Material Supplementary 8.DC1http://www.jimmunol.org/content/suppl/2010/07/06/jimmunol.100035

No abstract

Lines of mice were obtained by selective breeding for maximum (AIRmax) or minimum (AIRmin) acute inflammation. They present distinct neutrophil influx and show frequency disequilibrium of the solute carrier family 11a member 1 (Slc11a1) alleles. This gene is involved in ion transport at the endosomes within macrophages and neutrophils, interfering in their activation. Homozygous AIRmax and AIRmin sublines for the Slc11a1 gene were produced to examine the interaction of this gene with the acute inflammatory loci. The present work investigated wound-healing traits in AIRmax and AIRmin mice, in F(1) and F(2) intercrosses, and in Slc11a1 sublines. Two-millimeter ear punches were made in the mice and hole closure was measured during 40 days. AIRmax mice demonstrated significant tissue repair while AIRmin mice did not. Significant differences between the responses of male and female mice were also observed. Wound-healing traits demonstrated a correlation with neutrophil influx in F(2) populations. AIRmax( SS )showed higher ear-wound closure than AIRmax( RR ) mice, suggesting that the Slc11a1 S allele favored ear tissue repair. QTL analysis has detected two inflammatory loci modulating ear wound healing on chromosomes 1 and 14. These results suggest the involvement of the acute inflammation modifier QTL in the wound-healing phenotype.

We tested the role of aryl hydrocarbon receptor (Ahr) gene polymorphism in the inflammatory response and in skin and lung tumorigenesis in 2 lines of mice phenotypically selected for maximum or minimum acute inflammatory reaction (AIRmax and AIRmin, respectively). Following 7,anthracene (DMBA) treatment, AIRmin but not AIRmax mice showed early skin reactions and eventually developed malignant skin tumors and lung adenocarcinomas. In skin tissue, transcript levels of IL1b, Tnf, Il6, Tgfb1 and Cyp1b1 genes were upregulated in AIRmin but not AIRmax mice, consistent with the inflammatory responses to the carcinogen. These findings appeared to be related to the homozygosity status of the Ahr functional A375V polymorphism, which influences the binding capability of the receptor for DMBA: the 375A allele, encoding the high-affinity ligand-binding receptor (Ahr b1 ), segregated in AIRmin mice, whereas AIRmax mice carried the 375V, corresponding to the low-affinity binding receptor Key words: aryl hydrocarbon receptor; DMBA; inflammation; carcinogenesis; selected mice Two nonisogenic mouse lines derived by bidirectional selection based on the intensity of local acute inflammatory reactions (AIR) to polyacrylamide beads (Biogel) 1 have shown striking differences in resistance/susceptibility to chemical-induced carcinogenesis. Mice with the maximum acute inflammatory reaction (AIRmax) are significantly more resistant than minimum responders (AIRmin) to two-stage skin tumorigenesis induced by 7,12-dimethylbenz[a]anthracene (DMBA) followed by repeated doses of the promoter agent 12-O-tetradecanoyl-phorbol-13-acetate (TPA), 2 and also to lung carcinogenesis induced by urethane, 3 suggesting that a subset of loci affecting the inflammatory response also act as cancer modifier loci.DMBA and other polycyclic aromatic hydrocarbon compounds (PAHs) exert carcinogenic effects following metabolic activation mediated by the intracellular aryl hydrocarbon receptor (Ahr), a ligand-activated transcriptional factor regulating the induction of mono-oxygenase enzymes that convert the parent DMBA compound to a diol epoxide, the actual carcinogenic moiety. 4 Ahr gene presents functional polymorphism, which accounts for differences in responses of mouse strains to PAHs. 5 We investigated DMBA-induced skin contact reactions and skin and lung tumor development in AIR mice with respect to their allele status of the Ahr gene. Our results implicate Ahr in tumor susceptibility and in inflammatory response control in AIRmax and AIRmin mice. Material and methods Mice and tumor inductionAIRmax and AIRmin mice from the 38th generation of selective breedings were produced at Laboratory of Immunogenetics of Butantan Institute (São Paulo, Brazil). All experimental procedures were approved by the Animal Experimentation Ethics Committee of the Institute. Doses of 50 lg DMBA (Sigma, St. Louis, MO) in 0.1 ml acetone were applied to the shaved dorsal skin of mice during 5 consecutive days. Skin tumors were scored twice a week in groups of 15 AIRmax and 15 AIRmi...

We tested the possibility to map loci affecting the acute inflammatory response (AIR) in an (AIRmax  AIRmin) F2 intercross mouse population derived from non-inbred parents, by association analysis in the absence of pedigree information. Using 1064 autosomal single nucleotide polymorphisms (SNPs), we clustered the intercross population into 12 groups of genetically related individuals. Association analysis adjusted for genetic clusters allowed to identify two loci, inflammatory response modulator 1 (Irm1) on chromosome 7 previously detected by genetic linkage analysis in the F2 mice, and a new locus on chromosome 5 (Irm2), linked to the number of infiltrating cells in subcutaneous inflammatory exudates (Irm1: P ¼ 6.3  10 À7 ; Irm2: P ¼ 8.2  10 À5) and interleukin 1 beta (IL-1b) production (Irm1: P ¼ 1.9  10 À16 ; Irm2: P ¼ 1.1  10 À6 ). Use of a polygenic model based on additive effects of the rare alleles of 15 or 18 SNPs associated at suggestive genome-wide statistical threshold (Po3.4  10 À3 ) with the number of infiltrating cells or IL-1b production, respectively, allowed prediction of the inflammatory response of progenitor AIR mice. Our findings suggest the usefulness of association analysis in combination with genetic clustering to map loci affecting complex phenotypes in non-inbred animal species.