Fig 2: TTF affected the expression of mRNA of M1 and alternative macrophage markers. PM cells isolated from ApoE-/- mice were treated with 0.5 mg/ml TTF for 24 h followed by ox-LDL stimulating for 24 h (A) The cell survival rate of PM pretreated with TTF and IC50 of PM (B). *p < 0.05 and **p < 0.01 vs Control (TTF = 0 mg/ml) group (C–E) The mRNA expression of Fizz1, Arg1 and Mrc of alternative macrophage markers (F–H) The mRNA expression of TNFα, IL-1β and IL-6 of M1 markers. The relative mRNA level was measured by RT-qPCR analysis. Values are presented as means ± SEM (n = 3 per group). NS represents no significant with Control (PBS) group, *p < 0.05 vs Control (PBS) group, #p < 0.05 vs TTF (PBS) group, &p < 0.05 vs TTF (PBS) group (I) Western blotting showed the protein expression of CD68, Arg1, and IL-6 in PM cells treated with PBS, TTF-L (0.25 mg/ml), and TTF-L (0.5 mg/ml) stimulated with ox-LDL (80 μg/ml) (J) The quantitative analysis of grey ratio of Arg1/CD68 and TNFα/CD68. Values are presented as means ± SEM (n = 3 per group). *p < 0.05 vs Ctr + group, #p < 0.05 vs TTF-L group.

Fig 3: TTF functions through promoting PPARγ signal expression and inhibiting AKT/ERK pathway. PM cells were pretreated with 30 μM AKT activator (Recilisib), 25 nM ERK activator (PAD), or 50 μM PPARγ inhibitor (Mifobate) for 1 h, then treated with 0.5 mg/ml TTF and 80 μg/ml ox-LDL for 24 h. Total RNA was extracted and then relative mRNA level of Fizz1 (A), Arg1 (B), Mrc (C), TNF-α (D), IL-1β (E), and IL-6 (F) were detected by q-PCR. Values are presented as means ± SEM (n = 3 per group). *p < 0.05 vs group with ox-LDL only; #p < 0.05 vs group without ox-LDL and TTF; &p < 0.05 vs group with ox-LDL and TTF.

Fig 4: Immune cells, particularly M2-like, populating the corneal stroma at D28 post wounding are closely linked with perturbed EpBM. (A) Accumulation of Arg1+ macrophages (white) at sites of abnormal LN332 spill (green) in the central corneal stroma. (B) Colocalization of Arg1+β2 integrin+ macrophages (white/green) with disrupted EpBM α6 integrin (red). (C) A β2 integrin+ immune cell (red) with migratory morphology populating a crack within the corneal epithelium visualized by phalloidin staining (white). 3D structural rendering of the boxed region in (C) with transparent F-actin highlights an area of disconnected epithelial cells between which the immune cell traverses (Ci). (A,B) 1 & 3 µm projection; (C) single optical plane; and (Ci) 3D surface structure. Mag bars: 20 µm except for (Ci) 5 µm.

Fig 5: Macrophages migrating into the corneal stroma near erosion sites at D28 post wounding often exhibit an M2 phenotype. Shown are representative images of F4/80+ macrophages (green) at D28 co-labeled with M1 marker iNOS (red) and/or M2 markers including Arg1 (white) and CD206 (red). (A) shows the absence of iNOS and presence of Arg1 expression among F4/80+ macrophages in the central cornea of eyes with erosions. (B) shows variable expression of M2 phenotypic markers among macrophages recruited to the central and peripheral stroma of corneas with erosion. (C) compares the number of F4/80+Arg1+ macrophages found in the central and peripheral cornea. (Da) presents quantification of M2 vs. non-M2 subsets near erosion sites in the central cornea. Each datapoint in (C,Da) represents count from 20 FOVs in 5 non-consecutive sections per eye (n = 5). Bar plots show adjusted p-value after t-test (C) and one-way ANOVA with post-hoc Tukey (D) (**** p-value < 0.0001). (Db) shows percentage contribution of M2 and non-M2 subsets to the total recruited macrophages in the central cornea of each eye examined in (Da). All images are 2.5 µm projection. Mag bars: 20 µm. Endo corneal endothelium and epi corneal epithelium.