125 4 Proteomic signature of macrophages cultured on collagen type 1 were expressed significantly more by macrophages on globular collagen than by macrophages on fibrous collagen. However, the role of these proteins remains to be elucidated as their direct function in macrophages is not yet clear. Of those three, only translocation-associated membrane protein-1 (Tram1) was found to have some connection with fibrosis. This protein was shown to be involved in translocation of proteins through the endoplasmic reticulum and in a study comparing patients with IPF and healthy controls, higher levels of TRAM1 mRNA were found in IPF [34]. The authors hypothesized that TRAM1 is upregulated because IPF is characterized by endoplasmic reticulum stress [34]. Our study had some limitations. The number of proteins that was reproducibly quantified in all conditions was rather limited and only covered 17% (2.870 proteins) of the theoretical mouse proteome database (17.023 proteins). Consequently, GO enrichment analysis did not yield many hits. The absence of a cell cycle synchronization step may have contributed to this issue, but serum starvation is known to interfere with macrophage function and activation [35,36] and was therefore intentionally omitted. As explained before, we tried to limit the variability of macrophage responses to collagen types by using fetal liver-derived macrophages. However, this does make translation to primary alveolar macrophages still an open question. Furthermore, the current study does not provide insight into how monocyte-derived macrophages would respond in comparison to fetal liver-derived alveolar macrophages, which is a critical area of investigation. It is well-documented that in cases of lung injury, monocyte-derived macrophages can replace fetal liverderived alveolar macrophages in the lungs and that these two types of macrophages differ in their transcriptional and functional profiles [37,38]. In conclusion, this study shows that the presence as well as the morphology of collagen type I have pronounced effects on the proteomic signature of alveolar macrophages. Collagen type I induced expression of proteins associated with macrophage metabolism, specifically glycolysis, while also collagen morphology had specific effects on the macrophage proteome. Although the studied collagen morphologies are not directly translatable to a healthy or fibrotic tissue in vivo, it does indicate that changes in collagen morphology can have a major impact on macrophage behavior. Several protein candidates have previously been described to play a role in processes related to fibrosis. Further verification and exploration of collagen type I-induced metabolic changes and morphology-dependent effects on the macrophage proteome will provide better insight into the mechanisms behind macrophage-matrix interactions and possibly yield new targets for the treatment of fibrosis.
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