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Ricardo Lamy, Marie Wolf, Selene M Clay, Siyu Zheng, Peipei Pan, Matilda F Chan; Characterization of Restorative Macrophages After Corneal Wounding in Wild Type and Mmp12−/− Knockout Mice.. Invest. Ophthalmol. Vis. Sci. 2018;59(9):4338.
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© ARVO (1962-2015); The Authors (2016-present)
Macrophages are crucial for tissue repair and regeneration. We have previously shown that matrix metalloproteinase 12 (MMP12) decreases expression of chemokine CCL2 and reduces the accumulation of macrophages into wounded corneas. Following liver injury, restorative macrophages have been identified that are recruited to injured tissue, have anti-fibrotic properties, and have low Ly-6C expression and increased expression of MMPs, including Mmp12. The purpose of this study is to use flow cytometric analysis to better characterize the phenotype of the recruited restorative macrophages after corneal wounding in wild type and Mmp12−/− knockout mice.
Alkaline chemical injuries were performed on corneas of WT and Mmp12−/− mice by topical application of 0.1N NaOH. Unwounded and wounded corneas collected at 1, 2, and 6 days after injury were digested with collagenase. Macrophage subpopulations in wounded and unwounded corneas were assessed at each time point using flow cytometry with pre-conjugated antibodies (CD45, CD11B, F4/80, Ly-6C). Flow cytometric data analysis was performed using FlowJo10 software (Tree Star, Inc).
Dynamic changes during chemical injury resolution were observed in two subpopulations of recruited macrophages. Pro-inflammatory macrophages (CD45hi CD11Bhi F4/80int Ly-6Chi) were more abundant in the first 48 hours after chemical injury while the presence of restorative macrophages (CD45hi CD11Bhi F4/80int Ly-6Clo) increased during the later resolution phase. Six days after injury, corneas of WT mice had more restorative macrophages (CD45hi CD11Bhi F4/80int Ly-6Clo) compared with wounded corneas from Mmp12−/− mice.
These findings show that differential Ly-6C expression can be used to identify a recruited macrophage phenotype in corneal tissue, with the restorative subset (CD45hi CD11Bhi F4/80int Ly-6Clo) falling outside the M1/M2 classification. This restorative macrophage subset is reduced in wounded corneas of Mmp12−/− mice and correlates with the increased fibrosis noted in these corneas.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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