Hydroxychloroquine reduces microglial activity and attenuates experimental autoimmune encephalomyelitis
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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2020, International ImmunopharmacologyCitation Excerpt :Microglia are gliocytes capable of phagocyting and processing antigens in the CNS [7] that can be exaggeratedly activated, thus producing a variety of molecules that are harmful to neurons and oligodendrocytes, including free radicals and pro-inflammatory cytokines [8]. If the activation of microglia and astrocytes is persistent, these cell products can become toxic and worsen the process of progressive axonal degeneration in MS and in some of its animal models, such as the Experimental Autoimmune Encephalomyelitis (EAE) [5,9,10]. The traditional therapies for MS are still not diverse and do not benefit patients with progressive forms of the disease.
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2020, Journal of NeuroimmunologyCitation Excerpt :The use of hydroxychloroquine in COVID-19 remains controversial, and its application here was strictly off-label. Hydroxychloroquine use was based on data showing that pre-treatment of lipopolysaccharide-exposed microglia can attenuate the production of multiple cytokines in vitro (Koch et al., 2015). These data do not allow for extrapolation regarding hydroxychloroquine's influence on established cytokine release syndromes in severe COVID-19.
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Co-first authors.