Sex-Specific Differences in the Corneal Inflammatory Reparative Response via Estrogen Modulation of Phagocytosis

Abstract

Purpose: Clearance of apoptotic neutrophils from tissues by macrophages is a crucial and necessary component of inflammatory resolution. We previously demonstrated sex-specific differences in self-resolving corneal wound healing responses, which correlated with sex-specific differences in macrophage phenotypes. We also established that estrogen down-regulates intrinsic pro-resolving lipid mediators via ERβ. Pro-resolving lipid mediators such as lipoxin A4 regulate macrophage phagocytic capacity. Hence, we investigated if there is a sex-specific difference in macrophage phagocytosis and if this essential housekeeping function is regulated by estrogen.

Methods: Age-matched male and female mice underwent full corneal epithelial abrasion. Bone-marrow derived macrophages were used for in vitro studies. Neutrophils were collected from the peritoneum following zymosan A injection and allowed to apoptose before introduction to macrophages. Macrophage phagocytic capacity was measured using myeloperoxidase assay. Flow cytometry was run to determine macrophage phenotype.

Results: Following epithelial abrasion, there was a sex-specific difference in leukocyte dynamics (i.e. neutrophil recruitment and clearance) in the cornea. Males had higher levels of M2-type macrophages in the healing corneas compared to their female counterparts; however, estrogen did not alter macrophage polarization into either an M1 or M2 subtype in vitro. In contrast estrogen regulated macrophage function by inhibiting the pro-resolving actions of lipoxin A4 (48% inhibition of phagocytosis). Estrogen also regulated both neutrophil apoptosis and macrophage uptake/clearance of apoptotic neutrophils.

Conclusions: Estrogen orchestrates the inflammatory leukocyte response in the cornea, namely by regulating macrophage phagocytosis of apoptotic neutrophils. An essential function for healthy inflammation is limiting the innate immune response, the dysregulation of which can lead to activation of the adaptive immune response and subsequent chronic disease. This has potential ramifications in the cornea, where chronic inflammation can cause blindness or lead to autoimmune diseases.

Keywords: 765 wound healing • 557 inflammation • 480 cornea: basic science