Fig 2: IL-11 reduces infiltration of immune cells following corneal injury. (A) Schematic of the experimental design indicating dose and frequencies of IL-11 and MSA administration following injury. Corneas were harvested 24 hours postinjury, and single-cell suspensions were prepared to study immune cell infiltration using flow cytometry. (B) Representative dot plots (left) showing frequencies of total CD45+ cells in naive, MSA-treated, and IL-11–treated mice. Cumulative bar chart (right) quantifying frequencies of total CD45+ leukocytes in the cornea. (C) Representative dot plots (upper panel) indicating frequencies of CD11b+Ly6G+neutrophils and CD11b+ Ly6G− mononuclear cells (gated on CD45+ cells) in corneas of indicated treatment groups. Cumulative bar chart (lower panels) quantifying frequencies of neutrophils and CD11b+ mononuclear cells (macrophages and monocytes) in the corneas harvested from indicated groups of mice. (D) Bar chart depicting MPO levels in corneas harvested 24 hours postinjury. (E) Fold change in mRNA expression of TNFα in corneas of indicated mice groups. Fold change was calculated from naive mice. Data from three independent experiments are shown. Each experiment consisted of 4 to 6 animals per group. The values are expressed as mean ± SD (error bar). One-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 3: IL-11 downregulates activation of human macrophages. Human macrophages derived from THP-1 cell line were stimulated with IFNγ (10 ng/mL) and cultured with or without IL-11 (1 µg/mL) for 24 hours. Cells cultured in media alone served as control. (A) Representative histogram (left) showing expression of human macrophage activation marker HLA-DR. Bar chart (right) quantifying HLA-DR expression (MFI). (B) Representative dot plots (left) and bar chart (right) quantifying expression of TNFα in control and treatment groups. (C) Representative flow cytometry dot plots (left) and bar chart (right) depicting differential expression (MFI) of iNOS by macrophages in indicated groups. Representative data from three independent experiments are shown, expressed as mean ± SD (error bar). Data in each group are from triplicate wells. One-way ANOVA. ****P < 0.0001.

Fig 4: IL-11 expression in the cornea following injury. Corneal injury was induced by mechanical removal of the corneal epithelium and anterior stroma in BALB/c mice. At 24 hours postinjury, corneas were harvested. Corneal tissue harvested from naive mice served as control. (A) mRNA expression of IL-11 (normalized to Gapdh) by naive and injured corneas. (B) Protein levels of IL-11 in lysates of whole corneas collected from naive and injured mice. (C) Epithelial and stromal layers were separated from harvested corneas, and protein levels of IL-11 were quantified using ELISA. (D) HCFs and human monocytes were stimulated with IL-1β (100 ng/mL) for 24 hours. HCFs and monocytes cultured in media alone served as controls. Bar chart depicting protein levels of IL-11 in the culture supernatants of indicated groups. Data from three independent experiments are shown, and each experiment consisted of 4 to 6 animals/group. The values are shown as mean ± SEM (error bar). Student t-test. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 5: Topical supplementation of IL-11 accelerates wound healing. (A) Corneal fluorescein staining was performed to assess for epithelial defects (stained green). Area of epithelial defects was quantified using ImageJ software. Representative images of fluorescein-stained corneas (left), captured using slit-lamp biomicroscopy under cobalt blue light, and cumulative bar chart (right) showing the area of epithelial defect in pixel2 at indicated time points. (B) Single-cell suspensions of corneas harvested on day 6 postinjury were analyzed for immune cell infiltration using flow cytometry. Representative dot plots (left) showing frequencies of total CD45+ cells in naive, MSA-treated, and IL-11–treated mice. Cumulative bar chart (right) quantifying frequencies of total CD45+ leukocytes in corneas of different treatment groups. (C) H&E corneal cross sections showing corneal tissue architecture, edema, and inflammatory cells (black arrow). Data from three independent experiments are shown, and each experiment consisted of 6 to 8 animals/group. The values are shown as mean ± SD (error bar). Student t-test and one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001. Epi., epithelium; Stro., stroma.