105 4 Proteomic signature of macrophages cultured on collagen type 1 INTRODUCTION Fibrosis is defined as excessive accumulation of extracellular matrix (ECM), which eventually impairs the function of an affected organ. Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive lung disease with a short life expectancy of 2-3 years after diagnosis. Treatment options are limited, as only two drugs have been approved that slow down disease progression: nintedanib and pirfenidone [1]. The characteristic excessive deposition of ECM proteins in fibrosis, mostly by myofibroblasts, changes the biochemical composition of a tissue. However, it is becoming clear that ECM can also be structurally different because of aberrant post-translational modifications [2]. For example, fibrils of collagen type I, the most abundant ECM protein in fibrosis, are structurally different in lung tissue from patients with IPF compared to control lung tissue [3]. All changes in biochemical composition and post-translational modifications also affect the biophysical properties of fibrotic tissue, such as tissue stiffness and topography [4-6]. The resulting biochemically and biophysically altered ECM can be sensed by resident cells and subsequently change their behavior, resulting in constant two-way interactions [7]. Macrophages are important regulators of ECM homeostasis, as they can stimulate both the production and degradation of ECM proteins [8]. Their ability to prevent or resolve fibrosis in combination with the aberrant macrophage polarization observed in fibrosis, makes studying their possible role in the pathogenesis of fibrosis crucial. Although the effect of fibrosis-related soluble factors (such as cytokines) on macrophage polarization and function has been studied extensively [9,10], their ability to respond to fibrosis-related morphological changes in the ECM is relatively unexplored [10]. Previously, we have shown that changes in the morphology of collagen type I, either globular or fibrous, can affect the shape, marker expression and behavior of murine fetal liver-derived alveolar macrophages [11]. Higher expression of the mannose receptor (CD206), known to be upregulated on alveolar macrophages in IPF, was found when alveolar macrophages were cultured on globular collagen. Fibrous collagen led to higher expression of Ym1, a murine marker of pro-healing macrophages. Moreover, macrophage shape changed distinctly in response to fibrous and globular collagen, with a more amoeboid appearance on fibrous collagen and a more mesenchymal appearance with many filopodia on globular collagen. In parallel with these alterations in macrophage shape, transmigration was higher when macrophages were cultured on fibrous collagen compared to the uncoated condition [11]. However, the exact mechanisms behind these characteristic responses are still elusive. In this study, we applied differential proteomics and
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