Identification of novel loci controlling inflammatory bowel disease susceptibility utilizing the genetic diversity of wild-derived mice

Abstract

Inflammatory bowel disease (IBD) is a complex disorder that imposes a growing health burden. Multiple genetic associations have been identified in IBD, but the mechanisms underlying many of these associations are poorly understood. Animal models are needed to bridge this gap, but conventional laboratory mouse strains lack the genetic diversity of human populations. To more accurately model human genetic diversity, we utilized a panel of chromosome (Chr) substitution strains, carrying chromosomes from the wild-derived and genetically divergent PWD/PhJ (PWD) strain on the commonly used C57BL/6J (B6) background, as well as their parental B6 and PWD strains. Two models of IBD were used, TNBS- and DSS-induced colitis. Compared with B6 mice, PWD mice were highly susceptible to TNBS-induced colitis, but resistant to DSS-induced colitis. Using consomic mice, we identified several PWD-derived loci that exhibited profound effects on IBD susceptibility. The most pronounced of these were loci on Chr1 and Chr2, which yielded high susceptibility in both IBD models, each acting at distinct phases of the disease. Leveraging transcriptomic data from B6 and PWD immune cells, together with a machine learning approach incorporating human IBD genetic associations, we identified lead candidate genes, including Itga4, Pip4k2a, Lcn10, Lgmn, and Gpr65.

Figure 1.. PWD mice are resistant to DSS-induced colitis.

A 5% solution of DSS was administered to B6 and PWD mice in drinking water from days 0 – 5. Weight loss (A), disease activity index (B), were monitored as described in Materials and Methods. Disease activity index was calculated based on weight loss, stool consistency, and presence of blood in stool. Fecal LCN2 levels (C) were measured by ELISA. (D) Animals were euthanized on day 10, colons were fixed and processed for H&E sections, followed by semi-quantitative evaluation, as described in the Materials and Methods. Significance of differences between B6 and PWD were determined by two-way ANOVA with Holm-Sidak post-hoc comparisons in (A–C), and by Mann Whitney test in (D), and are indicated as follows: *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. Numbers of animals studied of each sex are provided in Table 1. These studies were performed in parallel with those in Fig. 3.

Figure 2.. PWD mice are highly susceptible to TNBS-induced colitis.

B6 and PWD mice received an intracolonic enema of 200 mg/kg TNBS in 50% ethanol, followed by monitoring of weight loss (A), disease activity index (B), and survival (C). Significance of differences were determined by two-way ANOVA with Holm-Sidak post-hoc comparisons in (A) and (B), and by Mantel-Cox test in (C), and are indicated as follows: *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. Numbers of animals studied were as follows: B6 (N=7, 4 males and 3 females) and PWD (N=6, 3 males and 3 females). These mice represent an independent cohort from those studied in Fig. 4.

Figure 3.. PWD loci regulate susceptibility to DSS-induced colitis.

DSS colitis was induced and evaluated in B6.Chr^PWD consomic strains and B6 controls as described in Fig. 1. (A) Survival is shown for B6 mice and any consomic strains that had less than 100% survival. (B) Average weight loss was calculated as the average % weight loss across all days that were measured. (C) Average disease activity was calculated as the average disease activity score across time points measured, excluding D0 (D3, D5, D7, D10). (D) Disease activity scores at the indicated time points. (E) LCN2 levels were measured by ELISA at the indicated time points. (F) Colonic tissue was collected from surviving animals on D10 and evaluated by semiquantitative histology as in Fig. 1. Significance of differences between each consomic strain and B6 controls was calculated using: Mantel-Cox test in (A); ordinary one-way ANOVA with Dunnet’s post-hoc comparisons in (B) (C), and (E); Kruskal-Wallis one-way ANOVA with Dunn’s post-hoc comparisons in (D), and (F), and are indicated as follows: and are indicated as follows: *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. Numbers of animals studied of each sex are provided in Table 2.

Figure 4.. PWD loci regulate susceptibility to TNBS-induced colitis.

DSS colitis was induced and evaluated in B6.Chr^PWD consomic strains and B6 controls as described in Fig. 2. (A) Survival is shown for all mice. (B) Average weight loss was calculated as the average % weight loss across all days that were measured. (C) Average disease activity was calculated as the average disease activity score across time points measured, excluding D0 (D4, D7, D10). (D) Disease activity scores at the indicated time points. (E) LCN2 levels were measured by ELISA at the indicated time points. (F) Colonic tissue was collected from surviving animals on D10 and evaluated by semi-quantitative histology as in Fig. 1. Significance of differences between each consomic strain and B6 controls was calculated using: Mantel-Cox test in (A); ordinary one-way ANOVA with Dunnet’s post-hoc comparisons in (B) (C), and (E); Kruskal-Wallis one-way ANOVA with Dunn’s post-hoc comparisons in (D), and (F), and are indicated as follows: and are indicated as follows: *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. Numbers of animals studied of each sex are provided in Table 2.

Figure 5.. B6.Chr12^PWD mice are highly susceptible to DSS-induced colitis.

A 5% solution of DSS was administered in drinking water as in Fig. 2, followed by monitoring of weight loss (A), disease activity index (B), and survival (C). Weights and disease activity are shown up until day 7, since after this point data for some mice would be missing due to mortality. Significance of differences were determined by two-way ANOVA with Holm-Sidak post-hoc comparisons in (A) and (B), and by Mantel-Cox test in (C), and are indicated as follows: **, P<0.01; ***, P<0.0001. Numbers of animals studied were as follows: B6 (N=6, 3 males and 3 females), Chr12^PWD (N=6, 4 males and 3 females). The B6 animals in this figure represent an independent cohort from those in Fig. 2, with colitis induced simultaneously with Chr12^PWD mice.

Figure 6.. Identification and prioritization of novel gene candidates associated with IBD susceptibility.

(A) Schematic illustrating the two distinct workflows that were used to identify and prioritize candidate genes. IBD 1:1 candidates were identified by the overlap of highly differentially expressed genes in B6 vs. PWD immune cells and IBD GWAS candidate genes (see C). Scores from tissue-specific “Intestine” and “Hemolymphoid” network SVMs that were trained with IBD GWAS genes were integrated with differential expression to identify functional IBD candidates (see B, D–F). (B) SVM using the intenstine network was integrated with the differentially expressed gene dataset from PWD and B6 Tregs. Significantly differentially expressed genes from chromosome 1 and 2 are plotted by normalized −log₁₀(p-value) on the x-axis and normalized SVM −log₁₀(false positive rate) (FPR)_SVM on the y-axis. FPR_SVM and p-values were normalized by their maximum value (see Methods). Orange points denote genes located on the Pareto front, represented by the blue line. Red points denote the top ten ranked genes based on the final score, which combines the count of the number of genes that have both a lower −log₁₀(p-value) and −log₁₀(FPR). Orange and red indicates top ranked genes that also fall on the Pareto front. SVM_FPR vs. differential expression plots for each of the five immune cell subtypes from the intestine and hemolymphoid network SVMs can be found in Fig. S4. Heatmaps of differential gene expression between PWD and B6 immune cell types (log2(fold change PWD/B6)) for the top candidate genes identified by the 1:1 IBD candidate approach (C) or the SVM approach (D). Purple and pink denote extreme differential expression values, and gray denotes genes whose differential expression was not significant (false discovery rate <0.05). (E) Overlap between genes identified using IBD 1:1 candidate approach and the intestine and hemolymphoid networks SVMs. (F) Functional connectivity of the top 1:1 IBD candidate genes on Chr1 and Chr2 with 10 most functionally connected predicted interactor genes was visualized using the FNTM tool. (G) Genes from the Ccc1 locus are plotted by their genomic location on the x-axis and normalized −log₁₀(FPR_SVM) on the y-axis. Each gene has a score from the intestine and hemolymphoid network SVM, and the size of each point indicates normalized significance of differential expression (“DE norm log₁₀(P)”). The names of top ten genes (ranked by SVM score) from each network are annotated.