Transcriptomics analysis of allergen-induced inflammatory gene expression in the Four-Core Genotype mouse model

Abstract

Sex differences in allergic inflammation have been reported, but the mechanisms underlying these differences remain unknown. Contributions of both sex hormones and sex-related genes to these mechanisms have been previously suggested in clinical and animal studies. Here, Four-Core Genotypes (FCG) mouse model was used to study the inflammatory response to house dust mite (HDM) challenge and identify differentially expressed genes (DEGs) and regulatory pathways in lung tissue. Briefly, adult mice (8-10 wk old) of the FCG (XXM, XXF, XYM, XYF) were challenged intranasally with 25 μg of HDM or vehicle (PBS-control group) 5 days/wk for 5 wk (n = 3/10 group). At 72 h after the last exposure, we analyzed the eosinophils and neutrophils in the bronchoalveolar lavage (BAL) of FCG mice. We extracted lung tissue and determined DEGs using Templated Oligo-Sequencing (TempO-Seq). DEG analysis was performed using the DESeq2 package and gene enrichment analysis was done using Ingenuity Pathway Analysis. A total of 2,863 DEGs were identified in the FCG. Results revealed increased eosinophilia and neutrophilia in the HDM-treated group with the most significantly expressed genes in XYF phenotype and a predominant effect of female hormones vs. chromosomes. Regardless of the sex hormones, mice with female chromosomes had more downregulated genes in the HDM group but this was reversed in the control group. Interestingly, genes associated with inflammatory responses were overrepresented in the XXM and XYF genotypes treated with HDM. Sex hormones and chromosomes contribute to inflammatory responses to HDM challenge, with female hormones exerting a predominant effect mediated by inflammatory DEGs.NEW & NOTEWORTHY Gene expression profiling helps to provide deep insight into the global view of disease-related mechanisms and responses to therapy. Using the Four-Core Genotype mouse model, our findings revealed the influence of sex hormones and sex chromosomes in the gene expression of lungs exposed to an aeroallergen (House Dust Mite) and identified sex-specific pathways to better understand sex disparities associated with allergic airway inflammation.

Keywords: Four-Core Genotypes; allergic inflammation; lung inflammation; sex chromosomes; sex hormones.

Graphical abstract

Figure 1.

Eosinophil and neutrophil counts in the FCG mouse model of airway inflammation in the FCG mice following chronic exposure to HDM, in FCG mice with male or female chromosomes, and in the FCG mice with male or female hormones. Changes in the different groups were not statistically significant (n = 7–10, P value < 0.05). A: nonsignificant increasing trend in eosinophil and neutrophil counts in the XYF genotype mice exposed to HDM compared with the other genotypes. B: nonsignificant increasing trend in eosinophils and neutrophils in the male and female chromosome HDM-induced mice compared with the control. C: nonsignificant increasing trend in eosinophil and neutrophil counts in the female hormone mice, induced with HDM compared with the male hormone mice. FCG, Four-Core Genotype; HDM, house dust mite.

Figure 2.

Numbers of DEGs in HDM-challenged mice over PBS-administered groups in the FCG mouse model (N = 3). Red part represents the upregulated DEGs, whereas the blue part represents the downregulated DEGs. More genes were upregulated in all the genotypes except in the XXM mice that have more genes downregulated (DESeq2 FDR < 0.05). DEG, differentially expressed gene; FCG, Four-Core Genotype; PBS, phosphate-buffered saline.

Figure 3.

A: numbers of significantly expressed genes in the HDM-challenged mice over the control. Venn diagram shows the number of genes that were significantly expressed comparing the HDM-challenged groups with the control mice in all the genotypes (N = 3). The diagram also shows the interception of these genes among the group. Only the XXF and the XXM had one gene, GM20831, in common while the XYF had the most significantly expressed genes. B: numbers of significantly expressed genes in the HDM-challenged mice. Venn diagram shows the number of genes that were significantly expressed in the HDM-challenged mice (N = 3). The different genotypes were paired and the HDM-challenged mice in XXM>XXF had the highest number of significantly expressed genes compared with the other pairs. Some genes were intercepted between the XXM vs. XXF and XYM vs. XXM pairs and with the XXM vs. XXF and XYM vs. XYF pairs. HDM, house dust mite.

Figure 4.

Volcano plots identifying the significantly expressed genes in the HDM-challenged over the control mice in the four core genotypes. A: significantly expressed genes in the HDM-challenged over the control mice in the XXF genotype. Volcano plot shows three genes that were significantly expressed in the XXF HDM-challenged mice compared with the control group (N = 3). One of the genes was downregulated while the other two were upregulated. B: significantly expressed genes in the XXM genotype. Volcano plot shows the 6 genes that were significantly expressed in the XXM HDM-challenged mice compared with the control group (N = 3). Only one of the genes was upregulated while the others were downregulated. C: significantly expressed genes in XYM genotype. Volcano plot shows the 6 genes that were significantly expressed in the XYM HDM-challenged mice compared with the control group (N = 3). All the significantly expressed genes in this genotype were upregulated. D: significantly expressed genes in the XYF genotype. Volcano plot shows the 6 genes that were significantly expressed in the XYF HDM-challenged mice compared with the control group (N = 3). Many of the significantly expressed genes in this genotype were upregulated while only three of them were downregulated. HDM, house dust mite.

Figure 5.

Volcano plots identifying the significantly expressed genes in the HDM-challenged mice when comparing the four core genotypes. A: significantly expressed DEGs in HDM-challenged XXF vs. XYF mice. Volcano plot shows the only gene, IGLC1, significantly downregulated in the HDM-challenged mice of the XXF vs. XYF genotypes (N = 3). B: significantly expressed DEGs in HDM-challenged XXM vs. XXF mice. This pair had the most significantly expressed genes (746 genes) with about one-third upregulated. C: DEGs in the HDM-challenged XYM vs. XXF mice. Numerous genes were identified but they were not statistically significantly expressed (DESeq2 FDR ≤ 0.05). D: DEGs in HDM-challenged XYM vs. XXM mice. Plot shows 40 DEGs in the HDM-challenged mice of the XYM and XXM genotypes (DESeq2 FDR ≤ 0.05). E: significantly expressed DEGs in HDM-challenged XYM vs. XYF mice. Plot shows 12 DEGs (downregulated). F: significantly expressed DEGs in the HDM-challenged female vs. male mice. Volcano plot shows the genes that were significantly expressed in the female and male mice after HDM exposure. Most of the genes were upregulated. In previous studies, some of the genes that were known to be anti-inflammatory were significantly downregulated in female mice. DEG, differentially expressed gene; HDM, house dust mite.

Figure 6.

Graphic summary of the pathways identified by IPA to be activated or inhibited relating to the DEGs of FCG mice model following HDM induction. A: identified pathways to be activated or inhibited in the XXM genotype exposed to HDM. B: identified pathways to be activated or inhibited in the XXF genotype exposed to HDM. C: identified pathways to be activated or inhibited in the XYM genotype exposed to HDM. D: identified pathways to be activated or inhibited in the XYF genotype exposed to HDM. DEG, differentially expressed gene; FCG, Four-Core Genotype; HDM, house dust mite; IPA, Ingenuity Pathway Analysis.