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Effects of methylmercury on the secretion of pro-inflammatory cytokines from primary microglial cells and astrocytes
NeuroToxicology 33 (2012) 229–234
Contents lists available at SciVerse ScienceDirect
NeuroToxicology
Effects of methylmercury on the secretion of pro-inflammatory cytokines from primary microglial cells and astrocytes Tyler Bassett, Paxton Bach, Hing Man Chan * Community Health Sciences Program, University of Northern British Columbia, Prince George, BC, Canada
A R T I C L E I N F O
A B S T R A C T
Article history: Received 17 June 2011 Accepted 13 October 2011 Available online 20 October 2011
Glial cells, including oligodendrocytes, astrocytes and microglia are important to proper central nervous system (CNS) function. Deregulation or changes to CNS populations of astrocytes and microglia in particular are expected to play a role in many neurodegenerative diseases, including Parkinson’s disease, amyotrophic lateral sclerosis (ALS) and Alzheimer’s disease (AD). Previous studies have reported methylmercury (MeHg) induced changes in glial cell function; however, the effects of MeHg on these cells remains poorly understood. This study aims to examine the effect of MeHg on the secretion of proinflammatory cytokines from microglia and astrocytes. The impact of the microglia/astrocyte ratio on cytokine secretion was also examined. Microglia and astrocytes were cultured from the brains of neonatal BALB/C mice and dosed with MeHg (0–1 mM) and stimulated with PAM(3)CSK(4) (PAM(3)), a tolllike receptor (TLR) ligand. After this, the secretion of interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-a) and interleukin-1-beta (IL-1b) was measured by ELISA. MeHg reduced the secretion of IL-6 in a dose dependant manner but did not effect the secretion of TNF-a. No change in IL-1b was observed in any treatments, indicating that PAM(3) cannot induce the secretion of this cytokine from glial cells. Additionally, the ratio of microglia/astrocyte had an effect on the secretion of IL-6 but not TNF-a. These results indicate that MeHg can modify the response of glial cells and the interactions with astrocytes can affect the response of the microglia cells in culture. These results are significant in understanding the potential relationship with MeHg and neurodegenerative diseases and for the interpretation of results of future in vitro studies using monoculture. ß 2011 Elsevier Inc. All rights reserved.
Keywords: Methylmercury Astrocytes Microglia IL-6 TNF-a
1. Introduction The roles of glial cells in influencing neuron function have been better understood in the last decade. Astrocytes and microglia demonstrate a great degree of plasticity and participate in many processes within CNS development and function (Freeman, 2010; Graeber, 2010). It is widely understood that these cells contribute an inflammatory response in the central nervous system (Henn et al., 2011; Silva et al., 2011; van Neerven et al., 2010). They are actively being investigated for any potential roles in neurodegenerative diseases such as Alzheimer’s disease (AD), Parkinson’s disease and amyotrophic lateral sclerosis (ALS) (Philips and Robberecht, 2011). It has been suggested that microglia may either play a contributing role or a protective role in the pathogenesis of Alzheimer’s disease (Streit, 2005; Town et al., 2005). For instance, the presence of an increased number of
* Corresponding author at: Center for Advanced Research in Environmental Genomics, University of Ottawa, 30 Marie Curie, Ottawa, ON, Canada K1H 1A1. Tel.: +1 613 562 5800x6349; fax: +1 613 562 5385. E-mail address: [email protected] (H.M. Chan). 0161-813X/$ – see front matter ß 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.neuro.2011.10.003
dystrophic microglia precedes the accumulation of neurofibrillary tangles associated with the pathology of AD. This implies that a loss of the neuroprotective functions of microglia contribute to the development of AD (Streit et al., 2009). In contrast, while microglial activation is thought to play a role in the clearance of beta-amyloid plaques associated with AD, their activation can lead to the release of neurotoxic factors that contribute to secondary neuronal cell death (D’Andrea et al., 2004). While the exact role of glial cells in the pathogenesis of neurodegenerative diseases is unclear, it is likely that they do play a role in the etiology of these conditions. Microglia and astrocytes can respond to pathogenic agents through interaction of common antigenic determinants with tolllike receptors (TLR) found on their surface. When bound, these receptors will activate downstream signalling pathways that will induce the expression of various pro-inflammatory cytokines (Henn et al., 2011; Zhou et al., 2008). TLRs are expressed on the surface of both microglia and astrocytes. Changes in TLR expression have been implicated in AD and multiple sclerosis (Okun et al., 2009). For example, mice with a mutation to the TLR4 gene show an increased susceptibility to the accumulation of amyloid-beta (Ab) plaques, while activation of TLR 2, 4 and 9 result in an increase in Ab clearance by a microglial cell (Tahara et al.,
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2006). While age related changes in TLR levels and activity likely play a role in the development of several age related neurodegenerative diseases, it would be interesting to consider whether environmental toxins might contribute to this also. Therefore, the objective of this study is to examine the effect of a well known environmental neurotoxin, MeHg on cytokine secretion from glial cells that have been stimulated with a TLR1/2 agonist, PAM(3). Due to its tendency to bioaccumulate and biomagnify along the aquatic food web, MeHg can be a health concern, especially in populations that sustain themselves on diets consisting of fish (Li et al., 2010). While the effects of acute exposure to toxic levels of MeHg are well known (Eto et al., 2010), the impacts of chronic exposure to lower concentrations of MeHg are less understood. Metals, including mercury, are known to cause immunomodulation in cells associated with the immune system, including lymphocytes, monocytes and macrophages (Hemdan et al., 2007; Lawrence and McCabe, 2002). In fact, when C6 glioma cells are challenged with short term exposure to high concentrations of MeHg, they can be induced to release IL-6 (Chang, 2007). However, microglia exposed to non-cytotoxic levels of MeHg did not have the same effect (Eskes et al., 2002). This study aims to determine whether a ubiquitous environmental toxicant, MeHg, can modulate the function of microglial cells and astrocytes through influencing their ability to produce pro-inflammatory cytokines. Additionally, we also hypothesize that co-culture of microglial cells with astrocytes can affect cytokine secretion. 2. Methods 2.1. Materials Balb/c mice were from Charles River (Montreal, QC). Methylmercury was obtained from Alfa Aesar (Ward Hill, MA). Minimum essential medium (MEM (10370–021)), fetal bovine serum (FBS (16000–044)), goat serum, Hanks Balanced Salt Solution (HBSS), F12 medium, Phosphate buffered saline (PBS), 1 Trypsin-EDTA, penicillin/streptomycin (10,000 U/mL), and L-glutamine (200 mM) were acquired from Gibco (Burlington, ON). CD11b microbeads and magnetic columns were obtained from Miltenyi Biotech (Auburn, CA). IC-07 hybridoma cells were acquired from HPA culture collections (Salisbury, UK) and A2B5 hybridoma cells were acquired from American Type Culture Collection (ATCC) (Manassas, VA). The rabbit complement was from Cedarlane (Burlington, ON). Anti-mouse CD11b antibody was from BD Pharmigen (Mississauga, ON). Anti-GFAP antibody, Cy3-strepavidin, DAPI, PAM(3)CSK(4), and MTT were from Invitrogen (Burlington, ON). FITC-anti rabbit IgG antibody was from Jackson Immunoresearch (West Grove, PA). IL-6, TNF-a and IL-1b ELISAs were obtained from eBioscience (San Diego, CA).
mice harvested. Cells were incubated in a humidified incubator (Thermo (Hudson, NH)) at 37 8C and 5% CO2. After 24 h, the nonadherent cells were removed and cells were incubated in culture medium for 10 days with a medium change for every 3–4 days. After 10 days, oligodendrocytes were removed by complementmediated cytolysis as previously described (Marek et al., 2008) and remaining cells were incubated in culture medium for 10 days without a medium change. Cells were then washed with PBS without Mg2+/Ca2+ and removed from the flask by incubating in 2 ml of 1 trypsin/EDTA solution at 37 8C for 5 min. Dislodged cells were separated by magnetic cell sorting using CD11b microbeads (Miltenyi Biotech, Auburn, CA) as per manufacturer’s directions. The positive fraction contained purified microglial cells while the negative fraction was enriched for astrocytes. Purified cells were re-suspended in culture medium at a final density of 2.5 105 cells/ml for plating. 2.3. MeHg dosing experiments Primary glial cells were plated at microglia/astrocytes proportions of 1, 0.6, 0.4, and 0 into a 96-well flat-bottom culture plate at a density of 25,000 cells/well and incubated at 37 8C, 5% CO2 overnight. The following day, the medium was removed and replaced with dosing medium (MEM containing 1% penicillin/ streptomycin, 0.2 mM L-glutamine, 5% fetal bovine serum and MeHg at a concentration of 0–1.0 mM). On day 3, medium was removed and replaced with dosing medium containing PAM(3)CSK(4) at a concentration of 1 mg/ml. After 48 h, 100 mL of supernatant was removed and stored at 808C for cytokine ELISAs. A 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed on the cells to examine viability. Briefly, 100 mL of MEM and 20 mL of MTT (5 mg/mL) was added to each well and cells were allowed to incubate at 378C for 4 h. After this, the medium was removed and replaced with 150 mL of DMSO. After 5 min of shaking, absorbance was measured at 595 nm on a Multiscan EX spectrophotometer (Thermo, Hudson, NH). 2.4. Enzyme-linked immunosorbant assay (ELISA) Supernatant from the dosing experiment was diluted 1:10 and ELISAs were performed to measure IL-6, TNF-a and IL-1b as per manufacturer’s instructions. 2.5. Immunofluorescence After separation of mixed glial cell cultures, astrocytes and microglia were plated into separate wells of a 12 well plate and a density of 2.5 105 cells/well. Cells were stained as previously described (Marek et al., 2008) and viewed on a confocal microscope (Olympus, Sacramento, CA).
2.2. Preparation of mixed glial cultures 2.6. Statistical analysis Research ethics approval for animal use was obtained from the Animal Care and Use Committee of the University of Northern British Columbia according to the guidelines of the Canadian Council on Animal Care (http://www.ccac.ca). Primary cell culture were prepared as previously described (Marek et al., 2008) with minor modification. Briefly, neonatal Balb/c mice were sacrificed by cervical dislocation within one day of birth and the brains harvested. The meninges were removed and the remaining tissue was placed in HBSS and dissociated by passing through a 20 gauge needle several times. Dissociated tissue was passed through a 70 mm nylon mesh and centrifuged (300 g for 10 min). The collected cells were re-suspended in culture medium (MEM; 1% penicillin/streptomycin; 0.2 mM L-glutamine; 10% FBS) and plated at a density of one 75 cm2 flask for every 2
Results are presented as means standard deviation of three independent experiments. One-way ANOVAs were performed, followed by Bonferroni multiple comparisons to examine differences between groups. Values of p < 0.5 were considered significant. All statistical analysis was done using GraphPad Prism software (GraphPad Software Inc., La Jolla, CA). 3. Results 3.1. Microglial and astrocyte purity Purified microglia and astrocytes from neonatal mouse brains were checked for purity by immunofluorescence using cell specific
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markers. Cultures were double-labelled with anti-CD11b, a microglial specific marker, and anti-GFAP, which is specific to astrocytes. The majority of cells within the purified microglial culture exhibited labelling for CD11b (>95%) (Fig. 1A). Similarly, most cells within the purified astrocyte culture were successfully labelled for GFAP (>90%) (Fig. 1B). The nuclei were also stained with counter stained DAPI (not pictured) and three fields of view were counted for each culture. 3.2. Effects of MeHg on IL-6 and TNF-a release from microglial monoculture Microglia show reduced secretion of IL-6, but not TNF-a upon exposure to MeHg in a concentration dependant manner. Microglial release of IL-6 was not significantly reduced for 0.1 mM MeHg exposure and reduced to 68% (p < 0.05) and 35% (p < 0.01) of the control for 0.5 mM and 1.0 mM MeHg concentrations respectively (Fig. 2A). TNF-a remained unchanged for the 0.1 mM treatment (Fig. 2B). IL-1b was also measured and was not expressed upon PAM(3) stimulation (data not shown). MTT assay exhibited no significant cytotoxicity for the 0.1 and 0.5 mM treatments compared to the control; however, and a reduction to 65% (p < 0.001) of the control was observed in the 1.0 mM dose (Fig. 2C). 3.3. Effects of MeHg on IL-6 and TNF-a release from microglial/ astrocyte co-culture Microglia/astrocyte co-culture show reduced secretion of IL-6, but not TNF-a upon exposure to MeHg in a concentration dependant manner. Glial release of IL-6 was significantly reduced to 87% (p < 0.05), 82% (p < 0.01) and 57% (p < 0.001) of the control for 0.1 mM, 0.5 mM and 1.0 mM exposures, respectively (Fig. 3A).
Fig. 1. Photo of glial cells under confocal microscope. Purified microglia (Panel A) and astrocytes (Panel B) from 1-day-old neonatal mice were plated onto treated culture slides and labelled with anti-CD11b (microglial marker; red) and anti-GFAP (astrocyte marker; green). Cultures were consistently pure (>95% for microglia; >90% for astrocytes). Three cultures from three separate litters were examined. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 2. PAM(3) stimulated microglia exhibited a concentration dependant reduction in IL-6 secretion (A) but not TNF-a secretion upon exposure to MeHg (B). Cells were incubated in 0.1, 0.5 and 1.0 mM concentrations of MeHg for 5 days and stimulated with PAM(3) after 72 h. MTT assay demonstrates that MeHg was not cytotoxic at lower exposures (C). Data are expressed as means S.D. from three independent experiments (n = 3). Groups statistically different from the control group are expressed as *p < 0.05 and ***p < 0.001.
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Fig. 3. PAM(3) stimulated microglia/astrocyte co-culture exhibited a concentration dependant reduction in IL-6 secretion (A) but not TNF-a secretion upon exposure to MeHg (B). A microglia/astrocyte proportion of 0.4 was plated and the cells were incubated in 0, 0.1, 0.5 and 1.0 mM concentrations of MeHg for 5 days and stimulated with PAM(3) after 72 h. MTT assay demonstrates that MeHg was not cytotoxic at lower exposures (C). Data are expressed as means S.D. from three independent experiments (n = 3). Groups statistically different from the control group are expressed as *p < 0.05, **p < 0.01, and ***p < 0.001.
TNF-a remained unchanged for all doses (98%, 102% and 104% for 0.1, 0.5, and 1.0 mM doses, respectively). The changes observed in TNF-a levels were not statistically significant. MTT assays showed a significant reduction to 90% cell viability in the 1 mM treatment (p < 0.01) (Fig. 3C). 3.4. Effects of microglia/astrocyte culture ratio on cell IL-6 and TNF-a release IL-6 secretion was increased upon increasing the relative proportion of astrocytes within the cell cultures (Fig. 4A). For the treatment without MeHg, IL-6 concentrations were found to be 2184, 3585 and 4386 pg/mL for the 100%, 60% and 40% microglial cultures respectively. In contrast, the 0% microglia culture (100% astrocytes) demonstrated a lower IL-6 concentration of 805 pg/mL. The amount of IL-6 released from the 100% microglia monoculture
was significantly different than the other three in the absence of MeHg (p < 0.001). Furthermore, when the 60% microglia culture was compared against the 40% microglial culture, the results demonstrated significance (p < 0.001). Additionally, IL-6 secretion showed a downward trend with respect to increasing MeHg exposure, with the exception of the astrocyte monoculture. Results were observed to be dose dependant with a total reduction at the 0.5 mM exposure of 68% of the no exposure treatment for the 100% microglia culture (p < 0.05); 80% of the no exposure treatment for the 60% culture (p < 0.05); and 82% of the no exposure treatment for the 40% culture (p < 0.01). Upon exposure to 1 mM MeHg, IL-6 secretion was even further suppressed to 35% (p < 0.001), 75% (p < 0.01), and 57% (p < 0.001) of the no exposure treatment for the 100%, 60% and 40% microglia cultures respectively. No significant changes were observed for the 0% microglia culture at any MeHg concentrations (Fig. 4A). TNF-a secretion was not
Fig. 4. IL-6 secretion is dependant (A) and TNF-a secretion is independent (B) of cell ratio and MeHg concentration. Glial cells were plated at varying ratios and incubated for 5 days. PAM(3) stimulation was conducted 48 h before supernatant was removed for measurement. Data are expressed as means S.D. from three independent experiments (n = 3).
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dependant on cell ratio or MeHg concentration. The astrocyte monoculture did not exhibit any TNF-a secretion (Fig. 4B). 4. Discussion Cytokines and chemokines are signalling proteins that are central to the immune response. IL-6 and TNF-a participate in immune function through promoting local inflammation, activating lymphocytes, and inducing fever. The inflammatory response, while important in fighting pathogens, can contribute to the development of cancer (Erkan et al., 2010), seizures (Zattoni et al., 2011) and Parkinson’s disease (Ferrari and Tarelli, 2011) as well as several other neurodegenerative conditions (Lee et al., 2010; Lue et al., 2010; Wee Yong, 2010; Worthmann et al., 2010). It is clear that any disruption to processes that control inflammation may have the potential to impact CNS function. We have demonstrated the effects of MeHg on the secretion of two pro-inflammatory cytokines, IL-6 and TNF-a by exposing primary microglial cells to MeHg followed by stimulation with PAM(3). Microglia cells were shown to exhibit reduced IL-6 secretion is a dose dependant manner (Fig. 2A). However, no significant change in TNF-a was observed in these same cells upon MeHg exposure (Fig. 2B). A similar trend was observed when microglia were co-cultured with astrocytes at ratio of 2:3 (Fig. 3). With regards to IL-6, it is apparent that the microglial monoculture was the most sensitive to MeHg, as it was reduced to 35% by the 1.0 mM MeHg exposure, whereas the co-culture was reduced to 57%. Furthermore, the MTT assays revealed that MeHg was more cytotoxic to the monoculture than the co-culture (Figs. 3 and 4). These results agree with the assessment that microglial are more sensitive to MeHg than astrocytes (Monnet-Tschudi et al., 1996; Ni et al., 2011). These data provide evidence that non-cytotoxic concentrations of MeHg can impact the secretion of specific cytokines. In addition, the ratio of glial cells present in culture influences the degree to which MeHg modulates the secretions of cytokines. It has been demonstrated that MeHg can induce the generation of reactive oxygen species (ROS) in microglial cells (Garg and Chang, 2006). Also, astrocytes have been shown to demethylate MeHg in an ROS-mediated manner (Shapiro and Chan, 2008). This would reduce the levels of organic mercury and increase the levels of inorganic mercury. Although both forms have been shown to be toxic to glial cells at similar concentrations (Monnet-Tschudi et al., 1996), their mechanisms to induce toxicity are likely different from one another. When examining the impact of the microglia/astrocyte ratio on the ability of these cells to secrete cytokines, IL-1b secretion was not observed in any of the culture, indicating that PAM(3) cannot induce the expression of this cytokine from microglial cells or astrocytes. IL-6 secretion was observed to increase as the ratio of microglia/astrocytes was reduced, with the highest expression observed in the 60% astrocyte treatment (Fig. 4A). However, IL-6 secretion was much lower in the astrocyte monoculture. PAM(3) has been shown to stimulate the release of IL-6 from microglia, but not astrocytes (Henn et al., 2011). However, astrocytes are known to secrete IL-6 upon stimulation with TNF-a (Van Wagoner et al., 1999). These results imply that glial cells are able to function synergistically in the production and secretion of IL-6. It is possible that the low amount of IL-6 observed within the astrocyte monoculture is attributed to a small number (>1%) of contaminating microglia. Furthermore, it is possible that a small number of oligodentrocytes remain in the cultures, although the methods used to obtain the cells have been shown to produce pure cultures (Marek et al., 2008). In contrast to IL-6, TNF-a secretion was not impacted by cell ratio, with the exception of the astrocyte monoculture which demonstrated no TNF-a release (Fig. 4B). These results are consistent with Zhou et al., who demonstrated
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that PAM(3) stimulated astrocytes lacked the ability to secrete TNF-a (Zhou et al., 2008). Interestingly, this study provides evidence that TNF-a release is not mediated through TLR1/2 binding in astrocytes but is in microglial cells. However, it is possible that microglial cells that have been activated by PAM(3) will promote the release of TNF-a from astrocytes. These results agree with those observed by Henn et al., that the responsiveness of astrocytes changes when they are exposed to activated microglial cells (Henn et al., 2011). These results also suggest the presence of a negative feedback mechanism that functions to maintain TNF-a levels, despite different cell populations. Further research must be done to determine the constituents of this pathway. Taken together, these results demonstrate that microglia and astrocytes are dependant on one another for function, and mixtures of these two cell types demonstrate increased IL-6 secretion and MeHg tolerance than when compared to the monocultures. Furthermore, low doses of MeHg can impair the secretion of IL-6, providing a plausible link between this toxin and reduced glial function associated with several neurodegenerative conditions (Streit et al., 2009; L’Episcopo et al., 2010). This is the first time that the complex relationships between glial cells and MeHg are reported. Future in vitro studies using monoculture model need to consider the interactive effects between cell types. Further research is needed to determine the mechanism of cell communications. In addition, implications of long term exposure to mercury on the pathogenesis of neurodegenerative diseases needs to be further studied and the roles of glial cells in mediating toxicity must be better defined. Conflict of interest statement No conflict of interest declared. Acknowledgment The research is funded by a Discovery Grant from the Natural Sciences and Engineering Research Council of Canada to HMC. References Chang JY. Methylmercury causes glial IL-6 release. Neurosci Lett 2007;416:217–20. D’Andrea MR, Cole GM, Ard MD. The microglial phagocytic role with specific plaque types in the Alzheimer disease brain. Neurobiol Aging 2004;25:675–83. Erkan M, Reiser-Erkan C, Michalski CW, Kleeff J. Tumor microenvironment and progression of pancreatic cancer. Exp Oncol 2010;32:128–31. Eskes C, Honegger P, Juillerat-Jeanneret L, Monnet-Tschudi F. Microglial reaction induced by noncytotoxic methylmercury treatment leads to neuroprotection via interactions with astrocytes and IL-6 release. Glia 2002;37:43–52. Eto K, Marumoto M, Takeya M. The pathology of methylmercury poisoning (Minamata disease). Neuropathology 2010;30:471–9. Ferrari CC, Tarelli R. Parkinson’s disease and systemic inflammation. Parkinsons Dis 2011;436813. Freeman MR. Specification and morphogenesis of astrocytes. Science 2010;330:774–8. Garg TK, Chang JY. Methylmercury causes oxidative stress and cytotoxicity in microglia: attenuation by 15-deoxy-delta 12,14-prostaglandin J2. J Neuroimmunol 2006;171:17–28. Graeber MB. Changing face of microglia. Science 2010;330:783–8. Hemdan NY, Lehmann I, Wichmann G, Lehmann J, Emmrich F, Sack U. Immunomodulation by mercuric chloride in vitro: application of different cell activation pathways. Clin Exp Immunol 2007;148:325–37. Henn A, Kirner S, Leist M. TLR2 hypersensitivity of astrocytes as functional consequence of previous inflammatory episodes. J Immunol 2011;186:3237–47. Lawrence DA, McCabe MJ Jr. Immunomodulation by metals. Int Immunopharmacol 2002;2:293–302. Lee YJ, Han SB, Nam SY, Oh KW, Hong JT. Inflammation and Alzheimer’s disease. Arch Pharm Res 2010;33:1539–56. L’Episcopo F, Tirolo C, Testa N, Caniglia S, Morale MC, Marchetti B. Glia as a turning point in the therapeutic strategy of Parkinson’s disease. CNS Neurol Disord Drug Targets 2010;9:349–72. Li P, Feng X, Qiu G. Methylmercury exposure and health effects from rice and fish consumption: a review. Int J Environ Res Publ Health 2010;7:2666–91. Lue LF, Kuo YM, Beach T, Walker DG. Microglia activation and anti-inflammatory regulation in Alzheimer’s disease. Mol Neurobiol 2010;41:115–28.
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Marek R, Caruso M, Rostami A, Grinspan JB, Das Sarma J. Magnetic cell sorting: a fast and effective method of concurrent isolation of high purity viable astrocytes and microglia from neonatal mouse brain tissue. J Neurosci Methods 2008;175:108–18. Monnet-Tschudi F, Zurich MG, Honegger P. Comparison of the developmental effects of two mercury compounds on glial cells and neurons in aggregate cultures of rat telencephalon. Brain Res 1996;741:52–9. Ni M, Li X, Yin Z, Sidoryk-Wegrzynowicz M, Jiang H, Farina M, et al. Comparative study on the response of rat primary astrocytes and microglia to methylmercury toxicity. Glia 2011;59:810–20. Okun E, Griffioen KJ, Lathia JD, Tang S, Mattson MP, Arumugam TV. Toll-like receptors in neurodegeneration. Brain Res Rev 2009;59:278–92. Philips T, Robberecht W. Neuroinflammation in amyotrophic lateral sclerosis: role of glial activation in motor neuron disease. Lancet Neurol 2011;10:253–63. Shapiro AM, Chan HM. Characterization of demethylation of methylmercury in cultured astrocytes. Chemosphere 2008;74:112–8. Silva SL, Osorio C, Vaz AR, Barateiro A, Falcao AS, Silva RF, et al. Dynamics of neuron-glia interplay upon exposure to unconjugated bilirubin. J Neurochem 2011;117:412–24. Streit WJ. Microglia and neuroprotection: implications for Alzheimer’s disease. Brain Res Brain Res Rev 2005;48:234–9. Streit WJ, Braak H, Xue QS, Bechmann I. Dystrophic (senescent) rather than activated microglial cells are associated with tau pathology and likely precede neurodegeneration in Alzheimer’s disease. Acta Neuropathol 2009;118:475–85.
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Contents lists available at SciVerse ScienceDirect
NeuroToxicology
Effects of methylmercury on the secretion of pro-inflammatory cytokines from primary microglial cells and astrocytes Tyler Bassett, Paxton Bach, Hing Man Chan * Community Health Sciences Program, University of Northern British Columbia, Prince George, BC, Canada
A R T I C L E I N F O
A B S T R A C T
Article history: Received 17 June 2011 Accepted 13 October 2011 Available online 20 October 2011
Glial cells, including oligodendrocytes, astrocytes and microglia are important to proper central nervous system (CNS) function. Deregulation or changes to CNS populations of astrocytes and microglia in particular are expected to play a role in many neurodegenerative diseases, including Parkinson’s disease, amyotrophic lateral sclerosis (ALS) and Alzheimer’s disease (AD). Previous studies have reported methylmercury (MeHg) induced changes in glial cell function; however, the effects of MeHg on these cells remains poorly understood. This study aims to examine the effect of MeHg on the secretion of proinflammatory cytokines from microglia and astrocytes. The impact of the microglia/astrocyte ratio on cytokine secretion was also examined. Microglia and astrocytes were cultured from the brains of neonatal BALB/C mice and dosed with MeHg (0–1 mM) and stimulated with PAM(3)CSK(4) (PAM(3)), a tolllike receptor (TLR) ligand. After this, the secretion of interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-a) and interleukin-1-beta (IL-1b) was measured by ELISA. MeHg reduced the secretion of IL-6 in a dose dependant manner but did not effect the secretion of TNF-a. No change in IL-1b was observed in any treatments, indicating that PAM(3) cannot induce the secretion of this cytokine from glial cells. Additionally, the ratio of microglia/astrocyte had an effect on the secretion of IL-6 but not TNF-a. These results indicate that MeHg can modify the response of glial cells and the interactions with astrocytes can affect the response of the microglia cells in culture. These results are significant in understanding the potential relationship with MeHg and neurodegenerative diseases and for the interpretation of results of future in vitro studies using monoculture. ß 2011 Elsevier Inc. All rights reserved.
Keywords: Methylmercury Astrocytes Microglia IL-6 TNF-a
1. Introduction The roles of glial cells in influencing neuron function have been better understood in the last decade. Astrocytes and microglia demonstrate a great degree of plasticity and participate in many processes within CNS development and function (Freeman, 2010; Graeber, 2010). It is widely understood that these cells contribute an inflammatory response in the central nervous system (Henn et al., 2011; Silva et al., 2011; van Neerven et al., 2010). They are actively being investigated for any potential roles in neurodegenerative diseases such as Alzheimer’s disease (AD), Parkinson’s disease and amyotrophic lateral sclerosis (ALS) (Philips and Robberecht, 2011). It has been suggested that microglia may either play a contributing role or a protective role in the pathogenesis of Alzheimer’s disease (Streit, 2005; Town et al., 2005). For instance, the presence of an increased number of
* Corresponding author at: Center for Advanced Research in Environmental Genomics, University of Ottawa, 30 Marie Curie, Ottawa, ON, Canada K1H 1A1. Tel.: +1 613 562 5800x6349; fax: +1 613 562 5385. E-mail address: [email protected] (H.M. Chan). 0161-813X/$ – see front matter ß 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.neuro.2011.10.003
dystrophic microglia precedes the accumulation of neurofibrillary tangles associated with the pathology of AD. This implies that a loss of the neuroprotective functions of microglia contribute to the development of AD (Streit et al., 2009). In contrast, while microglial activation is thought to play a role in the clearance of beta-amyloid plaques associated with AD, their activation can lead to the release of neurotoxic factors that contribute to secondary neuronal cell death (D’Andrea et al., 2004). While the exact role of glial cells in the pathogenesis of neurodegenerative diseases is unclear, it is likely that they do play a role in the etiology of these conditions. Microglia and astrocytes can respond to pathogenic agents through interaction of common antigenic determinants with tolllike receptors (TLR) found on their surface. When bound, these receptors will activate downstream signalling pathways that will induce the expression of various pro-inflammatory cytokines (Henn et al., 2011; Zhou et al., 2008). TLRs are expressed on the surface of both microglia and astrocytes. Changes in TLR expression have been implicated in AD and multiple sclerosis (Okun et al., 2009). For example, mice with a mutation to the TLR4 gene show an increased susceptibility to the accumulation of amyloid-beta (Ab) plaques, while activation of TLR 2, 4 and 9 result in an increase in Ab clearance by a microglial cell (Tahara et al.,
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2006). While age related changes in TLR levels and activity likely play a role in the development of several age related neurodegenerative diseases, it would be interesting to consider whether environmental toxins might contribute to this also. Therefore, the objective of this study is to examine the effect of a well known environmental neurotoxin, MeHg on cytokine secretion from glial cells that have been stimulated with a TLR1/2 agonist, PAM(3). Due to its tendency to bioaccumulate and biomagnify along the aquatic food web, MeHg can be a health concern, especially in populations that sustain themselves on diets consisting of fish (Li et al., 2010). While the effects of acute exposure to toxic levels of MeHg are well known (Eto et al., 2010), the impacts of chronic exposure to lower concentrations of MeHg are less understood. Metals, including mercury, are known to cause immunomodulation in cells associated with the immune system, including lymphocytes, monocytes and macrophages (Hemdan et al., 2007; Lawrence and McCabe, 2002). In fact, when C6 glioma cells are challenged with short term exposure to high concentrations of MeHg, they can be induced to release IL-6 (Chang, 2007). However, microglia exposed to non-cytotoxic levels of MeHg did not have the same effect (Eskes et al., 2002). This study aims to determine whether a ubiquitous environmental toxicant, MeHg, can modulate the function of microglial cells and astrocytes through influencing their ability to produce pro-inflammatory cytokines. Additionally, we also hypothesize that co-culture of microglial cells with astrocytes can affect cytokine secretion. 2. Methods 2.1. Materials Balb/c mice were from Charles River (Montreal, QC). Methylmercury was obtained from Alfa Aesar (Ward Hill, MA). Minimum essential medium (MEM (10370–021)), fetal bovine serum (FBS (16000–044)), goat serum, Hanks Balanced Salt Solution (HBSS), F12 medium, Phosphate buffered saline (PBS), 1 Trypsin-EDTA, penicillin/streptomycin (10,000 U/mL), and L-glutamine (200 mM) were acquired from Gibco (Burlington, ON). CD11b microbeads and magnetic columns were obtained from Miltenyi Biotech (Auburn, CA). IC-07 hybridoma cells were acquired from HPA culture collections (Salisbury, UK) and A2B5 hybridoma cells were acquired from American Type Culture Collection (ATCC) (Manassas, VA). The rabbit complement was from Cedarlane (Burlington, ON). Anti-mouse CD11b antibody was from BD Pharmigen (Mississauga, ON). Anti-GFAP antibody, Cy3-strepavidin, DAPI, PAM(3)CSK(4), and MTT were from Invitrogen (Burlington, ON). FITC-anti rabbit IgG antibody was from Jackson Immunoresearch (West Grove, PA). IL-6, TNF-a and IL-1b ELISAs were obtained from eBioscience (San Diego, CA).
mice harvested. Cells were incubated in a humidified incubator (Thermo (Hudson, NH)) at 37 8C and 5% CO2. After 24 h, the nonadherent cells were removed and cells were incubated in culture medium for 10 days with a medium change for every 3–4 days. After 10 days, oligodendrocytes were removed by complementmediated cytolysis as previously described (Marek et al., 2008) and remaining cells were incubated in culture medium for 10 days without a medium change. Cells were then washed with PBS without Mg2+/Ca2+ and removed from the flask by incubating in 2 ml of 1 trypsin/EDTA solution at 37 8C for 5 min. Dislodged cells were separated by magnetic cell sorting using CD11b microbeads (Miltenyi Biotech, Auburn, CA) as per manufacturer’s directions. The positive fraction contained purified microglial cells while the negative fraction was enriched for astrocytes. Purified cells were re-suspended in culture medium at a final density of 2.5 105 cells/ml for plating. 2.3. MeHg dosing experiments Primary glial cells were plated at microglia/astrocytes proportions of 1, 0.6, 0.4, and 0 into a 96-well flat-bottom culture plate at a density of 25,000 cells/well and incubated at 37 8C, 5% CO2 overnight. The following day, the medium was removed and replaced with dosing medium (MEM containing 1% penicillin/ streptomycin, 0.2 mM L-glutamine, 5% fetal bovine serum and MeHg at a concentration of 0–1.0 mM). On day 3, medium was removed and replaced with dosing medium containing PAM(3)CSK(4) at a concentration of 1 mg/ml. After 48 h, 100 mL of supernatant was removed and stored at 808C for cytokine ELISAs. A 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed on the cells to examine viability. Briefly, 100 mL of MEM and 20 mL of MTT (5 mg/mL) was added to each well and cells were allowed to incubate at 378C for 4 h. After this, the medium was removed and replaced with 150 mL of DMSO. After 5 min of shaking, absorbance was measured at 595 nm on a Multiscan EX spectrophotometer (Thermo, Hudson, NH). 2.4. Enzyme-linked immunosorbant assay (ELISA) Supernatant from the dosing experiment was diluted 1:10 and ELISAs were performed to measure IL-6, TNF-a and IL-1b as per manufacturer’s instructions. 2.5. Immunofluorescence After separation of mixed glial cell cultures, astrocytes and microglia were plated into separate wells of a 12 well plate and a density of 2.5 105 cells/well. Cells were stained as previously described (Marek et al., 2008) and viewed on a confocal microscope (Olympus, Sacramento, CA).
2.2. Preparation of mixed glial cultures 2.6. Statistical analysis Research ethics approval for animal use was obtained from the Animal Care and Use Committee of the University of Northern British Columbia according to the guidelines of the Canadian Council on Animal Care (http://www.ccac.ca). Primary cell culture were prepared as previously described (Marek et al., 2008) with minor modification. Briefly, neonatal Balb/c mice were sacrificed by cervical dislocation within one day of birth and the brains harvested. The meninges were removed and the remaining tissue was placed in HBSS and dissociated by passing through a 20 gauge needle several times. Dissociated tissue was passed through a 70 mm nylon mesh and centrifuged (300 g for 10 min). The collected cells were re-suspended in culture medium (MEM; 1% penicillin/streptomycin; 0.2 mM L-glutamine; 10% FBS) and plated at a density of one 75 cm2 flask for every 2
Results are presented as means standard deviation of three independent experiments. One-way ANOVAs were performed, followed by Bonferroni multiple comparisons to examine differences between groups. Values of p < 0.5 were considered significant. All statistical analysis was done using GraphPad Prism software (GraphPad Software Inc., La Jolla, CA). 3. Results 3.1. Microglial and astrocyte purity Purified microglia and astrocytes from neonatal mouse brains were checked for purity by immunofluorescence using cell specific
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markers. Cultures were double-labelled with anti-CD11b, a microglial specific marker, and anti-GFAP, which is specific to astrocytes. The majority of cells within the purified microglial culture exhibited labelling for CD11b (>95%) (Fig. 1A). Similarly, most cells within the purified astrocyte culture were successfully labelled for GFAP (>90%) (Fig. 1B). The nuclei were also stained with counter stained DAPI (not pictured) and three fields of view were counted for each culture. 3.2. Effects of MeHg on IL-6 and TNF-a release from microglial monoculture Microglia show reduced secretion of IL-6, but not TNF-a upon exposure to MeHg in a concentration dependant manner. Microglial release of IL-6 was not significantly reduced for 0.1 mM MeHg exposure and reduced to 68% (p < 0.05) and 35% (p < 0.01) of the control for 0.5 mM and 1.0 mM MeHg concentrations respectively (Fig. 2A). TNF-a remained unchanged for the 0.1 mM treatment (Fig. 2B). IL-1b was also measured and was not expressed upon PAM(3) stimulation (data not shown). MTT assay exhibited no significant cytotoxicity for the 0.1 and 0.5 mM treatments compared to the control; however, and a reduction to 65% (p < 0.001) of the control was observed in the 1.0 mM dose (Fig. 2C). 3.3. Effects of MeHg on IL-6 and TNF-a release from microglial/ astrocyte co-culture Microglia/astrocyte co-culture show reduced secretion of IL-6, but not TNF-a upon exposure to MeHg in a concentration dependant manner. Glial release of IL-6 was significantly reduced to 87% (p < 0.05), 82% (p < 0.01) and 57% (p < 0.001) of the control for 0.1 mM, 0.5 mM and 1.0 mM exposures, respectively (Fig. 3A).
Fig. 1. Photo of glial cells under confocal microscope. Purified microglia (Panel A) and astrocytes (Panel B) from 1-day-old neonatal mice were plated onto treated culture slides and labelled with anti-CD11b (microglial marker; red) and anti-GFAP (astrocyte marker; green). Cultures were consistently pure (>95% for microglia; >90% for astrocytes). Three cultures from three separate litters were examined. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 2. PAM(3) stimulated microglia exhibited a concentration dependant reduction in IL-6 secretion (A) but not TNF-a secretion upon exposure to MeHg (B). Cells were incubated in 0.1, 0.5 and 1.0 mM concentrations of MeHg for 5 days and stimulated with PAM(3) after 72 h. MTT assay demonstrates that MeHg was not cytotoxic at lower exposures (C). Data are expressed as means S.D. from three independent experiments (n = 3). Groups statistically different from the control group are expressed as *p < 0.05 and ***p < 0.001.
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Fig. 3. PAM(3) stimulated microglia/astrocyte co-culture exhibited a concentration dependant reduction in IL-6 secretion (A) but not TNF-a secretion upon exposure to MeHg (B). A microglia/astrocyte proportion of 0.4 was plated and the cells were incubated in 0, 0.1, 0.5 and 1.0 mM concentrations of MeHg for 5 days and stimulated with PAM(3) after 72 h. MTT assay demonstrates that MeHg was not cytotoxic at lower exposures (C). Data are expressed as means S.D. from three independent experiments (n = 3). Groups statistically different from the control group are expressed as *p < 0.05, **p < 0.01, and ***p < 0.001.
TNF-a remained unchanged for all doses (98%, 102% and 104% for 0.1, 0.5, and 1.0 mM doses, respectively). The changes observed in TNF-a levels were not statistically significant. MTT assays showed a significant reduction to 90% cell viability in the 1 mM treatment (p < 0.01) (Fig. 3C). 3.4. Effects of microglia/astrocyte culture ratio on cell IL-6 and TNF-a release IL-6 secretion was increased upon increasing the relative proportion of astrocytes within the cell cultures (Fig. 4A). For the treatment without MeHg, IL-6 concentrations were found to be 2184, 3585 and 4386 pg/mL for the 100%, 60% and 40% microglial cultures respectively. In contrast, the 0% microglia culture (100% astrocytes) demonstrated a lower IL-6 concentration of 805 pg/mL. The amount of IL-6 released from the 100% microglia monoculture
was significantly different than the other three in the absence of MeHg (p < 0.001). Furthermore, when the 60% microglia culture was compared against the 40% microglial culture, the results demonstrated significance (p < 0.001). Additionally, IL-6 secretion showed a downward trend with respect to increasing MeHg exposure, with the exception of the astrocyte monoculture. Results were observed to be dose dependant with a total reduction at the 0.5 mM exposure of 68% of the no exposure treatment for the 100% microglia culture (p < 0.05); 80% of the no exposure treatment for the 60% culture (p < 0.05); and 82% of the no exposure treatment for the 40% culture (p < 0.01). Upon exposure to 1 mM MeHg, IL-6 secretion was even further suppressed to 35% (p < 0.001), 75% (p < 0.01), and 57% (p < 0.001) of the no exposure treatment for the 100%, 60% and 40% microglia cultures respectively. No significant changes were observed for the 0% microglia culture at any MeHg concentrations (Fig. 4A). TNF-a secretion was not
Fig. 4. IL-6 secretion is dependant (A) and TNF-a secretion is independent (B) of cell ratio and MeHg concentration. Glial cells were plated at varying ratios and incubated for 5 days. PAM(3) stimulation was conducted 48 h before supernatant was removed for measurement. Data are expressed as means S.D. from three independent experiments (n = 3).
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dependant on cell ratio or MeHg concentration. The astrocyte monoculture did not exhibit any TNF-a secretion (Fig. 4B). 4. Discussion Cytokines and chemokines are signalling proteins that are central to the immune response. IL-6 and TNF-a participate in immune function through promoting local inflammation, activating lymphocytes, and inducing fever. The inflammatory response, while important in fighting pathogens, can contribute to the development of cancer (Erkan et al., 2010), seizures (Zattoni et al., 2011) and Parkinson’s disease (Ferrari and Tarelli, 2011) as well as several other neurodegenerative conditions (Lee et al., 2010; Lue et al., 2010; Wee Yong, 2010; Worthmann et al., 2010). It is clear that any disruption to processes that control inflammation may have the potential to impact CNS function. We have demonstrated the effects of MeHg on the secretion of two pro-inflammatory cytokines, IL-6 and TNF-a by exposing primary microglial cells to MeHg followed by stimulation with PAM(3). Microglia cells were shown to exhibit reduced IL-6 secretion is a dose dependant manner (Fig. 2A). However, no significant change in TNF-a was observed in these same cells upon MeHg exposure (Fig. 2B). A similar trend was observed when microglia were co-cultured with astrocytes at ratio of 2:3 (Fig. 3). With regards to IL-6, it is apparent that the microglial monoculture was the most sensitive to MeHg, as it was reduced to 35% by the 1.0 mM MeHg exposure, whereas the co-culture was reduced to 57%. Furthermore, the MTT assays revealed that MeHg was more cytotoxic to the monoculture than the co-culture (Figs. 3 and 4). These results agree with the assessment that microglial are more sensitive to MeHg than astrocytes (Monnet-Tschudi et al., 1996; Ni et al., 2011). These data provide evidence that non-cytotoxic concentrations of MeHg can impact the secretion of specific cytokines. In addition, the ratio of glial cells present in culture influences the degree to which MeHg modulates the secretions of cytokines. It has been demonstrated that MeHg can induce the generation of reactive oxygen species (ROS) in microglial cells (Garg and Chang, 2006). Also, astrocytes have been shown to demethylate MeHg in an ROS-mediated manner (Shapiro and Chan, 2008). This would reduce the levels of organic mercury and increase the levels of inorganic mercury. Although both forms have been shown to be toxic to glial cells at similar concentrations (Monnet-Tschudi et al., 1996), their mechanisms to induce toxicity are likely different from one another. When examining the impact of the microglia/astrocyte ratio on the ability of these cells to secrete cytokines, IL-1b secretion was not observed in any of the culture, indicating that PAM(3) cannot induce the expression of this cytokine from microglial cells or astrocytes. IL-6 secretion was observed to increase as the ratio of microglia/astrocytes was reduced, with the highest expression observed in the 60% astrocyte treatment (Fig. 4A). However, IL-6 secretion was much lower in the astrocyte monoculture. PAM(3) has been shown to stimulate the release of IL-6 from microglia, but not astrocytes (Henn et al., 2011). However, astrocytes are known to secrete IL-6 upon stimulation with TNF-a (Van Wagoner et al., 1999). These results imply that glial cells are able to function synergistically in the production and secretion of IL-6. It is possible that the low amount of IL-6 observed within the astrocyte monoculture is attributed to a small number (>1%) of contaminating microglia. Furthermore, it is possible that a small number of oligodentrocytes remain in the cultures, although the methods used to obtain the cells have been shown to produce pure cultures (Marek et al., 2008). In contrast to IL-6, TNF-a secretion was not impacted by cell ratio, with the exception of the astrocyte monoculture which demonstrated no TNF-a release (Fig. 4B). These results are consistent with Zhou et al., who demonstrated
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that PAM(3) stimulated astrocytes lacked the ability to secrete TNF-a (Zhou et al., 2008). Interestingly, this study provides evidence that TNF-a release is not mediated through TLR1/2 binding in astrocytes but is in microglial cells. However, it is possible that microglial cells that have been activated by PAM(3) will promote the release of TNF-a from astrocytes. These results agree with those observed by Henn et al., that the responsiveness of astrocytes changes when they are exposed to activated microglial cells (Henn et al., 2011). These results also suggest the presence of a negative feedback mechanism that functions to maintain TNF-a levels, despite different cell populations. Further research must be done to determine the constituents of this pathway. Taken together, these results demonstrate that microglia and astrocytes are dependant on one another for function, and mixtures of these two cell types demonstrate increased IL-6 secretion and MeHg tolerance than when compared to the monocultures. Furthermore, low doses of MeHg can impair the secretion of IL-6, providing a plausible link between this toxin and reduced glial function associated with several neurodegenerative conditions (Streit et al., 2009; L’Episcopo et al., 2010). This is the first time that the complex relationships between glial cells and MeHg are reported. Future in vitro studies using monoculture model need to consider the interactive effects between cell types. Further research is needed to determine the mechanism of cell communications. In addition, implications of long term exposure to mercury on the pathogenesis of neurodegenerative diseases needs to be further studied and the roles of glial cells in mediating toxicity must be better defined. Conflict of interest statement No conflict of interest declared. Acknowledgment The research is funded by a Discovery Grant from the Natural Sciences and Engineering Research Council of Canada to HMC. References Chang JY. Methylmercury causes glial IL-6 release. Neurosci Lett 2007;416:217–20. D’Andrea MR, Cole GM, Ard MD. The microglial phagocytic role with specific plaque types in the Alzheimer disease brain. Neurobiol Aging 2004;25:675–83. Erkan M, Reiser-Erkan C, Michalski CW, Kleeff J. Tumor microenvironment and progression of pancreatic cancer. Exp Oncol 2010;32:128–31. Eskes C, Honegger P, Juillerat-Jeanneret L, Monnet-Tschudi F. Microglial reaction induced by noncytotoxic methylmercury treatment leads to neuroprotection via interactions with astrocytes and IL-6 release. Glia 2002;37:43–52. Eto K, Marumoto M, Takeya M. The pathology of methylmercury poisoning (Minamata disease). Neuropathology 2010;30:471–9. Ferrari CC, Tarelli R. Parkinson’s disease and systemic inflammation. Parkinsons Dis 2011;436813. Freeman MR. Specification and morphogenesis of astrocytes. Science 2010;330:774–8. Garg TK, Chang JY. Methylmercury causes oxidative stress and cytotoxicity in microglia: attenuation by 15-deoxy-delta 12,14-prostaglandin J2. J Neuroimmunol 2006;171:17–28. Graeber MB. Changing face of microglia. Science 2010;330:783–8. Hemdan NY, Lehmann I, Wichmann G, Lehmann J, Emmrich F, Sack U. Immunomodulation by mercuric chloride in vitro: application of different cell activation pathways. Clin Exp Immunol 2007;148:325–37. Henn A, Kirner S, Leist M. TLR2 hypersensitivity of astrocytes as functional consequence of previous inflammatory episodes. J Immunol 2011;186:3237–47. Lawrence DA, McCabe MJ Jr. Immunomodulation by metals. Int Immunopharmacol 2002;2:293–302. Lee YJ, Han SB, Nam SY, Oh KW, Hong JT. Inflammation and Alzheimer’s disease. Arch Pharm Res 2010;33:1539–56. L’Episcopo F, Tirolo C, Testa N, Caniglia S, Morale MC, Marchetti B. Glia as a turning point in the therapeutic strategy of Parkinson’s disease. CNS Neurol Disord Drug Targets 2010;9:349–72. Li P, Feng X, Qiu G. Methylmercury exposure and health effects from rice and fish consumption: a review. Int J Environ Res Publ Health 2010;7:2666–91. Lue LF, Kuo YM, Beach T, Walker DG. Microglia activation and anti-inflammatory regulation in Alzheimer’s disease. Mol Neurobiol 2010;41:115–28.
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