Fig 1: IL-11 suppresses activation of neutrophils and monocytic cells. Bone marrow was harvested from BALB/c mice and single-cell suspensions were made. (A) Cells were stimulated with a neutrophil activator, fMLP (1 µM), in the presence or absence of IL-11 (1 µg/mL). Cells cultured in media alone served as control. Bar chart depicting MPO levels in supernatants of indicated cell cultures. (B) Representative histogram (left) and bar chart (right) depicting expression of neutrophil activation marker CD11b (MFI). (C–E) Bone marrow cells were stimulated with IFNγ (10 ng/mL) and cultured in the presence or absence of IL-11 (1 µg/mL). (C) Representative histogram (left) and bar chart (right) depicting MHC II expression (MFI) by mononuclear cells (gated on CD11b+Ly6G–). (D) Representative dot plots (left panel) and bar chart (right panel) quantifying mononuclear cells (gated on CD11b+Ly6G–) expression (MFI) of TNFα, a proinflammatory cytokine. (E) Representative flow cytometry dot plots (left) and bar chart (right) depicting differential expression (MFI) of iNOS by mononuclear cells (gated on CD11b+Ly6G–). 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 < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Fig 2: 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.
Fig 3: IL-11 receptor expression by infiltrating immune cells. Corneas were harvested 24 hours following injury and single-cell suspensions were prepared for flow cytometry analysis. Corneas harvested from naive mice served as controls. (A) Representative histograms (upper panel) showing frequencies of IL-11R expressing CD45+ total immune cells, CD11b+Ly6G+ neutrophils, and CD11b+Ly6G− monocytes in the cornea. Bar charts (lower panel) quantifying fold change in IL-11R expression (MFI, mean fluorescence intensity) by the indicated cells. (B) Representative dot plots (left) showing the gating strategies of bone marrow–isolated CD11b+Ly6G+ neutrophils and CD11b+Ly6G− mononuclear cells (macrophages, monocytes). Bar chart (right) quantifying expression (MFI) of IL-11R by CD11b+Ly6G+ neutrophils and CD11b+Ly6G− mononuclear cells (macrophages, monocytes). (C) Representative histogram (left) and bar chart (right) quantifying expression of IL-11R by human monocytes. Data from three independent experiments are shown, and each experiment consisted of 4 to 6 animals. Data are presented as mean ± SD (error bar). Student t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****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: 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.
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