Melatonin Modulates ZAP70 and CD40 Transcripts via Histone Modifications in Canine Ileum Epithelial Cells

Abstract

Melatonin (MLT), produced by the pineal gland and other tissues, is known for its anti-inflammatory effects, particularly in regulating inflammatory markers and cytokines in intestinal cells. Our study aimed to investigate how MLT influences the expression of inflammatory genes through histone modification in canine ileum epithelial cells (cIECs). In our experiment, cIECs were cultured and divided into a control group (CON) and an MLT-treatment group. MLT did not significantly affect cell growth or death in cIECs compared to the CON. However, MLT treatment led to an upregulation of CD40, ZAP70, and IL7R and a downregulation of LCK, RPL37, TNFRSF13B, CD4, CD40LG, BLNK, and CIITA at the mRNA expression level. Moreover, MLT significantly altered the NF-kappa B signaling pathway by upregulating genes, such as CD40, ZAP70, TICAM1, VCAMI, GADD45B, IRAK1, TRADD, RELA, RIPK1, and RELB, and downregulating PRKCB, LY96, CD40LG, ILIB, BLNK, and TNFRSF11A. Using ChIP-qPCR, we discovered that MLT treatment enhanced histone acetylation marks H3K9ac, H3K18ac, H3K27ac, and methylation marks H3K4me1 and H3K4me3 at the ZAP70 and CD40 gene loci (p < 0.05). Additionally, the enrichment of RNA polymerase II and phosphorylated Ser5 pol-II at these loci was increased in MLT-treated cells (p < 0.05), indicating heightened transcriptional activity. In conclusion, our findings suggest that MLT mitigates inflammation in cIECs by modulating the transcription of ZAP70 and CD40 through histone modifications, offering potential therapeutic insights for inflammatory bowel diseases.

Keywords: canine; histone modifications; ileum; melatonin; transcriptome.

Figure 1

MLT treatment does not affect cell survival and growth in canine epithelial cells. (A): MLT treatment did not significantly affect cell growth in canine ileum epithelial cells. (B) MLT treatment did not substantially affect cell death detected by caspase3/7 activities in canine ileum epithelial cells. (C,D) The whole-genome expression heat map and differential gene volcano plot of the CON and MLT group reflect the overall impact of MLT treatment on gene expression; the results show no significant differences between the two groups. (E) GSEA dynamic analysis to draw the top ranked pathways with significant differences in ES score. (F) There was no significant upregulation or downregulation of the cell cycle and proliferation genes due to MLT treatment in the cells (p > 0.05).

Figure 2

Functional annotation pathway enrichment result. (A–C) To “Enrichment of biological processes and pathways for differential genes, focusing on significant enrichment of primary immunodeficiency and NF-κB signaling pathways. (D) PPI (protein interaction) network diagram of significantly changed pathways and related genes enriched by KEGG. (E) Network diagram of the interaction between enriched pathways and cellular processes. Panels (D,E) highlight genes, pathways, and processes for further exploration.

Figure 3

The gene expression of primary immunodeficiency and NF-κB signaling pathway genes of cIECs affected by MLT treatment. Gene expression of primary immunodeficiency and NF-kappa B signaling pathway genes in cIECs affected by MLT treatment. (A) Functional network diagram of upregulated differential genes in inflammation- and immune-related pathways. (B) Functional network diagram of downregulated differential genes in inflammation- and immune-related pathways. (C) Heat map of core gene expression in primary immunodeficiency pathways. (D) Heat map of core gene expression in NF-κB signaling pathways. (E,F) Relative mRNA expression of inflammation- and immune-related pathway genes, consistent with sequencing results. Data are presented as means ± SEM (n = 3). * p < 0.05; ** p < 0.001.

Figure 4

Histone modifications on the ZAP70 and CD40 genes. (A) Demethylation pathway enrichment in MLT-treated cells using GSEA. (B) Heat map of core gene expression of demethylation enzymes. (C–E) Histone acetylated marks measured by ChIP-qPCR in the MLT-treated cells. (F,G) Histone methylated marks measured by ChIP-qPCR. (H,I) Histone hydroxybutyrylated marks measured by ChIP-qPCR. (J,K) RNA polymerase II and phosphorylation at Ser5 measured by ChIP-qPCR. Data are indicated as means ± SEM (n = 3). *** p < 0.0001.

Figure 5

Melatonin treatment could upregulate the expression of ZAP70 and CD40 and other core genes related to inflammation and immune response via histone modification.