The use of genetically humanized IL-10 mice to determine the molecular basis of allele-specific gene expression and disease susceptibility

Johns Hopkins University, Baltimore MD

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Abstract

DESCRIPTION (provided by applicant): The failure to properly control inflammatory responses is a common feature in human disease. IL-10 plays a central role in limiting inflammation and IL-10 levels are strongly linked to inflammatory disorders in humans. The levels of IL-10 production are reported to be influenced by single nucleotide polymorphisms (SNPs) in the IL10 promoter and these SNPs are also associated with disease susceptibility. This indicates that inter- individual differences in the regulation of IL-10 production are likely key factor which determines disease risk. However, the mechanisms that control human IL-10 (hIL-10) production remain unclear due to a lack of appropriate research tools. For that reason, we established a proof-of-principle system in the first funding period to transgenically model the regulation of hIL-10 expression in the absence of extraneous genetic and environmental influence. We used a large segment of human genomic DNA flanking the IL10 gene to assure that the regulatory information required to confer appropriate hIL-10 expression would be self-contained (hIL10BAC). In addition, because hIL-10 is functional in mice, we can use this model to establish the connection between hIL-10 regulation and disease outcomes in vivo. We have carefully validated hIL-10 expression in several well-characterized IL-10-dependent disease models as well as in primary human cells. We found that the hIL10BAC rescues Il10-/- mice from LPS toxicity and colitis which was associated with hIL-10 production from macrophages and CD4+FoxP3+ Tregs respectively. Interestingly, the hIL10BAC did not restore susceptibility to persistent L. donovani infection in Il10-/- mice. This was because only a small population of hIL-10+Th1 cells (which mediates this phenotype) were induced in hIL10BAC mice. We hypothesized that low T cell IL-10 was encoded by its promoter allele (ATA). To test this we generated new mice bearing a common variant promoter allele (GCC), previously associated with high IL-10. We confirmed these alleles encode for low/high IL-10 respectively and show that hIL10 alleles change the outcome to L. donovani infection. We now propose to extend these findings to other inflammatory disease of public health importance (sepsis and influenza) and to focus on the mechanisms which govern cell type- and allele-specific hIL-10 expression patterns and as a means to determine the molecular basis for hIL-10's role in human disease outcomes. In Aim 1, we will define allele-specific hIL-10 expression in different cell subsets of hIL10 mice, confirm findings in human cells, and determine how hIL10 SNPs effect disease. In Aim 2 we will identify the molecular mechanisms which control cell-specific hIL-10 expression. Together, these studies will clarify how cell- and allele-specific regulation of hIL-10 production contributes to human inflammatory diseases.