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Programmed Death-1 Pathway Blockade Reduces Pulmonary Fibrosis Following Bleomycin Administration
Description
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A2194 - Programmed Death-1 Pathway Blockade Reduces Pulmonary Fibrosis Following Bleomycin Administration
Author Block: G. Lee; Vanderbilt University School of Medicine, Nashville, TN, United States.
Rationale
Pulmonary fibrosis is a chronic disease characterized by progressive deposition of connective tissue components such as collagen in the lung. Previous studies have shown that immune dysfunction occurs in CD4+ T cells with Programmed Death-1 (PD-1) upregulation in human interstitial lung disease. In order to conduct an in vivo investigation, we performed intransal inoculation of bleomycin in C57BL6 wild-type (WT) mice as well as PD-1-/- mice with C57BL6 background, followed by lung harvesting at Day 14.
Methods
6 PD-1-/- mice (B6.Cg-Pdcd1tm1.1Shr/J) and 6 WT mice were purchased from the Jackson Lab (Bar Harbor, ME, USA). Bleomycin was administered via intranasal inoculation to WT and PD-1-/- mice at 7-9 weeks old (17-22 g). Lungs were harvested at day 14. Trichrome staining was used to visualize collagen involvement as a correlate of pulmonary fibrosis. In addition, fibrosis was confirmed through High Performance Liquid Chromatography (HPLC) assessment of hydroxyproline content. Pulmonary homogenates were acid hydrolyzed in 12M HCl at 120°C for 3 hours. HPLC was performed after evaporation of HCl. Mice were also weighed at baseline and at Day 14.
Results
There was no significant difference in weight between WT and PD-1-/- mice at baseline or 14 days post-treatment. Trichrome staining revealed significantly higher degree of fibrosis and lung area involvement as determined by Ashcroft scoring in WT mice compared to PD-1-/- mice (p=0.002, unpaired two-tailed t-test). HPLC also revealed significantly lower levels of hydroxyproline in PD-1-/- mice compared to the WT mice (41.7±17.0 versus 111.3±8.7 µg per lung, p=0.02, unpaired two-tailed t-test).
Conclusion
In this study, we found a significant difference in levels of fibrosis in PD-1-/- and WT mice following bleomycin administration. These in vivo findings identify a critical and previously unrecognized role for PD-1 upregulation in the induction of pulmonary fibrosis, supporting the use of readily available therapeutics that directly address fibrosis pathophysiology in interstitial lung disease. Future investigations will delineate the mechanisms by which PD-1 drives pulmonary fibrosis.
Author Block: G. Lee; Vanderbilt University School of Medicine, Nashville, TN, United States.
Rationale
Pulmonary fibrosis is a chronic disease characterized by progressive deposition of connective tissue components such as collagen in the lung. Previous studies have shown that immune dysfunction occurs in CD4+ T cells with Programmed Death-1 (PD-1) upregulation in human interstitial lung disease. In order to conduct an in vivo investigation, we performed intransal inoculation of bleomycin in C57BL6 wild-type (WT) mice as well as PD-1-/- mice with C57BL6 background, followed by lung harvesting at Day 14.
Methods
6 PD-1-/- mice (B6.Cg-Pdcd1tm1.1Shr/J) and 6 WT mice were purchased from the Jackson Lab (Bar Harbor, ME, USA). Bleomycin was administered via intranasal inoculation to WT and PD-1-/- mice at 7-9 weeks old (17-22 g). Lungs were harvested at day 14. Trichrome staining was used to visualize collagen involvement as a correlate of pulmonary fibrosis. In addition, fibrosis was confirmed through High Performance Liquid Chromatography (HPLC) assessment of hydroxyproline content. Pulmonary homogenates were acid hydrolyzed in 12M HCl at 120°C for 3 hours. HPLC was performed after evaporation of HCl. Mice were also weighed at baseline and at Day 14.
Results
There was no significant difference in weight between WT and PD-1-/- mice at baseline or 14 days post-treatment. Trichrome staining revealed significantly higher degree of fibrosis and lung area involvement as determined by Ashcroft scoring in WT mice compared to PD-1-/- mice (p=0.002, unpaired two-tailed t-test). HPLC also revealed significantly lower levels of hydroxyproline in PD-1-/- mice compared to the WT mice (41.7±17.0 versus 111.3±8.7 µg per lung, p=0.02, unpaired two-tailed t-test).
Conclusion
In this study, we found a significant difference in levels of fibrosis in PD-1-/- and WT mice following bleomycin administration. These in vivo findings identify a critical and previously unrecognized role for PD-1 upregulation in the induction of pulmonary fibrosis, supporting the use of readily available therapeutics that directly address fibrosis pathophysiology in interstitial lung disease. Future investigations will delineate the mechanisms by which PD-1 drives pulmonary fibrosis.
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