Exploiting RAG1-deficient chickens to construct a single-cell transcriptional atlas of chicken heterophils during...

Avian heterophils naturally lack myeloperoxidase (MPO), making chicken a unique evolutionary model for investigating MPO-independent innate immune strategies. However, the molecular programs governing heterophil maturation and activation remain largely unexplored.

Using single-cell RNA sequencing in a RAG1-deficient chicken model, we mapped heterophil maturation during emergency granulopoiesis and identified eight transcriptionally distinct subsets (H0-H7). We inferred a developmental trajectory spanning transcriptionally distinct heterophil subsets, from early subsets enriched for genes associated with translational activity, oxidative phosphorylation, and survival-related programs to terminal subsets enriched for effector-associated gene signatures.

Unlike mammalian neutrophils, heterophils did not sequentially deploy canonical granule gene expression; instead, maturation was marked by progressive MMP9 upregulation. Transcriptional profiling revealed a prominent enrichment of nitric oxide signaling pathway, with basal level of NADPH oxidase expression. Trajectory analysis indicated that cells transitioned from interferon-stimulated gene (ISG)-dominant states to effector profiles capable of degranulation, phagocytosis, and extracellular trap formation. Terminal subsets showed exhibited transcriptional signatures associated with enhanced extracellular matrix remodeling, acute phase responses, and nutritional immunity. Together, these findings provide a detailed single-cell atlas of avian heterophils, offering valuable evolutionary insights into the developmental trajectories and transcriptional heterogeneity of innate immune cells in a naturally MPO-independent species.