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Hydroxychloroquine reduces microglial activity and attenuates experimental autoimmune encephalomyelitis

Journal of the Neurological Sciences, Volume 358, Issue 1-2, November 2015, Pages 131 - 137

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.

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.

Keywords: Multiple sclerosis, Hydroxychloroquine, EAE, Microglia, Macrophage.

1. 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], and [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] and [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] and [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] and [14]. In addition to immunological actions, HCQ has antithrombotic, lipid-lowering and other metabolic actions [10] and [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.

2. Methods

2.1. 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% penicillin/streptomycin, 0.1% dextrose, nonessential amino acids, 10 μM glutamine and 1 mM sodium pyruvate (called complete MEM). Although microglia are known to be cells that react to perturbations in vivo, the human microglia in culture appear to be at a very low basal level of activation, as evident by the minimal levels of secreted cytokine molecules in control, unstimulated condition (see vehicle-treatment condition of Fig. 2).

Where indicated, adherent cells were treated with 100 ng/ml of the Toll-like receptor-4 agonist lipopolysaccharide (LPS), and with varying concentrations of HCQ. All treatments with HCQ were done 2 h prior to the addition of LPS, except where stated otherwise; this pretreatment period was necessary because LPS is a potent activator that triggers signaling cascades immediately after engagement of Toll-like receptors on cells. All chemicals were obtained from Sigma-Aldrich (St. Louis, USA). Cell-conditioned media were collected after 24 h and used for cytokine analyses.

2.2. Cytokine analyses

Tumor necrosis factor (TNF)-alpha concentrations in the microglia culture medium were measured with a single cytokine TNF-alpha ELISA (ELISA Kit KHC3011, Invitrogen, Carlsbad, USA). The concentration of 25 cytokines and chemokines was also measured simultaneously in the microglia medium with a multiplex human cytokine panel (Kit LHC0009, Invitrogen, Carlsbad, USA). The cytokines measured with the multiplex panel were granulocyte-macrophage colony stimulating factor, interferon (IFN)-alpha, IFN-gamma, interleukin (IL)-1 receptor antagonist (ra), IL-1beta, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IL-15 IL-17 and TNF-alpha. Chemokines measured were IFN-gamma-inducible protein 10 (IP10), monocyte chemoattractant protein 1 (MCP-1), monokine induced by IFN-gamma (MIG), macrophage inflammatory protein 1alpha (MIP-1alpha), MIP-1beta, RANTES and eotaxin.

2.3. EAE disease induction and analyses

EAE was induced in female C57BL/6 mice (Charles River, Montreal, Canada), ages 8–10 weeks, by subcutaneous injection of 50 μg of MOG35–55 in Freund's Complete Adjuvant (Thermo Fisher Scientific, Rockford, USA) supplemented with 4 mg/ml of Mycobacterium tuberculosis on day 0. Intraperitoneal pertussis toxin (0.1 μg/200 μl; List Biological Laboratories, Hornby, Canada) was administered at days 0 and 2. To increase the sensitivity of measurement, animals were assessed daily using a 15 point disease score scale [17] and [18] instead of the more commonly used 5 point scale. The 15 point scale score (0 to 15) is the sum of the disease state for the tail (scored from 0 to 2) and each limb (scored from 0 to 3); death is scored as 15. All animals were handled according to the policies outlined by the Canadian Council for Animal Care and the University of Calgary. HCQ was dissolved in phosphate buffered saline and injected intraperitoneally in a volume of 200 μl on the days and in the dosages noted.

2.4. Histological analysis

Animals were killed with an overdose of ketamine/xylazine (200 and 10 mg/kg, respectively). Spinal cords were removed, fixed in 10% buffered formalin and the lumbar region then embedded in paraffin. Longitudinal sections of 6 μm thickness were cut from the ventral to dorsal aspects of the lumbar spinal cord. Sections were stained with hematoxylin/eosin for general histology. To visualize microglia/macrophages, sections were stained with an antibody to Iba1 (Wako Chemicals, Richmond). To visualize demyelination, sections were stained with antibody to myelin basic protein (MBP) (Abcam, Cambridge, UK).

To quantify the extent of macrophage/microglia activation and the amount of demyelination, the longitudinal section with visible central canal, and stained respectively for Iba1 or MBP, was selected from each animal. Photographs of the stained lateral column were then taken using a 20 × objective, and an average of 12 non-overlapping images per animal was obtained. For Iba1, the picture was analyzed with Image J software (National Institutes of Health, USA) to contrast staining from background; the amount of staining was depicted as pixels. For MBP, the area devoid of myelin staining in the dorsal column was traced out, and the area was represented as pixels. The staining, capture of images and Image J quantitation were all conducted blind.

2.5. Statistical analyses

Statistical significance was taken to be at the two-sided 0.05 level. Group comparisons of cytokine levels in cell culture medium of human microglia were done with the Kruskal–Wallis test with Dunnett's post test for pairwise comparisons. Comparisons of histology between HCQ treated and control animals were done with Student's t-test. All statistical analyses were performed with the R statistical software package for Windows version 3.0.1 [19].

3. Results

3.1. 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 HCQ were not contributed by non-specific cytotoxicity since ATP luminescence assays (CellToter-Glo®, Promega, Madison, WI) did not detect cell death in any of the HCQ-treated conditions (data not shown).

gr1

Fig. 1

A) HCQ reduces the production of TNF-alpha in LPS-activated microglia in a concentration-dependent manner. B) The inhibition of activated microglia is stronger the longer the pretreatment interval with HCQ; the times indicated are hours prior to the addition of LPS. All samples (culture media) were collected for ELISA determination 24 h after the addition of LPS. Values are mean ± SEM of 4 wells of microglia per condition.

 

The timing of HCQ pretreatment also had an important effect on the reduction of TNF-alpha production by human microglia (Fig. 1B), with TNF-alpha production decreasing with longer times of pretreatment with HCQ 10 μM (all p < 0.0001). TNF-alpha levels reached approximate control levels at a time of 24 h of pretreatment.

To corroborate the TNF-alpha ELISA results, we proceeded to the multiplex human cytokine/chemokine array where TNF-alpha was one of the molecules measured. The multiplex analysis showed that treatment with 10 μM HCQ reduced the LPS stimulated production of multiple cytokines and chemokines (Fig. 2). Both pro-inflammatory (IL-6, IL-12, TNF-alpha) and anti-inflammatory (IL-10 and IL-1ra) cytokines elevated by LPS-activation were reduced by HCQ to approach control levels, as was the case for the chemokine MIP-1β; these results indicate that HCQ normalized activity of stimulated microglia to basal levels. Levels of other cytokines and chemokines were not significantly different between HCQ treated and untreated cultures (p > 0.05), or they were not detected in the culture media.

gr2

Fig. 2

Multiple cytokines and chemokines are decreased by HCQ (10 μM) in LPS-activated human microglia cultures. Values are mean ± SEM of 4 wells of microglia per condition.

 

3.2. EAE experiment

In the EAE experiment we explored the effect of two different HCQ doses on EAE development in animals pretreated from 10 days before EAE induction (Fig. 3). There was a dose–response in development of EAE symptoms, with animals treated with 50 mg/kg developing EAE on day 11 after EAE induction, while animals treated with 100 mg/kg developed no EAE symptoms until the end of the experiment on day 14 after EAE induction (Fig. 3). The spinal cords of animals in the control and the 100 mg/kg group were dissected for histological analysis. Representative sections of the lumbosacral spinal cord are shown in Fig. 4. The Iba-1 staining, which does not distinguish macrophages from microglia, shows that there are fewer Iba-1 positive cells in the spinal cords of HCQ treated animals. Moreover, the larger Iba-1 positive cells in vehicle-treated EAE-afflicted mice, indicative of highly activated macrophages/microglia, were not obvious in the spinal cord of HCQ-treated animals. Similarly, the demyelination seen in vehicle-treated EAE mice was not apparent in HCQ-treated animals (Fig. 4). The quantification of the decrease in macrophage/microglia activation or demyelination by HCQ is shown in Table 1.

gr3

Fig. 3

Effects of daily intraperitoneal HCQ pre-treatment on clinical scores of EAE in mice. Treatment was initiated 10 days before MOG immunization with either 50 (n = 8) or 100 (n = 6) mg/kg HCQ or vehicle (n = 7). The results are expressed as mean ± SEM.

 

gr4

Fig. 4

HCQ treatment reduces the histopathology of EAE in mice. These are representative images from the animals analyzed in Table 1, and include H&E of vehicle (A) or HCQ mice (B), Iba-1 of vehicle (C) and HCQ (D) animal, and MBP of vehicle (E) or HCQ (F) subject. Images were acquired originally with a 20 × lens.

 

Table 1

Extent of macrophage/microglia activation and demyelination in the spinal cord.

 

Vehicle HCQ (100 mg/kg) p (t-test)
Density of macrophages/microglia

× 105 pixels
28.2 ± 12.5 (5) 4.9 ± 1.0 (6) 0.014
Extent of demyelination

× 104 pixels
2.5 ± 1.7 (6) 0 (6) 0.017

Values are mean ± SD and the number in parentheses refers to the number of animals analyzed. Analyses were conducted blind.

4. 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 macrophages/microglia, even though they can have different functions [20].

Microglia is important for CNS immune responses and reacts to injury to protect the CNS. Macrophages that enter the CNS also have useful properties, for example the clearance of myelin debris [21], and activated microglia may even induce neurogenesis [22]. However, persistently activated microglia/macrophages are likely detrimental for the CNS that normally has low levels of immune surveillance. The detrimental roles of chronically activated microglia and macrophages include the production of toxins (free radicals, cytokines, glutamate, proteases, etc.) and antigen presentation [27]. Thus, macrophages/microglia have complex and often dichotomous functions and it may be beneficial to normalize the function of chronically activated macrophages/microglia in order to prevent tissue damage.

In this series of experiments, we provide in vitro and in vivo data on the effect of HCQ treatment on the production of microglial cytokines and on the development of EAE. Our in vitro experiments show that HCQ reduces the production of pro-inflammatory cytokines by human microglia, including TNF-alpha, IL-6, and IL-12. This is in keeping with in vitro experiments from the rheumatology literature investigating the effect of HCQ treatment of peripheral blood macrophages [14], mononuclear cells [24] and monocytes and T-cells [13]. These experiments similarly showed a reduction of TNF-alpha [14] and [24] and IL-6 [13] and [24] and relate this effect to the inhibition of the enzyme phospholipase A2 [14]. In our EAE experiments pre-treatment with HCQ dose-dependently delayed the onset of EAE. Our histological analysis showed that HCQ treated animals had fewer activated macrophages/microglia in their spinal cords compared to untreated animals. It is thus possible that HCQ affects macrophage and T-cell activation in the periphery while also reducing microglia/macrophage activation in the CNS.

In recent years, macrophages/microglia have been grossly divided into at least 2 subtypes (M1 and M2) based on their secretory products, molecules that they express, and functional roles. M1 cells are associated with the secretion of many pro-inflammatory cytokines including IL-1β and TNF-α; they express the cell surface markers CD86 and CD16/CD32 and have inducible nitric oxide synthase (iNOS) activity. The M2 subset, itself divided into 3 types, is associated with the secretion of anti-inflammatory cytokines such as IL-10, and expresses CD204, CD206 and the enzyme arginase-1. M2 subsets are also phagocytic and thought to facilitate regenerative processes, while M1 cells are thought to promote injurious processes. However, this over-simplification is dependent on the injury type and temporal sequence. In lysolecithin toxin-induced focal demyelination, both M1 and M2 subtypes are needed for repair: the early representation of M1 cells and their production of TNF-alpha facilitate the recruitment of OPCs while the later arriving M2 cells are needed for removal of inhibitory debris, and for secretion of growth factors to mature OPCs into oligodendrocytes [25]. In the EAE model of MS, activated macrophages/microglia are initially pro-inflammatory, but downregulate their M1 markers during recovery while elevating and maintaining expression of M2 markers [32]. Thus, in considering whether HCQ reduces either M1 or M2 microglia activities, our results indicate that both pro- and anti-inflammatory cytokines of activated microglia are reduced by HCQ. We propose that HCQ normalizes microglia activity, or prevents their activation, rather than shifting cells along either the M1 or M2 route. This normalization of activated microglia back to a baseline state is desirable, since cells that are shifted chronically to either an M1 pro-inflammatory or M2 anti-inflammatory state would represent an abnormal circumstance and likely have subsequent undesired impact on CNS physiology.

It is tempting to speculate that the reduction of pro-inflammatory cytokines through HCQ treatment could be a therapeutic avenue in MS. Several characteristics make HCQ an attractive agent to bring to clinical trial. HCQ is usually well tolerated and widely used as a chronic treatment in SLE and RA. One of the important properties of HCQ metabolism is its tendency to accumulate in tissues. HCQ concentrations in animal and human tissues after prolonged HCQ use are higher than in blood and increase in the sequence (from lowest to highest) blood, brain, muscle, skin, heart and liver [15], [28], and [29]. In rats, HCQ concentrations in the brain are two- to four times higher than in full blood [15] and [30].

While it is always difficult to extrapolate animal data to the human situation, there is some reason to believe that standard doses of HCQ as used in clinical practice may lead to meaningful concentrations in the brain. Two studies in clinical SLE populations showed that mean full blood concentrations of HCQ in SLE patients treated with standard doses of HCQ were around 750 to 1000 ng/ml [31] and [32]. This is equivalent to 2 to 3 μM in full blood, and if we assume, based on animal data, that brain concentrations are two- to fourfold higher than in full blood, this would translate into brain concentrations of between 4 and 12 μM. These concentrations are comparable to those used in our in vitro experiments (Fig. 1), which showed a significant effect of HCQ treatment on microglial cytokine production at concentrations of 3 μM and higher. Furthermore, the HCQ dose of 100 mg/kg we used in our EAE experiments translates (using the recommended mouse to human dose conversion) [33] and [34] to a human dose of around 8 mg/kg, which is very close to the standard human HCQ dose of 6 mg/kg [31] and [32].

In our EAE experiment, we used a pretreatment with HCQ. Given that MS, particularly of the relapsing–remitting form, is thought to constitute a condition with episodic assault upon the CNS, it can be argued that the pretreatment in our experiments represents an approach to prevent or ameliorate the next injurious event within the CNS, similar to current immunomodulatory drugs used in relapsing–remitting MS. However, this argument assumes that subsequent relapses in MS occur through the same mechanisms as the preceding relapse. Indeed, it would be desirable to assess the impact of HCQ on EAE mice when treatment is initiated in symptomatic animals.

In summary, our experiments show that HCQ treatment reduces the production of pro-inflammatory cytokines in human microglia in vitro, delays the onset of EAE, and reduces the representation of activated macrophages/microglia and demyelination in the spinal cord of mice. The pharmacological properties of HCQ and the fact that the doses used in our experiments correspond to standard doses used in clinical practice make HCQ a plausible candidate for further study in MS.

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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Footnotes

a Department of Clinical Neurosciences and Hotchkiss Brain Institute, University of Calgary, Calgary, AB, Canada

b Department of Community Health Sciences, University of Calgary, Calgary, AB, Canada

c Department of Neurological Sciences, University of Nebraska, Omaha, NE, USA

d Department of Medicine, University of Alberta, Edmonton, AB, Canada

e Department of Medical Genetics, University of Calgary, Calgary, AB, Canada

Corresponding author at: Department of Clinical Neurosciences and Hotchkiss Brain Institute, University of Calgary, Calgary, AB, Canada.

1 Co-first authors.


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