Quantitative lymphatic vessel trait analysis suggests Vcam1 as candidate modifier gene of inflammatory bowel disease

Abstract

Inflammatory bowel disease (IBD) is a chronic debilitating disease resulting from a complex interaction of multiple genetic factors with the environment. To identify modifier genes of IBD, we used an F2 intercross of IBD-resistant C57BL/6J-Il10(-/-) mice and IBD-susceptible C3H/HeJBir-Il10(-/-) (C3Bir-Il10(-/-)) mice. We found a prominent involvement of lymphatic vessels in IBD and applied a scoring system to quantify lymphatic vascular changes. Quantitative trait locus (QTL) analyses revealed a large-effect QTL on chromosome 3, mapping to an interval of 43.6 Mbp. This candidate interval was narrowed by fine mapping to 22 Mbp, and candidate genes were analyzed by a systems genetics approach that included quantitative gene expression profiling, search for functional polymorphisms, and haplotype block analysis. We identified vascular adhesion molecule 1 (Vcam1) as a candidate modifier gene in the interleukin 10-deficient mouse model of IBD. Importantly, VCAM1 protein levels were increased in susceptible C3H/HeJ mice, compared with C57BL/6J mice; systemic blockade of VCAM1 in C3Bir-Il10(-/-) mice reduced their inflammatory lymphatic vessel changes. These results indicate that genetically determined expression differences of VCAM1 are associated with susceptibility to colon inflammation, which is accompanied by extensive lymphatic vessel changes. VCAM1 is, therefore, a promising therapeutic target for IBD.

Figure 1

Lymphatic vessels are enlarged and tortuous in colitis; the phenotype differs between C3Bir- and B6-Il10^−/− mice. (a) Representative images of wild-type (WT) and Il10^−/− C3Bir or B6 mouse colon sections at different time points stained for LYVE1 (red) and counterstained with hematoxylin. The age of mice is indicated (w = weeks). Whereas WT mice had normal lymphatic vasculature, 4-week-old C3Bir-Il10^−/− mice showed signs of inflammation and a slightly denser lymphatic vasculature. At 6 and 8 weeks of age, lymphatics were dramatically enlarged and dense. In B6-Il10^−/− mice, the lymphatic vasculature first showed changes at 6 weeks, whereas at 8 weeks, changes were less pronounced than in age-matched C3Bir-Il10^−/− mice. Scale bars: 100 mm. (b) Quantification of the lymphatic vascular phenotype in the two parental Il10^−/− strains at different time points. Summary scores of the lymphatic vascular phenotype (y axis) are shown versus age (x axis). At 6 weeks, there was the most significant difference between the strains (Po0.001). Data are expressed as mean ± s.e.m. (N = 3–5 per strain and time point). The percentage of area covered by lymphatic vessels (c) and the average size of lymphatic vessels (d) in colon hot spots are normally distributed in F2 generation Il10^−/− mice.

Figure 2

Mapping of a large-effect QTL for lymphatic vessel inflammatory changes to chromosome 3. (a) The result of interval mapping for five lymphatic vessel subphenotypes (x axis: chromosomes and marker positions, y axis: LOD score). A single locus exceeding the genome-wide 5% significance levels gave a high peak on Chr 3 for all five traits (purple dashed line indicates the significance threshold determined by permutation test). (b) Detailed view of mapped QTL on Chr 3 showing LOD score curves for the five traits, with horizontal lines of matching color indicating the span of 95% approximate Bayesian credible intervals (x axis: marker position in cM). (c) Two-dimensional genome scan performed with 243 F2 mice and 110 microsatellite markers for the total score for lymphatic vessel changes revealed no interaction of the Chr 3 QTL with other loci and a high LOD score interaction between chromosome pairs X:19 and X:18. The upper left triangle contains the interaction model LOD scores, and the lower right triangle contains the conditional-interactive model LOD scores. The color bar indicates the LOD scores for the left and right triangle, respectively.

Figure 3

Fine mapping of the Chr 3 QTL interval narrows the candidate region. (a) Genetic map showing the position of the previously genotyped microsatellite markers and the newly genotyped 18 SNP markers on Chr 3 used for fine mapping (y axis: cM marker positions). (b) Genetic mapping with an increased number of markers and F2 mice provided higher resolution peaks on Chr 3 for all five traits. (c) Detailed view of mapped QTL on Chr 3 showing LOD score curves for the five traits, with horizontal lines indicating the span of 95% approximate Bayesian credible intervals (95% significance) (x axis: marker position in cM).

Figure 4

Gene expression levels in colitis-resistant and -susceptible mouse strains, assessed by high-throughput quantitative PCR. Each circle represents a gene from the Chr 3 QTL interval. The log2 ratio of expression in C3Bir mice, compared with B6 strain is shown on the x axis and log₁₀ of Benjamini-Hochberg corrected P-values are shown on the y axis. Genes that had a statistically significant change between the two strains are shown with open circles. The genes that were expressed at levels below the threshold are shown in black. Genes that were expressed at significantly higher levels in the C3H strain are shown on the right, whereas genes that were expressed at lower levels in the C3H strain are shown on the left (N per strain = 5).

Figure 5

Haplotype block analysis excludes a large part of QTL credible interval genes. On the basis of the identical-by-descent blocks of SNPs, approximately 40% of CI was excluded from the further analysis. C57BL/6J haplotype blocks are indicated by light grey and non-identical-by-descent C3H/HeJ haplotype blocks are indicated by dark grey. Areas that lack genotype information are shown in black. Sequence coordinates of the Chr 3 CI (in Mbp) are provided at the bottom line of the image.

Figure 6

VCAM1 is expressed at higher levels in colon cells of C3H/HeJ, compared with C57BL/6J mice. Whole-cell suspensions of colon (a) were used to compare the expression levels of VCAM1 between wild-type C3H/HeJ and C57BL/6J mice. To distinguish between different cell types, 4-color FACS analysis was performed with colon cells. VCAM1 mean fluorescence intensity (MFI) values are shown for colon lymphatic endothelial cells (CD45⁻CD31⁺ podoplanin⁺) (b), and colon blood vascular endothelial cells (CD45⁻ CD31⁺ podoplanin⁻) (c). A higher expression level of VCAM1 was observed in all C3H/HeJ cell types. Data are shown from four separate experiments. Each box plot shows the median, upper, and lower quartiles as well as minimum and maximum MFI values. The paired t-test was used to calculate the P-values.

Figure 7

Systemic blockade of VCAM1 reduces lymphatic vessel inflammatory changes and distal colon inflammation in a mouse model of IBD. (a) Representative images showing the extent of inflammatory lymphatic vessel changes in colon sections from C3Bir-Il10^−/− mice injected with control (IgG) or anti-VCAM1 antibodies, immunostained for LYVE1 (red). Scale bars: 100 μm. (b) Quantitative lymphatic vessel and colitis analysis was performed on colon sections of anti-VCAM1-treated mice (filled triangles) and control IgG-treated mice (filled squares). A significant reduction of the total score for lymphatic vessel changes in the colon wall area and distal colon inflammation was observed (*P<0.05). Horizontal lines represent the mean values (N mice IgG group = 8, N mice anti-VCAM1 group = 6).