M2 Macrophages Mitigate Ocular Surface Inflammation and Promote Recovery in a Mouse Model of Dry Eye.

Abstract

Dry eye disease (DED) is a chronic, progressive, multifactorial condition characterized by tear film instability and ocular surface damage. Ocular surface inflammation, triggered by multiple pathogenic factors, represents one of the key mechanisms in DED pathogenesis. This study aims to investigate the therapeutic effects of anti-inflammatory M2 macrophages conditioned medium (M2-CM) on ocular surface inflammation and their potential mechanisms in improving dry eye symptoms in a mouse model. Mouse macrophages (RAW264.7) were polarized into M2 macrophages by IL-4 under different osmolarities, and M2-CM was collected. Flow cytometry and ELISA were applied to measure the cytokine expression of the M2 macrophages. Primary mouse corneal epithelial cells (CECs) were co-cultured with RAW264.7 and M2 macrophages using a Transwell system. The viability and migration of CECs were assessed using CCK-8 and scratch assays. Mouse DED was established by subcutaneous injection of scopolamine, and the therapeutic effects of M2-CM were evaluated by phenol red thread test, fluorescein staining, and tear film breakup time (TBUT). PCR and immunofluorescence staining were applied to observe inflammatory factors and cells on the ocular surface. M2 macrophages enhanced CEC viability, proliferation, and migration, but hyperosmolarity inhibited M2 macrophage polarization. In the DED model, M2-CM improved ocular surface conditions, reduced pro-inflammatory cytokine expression, and increased anti-inflammatory factors. Immunofluorescence revealed reduced pro-inflammatory cells (M1 macrophages, Th1, and Th17) and increased M2 macrophages in the ocular tissues after M2-CM treatment. These results suggest that M2-CM ameliorates ocular surface inflammation and promotes recovery in DED, offering a potential therapeutic strategy for DED.