We used the Il10−/− mouse model of colon inflammation to identify genes that modify susceptibility to IBD, a disease with rising incidence in developed countries. Genetic mapping, together with lymphatic vessel phenotyping, enabled us to identify a large-effect QTL on Chr 3. We identified VCAM1 as a candidate modifier gene for IBD by combining a systems genetics approach, quantitative analysis of protein expression levels, and an in vivo function-blocking approach.
Several mouse models of IBD have been proposed.1
model faithfully reflects the human IBD processes in several aspects, namely spontaneous chronic disease development because of immune dysregulation, Th1/Th17 polarized response, and intestinal microflora phenotype dependence.22
Moreover, a SNP near the 3′ UTR of the Il10
gene was consistently replicated in human genome-wide ulcerative colitis association studies and low ileal IL10 was found to be predictive of Crohn's disease recurrence.5,35
model is also valuable because the phenotype ranges from mild to severe, depending on the genetic background of the mouse.23
A colitis QTL (cytokine deficiency-induced colitis susceptibility; Cdcs1
) was previously mapped to the distal part of Chr 3 by scoring colon mucosal inflammation.28,29
We found a high degree of correlation between the mucosal inflammation phenotype and the lymphatic vessel phenotype in colons of F2 mice. Importantly, the Cdcs1
interval spans between 62 and 70 cM with the peak marker at 61.8 cM,28,36
thus overlapping at its proximal end with the interval mapped scoring the lymphatic vessel traits. Further subcongenic analysis revealed high genetic complexity of Cdcs1 with likely more than one gene contributing to the phenotype.37
In this study, we have evaluated the thus-far neglected inflammatory changes that occur in the lymphatic vasculature, in contrast to the earlier investigations of the `classical' colitis symptoms of epithelial damage and inflammatory cell accumulation. This new approach of scoring related, but distinct quantitative traits enabled us to identify Vcam1
, within the Cdcs1
interval, as a modifier of lymphatic vessel inflammatory changes, underlining the complexity of Cdcs1
Lymphatic vessels undergo significant changes during inflammation, and a higher density of lymphatic vessels was also observed in the colons of IBD patients.26
Moreover, when normal and diseased human bowel was studied in Crohn's disease patients, lymphatic failure was found in some apparently unaffected areas, and further examination of these areas confirmed the presence of early IBD.38
Pathologists have also noted that marked edema of the submucosa with enlarged lymphatic capillaries is present without mucosal ulceration, pointing to the involvement of lymphatic vessels in the early stages of human IBD.39
There are two possible reasons for lymphatic vessel enlargement in inflamed tissue. Lymphatic endothelial cells might, due to the presence of activating inflammatory signals, start to proliferate, resulting in enlarged vessel diameters.9
On the other hand, lymphatics might be enlarged because they are dilated within the edematous tissue and have lost their draining function. This could be caused by increased blood vessel permeability or by an obstruction further down the collecting lymphatic vessels that is created by cell, bacteria, and protein debris, which are abundant in chronically inflamed tissues.17,38
In the normal colon, lymphatic vessels are located longitudinally, in a narrow area beneath the muscularis mucosae, with rare branches that reach the most basal aspect of the crypts and with perpendicular branches that reach deep into submucosa.26
As an additional trait, we analyzed the invasion of lymphatic vessels into the lamina propria between the epithelial crypts. The observation of lymphatics in the mucosa of inflamed colon clearly shows active lymphangiogenesis, because under normal physiological conditions, no lymphatics are found at that site.
Angiogenesis has been reported to have an important function in the pathogenesis of IBD.6
Surprisingly, we found that lymphatic vessel changes observed in mice with colitis were more dramatic than the blood vessel changes observed; area covered by blood vessel endothelium did not change significantly in colitis. These findings indicate that the numerous earlier studies that investigated the CD31+
vasculature in IBD overlooked the fact that lymphatic vessels also express CD31.40
Thus, some of the earlier reported blood vascular changes might indeed represent alterations of the lymphatic vasculature.41,42
The scoring of the lymphatic inflammatory changes observed in Il10−/− mice proved to be a very sensitive method of analysis; it enabled us to map a large-effect QTL that spanned 43.6 Mbp on Chr 3. Using SNP markers and a larger number of F2 mice, we were able to narrow the CI to only 22 Mbp on Chr 3.
Nonetheless, the identification of genes that determine IBD susceptibility remained a challenging task that required a systems genetics approach. High-throughput gene expression analysis is a suitable complementary approach to QTL analysis;43
we performed low-density array qPCR analysis, which enabled us to simultaneously quantify the expression of 87 Chr 3 QTL interval genes and to identify Vcam1
as the most plausible candidate; its expression was significantly higher in colons of C3Bir-Il10−/−
compared with B6-Il10−/−
Recently, a sequence-based variation map of 8.27 million SNPs in inbred mouse strains became available, leading to the generation of a mouse haplotype map.32
Laboratory mice have been inbred for the relatively short time of 100 years, so an estimated 97% of polymorphisms are ancestral, providing a 3% chance that a causative QTL lies inside an identical-by-descent block.20,43
Using the haplotype block predictor tool, we were thus able to exclude a large part of the Chr 3 QTL CI, reducing the number of genes in this region from 220 to 89.
The in silico search for polymorphic SNPs that might influence protein abundance, function, or stability led to the identification of four functional SNPs associated with Vcam1. As the polymorphisms reported for the C3H/HeJ strain might not necessarily be present in the C3Bir substrain, we investigated by sequencing the most promising candidate SNP in the Vcam1 gene and confirmed its effect on the secondary protein structure by a prediction tool.
This is the first report of differential mRNA and protein expression levels of VCAM1 in the C3H versus the C57BL/6J mouse strain. Although with the chosen dosage and timing of the blocking antibody treatment only distal colon inflammation was significantly diminished, the ability of a VCAM1 neutralizing antibody to reduce the lymphatic vessel changes associated with colitis in C3Bir-Il10−/−
mice supports the concept that VCAM1 has a function in IBD pathogenesis, along with results from genome-wide association studies of human IBD44
and other models.45,46
VCAM1 expression by blood vessel endothelium is required for leukocyte entry into sites of inflammation.7,47
Therefore, reagents designed to block VCAM1 function in vivo
could reduce the number of immune effector cells that localize to the colon and diminish IBD severity. Importantly, however, VCAM1 is also upregulated on the lymphatic endothelium during inflammation, which was recently shown to occur in inflamed human small intestine and a mouse model of skin contact hypersensitivity,13,48
and seems to be required for the process of leukocyte transmigration across lymphatic endothelium to the draining lymph nodes.13,49
There are several possibilities how VCAM1 may contribute to the lymphatic vessel phenotype in IBD. Elevated expression of VCAM1 in susceptible individuals could allow for a higher rate of leukocyte infiltration into the tissue, resulting in higher levels of lymphangiogenic factors. In chronic inflammatory states, such as IBD, the persistence of mucosal antigens supports a switch from the innate to the adaptive immune response and a subsequent change in the inflammatory infiltrate. A crucial step allowing for this switch is the transport of antigen-presenting cells from inflamed tissue, via lymphatic vessels, to the draining lymph nodes in which lymphocyte priming occurs. Thus, it is likely that because of the role of VCAM1 in regulating dendritic cell egress from the tissue,13
its elevated expression on lymphatic vessels supports a higher rate of lymphocyte priming in the draining lymph nodes. Increased levels of circulating VCAM1 were shown in Crohn's disease patients and VCAM1 upregulation was reported in bowel EC isolated from IBD patients.50,51
Further support for the role of VCAM1 in IBD pathogenesis comes from studies showing the efficacy of natalizumab, a humanized antibody against the a4 integrin subunit, in treatment of patients with IBD;52
the integrins α4b1 and α4b7 are expressed by leukocytes and are VCAM1 ligands.
We show that a mouse systems genetics approach represents a powerful method for studying complex diseases; we used it to identify VCAM1 as a modifier gene for IBD. Our findings also reveal the importance of lymphatic vessel changes for the pathogenesis of IBD. The molecular evidence we present indicates that reagents designed to target adhesion molecules that mediate leukocyte adhesion to the activated lymphatic vasculature might be effective in treating patients with IBD.