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Lars Bellner, Giuseppina Marrazzo, Kirkland Castellano, Nico van Rooijen, Michael W. Dunn, Michal L. Schwartzman; Macrophage Dysfunction and Impaired Wound Healing in Corneas of Heme Oxygenase (HO)-2 Deficient Mice. Invest. Ophthalmol. Vis. Sci. 2012;53(14):3536. doi: https://doi.org/.
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We have shown that in HO-2 null mice epithelial injury leads to massive infiltration of inflammatory cells, chronic inflammation, ulceration, perforation and neovascularization. We examined the effect of systemic depletion of macrophages or neutrophils on corneal wound healing and neovascularization. We used in vitro cultured macrophages to determine the effect of HO-2 silencing, or HO-induction on macrophage phagocytic capacity.
Clodronate liposomes or a neutrophil specific antibody (Gr-1) were injected into the peritoneum starting one day prior to injury and every other day thereafter. The corneal epithelium was removed using an Alger Brush on anesthetized mice. Re-epithelialization was assessed by fluorescein staining. Inflammatory response was quantified by histology and MPO activity. Levels of mRNA were measured by RT-PCR. RAW 264.7 macrophages were transfected with HO-2 shRNA or treated with SnCl2 and phagocytic capacity was assessed by incubation with fluorescence labeled Zymosan particles.
Systemic depletion of neutrophils increased corneal wound healing rate in both WT and HO-2 null mice as compared to respective control. Macrophage depletion of WT mice delayed corneal re-epithelialization when compared with the control treated group by 15% at day 4 after injury (p<0.05). Corneal wound healing in HO-2 null mice was significantly delayed as compared to WT at day 4 after injury (p<0.05). Surprisingly, macrophage depletion of HO-2 null mice had no additional effect on an already impaired corneal wound healing. HO-2 silencing of RAW 264.7 macrophages in vitro reduced phagocytic capacity by 40% (p<0.05), whereas HO-induction of RAW 264.7 macrophages increased phagocytic capacity by 85% (p<0.05).
This study shows that neutrophil depletion enhances wound healing rate in both WT and HO-2 null mice, whereas macrophage depletion has a negative impact on wound healing rate in WT, but not in HO-2 null mice. Further, silencing of HO-2 in vitro inhibited macrophage phagocytic capacity, whereas pharmacological induction of the HO-system increased macrophage phagocytic capacity in vitro. Together these data shows an important link between the heme oxygenase system and macrophage function, including phagocytosis.
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