Pro-resolution mediators such as resolvins and axonal guidance receptor-ligand interactions are increasingly recognized to regulate leukocyte trafficking into sites of inflammation. The implication of this biology in chronic allograft rejection is as yet unknown. We developed microfluidic platforms that mimic the human allograft microenvironment for the identification and screening of molecules that enhance pro-resolution. In these studies, we evaluated the effects of the axonal guidance cues Netrin-1 and Slit-2 on fMLP- or IL-8-induced neutrophil migration. When infused into the device, human leukocytes can migrate either towards or away from inflammatory foci through micro-channels and/or through mazes that model chemo-physical encounters in vivo. Slit-2 (1.5[micro]g/mL) significantly reduced the migratory response induced by fMLP from 76.9±3.9 % to 25.8±6.9 % of migrating cells (p<0.001). Netrin-1 (0.5[micro]g/mL) also reduced migration in response to IL-8 from 19.6±2.2 % migrating cells to 11.1±1.7 % (p<0.05). Interestingly, although both cues are reported as chemorepellants, we observed a striking pattern of chemoinhibition in response to pro-migratory chemokines. To study patrolling/surveillance mechanisms, we analyzed neutrophil migration in mazes resembling the interstitium of a graft. LTB4 was potent to elicit directional migratory responses towards the inflammatory focus (average ~95% maze area traversed). In contrast, C5a induced an exploratory migratory pattern within the maze without a directional response (~40% traversed, p<0.001). Thus, classic chemoattractants induce diverse migratory phenotypes. We also evaluated the effects of chemorepellents on T-lymphocyte trafficking. High concentrations of SDF-1 (10 [micro]g/mL) were potent to repel pooled populations of T-lymphocytes (25.7±3.1 % repelled, 13.3±2.0 % attracted; p<0.001). In contrast, SDF-1 had no repellent effects on CD4+ T-reg migration (5.7±2.0 % repelled, 5.1±1.8 % attracted). This suggests that local release of SDF-1 within allografts has high potential to inhibit T effector cell infiltration, but has no effect on T-reg trafficking and/or local immune regulation. We conclude that pro-resolving mediators represent novel families of molecules with high potential to regulate leukocyte trafficking into allografts. Allograft-on-a-chip microfluidics supports the translation of these discoveries and the screening of novel anti-inflammatory agents.

CITATION INFORMATION: Boneschansker L, Irimia D, Briscoe D. Microfluidics to Characterize Leukocyte Trafficking into Allografts and the Resolution of Inflammation. Am J Transplant. 2016;16 (suppl 3).

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