Hydroxychloroquine reduces microglial activity and attenuates experimental autoimmune encephalomyelitis

https://doi.org/10.1016/j.jns.2015.08.1525Get rights and content

Highlights

  • HQC pretreatment attenuates the activation of human microglia in vitro.

  • HQC pretreatment reduces the production of pro- and anti-inflammatory cytokines in vitro.

  • HQC pretreatment delays the onset of EAE.

  • HQC pretreatment reduces microglia/macrophages in the spinal cord in EAE.

  • HQC pretreatment reduces signs of demyelination in the spinal cord in EAE.

Abstract

Background

Microglial activation is thought to be a key pathophysiological mechanism underlying disease activity in all forms of MS. Hydroxychloroquine (HCQ) is an antimalarial drug with immunomodulatory properties that is widely used in the treatment of rheumatological diseases. In this series of experiments, we explore the effect of HCQ on human microglial activation in vitro and on the development of experimental autoimmune encephalitis (EAE) in vivo.

Methods

We activated human microglia with lipopolysaccharide (LPS), and measured concentrations of several pro- and anti-inflammatory cytokines in untreated and HCQ pretreated cultures. We investigated the effect of HCQ pretreatment at two doses on the development of EAE and spinal cord histology.

Results

HCQ pretreatment reduced the production of pro-inflammatory (TNF-alpha, IL-6, and IL-12) and anti-inflammatory (IL-10 and IL-1 receptor antagonist) cytokines in LPS-stimulated human microglia. HCQ pretreatment delayed the onset of EAE, and reduced the number of Iba-1 positive microglia/macrophages and signs of demyelination in the spinal cords of HCQ treated animals.

Conclusion

HCQ treatment reduces the activation of human microglia in vitro, delays the onset of EAE, and decreases the representation of activated macrophages/microglia and demyelination in the spinal cord of treated mice. HCQ is a plausible candidate for further clinical studies in MS.

Introduction

Multiple sclerosis (MS) is a chronic inflammatory and neurodegenerative disease of the brain and spinal cord that leads to disability and functional loss due to demyelination and neuronal injury [1]. Although the cause of MS is unknown, pathological research has shown that inflammation is the hallmark of all forms of MS, and that activated microglia and phagocytic macrophages are important participants in this pathology. Microglial activation is present in all types of MS plaques [2] and also in the extralesional normal appearing white matter (NAWM) [3], [4], [5]. While there are beneficial activities of microglia [6], abnormally activated microglia produce a variety of molecules that can destroy neurons and oligodendrocytes, including free radicals, proteases and glutamate [7]. Indeed, the chronic activation of microglia and the persistence of their toxic products are thought to drive the progressive destruction of axons in MS and its animal models [4], [8].

Current treatments for MS only impact the most common subtype of MS, relapsing–remitting MS (RRMS), while no treatments so far have shown a convincing effect on primary and secondary progressive MS. One strategy in the search for treatments for all forms of MS, but in particular for the currently untreatable progressive forms of MS, is the application of generic drugs to MS. The underlying thought of this approach is to screen an available generic drug for its effect on a pathophysiological mechanism thought to be important in MS, and to test this generic drug in a clinical trial. For example, a recent effort to screen generic drugs for their influence on oligodendrocyte differentiation and remyelination led to the identification of the generic antihistamine clemastine as a candidate drug to promote remyelination [9], and to a phase II trial of this agent in RRMS (Clinicaltrials.gov reference NCT02040298). Current treatments for MS do not directly target microglia. However, given the prominence of microglial activation in the pathology of RRMS as well as progressive MS, drugs that target microglial activation could have an important impact on the pathophysiology of all forms of MS.

Hydroxychloroquine (HCQ) is an antimalarial drug with immunomodulatory effects that is widely used in combination with other disease suppressing medications in the treatment of rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) [10], [11]. Long term use of HCQ as maintenance or adjunct treatment in SLE has been shown to reduce occurrence of disease exacerbations, and it may delay development of neuropsychiatric features [12]. While its precise mode of action in these diseases is uncertain it is notable that HCQ can inactivate macrophage phospholipase A2 and reduce production of pro-inflammatory cytokines by macrophages and lymphocytes [13], [14]. In addition to immunological actions, HCQ has antithrombotic, lipid-lowering and other metabolic actions [10], [11]. Interestingly, with prolonged treatment HCQ tends to accumulate in tissues including the brain [15], and it might therefore be effective in the treatment of MS, where the blood–brain barrier often forms an obstacle to achieving sufficient drug levels. We conducted a series of experiments to test HCQ as an inhibitor of microglial activation in vitro, and to investigate its effects on experimental autoimmune encephalomyelitis (EAE), an animal model of MS.

Section snippets

Preparation and treatment of human microglia

Human microglia of over 95% purity was isolated from the brains of adult humans undergoing resection to treat intractable epilepsy, as previously described [16]. The use of these specimens was approved by the University of Calgary Research Ethics Board. Cells were plated in 96-well flat-bottomed plates (BD Pharmingen, San Jose, USA) at a density of 10,000 cells per well. The feeding medium used was minimum essential medium (MEM) supplemented with 10% fetal bovine serum, 1%

Effect of HCQ on human microglial cytokine production

We measured LPS stimulated TNF-alpha production by human microglia as a general measure of their activation. HCQ reduced the production of TNF-alpha in a concentration-dependent fashion (Fig. 1A). There was no significant effect of HCQ in a concentration of 1 μM (p > 0.05), but highly significant reduction in TNF-alpha production occurred at HCQ concentrations ranging from 3 to 50 μM (all p < 0.0001). TNF-alpha levels reached approximate control levels at a HCQ concentration of 50 μM. These effects of

Discussion

In the healthy CNS, resting microglia are characterized by many ramified processes, surveying the parenchyma for any possible threats. Upon CNS injury, microglia become activated and take on an amoeboid shape, characterized by retracted processes. Monocytes also infiltrate the CNS upon neural injury and become amoeboid-shaped macrophages that express many of the same antigenic markers as microglia. Due to the difficulty in distinguishing these cells, many authors refer to them collectively as

Conflict of interest statement

The authors declare that there is no conflict of interest.

Acknowledgments

This study was funded by operating grants from the Canadian Institutes of Health Research (grant number #133477), the Multiple Sclerosis Scientific Research Foundation of the Multiple Sclerosis Society of Canada (grant number EGID678), and the Alberta Innovates — Health Solutions' CRIO Team program (grant number #3769). We thank Yan Fan, Tammy Wilson, Janet Wang, Brooke Verhaeghe and Claudia Silva for skilled technical assistance.

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