In Vitro and In Vivo Models of Cerebral Ischemia Show Discrepancy in Therapeutic Effects of M2 Macrophages

Virginie Desestret,Denis Angoulvant,Serge Nataf,Marilena Marinescu,Emilie Devillard,Catherine Rey,Marlène Wiart,Tae-Hee Cho,Marie Chanal,Norbert Nighoghossian,René Ferrera,Adrien Riou,Fabien Chauveau,Yves Berthezène,Jérôme Honnorat

doi:10.1371/journal.pone.0067063

Abstract

The inflammatory response following ischemic stroke is dominated by innate immune cells: resident microglia and blood-derived macrophages. The ambivalent role of these cells in stroke outcome might be explained in part by the acquisition of distinct functional phenotypes: classically (M1) and alternatively activated (M2) macrophages. To shed light on the crosstalk between hypoxic neurons and macrophages, an in vitro model was set up in which bone marrow-derived macrophages were co-cultured with hippocampal slices subjected to oxygen and glucose deprivation. The results showed that macrophages provided potent protection against neuron cell loss through a paracrine mechanism, and that they expressed M2-type alternative polarization. These findings raised the possibility of using bone marrow-derived M2 macrophages in cellular therapy for stroke. Therefore, 2 million M2 macrophages (or vehicle) were intravenously administered during the subacute stage of ischemia (D4) in a model of transient middle cerebral artery occlusion. Functional neuroscores and magnetic resonance imaging endpoints (infarct volumes, blood-brain barrier integrity, phagocytic activity assessed by iron oxide uptake) were longitudinally monitored for 2 weeks. This cell-based treatment did not significantly improve any outcome measure compared with vehicle, suggesting that this strategy is not relevant to stroke therapy.

Highlights

It is acknowledged that inflammation plays a major pathophysiological role in ischemic stroke [1]
Macrophages Prevent Hypoxia-induced Neuron Cell Death in vitro In organotypic hippocampal slice cultures (OHCs) subjected to oxygen-glucose deprivation (OGD), neuron cell loss could be demonstrated in different hippocampal neuron populations, as assessed by neuronal nuclei (NeuN) staining (Figure 2 A–D) or DAPI coloration
The cytokine profile of the macrophage co-cultures was assessed at the mRNA level by Q-RTPCR examination of transcripts coding M1-type cytokines (TNF-a, IL-6, inductible nitric oxide synthase (iNOS)) or Arginase 1, a molecule expressed by alternatively-activated macrophages (Figure 3)

Summary

Introduction

It is acknowledged that inflammation plays a major pathophysiological role in ischemic stroke [1]. The inflammatory response is dominated by innate immune cells of myeloid lineage: resident microglia and blood-derived macrophages It remains to be shown whether the impact of macrophages on the outcome of brain ischemia is deleterious [2] or beneficial [3]. As they penetrate tissue, they respond to microenvironmental signals, acquiring distinct functional phenotypes: classically (M1) or alternatively activated (M2) macrophages. M2 macrophages are important in resolving the inflammatory response and play a part in debris scavenging, tissue remodeling, and angiogenesis [4] They were shown to support neurorepair in various models of central nervous system lesion [5,6,7]. The majority of M2-type genes began to decrease at 7 days and returned to pre-injury levels by day 14 [9]

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Results

Conclusion