Increasing data indicates that axonal guidance cues play a critical role in immunity, both in activation, migration as well as in the process of inflammation resolution. One such molecule, Netrin-1, has been found to have anti-inflammatory effects in models of ischemia-reperfusion injury and acute inflammation, mediated through a receptor called UNC5B expressed on PBMC, neutrophil and macrophages. However, Netrin-1 also has proinflammatory effects in models of chronic disease and can bind to a chemoattractant receptor called Neogenin, suggesting that its biology is complex. In this study, we wished to evaluate whether Netrin-1/Neogenin interactions have potential to elicit migratory responses in lymphocytes and in T cell-dependent immunity. By qPCR, Western blot analysis and FACS, we profiled seven Netrin-1 receptors on unactivated and mitogen-activated (anti-CD3/CD28) human CD4+ T cells. We find that CD4+ T cells express UNC5A and UNC5B at low levels in both unactivated and activated cells. In contrast, Neogenin expression is found at low levels at baseline, but is markedly upregulated following 6-48h activation (mean 6-fold). Since Neogenin classically promotes guidance of axons, we speculated that its function relates to chemoattraction. To evaluate migration, we used our microfluidic platform that allows for the simultaneous analysis of chemoattraction and chemorepulsion at the single cell level. We found that 39.7±5.0% of mitogen-activated CD4+ T cells respond to Netrin-1 (100ng/mL). In contrast, unactivated and neogenin^low CD4+ T cells do not respond above controls. Of note, we found that Netrin-1 elicited both chemoattraction and chemorepulsion in 21.5±2.1% and in 18.2±3.0% of cells respectively, and both responses were inhibited following neogenin lentiviral shRNA knockdown in T cells. This indicates that Netrin-1 functions as a chemokinetic agent through Neogenin. Finally, to evaluate function in vivo we injected netrin-1 (5¯o;g) into human skin that was engrafted onto humanized SCID mice. After 14 days, we found that Netrin-1 elicited a diffuse inflammatory response with an overall ∼1.5 fold increase in infiltrates by day 14 vs. control (P<0.01) and a ∼2 fold increase in infiltrating CD3+ T cells vs. controls (P<0.01). Collectively, these novel findings demonstrate that Neogenin is expressed on activated CD4+ T cells where it functions to elicit a marked chemokinetic response in vitro and in vivo.

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