Genes and Immunity (2010) 11, 219–231 & 2010 Macmillan Publishers Limited All rights reserved 1466-4879/10 $32.00 www.nature.com/gene ORIGINAL ARTICLE Quantitative lymphatic vessel trait analysis suggests Vcam1 as candidate modifier gene of inflammatory bowel disease G Jurisic1, JP Sundberg2, A Bleich3, EH Leiter2, KW Broman4, G Buechler3, L Alley2, D Vestweber5 and M Detmar1 1Institute of Pharmaceutical Sciences, Swiss Federal Institute of Technology, ETH Zurich, Zurich, Switzerland; 2The Jackson Laboratory, Bar Harbor, ME, USA; 3Institute for Laboratory Animal Science and Central Animal Facility, Hannover Medical School, Hannover, Germany; 4Department of Biostatistics and Medical Informatics, University of Wisconsin, Madison, WI, USA and 5Max Planck Institute of Molecular Biomedicine, Mu¨nster, Germany 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-Il10À/À mice and IBD-susceptible C3H/HeJBir-Il10À/À (C3Bir-Il10À/À) 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-Il10À/À 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. Genes and Immunity (2010) 11, 219–231; doi:10.1038/gene.2010.4; published online 11 March 2010 Keywords: lymphangiogenesis; inflammation; systems genetics; colitis Introduction inflammatory cytokines such as tumor necrosis factor, endothelial cells lining the blood vessels become acti- Inflammatory bowel disease (IBD) is a chronic inflam- vated and upregulate the expression of cell adhesion matory condition that is believed to be caused by an molecules, promoting the transmigration of leukocytes inadequate reaction of a genetically susceptible host to into the inflamed tissue.7 In contrast, the function of the normal intestinal microflora. Genome-wide associa- lymphatic vessels in inflammation became a focus of tion studies of the two major forms of human IBD, research only recently. Lymphatic vessels have a major Crohn’s disease and ulcerative colitis, have identified function in the maintenance of tissue fluid homeostasis, several possible susceptibility genes, which are involved immune surveillance, and intestinal lipid absorption.8 in innate immunity, autophagy, and immune regula- Research into the lymphatic system was hampered by tion.1–5 However, IBD pathogenesis likely involves the lack of tools to reliably distinguish these vessels from complex interactions of multiple genes with the environ- blood vessels. However, the recent discovery of mole- ment, and the exact molecular mechanisms that lead to cular lymphatic vessel markers (for example lymphatic IBD have remained elusive. vessel endothelial hyaluronan receptor 1, LYVE1; Previously, vascular changes have been suggested to PROX1; podoplanin) has opened new avenues to contribute to IBD pathogenesis.6 As a response to detailed investigations of the lymphatic vasculature in inflammation. There is growing evidence that lymphatic vessels Correspondence: Dr M Detmar, Institute of Pharmaceutical actively participate in the inflammatory response. Pro- Sciences, Swiss Federal Institute of Technology, ETH Zurich, nounced lymphangiogenesis has been found in mouse Wolfgang-Pauli-Strasse 10, HCI H303, Zurich CH-8093, Switzer- models of chronic airway inflammation, rheumatoid land. 9–11 E-mail: [email protected] arthritis, and psoriasis. Activation of lymphatic Received 28 August 2009; revised 10 November 2009; accepted 2 vessels might support inflammation by promoting December 2009; published online 11 March 2010 inflammatory cell transport to draining lymph nodes.12 Vcam1 is a candidate modifier gene in IBD G Jurisic et al 220 This process was shown to involve active interaction Results with lymphatic vessels and upregulation of intercellular À/À adhesion molecule 1 and vascular adhesion molecule 1 Pronounced lymphatic vessel abnormalities in the Il10 13 mouse model of IBD (VCAM1) on lymphatic endothelium in vivo. On the À/À other hand, the resolution of chronic inflammation might Il10 mice spontaneously develop inflammation of the colon, caused by their inability to control immune be promoted by lymphatic vessel drainage, and thereby 22,29 removal of accumulated fluid, immune cells, and responses against the normal bacterial flora. In line with earlier reports,22,29 histological examination of tissue inflammatory cytokines from the sites of inflamma- À/À tion.14,15 Indeed, impairment of the lymphatic vascular sections from 7-week-old C3Bir-Il10 mice revealed network and flow can exacerbate inflammation, and epithelial hyperplasia, focal erosions and ulceration, blockade of lymphangiogenic factors in inflammation infiltration by leukocytes, and enlarged vessels (Supple- mentary Figure S1). Importantly, the degree of inflam- results in delayed antigen clearance and inflammation À/À resolution.16–19 mation was much more severe in C3Bir-Il10 mice than in age-matched B6-Il10À/À mice (Supplementary Figure Genetic variation among mouse inbred strains most À/À commonly results in quantitative traits, measurable S1). To further investigate the vascular reaction in Il10 continuous phenotypic variations.20 Quantitative traits mice colons, we used antibodies for the panendothelial marker CD31 and for the lymphatic vessel-specific are influenced by multiple genes and environmental 30 factors and their analysis in mice is being successfully marker LYVE1. Unexpectedly, we found that lymphatic used to gain insight into the genetics of complex human vessels were prominently enlarged in the submucosal diseases such as IBD.21 To identify genes that determine area of inflamed colons and that they invaded the lamina susceptibility to IBD, we used interleukin 10 (IL10)- propria (Figure 1a; Supplementary Figures S2 and S7). deficient (Il10À/À) mice. Il10À/À mice develop a form of These changes were much more pronounced in C3Bir colitis that is similar to human IBD, because of impaired mice than in C57BL/6J mice. Although there was a dramatic increase in the area covered by lymphatic regulation of the immune response to microbial antigens À/À in the colon.22 Importantly, the extent of the IBD vessel endothelium in Il10 mouse colons compared phenotype in Il10À/À mice is highly influenced by the with wild-type mouse colons, there was no significant genetic background: C57BL/6J-Il10À/À (B6-Il10À/À) mice change in the colon area covered by blood are relatively resistant to colitis, whereas C3H/HeJBir- vessel endothelium in inflammation (Supplementary À/À À/À Figure S2). Il10 (C3Bir-Il10 ) mice are highly susceptible (for full À/À designation of the strains, see Materials and methods).23 In C3Bir-Il10 mice, the first inflammatory changes In a pilot study, we found that in C3Bir-Il10À/À mice, in the colon were visible at 4 weeks of age, along with colitis is accompanied by increased numbers of enlarged marked lymphangiogenesis and lymphatic vessel en- and tortuous lymphatic vessels and by pronounced largement (from a normal average size of approximately m 2 4 m 2 submucosal edema. Moreover, it has been reported that 500 m to 5000 m ), whereas no such changes were observed in B6-Il10À/À mice (Figure 1a). At 6 weeks, obstruction of lymphatic vessels in the intestine leads to À/À Crohn’s disease-like symptoms in experimental ani- C3Bir-Il10 mice had further increases in the number of 24,25 enlarged lymphatic vessels in the submucosal area of mals. A higher density of lymphatic vessels was also À/À observed in the colons of human IBD patients.26 More- inflamed colons, whereas B6-Il10 mice had only mild over, a number of eminent pathologists during the early colitis without any major lymphatic abnormalities. The extent of colitis was severe at 8 weeks of age in C3Bir- 20th century pointed to the important function of the À/À lymphatic vasculature in the etiology of Crohn’s dis- Il10 mice and included a dramatic enlargement of the ease.27 On the basis of these findings, we developed and lymphatic vessels and their invasion into the lamina propria (Figure 1a; Supplementary Figure S7). In B6- applied a scoring system to quantify lymphatic vessel À/À traits in an F2 intercross between B6-Il10À/À and C3Bir- Il10 mice, the changes were milder with only Il10À/À mice. This cross was earlier established for the moderately enlarged lymphatic vessels (Figure 1a). purpose of mapping susceptibility loci for colon mucosal Quantitative analyses of five lymphatic vessel traits inflammation.28 Quantitative trait locus