Novel Method to Assess Macrophage Phenotype Using Eluted Media.

Introduction: Macrophages exist on a spectrum from pro-inflammatory (M1) to pro-healing (M2). Characterization of macrophage phenotype is important to understand tissue healing and response. The gold standard for assessing macrophage phenotypes is immunocytochemistry (ICC), which stains inducible Nitric Oxide Synthase (iNOS) and arginase (Arg1), the proteins secreted before nitrite and urea production. The ICC method is an endpoint assay, time-consuming, and costly. Therefore, a more effective method to assess the phenotype of macrophages in vitro is needed. Based on the phenotype of the macrophage, arginine gets metabolized differently. If arginine is metabolized by M1 macrophages it produces nitrite and if it is metabolized by M2 macrophages it produces urea. A method that leverages arginine metabolism through secreted products (urea and nitrite) has the potential to determine macrophage phenotype in real-time.

Methods: Rat bone marrow-derived macrophages were cultured to be naïve or polarized to M1-like or M2-like. The ICC method was used to determine the intensity of the iNOS and Arg1 staining. Nitrite and urea kits were utilized to measure the concentration of nitrite and urea in the media eluted from the various phenotypes. Nitrite and urea concentrations were compared to ICC results to validate the new method.

Results: ICC revealed the iNOS staining was significant and 2.5 folds higher in M1-like macrophages and the Arg1 staining was significant and 1.5 folds higher in the M2-like macrophages. The nitrite concentration was significant and 4 folds higher in the M1-like macrophage. The urea concentration was significant and 2.5 folds higher in the M2-like macrophage media. A correlation analysis showed that iNOS staining intensity and nitrite concentration levels had a linear correlation as well as Arg1 staining intensity and urea concentration levels.

Conclusion: Data confirm that the determination of nitrite and urea concentration can be utilized to assess macrophage phenotypes.