Elsevier

Neurochemistry International

Volume 108, September 2017, Pages 472-480
Neurochemistry International

Type-I interferon signalling through IFNAR1 plays a deleterious role in the outcome after stroke

https://doi.org/10.1016/j.neuint.2017.06.009Get rights and content

Highlights

  • The role of type-1 IFN signalling as a significant contributor to brain damage following stroke.

  • The specific anti-IFNAR1 blocking monoclonal antibody, MAR-1, significantly improves the outcome in a mouse model of stroke.

  • IFNAR2 mice do not respond as IFNAR1 mice raising the issue of differential type-I IFN signalling in the brain.

Abstract

Neuroinflammation contributes significantly to the pathophysiology of stroke. Here we test the hypothesis that the type I interferon receptor (IFNAR1) plays a critical role in neural injury after stroke by regulating the resultant pro-inflammatory environment. Wild-type and IFNAR1-/- primary murine neurons and glia were exposed to oxygen glucose deprivation (OGD) and cell viability was assessed. Transient cerebral ischemia/reperfusion injury was induced by mid-cerebral artery occlusion (MCAO) in wild-type and IFNAR1-/- and IFNAR2-/- mice in vivo, and infarct size, and molecular parameters measured. To block IFNAR1 signalling, wild-type mice were treated with a blocking monoclonal antibody directed to IFNAR1 (MAR-1) and MCAO was performed. Quantitative PCR confirmed MCAO in wild-type mice induced a robust type-I interferon gene regulatory signature. Primary cultured IFNAR1-deficient neurons were found to be protected from cell death when exposed to OGD in contrast to primary cultured IFNAR1-deficient glial cells. IFNAR1-/- mice demonstrated a decreased infarct size (24.9 ± 7.1 mm3 n = 8) compared to wild-type controls (65.1 ± 4.8 mm3 n = 8). Western blot and immunohistochemistry showed alterations in Akt and Stat-3 phosphorylation profiles in the IFNAR1-/- brain. MAR-1 injection into WT mice (i.v. 0.5 mg 60 min prior to MCAO) resulted in a 60% decrease in infarct size when compared to the IgG control. IFNAR2-/- mice failed to display the neuroprotective phenotype seen in IFNAR1-/- mice after MCAO. Our data proposes that central nervous system signalling through IFNAR1 is a previously unrecognised factor that is critical to neural injury after stroke.

Introduction

The immune system and the central nervous system (CNS) have long been considered separate systems with minimal interaction. However, the immune molecular mechanisms and pathways that exist in the periphery are also present in the CNS. These same pathways in the CNS become activated in the event of a cerebral ischemia/reperfusion injury or stroke, resulting in neuroinflammation, which further exacerbates primary brain damage. It is becoming increasingly clear that the regulation of inflammation after stroke is multifaceted and comprises vascular effects, distinct cellular responses, apoptosis, and chemotaxis. There are many cell types that are affected including neurons, glial cells, microglia, endothelial cells and they all respond to the resultant neuroinflammation in different ways.

Hallmark features of neuroinflammation including activated microglia, astrogliosis, and increased cytokine and chemokine levels (Downes and Crack, 2010, Downes et al., 2013) have all been reported in animal models and postmortem human brain samples of stroke. With the development of highly specific molecular, biochemical, and immunohistochemical techniques, the presence of numerous inflammatory mediators in and around ischaemic brain tissue has also been documented. In brief, the presence of inflammatory cytokines, chemokines and adhesion molecules has triggered intense research on strategies for blocking their action. However, it is likely that some inflammatory cell–derived mediators will be critical in the proper repair and recovery of neuronal networks and in enhancing plasticity and reformatting of circuits necessary for taking over tasks for which the lost brain tissue was responsible. This is evidenced by a recent study using a brain slice model showing microglia playing a neuroprotective role in acute brain injury (Neumann et al., 2006). The timing, as well as the specific cytokine or factor to be targeted are important variables in determining whether inflammation helps or hinders improved outcomes after stroke.

In the periphery type-I interferons (IFNs) are key regulators in the production of cytokines (de Weerd et al., 2007, Kawai and Akira, 2006) however their role in the generation of neuroinflammation is not well described. In view of the potential proinflammatory effects of type I IFNs, we investigated the impact of this family of cytokines in the acute neural injury model of murine stroke. Since we have had considerable interest in the factors that regulate the pathogenesis of stroke for some years, and IFNs have been implied in brain pathology, we investigated the impact of type I interferon receptor (IFNAR1) deficient mice in the MCAO model of ischaemic brain injury. Our data presented in this study proposes that signalling through IFNAR1 is a previously unrecognised factor that is critical to neural injury after stroke.

Section snippets

Animals

All animal experiments complied with the regulatory standards of, and were approved by, the University of Melbourne, Medicine Dentistry and Health Sciences Animal Ethics Committee. IFNAR1-/- and IFNAR2-/- mice were on a C57Bl6 background, male, 8–10 weeks of age and backcrossed to 15 generations and were previously generated in the laboratory of Prof Hertzog (de Weerd et al., 2013, Hwang et al., 1995, Owczarek et al., 1997). All surgery was performed under isoflurane anesthesia, and all efforts

Type-I interferon expression is elevated after MCAO

In view of the proinflammatory nature of the environment in the brain after stroke we examined type-I interferon responses in wild-type mice in the MCAO model using quantitative PCR. The expression of IFNα was induced at 8 h post-arterial occlusion and several interferon-regulated genes (IRGs); CCL2, CCL3, CXCL1, SAA3, SOCs3 and IRF7 also induced 2–8 h after stroke (Fig. 1). Since neither IFNβ, nor the newly identified IFNε, nor IFNγ was detectable at these times (data not shown), it suggests

Discussion

Over the past 20 years, researchers examining brain tissue at various time intervals after stroke have observed the presence of inflammatory cells, neutrophils and monocytes at the site of injury, as well as the activation of endogenous glia and microglia (Le Thuc et al., 2015). These observations have led to the hypothesis that this resultant neuroinflammation plays a role in the progression and exacerbation of the neural injury that results from stroke. Here we describe that blocking

Conflict of interest statement

The authors report that there are no conflicts of interest concerning this paper.

Author contributions

MZ, CED, CHYW, KMB, PLGA, JG, RA carried out experiments, PJH contributed critical reagents and contributed to experimental design, JMT and PJC designed experiments, analysed data and wrote the manuscript.

Acknowledgements

This study was supported by grants from the National Health and Medical Research Council (NHMRC) of Australia. PJC was supported by an Australian Research Council (ARC) Future Fellowship. PJH is a Senior Principle Research Fellow of the NHMRC.

References (30)

  • C.E. Downes et al.

    Neural injury following stroke: are Toll-like receptors the link between the immune system and the CNS?

    Br. J. Pharmacol.

    (2010)
  • C.E. Downes et al.

    MyD88 is a critical regulator of hematopoietic cell-mediated neuroprotection seen after stroke

    PLoS One

    (2013)
  • M.A. Hamner et al.

    Ischemic preconditioning in white matter: magnitude and mechanism

    J. Neurosci.

    (2015)
  • S.Y. Hwang et al.

    A null mutation in the gene encoding a type I interferon receptor component eliminates antiproliferative and antiviral responses to interferons alpha and beta and alters macrophage responses

    Proc. Natl. Acad. Sci. U. S. A.

    (1995)
  • A.R. Inacio et al.

    Endogenous IFN-beta signaling exerts anti-inflammatory actions in experimentally induced focal cerebral ischemia

    J. Neuroinflammation

    (2015)
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