Bleomycin-Treated Chimeric Thy1-Deficient Mice with Thy1-Deficient Myofibroblasts and Thy-Positive Lymphocytes Resolve Inflammation without Affecting the Fibrotic Response

Figure 3

Assessment of fibrotic lung injury following bleomycin intratracheal instillation (IT) in wild-type mice with Thy1⁺ lymphocytes and Thy1⁺ myofibroblasts (WT), and chimeric Thy1-deficient mice with Thy1⁺ WT lymphocytes and Thy1⁻ myofibroblasts (chimeric). Thy1-deficient mice (DEF) were provided by Prof. R.J. Morris. Chimeric mice were created by whole-body irradiation (750 rad) of the Thy1-deficient mice, followed by transplantation with WT Thy1⁺ lymphoid cells (chimeric). Wild-type mice underwent whole-body irradiation and reconstitution with WT Thy1⁺ lymphoid cells as control (WT). (A) Flow cytometry analysis to detect Thy1⁺ immune cells in Thy1-deficient mice (DEF), wild-type mice (WT), and chimeric Thy1-deficient mice with transplanted WT Thy1⁺ lymphocytes. Thy1⁺ immune cells were identified in blood cells by double staining with PE-conjugated anti-Thy1 and FITC-conjugated anti-CD3. (B) Quantitative image analysis of pathological sections in chimeric Thy1-deficient mice (Thy1⁺ lymphocytes/Thy1– myofibroblasts, empty bars) and control WT mice (grey bars) 14 d following IT bleomycin. WT and 8 chimeric 2 (C) Analysis of collagen content in lungs using the Sircol assay 14 d following IT bleomycin in chimeric Thy1-deficient (empty bars) and control WT mice (grey bars). (D) Total BAL cell count in lungs 14 d following IT bleomycin in chimeric (empty bars) and control WT mice (grey bars) () (E1, F1) αSMA and sirius staining in lung tissue sections 14 d following IT bleomycin of chimeric Thy1-deficient (empty bars) and control WT mice (grey bars). (E2, F2) 10 fields of every IHC-slide were digitized using the Ariol machine and semiquantitatively analyzed with the Ariol system. The relative αSMA and sirius staining ratios represent the ratio between numbers of stained and unstained cells in a certain analyzed area. Results were similar in 2 different experiments.

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