The long-term goal of the Shlomchik lab is to understand the role of TLRs in SLE. Prior work by us and others revealed that, surprisingly, disease was markedly exacerbated in the absence of TLR9, even though TLR9 was required for anti-DNA and anti-chromatin autoantibody production. The opposite roles for these two similar TLRs is likely a fundamental feature of TLR biology and of SLE, yet it is poorly understood. Given the large number of SLE risk alleles that map to the TLR signaling pathway, we will elucidate this fundamental biology and how it applies to disease mechanisms and therapy. Experiments proposed in the original application and which will be continued during the MERIT extension will directly address how TLR7 and TLR9 have divergent roles, how they cross-regulate each other, and how this promotes and regulates disease. Our lab has pioneered genetic in vivo methods for the study of lupus mechanism in animal models, in part leading to the recognition of the roles of TLR7 and TLR9, in studies supported in the prior period of this PPG. We have created novel models and used genetic approaches in order to dissect the cells and molecular signaling pathways required to promote disease in vivo. In the first three years we have made and analyzed multiple novel models, which have already greatly informed our insights into how TLR7 and 9 function differently and control lupus. Additional models are needed, and the mice we have recently made will require substantial in depth analysis; we must wait 6 months for age-dependent lupus to evolve in these mice. In addition to in vivo studies, we are now on the verge of determining biochemical and signaling properties of the mutants, which will provide very novel insights into TLR7 and 9 biology. Our mutant mice allow us to study this in primary B cells, which our data from Aim 1 shows us are the key cell type. In Aim 1 we will test the hypothesis that tissue-specific expression of TLR7 and TLR9 explains some or all of their divergent phenotypes. We will use our new TLR7 floxed mice to examine tissue-specific TLR7 expression to both macrophages and neutrophils given our recent results that TLR7 expression in B cells is not the whole story. We will also now extend our studies to TLR8. In Aim 2 we will address the hypothesis that TLR9 regulates TLR7 signals in a cell intrinsic way. To test this, we are making a series of mutant mice, whose phenotypes need to be determined together in order to determine how TLR7 and 9 differ and how they cross-regulate. We propose new imaging, biochemical and cell biological experiments that will illuminate how TR7 vs TLR9 function in primary B cells. Finally, in Aim 3, we will test the therapeutic potential of TLR inhibition using an inducible Cre to overexpress TLR9. Last, we will test TLR9 agonists to determine if TLR9 can be used therapeutically to treat disease. RELEVANCE (See instructions):