# Impact of low and high lipopolysaccharide doses on the early expression of immune related genes and transcription factors in chicken macrophage-like HD11 cells

## TL;DR

This study shows how different doses of LPS affect gene expression in chicken macrophage-like cells, revealing key differences in immune response pathways.

## Contribution

The study identifies specific transcription factors and pathways that differ between low and high LPS doses in chicken macrophage-like cells.

## Key findings

- High LPS doses downregulate five immune-related transcription factors not affected at low doses.
- Three pathways (toll-like receptor, cytokine-cytokine receptor interaction, C-type lectin receptor signaling) are more affected at high LPS doses.
- Using multiple LPS doses is recommended for studying inflammation in chicken macrophage models.

## Abstract

This study aimed to investigate how early transcriptional reprogramming of the chicken macrophage-like HD11 cell line differs when exposed to a low and high dose of lipopolysaccharide (LPS). Transcriptome analysis was performed on cells stimulated with 0, 3 and 300 ng/ml E. coli LPS for 5 h. After the processing steps, 10796 genes remained. Compared to the negative control, 11.8 and 16.3 % were upregulated at 3 and 300 ng and 25.8 and 44.1 % of the genes were downregulated. Subsequently, gene set enrichment analysis of Kyoto Encyclopedia of Genes and Genomes pathways demonstrated that, compared to the negative control, LPS at 3 and 300 ng/ml upregulated and downregulated similar pathways, out of which only three were significantly more affected at 300 than at 3 ng/ml LPS. These three enriched pathways were the toll-like receptor, cytokine-cytokine receptor interaction and C-type lectin receptor signalling pathways. Five transcription factors related to immune genes (PITX2, TCF7L2, NFIL3, NFATC1 and FOXO1) were downregulated at the high dose of LPS only and retrieved in the regulatory network between the differentially expressed immune related genes and transcription factors. Six other transcription factors were only retrieved in the 300 vs 3 ng/ml network: HMGB1, MEF2C, IKZF1, and PITXA, which were downregulated, and SRF and FOS, which were upregulated. These 11 transcription factors may account for the differences in the intensity of the cell response to low and high doses of LPS. Our results indicate that while the expression of transcription factors is largely similar, there are some differences between low and high doses of LPS. These differences could have implications when using LPS-stimulated HD11 cells as a model to study potential feed additives in poultry. Based on this knowledge we recommend considering the use of multiple LPS doses when using HD11 cells as an in vitro model to study the effects of dietary or pharmaceutical compounds on inflammation in chicken macrophages.