Project Summary/Abstract The precise control of granulocyte migration is critical for fighting off infection and for avoiding excessive tissue damage. In preliminary work we have obtained evidence for a new chemoattractant receptor, the metabolite-responsive receptor GPR35, having an important role in the trafficking of two types of granulocyte to sites of inflammation. Neutrophils are the major early responders of the innate immune system. Recruitment of neutrophils to the inflamed peritoneum has served as an important model for studying neutrophil trafficking. Neutrophils move from blood into the peritoneum via specialized blood vessels in the omentum. We have found that GPR35- deficient neutrophils are less efficiently recruited to the inflamed peritoneum and this reflects less efficient entry into this abdominal tissue site. We propose that neutrophils respond to GPR35 ligands soon after attachment to the inflamed omental endothelial and that GPR35 signaling contributes to transmigration across the endothelium. We will test this hypothesis using whole mount and real time imaging studies of omentum. Our preliminary data indicate that GPR35 function in thioglycolate elicited peritoneal inflammation depends on type I IFN. We will determine what cell types must respond to type I IFN for GPR35 function using IFNAR floxed mice and various Cre-driver mouse lines. This work will help determine which cell types are a source of GPR35 ligand(s). Our preliminary data show a similar GPR35-dependence of neutrophil recruitment to inflamed lymph nodes (LNs). Similar approaches will be used to study how GPR35 contributes to neutrophil LN homing. A second granulocyte type that highly expresses GPR35 is eosinophils. Eosinophils are recruited to sites of type 2 inflammation whether they can have profound influences on tissue homeostasis. Cryptococcal neoformans is an opportunistic fungal pathogen that can cause fatal disease in severely immune compromised individuals. Protection from Cryptococcal growth in the lungs is mediated by type 1 immunity. By contrast, type 2 immune responses and recruitment of eosinophils is associated with exacerbation of disease. We have found that GPR35-deficient mice have improved protection from Cryptococcal infection. In preliminary experiments we find that GPR35-deficiency is associated with lower recruitment of eosinophils to the infected lung. In Aim 2 of this exploratory study we will begin to define the basis for the improved Cryptococcal clearance in GPR35-deficient mice and test for intrinsic roles of GPR35 in eosinophils. The work will define the step that GPR35 functions at during neutrophil recruitment, it will begin to define the sources of GPR35 ligand, and it will elucidate how GPR35 negatively impacts on Cryptococcal clearance from the lung. These findings will provide a framework for more broadly understanding GPR35 function in vivo, and they will establish a rationale for the therapeutic development of GPR35 an...