Fibronectin aggregates promote features of a classically and alternatively activated phenotype in macrophages

Background: Means to promote endogenous remyelination in multiple sclerosis (MS) benefit from insights into the role of inhibitory molecules that preclude remyelination. Fibronectin assembles into aggregates in MS, which impair oligodendrocyte differentiation and remyelination. Microglia and macrophages are required for complete remyelination and normally switch from a pro-inflammatory classical phenotype upon demyelination to a supportive alternative phenotype during remyelination. Here, we investigated the role of fibronectin aggregates in modulating microglia and macrophage behavior and phenotypes. Methods: Bone marrow-derived macrophages and microglia from newborn rats were exposed to (a) plasma fibronectin coatings; (b) coatings of deoxycholate-insoluble fibronectin aggregates; (c) interferon-gamma (IFN gamma) treatment, as an inducer of the pro-inflammatory classically activated phenotype; (d) interleukin-4 (IL-4) treatment, to promote the pro-regenerative anti-inflammatory alternatively activated phenotype; or (e) left unstimulated on uncoated plastic. To examine the in vitro effects of the different stimulations on cell behavior and phenotype, proliferation, phagocytosis, morphology, and pro- and anti-inflammatory features were assessed. Results: In line with a classically activated phenotype, exposure of microglia and macrophages to both plasma fibronectin and fibronectin aggregates induced an amoeboid morphology and stimulated phagocytosis by macrophages. Furthermore, as observed upon IFN gamma. treatment, coatings of aggregated, but not plasma fibronectin, promoted nitric oxide release by microglia and macrophages. Remarkably, fibronectin aggregates induced nitric oxide release in an integrin-independent manner. In addition, fibronectin aggregates, but not plasma fibronectin, increased the expression of arginase-1, similarly as observed upon treatment with IL-4. Proteomic analysis revealed that aggregates of fibronectin act as a scaffold for other proteins, including Hsp70 and thrombospondin-1, which may clarify the induction of both pro-inflammatory and anti-inflammatory features in macrophages cultured on fibronectin aggregate, but not plasma fibronectin coatings. Conclusions: Macrophages and microglia grown on aggregated fibronectin coatings adopt a distinct phenotype compared to plasma fibronectin coatings, showing pro-inflammatory and anti-inflammatory features. Therefore, the pathological fibronectin aggregates in MS lesions may impair remyelination by promoting and/or retaining several classically activated phenotypic features in microglia and macrophages

Additional file 1: of Fibronectin aggregates promote features of a classically and alternatively activated phenotype in macrophages

Figure S1. Fibronectin aggregates do not induce cell cytotoxicity as determined by lactate dehydrogenase release and an MTT assay. Microglia (a, b) or bone marrow-derived macrophages (BMDMs, c, d) were left unstimulated (ctrl) or cultured on fibronectin aggregates (aFn; 2, 5, or 10 μg, respectively) for 24 h, followed by measurements of lactate dehydrogenase (LDH) release in the culture medium (a, c) and of MTT reduction (b, d). Bars represent means versus control (set at 1 for each independent experiment, horizontal line). Error bars show the standard error of the mean. Statistical analyses were performed using the one-sample t test when compared to control (**p < 0.01). (TIF 11900 kb