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Alexander J. Leedom, Jeff LeDue, Samantha Wang, Kari Lam, Karsten Gronert; RvD1 Regulates Leukocyte Dynamics And Phenotype In The Cornea. Invest. Ophthalmol. Vis. Sci. 2011;52(14):1117.
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© ARVO (1962-2015); The Authors (2016-present)
The dynamic behavior and phenotype of leukocytes are critical to the resolution of healthy inflammation but are poorly defined in living tissues. We developed an in vivo approach to study leukocyte populations and behavior using multi-photon microscopy. Intrinsic lipid circuits are an integral component of inflammatory programs in the cornea. We investigated the role of an endogenous DHA-derived protective signals (RvD1) in regulating dynamic leukocyte behavior and phenotypes during chronic inflammation.
Mice were placed on a w-3 PUFA or w-6 PUFA diet for 3 months and a silk suture was placed in corneas of Lys-EGFP+ mice for 7d to induce chronic inflammation. Mice on a standard balanced diet were treated with RvD1 (100 ng, tid) or saline for 7d after placing the suture or pulse treated once after 7d of suture injury. Inflamed eyes were imaged in vivo for 30 min using a multi-photon microscope using a near-infrared Ti:Al2O3 laser as the excitation source. 3-D/4-D movement of GFP+ leukocytes was quantified and analyzed by Imaris and MATLAB. Established inflammatory and macrophage markers were assessed by QPCR and/or immuno-histochemistry. Lipid autacoid profiles were analyzed by mass spectrometry-based lipidomics employing a triple quadrupole linear ion trap LC/MS/MS system.
In vivo imaging of leukocytes revealed that RvD1 treatment for 7d increases overall leukocyte velocity and directionality. More importantly, a single pulse treatment of RvD1 to 7d injured corneas with characteristic granuloma-like clusters of leukocytes induced rapid and dramatic leukocyte swarming. Velocity and directionality of leukocytes increased 3 fold with addition of RvD1 compared to saline alone. Treatment with RvD1 also shifted the macrophages from an inflammatory (M1) to a wound healing (M2) phenotype. Of interest, w-3 PUFA diet significantly increased the number macrophages and production of RvD1 in the injured cornea, correlating with reduced inflammation.
These findings provide the first insight into the dynamic leukocyte behavior during a corneal inflammatory response and demonstrate that RvD1, an endogenous protective DHA-derived signal, regulates this fundamental response and macrophage phenotype.
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