111 4 Proteomic signature of macrophages cultured on collagen type 1 Pathway analysis: Enrichment of biological processes and pathways was analyzed by performing GO enrichment analysis with the annotation data sets ‘Panther GO-Slim Biological Process’ and ‘Panther Pathways’, using a Fisher’s Exact test to determine the p-value with a Benjamini-Hochberg correction to compensate for the false discovery rate. Results with a corrected p-value <0.05 were considered to be significant. All other statistics: Experimental groups for other parameters were compared using GraphPad Prism 8.0 (GraphPad Software, La Jolla, USA). Control macrophages cultured on plastic were compared to macrophages cultured on any type of collagen using a Mann-Whitney U test, while the effect of collagen morphology was investigated by comparing macrophages grown on fibrous or globular collagen using a Wilcoxon matched-pairs signed-rank test. A p-value <0.05 was considered significant. RESULTS Proteome analysis To investigate the effect of collagen morphology on the proteome signature of macrophages, fetal liver-derived alveolar macrophages were cultured on tissue culture plastic coated with either fibrous or globular collagen, or left uncoated (control) for 72 hours. The reason for using this model instead of primary alveolar macrophages was to eliminate any potential interference of transplantation effects that primary alveolar macrophages may experience in cell culture [12]. Primary alveolar macrophages that are removed from their lung-specific environment, which includes exposure to air and cyclic stretch, may experience changes that could affect their responses to collagen sensing. Cell lysates were analyzed by LC-MS in DIA mode. After data processing using our fetal liver-derived alveolar macrophagespecific protein library, 2.870 unique proteins were reproducibly quantified in the three distinct conditions. Partial least squares discriminant analysis indicated that all conditions could be separated based on their proteomic profile (Figure 1a) after data normalization (Supplementary Figure 1).
RkJQdWJsaXNoZXIy MTk4NDMw