Abstract
Purpose :
Macrophages exhibit a spectrum of phenotypes with differential biological functions including phagocytic, proinflammatory or wound healing, profibrotic. Cornea and conjunctiva contain a significant resident macrophage population. Injury to the cornea and systemic diseases such as graft-versus-host disease can cause ocular surface fibrosis and myofibroblast formation. The present study was designed to investigate whether macrophage polarization can modulate profibrotic signaling in corneal and conjunctival fibroblasts.
Methods :
Bone marrow-derived macrophages (M0) were cultured from femur and tibia bone marrow cells of BALB/c mice in the presence of macrophage colony stimulating factor (m-CSF). Macrophage polarization was induced by adding LPS + IFN-γ (M1 spectrum) or IL-4 (M2 spectrum). Flow cytometry was used to characterize the macrophage subtypes by staining for CD11b, F4/80 (Pan markers), CD86 (M1) and CD206 (M2) markers. Cultured murine corneal and conjunctival fibroblasts were exposed to M0, M1 or M2 macrophage supernatants. Immunostaining and real-time PCR were performed to quantify alpha-smooth muscle actin (α-SMA) as a marker for transdifferentiation of the fibroblast to myofibroblasts. The cDNA of supernatant exposed fibroblasts was used to quantify gene expression changes in profibrotic cytokines (TGF-β1, PDGF, CTGF), renin angiotensin system (RAS) components (angiotensin converting enzyme (ACE) and angiotensinogen), and the macrophage proliferation cytokine (m-CSF) using real-time PCR.
Results :
Supernatants from M0 and M2 macrophages caused a 2- to 4-fold increase in expression of α-SMA in corneal and conjunctival fibroblasts. Further, > 60% fibroblasts exposed to supernatant of M0 and M2 macrophages showed α-SMA staining. Supernatant from all the three macrophage phenotypes significantly increased the expression of cytokines (TGF-β1 and PDGF), RAS components (ACE and angiotensinogen), and m-CSF and the changes were phenotype specific. Supernatant of M1 macrophages had more prominent effect on RAS components whereas cytokine expression was increased more robustly by supernatant of M0 and M2 macrophage subtypes.
Conclusions :
Polarized macrophages cause transdifferentiation of corneal/conjunctival fibroblasts to myofibroblasts and increase the expression of profibrotic mediators in the fibroblasts in a phenotype-specific manner.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.