# Integrated Single-Cell and Bulk Transcriptomics Unveils Immune Profiles in Chick Erythroid Cells upon Avian Pathogenic Escherichia coli Infection

## TL;DR

This study explores how chick erythroid cells respond to E. coli infection, revealing immune-related genes and cell subtypes that could help improve poultry disease resistance.

## Contribution

The first systematic characterization of immune profiles in chick erythroid cells during APEC infection using single-cell and bulk transcriptomics.

## Key findings

- Identified 10 distinct chick erythroid cell subpopulations with significant compositional changes after APEC infection.
- Discovered 62 immune-related genes and key transcription factors like Fos, Srf, and Stat3 linked to immune responses.
- Found activation of MHC class I antigen presentation and suppression of MAPK and NLR signaling pathways in infected cells.

## Abstract

In addition to their primary role in oxygen transport, chicken erythroid cells have been shown to participate in immune responses. This study utilized advanced single-cell analysis alongside bulk RNA sequencing to explore the immune mechanisms of erythroid cells in chicks infected with avian pathogenic Escherichia coli (APEC). Through integrated multi-omics analysis, we identified immune-related genes and signaling pathways associated with antiviral defense. Preliminary single-cell analysis revealed heterogeneity within the erythroid cell population, unveiling four erythrocyte subtypes that exhibited significant quantitative changes following APEC infection, as well as key transcription factors potentially involved in pathogen defense. This research represents the first systematic characterization of the immune landscape of chick erythroid cells during infection, enhancing our understanding of avian immune mechanisms and providing a theoretical foundation for the development of novel disease-resistance strategies in poultry, thereby contributing to sustainable poultry production.

Nucleated erythroid cells (NECs) have emerged as active participants in immune responses in addition to their canonical oxygen transport function. The subpopulations and immune heterogeneity of chick erythroid cells (ch-ECs) upon infection have not been fully characterized. Single-cell RNA sequencing (scRNA-seq) was used to profile ch-ECs in chicks infected with avian pathogenic Escherichia coli (APEC). Unsupervised clustering uncovered ten distinct ch-EC subpopulations (C1–C10), with significant compositional shifts between infected and control groups. Pseudotime analysis revealed a developmental continuum: C1, C3, C5, and C9 as early progenitors; C2, C4, C6, C7, and C10 as mature erythroid cells; and C8 as a naive population. We revealed 62 immune-related genes, including protein kinases and heat shock proteins, and subpopulation-specific differentially expressed genes (DEGs) linked to immune functions. SCENIC analysis revealed Fos, Srf, and Stat3 as key transcription factors with elevated regulon activity and specificity following infection. Subpopulations C2, C4, C6, and C7, which exhibited marked abundance changes, were scrutinized for immune relevance through integrated multi-omics analysis. Immune-related genes including FOS, AKAP9, HS6ST1, GAB3, TFRC, HSPA8, HSP90AA1, and DNAJB6 were identified. Enrichment analysis indicated activation of the MHC class I antigen presentation pathway, while pathways such as Mitogen-Activated Protein Kinase (MAPK) signaling, NOD-like receptor (NLR) signaling, and the heat shock response were found to be suppressed. In conclusion, this study delineates the immune gene repertoire and signaling networks of ch-ECs during APEC infection, offering new perspectives on NEC immunoregulatory functions.