Epigenomic insights into the immune regulatory mechanism of GBP4L in poultry

Abstract

High-temperature environments induce heat stress in poultry, leading to compromised immune function and reduced disease resistance. In the Dexamethasone-induced stress-related immune suppression model in chickens, both transcriptomic and proteomic analyses revealed significantly low expression of GBP4L, highlighting its potential as a key target for combating stress. It is currently unclear whether GBP4L can alleviate the heat stress response in the body. We conducted this study to explore the role of GBP4L in stress resistance. First, to evaluate the role of GBP4L in immune function, we established an inflammatory cell model via Lipopolysaccharide (LPS) treatment and then generated both a pcDNA3.1-EGFP-GBP4L plasmid and a si-GBP4L interference fragment for transfection into HD11 model cells. Next, we exposed 28-day-old Gushi roosters to high heat (32 ± 1 °C) for 4 weeks to establish a heat stress model. We administered a virus carrying pAAV-GBP4L-3FLAG via subcutaneous injection, evaluated immune marker levels and conducted ATAC-seq on spleen tissues to investigate the effect of GBP4L on chromatin accessibility. The results revealed that GBP4L overexpression reduced the expression of proinflammatory factors, promoted the polarization of HD11 cells from the M1 phenotype to the M2 phenotype, reduced the LPS-induced expression of IL-8 (P < 0.05), alleviated inflammation, increased cell proliferation (P < 0.05), and inhibited apoptosis (P < 0.05). In the animal model, increasing the expression of GBP4L alleviated heat stress-induced inflammation, improved blood biochemistry, enhanced immune function in the spleen and bursa of Fabricius, and preserved the structure of the spleen. ATAC-seq revealed that GBP4L reduced chromatin accessibility in the promoter regions of 34 heat stress-induced genes. Furthermore, the expression of SP9 was significantly increased in animals under heat stress but was decreased in animals overexpressing GBP4L under heat stress. In conclusion, GBP4L alleviates heat stress-induced inflammation, enhances immune status, and reduces spleen tissue damage. Mechanistically, GBP4L overexpression may enhance heat stress resistance by altering chromatin spatial structure to regulate SP9 expression. The research findings offer valuable insights into the mechanisms of heat stress resistance in poultry, contributing to the development of strategies to enhance heat tolerance. These results also expand the epigenetic regulation theory of heat tolerance and support the breeding of heat-tolerant chicken varieties.

Keywords: ATAC-seq; Chromatin accessibility; GBP4L; Heat stress; SP9.

Fig. 1

The effect of GBP4L on inflammatory cytokines. (A and B) Detection of transfection efficiency of pcDNA3.1-EGFP-GBP4L and si-GBP4L. (C and D) Effect of overexpression of GBP4L and interfering with GBP4L on inflammatory factors in HD11 cells. Data are mean ± SEM. “*” means P < 0.05, “**” means P < 0.01, “***” means P < 0.001.

Fig. 2

The effects of GBP4L on the proliferation and apoptosis of HD11 cells. (A - F) Effects of Gallus GBP4L on HD11 cells proliferation by CCK8 assay, qPCR and EDU. (G and H) Effects of Gallus GBP4L on HD11 cells cycle. (I - L)Effects of Gallus GBP4L on HD11 cells apoptosis. Data are mean ± SEM. “*” means P < 0.05, “**” means P < 0.01, “***” means P < 0.001.

Fig. 3

GBP4L improves LPS induced inflammatory reactions. (A) The expression of IL-8 stimulated by different concentrations of LPS for 9 h. (B) The expression of IL-8 stimulated by 1 μg/mL LPS at different times. (C - F) After transfecting HD11 cells with the pcDNA3.1-EGFP-GBP4L plasmid and si-GBP4L interference fragment for 24 hours, the cells were stimulated with 1 μg/mL LPS for 9 hours, and the expression levels of GBP4L and IL-8 were measured. Data are mean ± SEM. “*” means P < 0.05, “**” means P < 0.01, “***” means P < 0.001.

Fig. 4

The impact of GBP4L on the immune-related factors of heat-stressed chickens. (A) The expression results of recombinant GBP4L adeno-associated virus in vivo. (B - K) Effect of GBP4L on immune-related factors in heat-stressed chickens. l Effect of GBP4L on spleen tissue structure of heat-stressed chicken. Data are mean ± SEM. “*” means P < 0.05, “**” means P < 0.01, “***” means P < 0.001.

Fig. 5

Identification and distribution of open chromatin regions. (A) Tn5 transposable enzyme length distribution map. (B) TSS signal distribution from TES. (C) Peak histogram identified in spleen chromatin after three different treatments. (D) Pie chart of Peak distribution in the genome after three different treatments. (E) The openness of the promoter (≤3 kb) region after three different treatments.

Fig. 6

Identification and distribution of open chromatin regions. (A - C) Differential chromatin regions in spleen after different treatments. (D - F) The distribution of differential chromatin regions in the spleen. (G) Distribution of promoters in the splenic region of differential chromatin. (H) Compared with the control group, chromatin openness in the promoter region was enhanced in the remaining two groups.

Fig. 7

Differential transcription factors were predicted and their activity tested in the three treatments. (A) The top thirty differentially expressed TFs identified in regions of increased chromatin accessibility among the three groups are presented below. (B) The top thirty differentially expressed TFs identified in regions of decreased chromatin accessibility among the three groups are listed below. (C) Analysis of transcription factor (TF) footprints across the three groups following different treatments. (D) SP9 motif diagram. (E) Vitality scores for SP9 across the three groups.