Single-Cell sequencing Reveals Dynamic Intraepithelial Lymphocyte Responses to Salmonella Enteritidis Infection in Chickens 2257021

Abstract Introduction Intraepithelial lymphocytes (IELs) represent a heterogeneous population of immune cells located at the interface between the intestinal lumen and the lamina propria. Due to this unique anatomical location, IELs play a crucial role in maintaining intestinal homeostasis and providing mucosal immune defense against innocuous and pathogenic agents. However, the cellular mechanisms and functional roles of IELs during enteric infections in chickens remain poorly understood. This study aimed to elucidate the cellular and molecular mechanisms underlying IEL responses during Salmonella infection in chickens. Salmonella is a major foodborne pathogen of global concern, posing significant threats to both poultry production and public health. Methods In this study, sixty 21-day-old SPF chicks were orally challenged with Salmonella Enteritidis (1.6 × 108 CFU/mL; n = 30), while another 30 birds served as uninfected controls. Ileal samples were collected from eight birds per group at 2- and 6-days post-infection (dpi), and IELs were isolated for flow cytometric analysis. Enriched IELs from 2 dpi were further analyzed using single-cell RNA sequencing. Results At 2 dpi, infected group exhibited a significant increase in IEL frequency, particularly the CD8αα+TCRαβ+ and CD8αα+TCRγδ+ subsets, whereas IEL frequencies were comparable between groups at 6 dpi. Preliminary transcriptomic analysis identified 12 distinct cell clusters, including 5 lymphoid, 3 myeloid, and 4 epithelial populations. Within the lymphoid clusters, upregulated genes were associated with cell cycle progression, immune signaling, metabolic reprogramming, leukotriene signaling, granule release, proinflammatory responses, immune cell recruitment, apoptosis, cytotoxicity, and cell proliferation. Conclusion These results provide new insights into the cellular heterogeneity and transcriptional profiles of chicken IELs, highlighting their dynamic role in coordinating intestinal immune responses during Salmonella infection. Funding Source n/a Topic Categories Veterinary and Comparative Immunology (VET)