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

Arend H Sikkema,Jeffrey J Bajramovic,Josephine M J Stoffels,Robbert Bulsink,Wia Baron,Peng Wang,Frederike J Basedow

doi:10.1186/s12974-018-1238-x

Abstract

BackgroundMeans 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.MethodsBone marrow-derived macrophages and microglia from newborn rats were exposed to (a) plasma fibronectin coatings; (b) coatings of deoxycholate-insoluble fibronectin aggregates; (c) interferon-γ (IFNγ) 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.ResultsIn 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 IFNγ 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.ConclusionsMacrophages 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.

Highlights

Means to promote endogenous remyelination in multiple sclerosis (MS) benefit from insights into the role of inhibitory molecules that preclude remyelination
Fibronectin aggregates and plasma fibronectin tend to promote proliferation of microglia, but not of bone marrow-derived macrophages During central nervous system (CNS) demyelination, microglia and infiltrating macrophages increase their numbers by proliferation [30], thereby presumably maximizing their effector functions
To assess whether aggregated fibronectin (aFn), which is typically present in MS lesions [16], contributes to such an expansion, we analyzed the effect of aFn and plasma Fn (pFn) coatings on proliferation of microglia and macrophages, using a BrdU incorporation assay (Fig. 1a, c)

Summary

Introduction

Means to promote endogenous remyelination in multiple sclerosis (MS) benefit from insights into the role of inhibitory molecules that preclude 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. As chronic demyelination leads to neurological disability in MS [5], promoting endogenous remyelination is an attractive therapeutic strategy for structural and functional recovery of MS lesions. Upon demyelination in the CNS, microglia and macrophages acquire a predominantly classical phenotype, which leads to an increase of phagocytosis as well as expression of pro-inflammatory features, among others, TNFα, IL-1β, and inducible nitric oxide synthase (iNOS, [11,12,13]). At later stages of remyelination, oligodendrocyte differentiation benefits from a switch to the alternative regenerative phenotype, characterized by expression of arginase-1 and the mannose receptor [11,12,13]

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Discussion

Conclusion