Quantitative trait loci in a bacterially induced model of inflammatory bowel disease

Abstract

Inflammatory bowel diseases (IBDs) are complex disorders caused by a combination of environmental, microbial, and genetic factors. Genome-wide association studies in humans have successfully identified multiple genes and loci associated with disease susceptibility, but the mechanisms by which these loci interact with each other and/or with environmental factors (i.e., intestinal microbiota) to cause disease are poorly understood. Helicobacter hepaticus-induced intestinal inflammation in mice is an ideal model system for elucidating the genetic basis of IBD susceptibility in a bacterially induced system, as there are significant differences in H. hepaticus-induced disease susceptibility among inbred mouse strains. Infected A/J mice develop acute overexpression of proinflammatory cytokines followed 2-3 months later by chronic cecal inflammation, whereas infected C57BL/6 mice fail to develop cecal inflammation or increased cytokine expression. The goal of this project was to use quantitative trait locus (QTL) mapping to evaluate genetic factors that contribute to the differential disease susceptibility between these two mouse strains. Using acute cecal IL-12/23p40 expression as a biomarker for disease susceptibility, QTL analysis of H. hepaticus-infected F(2) mice revealed involvement of multiple loci. The loci with the strongest association were located on Chromosome 3 and Chromosome 17, with logarithm of odds (LOD) scores of 6.89 and 3.09, respectively. Cecal expression of IL-12/23p40 in H. hepaticus-infected C57BL/6J-Chr3(A/J)/NaJ chromosome substitution mice had an intermediate phenotype, significantly higher than in resistant C57BL/6 but lower than in susceptible A/J mice, confirming the importance of this locus to the immune response to H. hepaticus infection.

Fig. 1

Susceptibility to Helicobacter hepaticus in F₁ crosses at 4 and 90 days post-inoculation. a Consistent with previous studies, A/J mice expressed significantly higher mean levels of IL-12/23p40 normalized to HPRT (p < 0.05) than did C57BL/6 mice at 4 days post-inoculation with Helicobacter hepaticus. Mean expression levels of IL-12/23p40 in AB6 F₁ mice were similar to those seen in C57BL/6 mice. In contrast, B6A F₁ mice had a slight but significant (p < 0.05) increase in p40 expression when compared to C57BL/6 controls. At 90 days post-inoculation, A/J mice had significantly higher lesion scores (b) and IL-12/23p40 expression (c) when compared to all other strains. Female A/J mice had higher lesion scores and cytokine expression than males. F female, M male

Fig. 2

Early expression of IL-12/23p40 in F₂ mice and QTL analysis. Expression of IL-12/23p40 at 4 days post-inoculation was used as a biomarker for disease susceptibility in 314 Helicobacter hepaticus-infected F₂ mice. a Distribution of phenotypes in the F₂ samples ranged from high (A/J-like) to low IL-12/23p40 expression (C57BL/6-like). b QTL analysis of 314 F₂ mice revealed significant QTLs on Chromosome 3 at mCV23483645 and Chromosome 17 at rs6239530, with LOD scores of 6.89 and 3.09, respectively, associated with increased expression of IL-12/23p40 upon exposure to H. hepaticus. Hatch marks along the X axis indicate the position of individual markers and vertical dotted lines indicate chromosomal boundaries. Loci were considered significant if the LOD score reached an α threshold >0.05

Fig. 3

Influence of sex on IL-12/23p40 expression-associated QTL. a Linkage analysis of the 97 F₂ female mice revealed two QTLs with significant LOD scores: a QTL on Chromosome 3 at mCV23483645 with a LOD score of 4.23 and a QTL on Chromosome 11 at rs13481045 with a LOD score of 3.16. b Analysis of 88 F₂ male mice revealed two QTLs on Chromosome 3 at D3Mit98 and D3Mit57 with LOD scores of 3.13 and 3.09, respectively. In addition, a QTL was identified on Chromosome 17 at marker rs6239530 with a LOD score of 3.47

Fig. 4

Early expression of IL-12/23p40 in subgroups of F₂ mice and QTL analysis. QTL analysis was performed on F₂ subsamples partitioned according to the maternal cross from which they were derived. a The mean IL-12/23p40 expression in F₂ mice derived from a B6A F₁ mother was significantly higher than the mean expression in F₂ mice derived from an AB6 F₁ mother. Analysis of individual F₂ crosses of b AB6 × AB6 F₂ mice and c B6A × B6A F₂ mice which consistently identified the locus on Chromosome 3 but not the Chromosome 17 QTL. Analysis of the B6A × B6A F₂ cross identified a QTL on Chromosome 7 that was not identified in the original scan

Fig. 5

Response of C57BL/6-Chr3^A/J mice to Helicobacter hepaticus at 4 days and 90 days post-inoculation. To test the effect of the QTL identified on Chromosome 3, C57BL/6J-Chr3^A/J/NaJ chromosome substitution mice (B6.A-Chr3) were inoculated with Helicobacter hepaticus and assessed for IL-12/23p40 expression at 4 and 90 days post-inoculation and cecal inflammation 90 days post-inoculation. a At 4 days post-inoculation, cecal IL-12/23p40 expression was significantly increased in B6.A-Chr3 mice compared to the resistant C57BL/6 controls. Expression of IL-12/23p40 was also significantly lower in the B6.A-Chr3 mice than in the susceptible A/J controls. b Expression of IL-12/23p40 90 days post-inoculation was increased in B6.A-Chr3 mice relative to C57BL/6 controls, but was not increased to the levels of the susceptible A/J controls. c While IL-12/23p40 expression was greater in B6.A-Chr3 mice than in C57BL/6 mice, the increase was not sufficient to increase the overall inflammation in the ceca of infected animals. Inflammation scores in the B6.A-Chr3 mice were not significantly different than in the resistant C57BL/6 controls