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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
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.
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.
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.
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.
- 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.
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
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
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)  and . 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
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
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  and  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
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 (
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 (
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 (
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 (
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.
HCQ treatment reduces the histopathology of EAE in mice. These are representative images from the animals analyzed in
Extent of macrophage/microglia activation and demyelination in the spinal cord.
× 105 pixels
× 104 pixels
Values are mean ± SD and the number in parentheses refers to the number of animals analyzed. Analyses were conducted blind.
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
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
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
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
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 , , and . In rats, HCQ concentrations in the brain are two- to four times higher than in full blood  and .
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  and . 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 (
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.
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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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.
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