Flow cytometric analysis of the leukocyte landscape during bleomycin-induced lung injury and fibrosis in the rat

Bleomycin-induced lung injury and fibrosis is a well-described model to investigate lung inflammatory and remodeling mechanisms. Rat models are clinically relevant and are also widely used, but rat bronchoalveolar lavage (BAL) cells are not fully characterized with flow cytometry due to the limited availability of antibodies for this species. We optimized a comprehensive time-dependent flow cytometric analysis of cells after bleomycin challenge, confirming previous studies in other species and correlating them to histological staining, cytokine profiling, and collagen accumulation analysis in rat lungs. For this purpose, we describe a novel panel of rat surface markers and a strategy to identify and follow BAL cells over time. By combining surface markers in rat alveolar cells (CD45 ), granulocytes and other myeloid cells, monocytes and macrophages can be identified by the expression of CD11b/c. Moreover, different activation states of macrophages (CD163 ) can be observed: steady state (CD86 MHC-II ), activation during inflammation (CD86 ,MHC-II ), activation during remodeling (CD86 MHC-II ), and a population of newly recruited monocytes (CD163 α-granulocyte ). Hydroxyproline measured as marker of collagen content in lung tissue showed positive correlation with the reparative phase (CD163 cells and tissue inhibitor of metalloproteinases (TIMP) and IL-10 increase). In conclusion, after a very early granulocytic recruitment, inflammation in rat lungs is observed by activated macrophages, and high release of IL-6 and fibrotic remodeling is characterized by recovery of the macrophage population together with TIMP, IL-10, and IL-18 production. Recruited monocytes and a second peak of granulocytes appear in the transitioning phase, correlating with immunostaining of arginase-1 in the tissue, revealing the importance of events leading the changes from injury to aberrant repair.