophages were further divided into two functionally distinct groups: Macro_cxcl19_NEW1, an M2-like subset with anti-inflammatory and immunosuppressive features, and Macro_cxcl19_NEW2, an immune-activating subset. Pseudotime analysis indicated a differentiation trajectory from NEW2 to NEW1 and highlighted candidate regulators, including cfbl (complement factor B), npm1a (nucleophosmin), prkaa1.1 (AMPK catalytic subunit), and selenow1. These findings were confirmed by proteomics, demonstrating significant downregulation of the CFBL protein in infected fish.

This study provides, for the first time at single-cell resolution, a systematic map of head kidney immune responses to bacterial infection in Common carp. This revealed substantial macrophage heterogeneity and a central role for these cells in immune regulation. The identified subpopulations and regulatory genes offer new insights into fish cellular immunity and establish a molecular foundation for targeted strategies against Aeromonas hydrophila, including immune enhancers and disease-resistant breeding.