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Mechanisms of Ageing and Development 173 (2018) 84–91
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Infiltrating macrophages contribute to age-related neuroinflammation in C57/BL6 mice H. Wolfe, A.M. Minogue, S. Rooney, M.A. Lynch
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Trinity College Institute for Neuroscience, Trinity College, Dublin 2, Ireland
A R T I C LE I N FO
A B S T R A C T
Keywords: Age Microglial activation Macrophage infiltration Inflammatory cytokines
The recognized role of neuroinflammation in the age-related deterioration of neuronal function highlights the importance of understanding the factors that control microglial activation. Microglia, as the immune cells of the brain, are the arbiters of the inflammatory profile in the brain. Normally they are maintained in a quiescent state by means of ligand-receptor interactions with neurons, within a prevailing anti-inflammatory microenvironment. The evidence indicates that, as the ageing process continues, microglia become activated, shift towards an inflammatory phenotype and alter the milieu in the brain. Although there has been progress in identifying factors that contribute to age-related microglial activation, our understanding remains incomplete. Here we report that there was an age-related increase in circulating inflammatory cytokines, accompanied by microglial activation. Neutrophils, and to a greater extent, macrophages, infiltrate the brain with age, perhaps as a result of increased chemokine expression in the brain, specifically CXCL1 and CCL2. We sought to determine whether macrophages might trigger microglial activation and the evidence shows that conditioned medium obtained from interferon-γ (IFNγ)-stimulated macrophages potently activated microglia. The data suggest that infiltrating macrophages may be one factor that contributes to age-related microglial activation.
1. Introduction With age, a host of dynamic changes in the immune system occurs. The upshot of this is that low-grade inflammation gradually leads to chronic inflammation; this has been referred to as inflammaging. In addition to peripheral changes, inflammatory changes also occur in the brain with age and microglia, as the primary immune cells in the brain, provide the driving force for neuroinflammation. Thus, neuroinflammation is characterized by increased microglial activation with evidence indicating increased expression of cell surface markers of activation, like CD11b, CD68 and MHCII and increased expression of inflammatory cytokines, like tumour necrosis factor-α (TNFα), interleukin (IL)-1β and IL-6. The implication is that, with age, microglia that are vital for neuroprotection gradually morph from their neuroprotective and anti-inflammatory phenotype to an inflammatory phenotype (Lee et al., 2013), which can ultimately become detrimental to neurons (Kielian, 2014; Ransohoff, 2016). Likewise, age tends to initiate a shift in macrophages from the so-called M2 phenotype to the inflammatory M1 phenotype; for example lipopolysaccharide (LPS) and interferon-γ (IFNγ) triggered a greater release of inflammatory cytokines in macrophages from old, compared with young, rats (Barrett et al., 2015a; Dimitrijevic et al., 2016). However, in contrast to the consistent
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Corresponding author. E-mail address: [email protected] (M.A. Lynch).
https://doi.org/10.1016/j.mad.2018.05.003 Received 21 March 2018; Received in revised form 24 April 2018; Accepted 9 May 2018 0047-6374/ © 2018 Elsevier B.V. All rights reserved.
findings in microglia, there are conflicting reports with respect to macrophages and some findings indicate that their response to inflammatory stimuli is reduced (Rawji et al., 2016; Shaw et al., 2013) as opposed to increased with age. In addition to age-related changes, microglial activation and the associated neuroinflammation is a characteristic of neurodegenerative diseases including Alzheimer's disease (AD) and Parkinson's disease (Molteni and Rossetti, 2017) and recent evidence from genome-wide association studies identified polymorphisms in several genes known to play a role in inflammation and immune modulation as risk factors in AD (Karch and Goate, 2015). The particular significance of this finding is that it pointed to altered microglial activation as a possible contributor to AD rather than being a consequence of the pathogenic processes associated with the disease. This finding highlights the need to identify the factors that trigger inappropriate microglial activation. Microglia react to a plethora of stimuli and adopt an inflammatory signature in response to inflammatory stimuli. Under normal circumstances, several factors converge to maintain an anti-inflammatory milieu in the brain. Resting concentrations of inflammatory mediators are low, while there is a relatively higher concentration of anti-inflammatory cytokines like IL-10 and TGFβ (Aloisi, 2001). In addition, microglia are maintained in a relatively quiescent state by interacting
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with Live/Dead stain (1 ml; 1:1000 in PBS, Life Technologies, US; 30 min) and blocking solution (1:50; 50 μl; purified rat anti-mouse CD16/CD32, BD Pharmingen, US; 15 min) with intermediate washing and centrifuging (1200 rpm; 5 min; 22 °C). The antibody Master Mix (50 μl; 15 min) was added and samples were incubated with the cell surface antibodies for 15 min; this included CD11b (PE-Cy7; 1:100; Biolegend, US), CD45 (APC-Cy7; 1:80, Biolegend US), CCR1 (APC; 1:20; R&D Systems, UK), CXCR2 (PE1:20; R&D Systems) and Ly6G (FITC; 1:100; Biolegend, US). Propidium iodide (PI; Sigma-Aldrich, UK) was added (1:100) immediately before reading the samples and was used as a live/dead stain. The appropriate compensation controls and FMO (fluorescence minus one) controls were also prepared. Flow cytometric analysis was performed using a DAKO CyAn-ADP 7 colour flow cytometer (Beckman Coulter, US) and analysed with FlowJo v7.6.5 software (Tree Star Inc., US). Microglia were identified as being CD11b+CD45low cells and macrophages as CD11b+CD45high cells (Becher and Antel, 1996; Sedgwick et al., 1991).
with other cells, for example neurons through CD200-CD200R and CX3CL1-CX3CR1 interactions (Lyons et al., 2007; Lyons et al., 2009). With age the neuron-microglial interactions are less robust (Lyons et al., 2007; Lyons et al., 2009) probably contributing to microglial activation but in addition, inflammatory cytokine concentration increases, perhaps as a consequence of infiltrating cells, including macrophages (Minogue et al., 2014). Here we set out to investigate the impact of infiltrating macrophages on microglia in the brain of aged mice and show that the increase in circulating cytokines is accompanied by increased infiltration of neutrophils and, to a greater extent, macrophages. Conditioned medium (CM) from stimulated macrophages markedly increased microglial activation suggesting that infiltrating macrophages may be one factor that contributes to the age-related microglial activation. 2. Materials and methods 2.1. Animals
2.4. Preparation of BMDM Groups of young (2–3 months; n = 8) and aged (16-18 months; n = 10) C57/BL6 mice were used to assess microglial activation; half of these were used for FACS analysis and the other half for PCR. A second group of 5 young C57/BL6 mice was used to prepare bone marrowderived macrophages (BMDM) and neonatal mice were used to prepare microglia for cell culture. They were maintained under veterinary supervision in a specific pathogen-free environment in the Comparative Medicine Unit, Trinity College Dublin, and were housed in groups of 2–4 per cage, at 20–22 °C with a 12 h light/dark cycle. Mice had free access to food and water and were fed a standard laboratory diet. All mice were maintained according to European Union regulations, and experiments were performed under license from the Department of Health (HPRA) and with approval from the Trinity College Dublin Ethics Committee.
BMDMs were prepared as described (Barrett et al., 2015b). The marrow from the femurs and tibias was flushed into a sterile falcon tube in DMEM containing heat-inactivated foetal bovine serum (10%) and penicillin-streptomycin (1%; both Gibco, UK), the cell suspension was triturated, filtered into a sterile tube and centrifuged (400 x g, 5 min). The pellet was resuspended in red blood cell lysis buffer (Sigma Aldrich, UK) for 1 min, DMEM was added to terminate the lysis, the suspension was centrifuged (400 × g, 5 min) and the cells were washed, centrifuged (400 × g, 5 min) and resuspended in DMEM supplemented with L929 conditioned media (20%). Cells were seeded in sterile cell culture T75cm2 flasks, media was replaced after 2 days and cells were cultured for 6 days, which included a media change. Cells were transferred to 6-well plates (0.5 × 106 cells per well), cultured for a further 2 days, incubated in the presence/absence of IFNγ (50 ng/ml) for 4 h after which time the media was replaced with control media and incubation continued for 20 h in the absence of IFNγ. Cells were harvested for assessment of TNFα and iNOS mRNA and conditioned medium was collected to assess its effect on microglia. In a separate set of experiments, BMDMs were exposed to CM from IFNγ-stimulated microglia for 24 h and, at the end of this period, cells were harvested for analysis of TNFα mRNA and iNOS mRNA and the supernatant was collected for assessment of nitrite and TNFα concentration.
2.2. Blood sampling and plasma analysis Blood samples were obtained from mice under euthatal-induced anaesthesia by cardiac puncture using EDTA-coated syringes, collected in 1.5 ml sterile Eppendorf tubes and centrifuged (2000 × g; 10 min; 22 °C). The resultant plasma fraction was aliquoted and stored at −80 °C until further use. The concentration of circulating inflammatory mediators was investigated using the mouse Proinflammatory Panel 1 Kit V-PLEX (Meso Scale Discovery, US) as per the manufacturer’s instructions. Briefly, standards and samples were added in duplicate to the plate in Diluent 41 reagent (50 μl; samples diluted 1:2) and incubated for 2 h on a shaker at room temperature (Orbital Shaker SO3, Stuart Scientific, UK). The plate was washed, detection antibody was added (25 μl), samples were incubated for 2 h at room temperature and washed. Read buffer T was added, the plate was read using a Mesoscale Sector Imager and cytokine/chemokine concentrations were calculated relative to the standard curve (expressed as pg/ml).
2.5. Preparation of primary glial cultures Microglia were prepared according to a previously-described method (Costello et al., 2015). In short, mixed glia from cortical tissue of neonatal mice, were cultured in T25 cm2 flasks in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen, UK) containing macrophage colony stimulating factor (100 ng/ml) and granulocyte macrophage colony stimulating factor (100 ng/ml; both R&D Systems, UK) for 10–12 days. Non-adherent microglia were isolated and seeded in 6-well plates (3 × 105cells/well) and cultured for 2 days. Cells were stimulated with CM from vehicle or IFNγ-treated BMDMs for 24 h after which time cells were harvested for assessment of TNFα and iNOS mRNA expression. In a separate series of experiments, cells were washed, incubated for 6 h in fresh cDMEM ± IFNγ (50 ng/ml), media was replaced by fresh media and incubation continued in the absence of IFNγ for 18 h. CM was collected at the end of this period and macrophages were exposed to the CM as described above.
2.3. Preparation of cells for flow cytometry While under anaesthesia, mice were perfused with ice-cold 1X PBS, brain tissue was removed and CNS mononuclear cells and neutrophils were isolated as described (McManus et al., 2014). Brain tissue was cross-chopped, mechanically dissociated in a hand-held homogeniser, passed through a cell strainer (70 μm) and the flow-through centrifuged (200 × g; 10 min; 4 °C). The pellet was re-suspended in 75% Percoll (10 ml), overlaid with 25% Percoll (10 ml) and 1 X PBS (6 ml), and centrifuged (800 x g; 30 min; 4 °C) yielding the mononuclear cells at the 25–75% interface which were collected, transferred to 50 ml Falcon tubes and centrifuged (200 × g; 10 min; 4 °C). The pellet containing the cells was transferred to FACS tubes, centrifuged (1200 rpm; 5 min; 22 °C) and incubated sequentially, in the dark at room temperature,
2.6. PCR analysis Samples of hippocampal tissue were snap-frozen in liquid nitrogen and harvested macrophages and microglia were assessed for expression of 85
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Fig. 1. Age-related changes in circulating cytokines. Blood samples, collected from 3 and 18 month-old euthatal-anaesthetized mice by cardiac puncture, were centrifuged (2000 × g; 10 min; 22 °C) to obtain plasma, which was assessed for cytokines and chemokines using the mouse Proinflammatory Panel 1 Kit V-PLEX (Meso Scale Discovery, US). The data indicate that the concentrations of IL-1β, TNFα, IL-6, IL-12p70, CXCL1 and the anti-inflammatory cytokine, IL-10 were increased in samples from aged, compared with young, mice (*p < 0.05; Student’s t-test for independent means). Data are expressed as the mean ± SEM (n = 4 (young) and 5 (aged)).
2.8. Greiss assay
markers of activation. RNA was isolated from cells (Nucleospin® RNAII kit; Macherey-Nagel GmbH, Germany) and reverse transcribed into cDNA (HighCapacity cDNA Archive kit; Applied Biosystems, UK) according to the manufacturer’s instructions. Assay ID’s for the genes examined were: β-actin (Mm00407939_s1), TNFα (Mm00443258_m1), iNOS (Mm0040502_m1), IL-6 (Mm00446190_m1), CXCL1 (Mm00433859_m1), CCL2 (Mm00441242_ m1), CCL3 (Mm01302427_m1), CD11b (Mm00434455_m1), MHCII (Mm00482914_m1) CD68 (Mm03047343_m1). Real-time PCR was performed using an ABI Prism 7300 instrument (Applied Biosystems, UK), βactin was the endogenous control and relative gene expression was calculated with reference to untreated cells using the ΔΔCT method with Applied Biosystems RQ software (Applied Biosystems, UK).
Supernatant samples from control- and IFNγ-treated macrophages were assessed for nitrite using the Greiss assay. Samples or standards (serial dilutions of sodium nitrite) were added to a 96-well plate and incubated (10 min, room temperature) in the presence of Greiss Reagent I (25 μl; 1% sulphanamide, orthophosphoric acid, H2O2). Greiss Reagent II (25 μl; 0.1% N-1-naphthyl-ethlenediamine dihydrochloride, distilled water) was added, incubation continued (10 min, room temperature), and absorbance was read at and nitrite concentrations were calculated with reference to the standard curve. 2.9. Statistical analysis
2.7. Analysis of TNFα concentration by ELISA
Data were analysed using a Student's t-test for independent means and are expressed as means + SEM.
Supernatant concentration of TNFα (R&D Systems, UK) obtained from macrophages was measured using ELISA. Briefly, standards or samples (100 μl) were added to antibody-coated 96-well plates and incubated for 2 h at room temperature, plates were washed and samples were incubated in detection antibody for 2 h. Plates were washed and incubated in horseradish peroxidase-conjugated streptavidin (1:200 in PBS containing 1% BSA) for 20 min at room temperature. Substrate solution (tetramethylbenzidine, Sigma-Aldrich, UK) was added, incubation continued at room temperature in the dark for 30 min and the reaction was stopped using 1 M h2SO4. Absorbance measurements were read at 450 nm using a microplate reader (Bio Tek Instruments, US). Concentrations were calculated relative to the appropriate standard curve.
3. Results Analysis of plasma from aged, compared with young mice revealed marked increases in the inflammatory cytokines, IL-1β, TNFα and IL-6, but no change in IL-12p70 (Fig. 1A–D; *p < 0.05; Student's t-test for independent means; n = 4 or 5). Significant increases in the chemokine, CXCL1 and the anti-inflammatory cytokine, IL-10, were also observed (*p < 0.05). It has been suggested that peripheral cells can infiltrate the brain with age (Barrett et al., 2015a; Minogue et al., 2014) and here we show that there was a small but significant increase in the number of CD11b+ Ly6G+ neutrophils (Fig. 2A,B) and a greater infiltration of CD11b+ 86
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Fig. 2. Macrophages and neutrophils infiltrate the brain with age. A single cell suspension, prepared from perfused brain tissue of 3 and 18 month-old euthatal-anaesthetized mice, was assessed by flow cytometry, as described in the Methods. A,B. Neutrophils, identified as CD11b+Ly6G+ cells, were increased in the brains of aged mice compared with their younger counterparts as indicated by the mean data and sample plot (*p < 0.05; Student’s t-test for independent means). D,E. Macrophages, identified CD11b+CD45high cells, were also increased in the brains of aged, compared with their young, mice (*p < 0.05; Student’s t-test for independent means). C,F. Hippocampal expression of CXCL1 mRNA and CCL2 mRNA was increased in aged, compared with young, mice (*p < 0.05; **p < 0.01; Student’s t-test for independent means). Data are expressed as the mean, expressed as a % of the total cell number ± SEM (n = 4 (young) and 5 (aged)).
CD45high macrophages (Fig. 2D,E) in the brain of aged, compared with young mice (*p < 0.05; ***p < 0.001; n = 4 or 5). These changes were associated with increased expression of CXCL1 (**p < 0.01; Fig. 2C) and CCL2 (*p < 0.05; Fig. 2F) in the hippocampus, which may provide the chemotactic signals for neutrophils and macrophages respectively. The data indicated that there was an increase in CD11b+ CD45low CXCR2+ and CD11b+ CD45low CCR1+ microglia (*p < 0.05; Fig. 3A–D; n = 4 or 5). CXCR2 is one of the receptors that is activated by CXCL1 which is increased in hippocampus of aged mice (Fig. 2C) while CCR1 binds many chemokines including CCL3, expression of which is increased in hippocampus from aged, compared with young, mice (Fig. 3E). These changes were associated with evidence of increased microglial activation including increased expression of CD11b, MHCII, CD68, TNFα and IL-6 (*p < 0.05; **p < 0.01; Fig. 4A–E; n = 4 or 6). To assess the possibility that infiltrating macrophages are responsible for driving the increase in microglial activation, an in vitro model was used. We stimulated BMDMs with IFNγ for 4 h and in IFNγfree medium for 20 h and show that this increased expression of TNFα and iNOS (*p < 0.05; **p < 0.01; Fig. 5A,B; n = 4). The CM from control-stimulated and IFNγ-stimulated macrophages was collected, microglia were incubated in this CM and the data show that mRNA expression of TNFα and iNOS was significantly increased in microglia that were incubated with CM prepared from IFNγ-stimulated, compared with control-stimulated, macrophages (***p < 0.001; Fig. 5C,D; n = 5). This suggests that inflammatory macrophages that infiltrate the brain may contribute to microglial activation. As shown here and in other studies, microglia in the brain of aged animals are inflammatory, contributing to the neuroinflammation that characterizes the aged brain. Therefore infiltrating macrophages
encounter this microenvironment. We used an in vitro approach to model the impact of inflammatory microglia on macrophages. Microglia were incubated with or without IFNγ (50 ng/ml) for 6 h and in IFNγ-free DMEM for 18 h. Cells were harvested for analysis of TNFα and iNOS mRNA; IFNγ significantly increased TNFα mRNA from 0.82 ± 0.09 (mean ± SEM expressed in RQ relative to β-actin) to 7.68 ± 1.08 (p < 0.001; student’s t-test for independent means; n = 5). The equivalent changes in iNOS mRNA were 0.88 ± 0.18 (control-treated) and 19.41 ± 4.92 (IFNγ-treated; p < 0.001; student’s t-test for independent means; n = 5). The supernatant was removed and incubated with macrophages for 24 h. The data show that mRNA expression of TNFα and iNOS and TNFα, as well as supernatant concentrations of TNFα and nitrite were increased in macrophages exposed to CM from IFNγ-treated microglia (*p < 0.05; Fig. 6A–D; n = 3–6), suggesting that there is a positive feedback loop operating between macrophages and microglia such that when stimulated with an inflammatory stimulus, a self-perpetuating loop of inflammation is initiated. 4. Discussion Inflammation is a recognized age-related change and here we show that this is manifest by increased circulating concentrations of inflammatory cytokines as well as microglial activation and increased expression of inflammatory markers in the hippocampus. The evidence presented suggests that one possible contributor to microglial activation is the infiltration of macrophages. The age-related increases in circulating concentrations of IL-1β, TNFα and IL-6 observed here are consistent with the concept of inflammaging and are similar to the findings of recent reports that show 87
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Fig. 3. Expression of CXCR2 and CCR1 is increased on microglia from aged, compared with young, mice. A single cell suspension, prepared from perfused brain tissue of 3 and 18 month-old euthatal-anaesthetized mice, was assessed by flow cytometry, as described in the Methods. The expression of CXCR2 (A,B) and CCR1 (C,D) on CD11b+CD45low microglia was significantly greater in samples from aged, compared with young, mice (*p < 0.05; ***p < 0.001; Student’s t-test for independent means). Representative FACS plots are shown. Data are expressed as the mean, expressed as a % of the total cell number ± SEM (n = 4–5). E. Hippocampal expression of CCL3 mRNA was increased in aged, compared with young, mice (*p < 0.05; Student’s t-test for independent means). Data are expressed as the mean (RQ) ± SEM (n = 4 (young) and 5 (aged)). Fig. 4. Microglial activation is increased with age. Hippocampal tissue from 3 and 18 month-old euthatal-anaesthetized mice was prepared for analysis of markers of microglial activation by RT-PCR as described in the Methods. A–F. mRNA expression of CD11b (A) MHCII (B) and CD68 (C), as well as TNFα (D) and IL-6 (E) was increased in hippocampal tissue from aged, compared with young, mice (*p < 0.05; **p < 0.01; Student’s t-test for independent means). Data are expressed as the mean (RQ) ± SEM (n = 4 (young) and 6 (aged)).
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Fig. 5. Conditioned medium from IFNγ-stimulated macrophages increases microglial activation. A,B. BMDMs were incubated in the presence or absence of IFNγ (50 ng/ml) for 4 h and in IFNγ-free medium for 20 h which significantly increased TNFα mRNA and iNOS mRNA (*p < 0.05; **p < 0.01; n = 4). C,D. CM from IFNγ-stimulated macrophages significantly increased TNFα mRNA and iNOS mRNA in microglia, compared with CM from control-stimulated macrophages (***p < 0.001; Student’s t-test for independent means). Data are expressed as the mean (RQ) ± SEM (n = 5).
Fig. 6. Conditioned medium from IFNγ-stimulated microglia increases TNFα and iNOS in macrophages. Microglia were incubated in the presence or absence of IFNγ (50 ng/ml) for 6 h and in IFNγ-free medium for 18 h (*p < 0.05; **p < 0.01). BMDMs were incubated with CM from control- or IFNγ-treated microglia for 24 h. CM from IFNγ-treated microglia significantly increased TNFα mRNA (A) and protein (B) as well as iNOS mRNA (C) and nitrite (D; *p < 0.05; ***p < 0.001; Student’s t-test for independent means). Data are expressed as the mean ± SEM (n = 3–6).
similar age-related increases in inflammatory cytokines in mice (Puchta et al., 2016; Scott et al., 2017). In contrast to these findings of agerelated increases in inflammatory cytokines, it has been reported that the concentrations of TNFα and IL-12p70 were decreased in plasma from 24 month-old, compared with 3 month-old, rats (Katharesan et al., 2016). Interestingly, age-related increased circulating TNFα and IL-6 is also a feature in humans (Scott et al., 2017). We show that neutrophils infiltrated the brains of older mice, albeit to a lesser extent than macrophages. Infiltration of neutrophils has also been reported in APP/PS1 mice, where it is associated with increased amyloid pathology and neuroinflammation (Minogue et al., 2014). More recently, infiltration was observed in 2 other models of AD and the cells were shown to release the inflammatory cytokine, IL-17, and neutrophil extracellular traps (Zenaro et al., 2015). When neutrophil infiltration was inhibited by blocking LFA-1 integrin, microgliosis and amyloid pathology were reduced and improvements in cognition were observed in the mice (Zenaro et al., 2015). The neutrophil infiltration described here was associated with increased expression of CXCL1, which is a major neutrophil chemotactic factor (De Filippo et al., 2008), in the hippocampus. Indeed increased CXCL1, which is evident in several areas of the brain including hippocampus 3 h after seizure, directly precedes and positively correlates with neutrophil infiltration (Johnson et al., 2011). The data indicate that macrophages infiltrate the brain in 18 monthold mice, confirming our earlier findings in rats (Barrett et al., 2015a). Infiltration of macrophages has also been observed in APP/PS1 compared with WT mice (Barrett et al., 2015b; Kelly et al., 2013; Minogue et al., 2014) and also in the ArcAβ model of amyloidosis (Ferretti et al., 2016). The infiltration of cells has been shown to be exacerbated with infection (McManus et al., 2014).
We have consistently found that microglial activation is evident in circumstances of macrophage infiltration, for example in CD200-deficient mice (Denieffe et al., 2013), and APP/PS1 mice (Minogue et al., 2014), particularly following Bordetella pertussis infection (McManus et al., 2014). Here we provide further data indicating a correlation between infiltrating macrophages and microglial activation perhaps implying a causal relationship between the changes. Expression of several markers of microglial activation was increased in aged, compared with young, mice including cell surface markers CD11b, CD40 and MHCII as well as TNFα and IL-6 and these changes mirror those that have been previously reported (Minogue et al., 2014; Murphy et al., 2012). There are number of factors that act as chemotactic signals for macrophages; one of these is CCL2 (Deshmane et al., 2009) which, in the present study, was increased in hippocampus of aged, compared with young, mice and which is produced by several cells including microglia. The data indicate that microglia expressed CXCR2 and CCR1, receptors for several chemokines including CXCL1 and CCL3, both of which are increased with age in the hippocampus and increase microglial activation (Skuljec et al., 2011). These chemokines may therefore contribute to the age-related changes in microglia, although it is also possible that infiltrating macrophages may be a contributory factor. At present there is no consensus about the impact of infiltrating macrophages with evidence suggesting beneficial effects in some circumstances but not others (Herz et al., 2017). Here we used an in vitro approach to model the impact of activated macrophages on 89
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microglia incubating microglia in the presence of CM from control-treated or IFNγ-treated macrophages. The data show that CM from IFNγ-stimulated macrophages markedly increased TNFα and iNOS indicative of increased microglial activation and suggesting that inflammatory macrophages may contribute to the neuroinflammation that is observed with age. It has been proposed that ageing affects macrophages and microglia differently; microglia become sensitized to inflammatory stimuli perhaps as a result of priming (Perry and Holmes, 2014) but it appears that macrophages become de-sensitized and production of inflammatory mediators is reduced (Rawji et al., 2016; Shaw et al., 2013). However exactly when this desensitization becomes evident during the ageing process remains to determined and, at least in our hands, BMDMs from 16 month-old rats responded more to IFNγ in terms of TNFα release than BMDMs from 3 to 4 month-old rats (Barrett et al., 2015a). With respect to microglia, there is a great deal of data that indicate they are sensitized to inflammatory stimuli at least in middle-age; specifically, microglia from 14 to 16 month-old mice were more responsive to LPS and PamCSK3 than microglia from 1–2 month-old mice (Njie et al., 2012) and a similar LPS-induced responsiveness was observed in 18 month-old compared with 2 month-old mice (Sierra et al., 2007), while IL-6 expression was constitutively higher in hippocampal tissue and glia from 24 month-old mice compared with adult or neonatal mice (Ye and Johnson, 1999). It is important to note that the selection of mouse strain impacts on readouts (Koks et al., 2016) and, for example, genetic background results in distinct inflammatory signatures in models of inflammatory angiogenesis (Marques et al., 2011) and fat/ethanol-induced liver inflammation (Bavia et al., 2015), while it also affects neuroinflammatory changes resulting from infection with Toxoplasma gondii (Brandao et al., 2011). These differences in mouse strain serve to emphasise the difficulty in extrapolating from animal studies into the human context. The evidence presented indicates that the age-related inflammatory markers in plasma are reflected by neuroinflammatory changes in the brain and that infiltrating macrophages may play a role in driving the neuroinflammatory changes. It is likely that macrophage infiltration is facilitated by chemotactic signals including increased CCL2 in hippocampus, an additional factor may be the increased permeability of the blood brain barrier which occurs with age (Minogue et al., 2014) and is negatively impacted by inflammatory cytokines (Varatharaj and Galea, 2017).
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Acknowledgements This work was funded by a Government of Ireland Postgraduate Scholarship (Irish Research Council) to Hannah Wolfe (GOIPG/2013/ 331) and by a Science Foundation Ireland PI award to MAL (15/iA/ 3052); the authors gratefully acknowledge this support. The authors declare that there are no conflicts of interest. References Aloisi, F., 2001. Immune function of microglia. Glia 36, 165–179. Barrett, J.P., Costello, D.A., O’Sullivan, J., Cowley, T.R., Lynch, M.A., 2015a. Bone marrow-derived macrophages from aged rats are more responsive to inflammatory stimuli. J. Neuroinflamm. 12, 67. Barrett, J.P., Minogue, A.M., Jones, R.S., Ribeiro, C., Kelly, R.J., Lynch, M.A., 2015b. Bone marrow-derived macrophages from AbetaPP/PS1 mice are sensitized to the effects of inflammatory stimuli. J. Alzheimers Dis. 44, 949–962. Bavia, L., de Castro, I.A., Isaac, L., 2015. C57BL/6 and A/J mice have different inflammatory response and liver lipid profile in experimental alcoholic liver disease. Mediators Inflamm. 2015, 491641. Becher, B., Antel, J.P., 1996. Comparison of phenotypic and functional properties of immediately ex vivo and cultured human adult microglia. Glia 18, 1–10. Brandao, G.P., Melo, M.N., Caetano, B.C., Carneiro, C.M., Silva, L.A., Vitor, R.W., 2011. Susceptibility to re-infection in C57BL/6 mice with recombinant strains of Toxoplasma gondii. Exp. Parasitol. 128, 433–437.
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normal and inflamed central nervous system. Proc. Natl Acad. Sci. U. S. A 88, 7438–7442. Shaw, A.C., Goldstein, D.R., Montgomery, R.R., 2013. Age-dependent dysregulation of innate immunity. Nat. Rev. Immunol. 13, 875–887. Sierra, A., Gottfried-Blackmore, A.C., McEwen, B.S., Bulloch, K., 2007. Microglia derived from aging mice exhibit an altered inflammatory profile. Glia 55, 412–424. Skuljec, J., Sun, H., Pul, R., Benardais, K., Ragancokova, D., Moharregh-Khiabani, D., Kotsiari, A., Trebst, C., Stangel, M., 2011. CCL5 induces a pro-inflammatory profile in microglia in vitro. Cell. Immunol. 270, 164–171.
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Mechanisms of Ageing and Development journal homepage: www.elsevier.com/locate/mechagedev
Infiltrating macrophages contribute to age-related neuroinflammation in C57/BL6 mice H. Wolfe, A.M. Minogue, S. Rooney, M.A. Lynch
T
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Trinity College Institute for Neuroscience, Trinity College, Dublin 2, Ireland
A R T I C LE I N FO
A B S T R A C T
Keywords: Age Microglial activation Macrophage infiltration Inflammatory cytokines
The recognized role of neuroinflammation in the age-related deterioration of neuronal function highlights the importance of understanding the factors that control microglial activation. Microglia, as the immune cells of the brain, are the arbiters of the inflammatory profile in the brain. Normally they are maintained in a quiescent state by means of ligand-receptor interactions with neurons, within a prevailing anti-inflammatory microenvironment. The evidence indicates that, as the ageing process continues, microglia become activated, shift towards an inflammatory phenotype and alter the milieu in the brain. Although there has been progress in identifying factors that contribute to age-related microglial activation, our understanding remains incomplete. Here we report that there was an age-related increase in circulating inflammatory cytokines, accompanied by microglial activation. Neutrophils, and to a greater extent, macrophages, infiltrate the brain with age, perhaps as a result of increased chemokine expression in the brain, specifically CXCL1 and CCL2. We sought to determine whether macrophages might trigger microglial activation and the evidence shows that conditioned medium obtained from interferon-γ (IFNγ)-stimulated macrophages potently activated microglia. The data suggest that infiltrating macrophages may be one factor that contributes to age-related microglial activation.
1. Introduction With age, a host of dynamic changes in the immune system occurs. The upshot of this is that low-grade inflammation gradually leads to chronic inflammation; this has been referred to as inflammaging. In addition to peripheral changes, inflammatory changes also occur in the brain with age and microglia, as the primary immune cells in the brain, provide the driving force for neuroinflammation. Thus, neuroinflammation is characterized by increased microglial activation with evidence indicating increased expression of cell surface markers of activation, like CD11b, CD68 and MHCII and increased expression of inflammatory cytokines, like tumour necrosis factor-α (TNFα), interleukin (IL)-1β and IL-6. The implication is that, with age, microglia that are vital for neuroprotection gradually morph from their neuroprotective and anti-inflammatory phenotype to an inflammatory phenotype (Lee et al., 2013), which can ultimately become detrimental to neurons (Kielian, 2014; Ransohoff, 2016). Likewise, age tends to initiate a shift in macrophages from the so-called M2 phenotype to the inflammatory M1 phenotype; for example lipopolysaccharide (LPS) and interferon-γ (IFNγ) triggered a greater release of inflammatory cytokines in macrophages from old, compared with young, rats (Barrett et al., 2015a; Dimitrijevic et al., 2016). However, in contrast to the consistent
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Corresponding author. E-mail address: [email protected] (M.A. Lynch).
https://doi.org/10.1016/j.mad.2018.05.003 Received 21 March 2018; Received in revised form 24 April 2018; Accepted 9 May 2018 0047-6374/ © 2018 Elsevier B.V. All rights reserved.
findings in microglia, there are conflicting reports with respect to macrophages and some findings indicate that their response to inflammatory stimuli is reduced (Rawji et al., 2016; Shaw et al., 2013) as opposed to increased with age. In addition to age-related changes, microglial activation and the associated neuroinflammation is a characteristic of neurodegenerative diseases including Alzheimer's disease (AD) and Parkinson's disease (Molteni and Rossetti, 2017) and recent evidence from genome-wide association studies identified polymorphisms in several genes known to play a role in inflammation and immune modulation as risk factors in AD (Karch and Goate, 2015). The particular significance of this finding is that it pointed to altered microglial activation as a possible contributor to AD rather than being a consequence of the pathogenic processes associated with the disease. This finding highlights the need to identify the factors that trigger inappropriate microglial activation. Microglia react to a plethora of stimuli and adopt an inflammatory signature in response to inflammatory stimuli. Under normal circumstances, several factors converge to maintain an anti-inflammatory milieu in the brain. Resting concentrations of inflammatory mediators are low, while there is a relatively higher concentration of anti-inflammatory cytokines like IL-10 and TGFβ (Aloisi, 2001). In addition, microglia are maintained in a relatively quiescent state by interacting
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with Live/Dead stain (1 ml; 1:1000 in PBS, Life Technologies, US; 30 min) and blocking solution (1:50; 50 μl; purified rat anti-mouse CD16/CD32, BD Pharmingen, US; 15 min) with intermediate washing and centrifuging (1200 rpm; 5 min; 22 °C). The antibody Master Mix (50 μl; 15 min) was added and samples were incubated with the cell surface antibodies for 15 min; this included CD11b (PE-Cy7; 1:100; Biolegend, US), CD45 (APC-Cy7; 1:80, Biolegend US), CCR1 (APC; 1:20; R&D Systems, UK), CXCR2 (PE1:20; R&D Systems) and Ly6G (FITC; 1:100; Biolegend, US). Propidium iodide (PI; Sigma-Aldrich, UK) was added (1:100) immediately before reading the samples and was used as a live/dead stain. The appropriate compensation controls and FMO (fluorescence minus one) controls were also prepared. Flow cytometric analysis was performed using a DAKO CyAn-ADP 7 colour flow cytometer (Beckman Coulter, US) and analysed with FlowJo v7.6.5 software (Tree Star Inc., US). Microglia were identified as being CD11b+CD45low cells and macrophages as CD11b+CD45high cells (Becher and Antel, 1996; Sedgwick et al., 1991).
with other cells, for example neurons through CD200-CD200R and CX3CL1-CX3CR1 interactions (Lyons et al., 2007; Lyons et al., 2009). With age the neuron-microglial interactions are less robust (Lyons et al., 2007; Lyons et al., 2009) probably contributing to microglial activation but in addition, inflammatory cytokine concentration increases, perhaps as a consequence of infiltrating cells, including macrophages (Minogue et al., 2014). Here we set out to investigate the impact of infiltrating macrophages on microglia in the brain of aged mice and show that the increase in circulating cytokines is accompanied by increased infiltration of neutrophils and, to a greater extent, macrophages. Conditioned medium (CM) from stimulated macrophages markedly increased microglial activation suggesting that infiltrating macrophages may be one factor that contributes to the age-related microglial activation. 2. Materials and methods 2.1. Animals
2.4. Preparation of BMDM Groups of young (2–3 months; n = 8) and aged (16-18 months; n = 10) C57/BL6 mice were used to assess microglial activation; half of these were used for FACS analysis and the other half for PCR. A second group of 5 young C57/BL6 mice was used to prepare bone marrowderived macrophages (BMDM) and neonatal mice were used to prepare microglia for cell culture. They were maintained under veterinary supervision in a specific pathogen-free environment in the Comparative Medicine Unit, Trinity College Dublin, and were housed in groups of 2–4 per cage, at 20–22 °C with a 12 h light/dark cycle. Mice had free access to food and water and were fed a standard laboratory diet. All mice were maintained according to European Union regulations, and experiments were performed under license from the Department of Health (HPRA) and with approval from the Trinity College Dublin Ethics Committee.
BMDMs were prepared as described (Barrett et al., 2015b). The marrow from the femurs and tibias was flushed into a sterile falcon tube in DMEM containing heat-inactivated foetal bovine serum (10%) and penicillin-streptomycin (1%; both Gibco, UK), the cell suspension was triturated, filtered into a sterile tube and centrifuged (400 x g, 5 min). The pellet was resuspended in red blood cell lysis buffer (Sigma Aldrich, UK) for 1 min, DMEM was added to terminate the lysis, the suspension was centrifuged (400 × g, 5 min) and the cells were washed, centrifuged (400 × g, 5 min) and resuspended in DMEM supplemented with L929 conditioned media (20%). Cells were seeded in sterile cell culture T75cm2 flasks, media was replaced after 2 days and cells were cultured for 6 days, which included a media change. Cells were transferred to 6-well plates (0.5 × 106 cells per well), cultured for a further 2 days, incubated in the presence/absence of IFNγ (50 ng/ml) for 4 h after which time the media was replaced with control media and incubation continued for 20 h in the absence of IFNγ. Cells were harvested for assessment of TNFα and iNOS mRNA and conditioned medium was collected to assess its effect on microglia. In a separate set of experiments, BMDMs were exposed to CM from IFNγ-stimulated microglia for 24 h and, at the end of this period, cells were harvested for analysis of TNFα mRNA and iNOS mRNA and the supernatant was collected for assessment of nitrite and TNFα concentration.
2.2. Blood sampling and plasma analysis Blood samples were obtained from mice under euthatal-induced anaesthesia by cardiac puncture using EDTA-coated syringes, collected in 1.5 ml sterile Eppendorf tubes and centrifuged (2000 × g; 10 min; 22 °C). The resultant plasma fraction was aliquoted and stored at −80 °C until further use. The concentration of circulating inflammatory mediators was investigated using the mouse Proinflammatory Panel 1 Kit V-PLEX (Meso Scale Discovery, US) as per the manufacturer’s instructions. Briefly, standards and samples were added in duplicate to the plate in Diluent 41 reagent (50 μl; samples diluted 1:2) and incubated for 2 h on a shaker at room temperature (Orbital Shaker SO3, Stuart Scientific, UK). The plate was washed, detection antibody was added (25 μl), samples were incubated for 2 h at room temperature and washed. Read buffer T was added, the plate was read using a Mesoscale Sector Imager and cytokine/chemokine concentrations were calculated relative to the standard curve (expressed as pg/ml).
2.5. Preparation of primary glial cultures Microglia were prepared according to a previously-described method (Costello et al., 2015). In short, mixed glia from cortical tissue of neonatal mice, were cultured in T25 cm2 flasks in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen, UK) containing macrophage colony stimulating factor (100 ng/ml) and granulocyte macrophage colony stimulating factor (100 ng/ml; both R&D Systems, UK) for 10–12 days. Non-adherent microglia were isolated and seeded in 6-well plates (3 × 105cells/well) and cultured for 2 days. Cells were stimulated with CM from vehicle or IFNγ-treated BMDMs for 24 h after which time cells were harvested for assessment of TNFα and iNOS mRNA expression. In a separate series of experiments, cells were washed, incubated for 6 h in fresh cDMEM ± IFNγ (50 ng/ml), media was replaced by fresh media and incubation continued in the absence of IFNγ for 18 h. CM was collected at the end of this period and macrophages were exposed to the CM as described above.
2.3. Preparation of cells for flow cytometry While under anaesthesia, mice were perfused with ice-cold 1X PBS, brain tissue was removed and CNS mononuclear cells and neutrophils were isolated as described (McManus et al., 2014). Brain tissue was cross-chopped, mechanically dissociated in a hand-held homogeniser, passed through a cell strainer (70 μm) and the flow-through centrifuged (200 × g; 10 min; 4 °C). The pellet was re-suspended in 75% Percoll (10 ml), overlaid with 25% Percoll (10 ml) and 1 X PBS (6 ml), and centrifuged (800 x g; 30 min; 4 °C) yielding the mononuclear cells at the 25–75% interface which were collected, transferred to 50 ml Falcon tubes and centrifuged (200 × g; 10 min; 4 °C). The pellet containing the cells was transferred to FACS tubes, centrifuged (1200 rpm; 5 min; 22 °C) and incubated sequentially, in the dark at room temperature,
2.6. PCR analysis Samples of hippocampal tissue were snap-frozen in liquid nitrogen and harvested macrophages and microglia were assessed for expression of 85
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Fig. 1. Age-related changes in circulating cytokines. Blood samples, collected from 3 and 18 month-old euthatal-anaesthetized mice by cardiac puncture, were centrifuged (2000 × g; 10 min; 22 °C) to obtain plasma, which was assessed for cytokines and chemokines using the mouse Proinflammatory Panel 1 Kit V-PLEX (Meso Scale Discovery, US). The data indicate that the concentrations of IL-1β, TNFα, IL-6, IL-12p70, CXCL1 and the anti-inflammatory cytokine, IL-10 were increased in samples from aged, compared with young, mice (*p < 0.05; Student’s t-test for independent means). Data are expressed as the mean ± SEM (n = 4 (young) and 5 (aged)).
2.8. Greiss assay
markers of activation. RNA was isolated from cells (Nucleospin® RNAII kit; Macherey-Nagel GmbH, Germany) and reverse transcribed into cDNA (HighCapacity cDNA Archive kit; Applied Biosystems, UK) according to the manufacturer’s instructions. Assay ID’s for the genes examined were: β-actin (Mm00407939_s1), TNFα (Mm00443258_m1), iNOS (Mm0040502_m1), IL-6 (Mm00446190_m1), CXCL1 (Mm00433859_m1), CCL2 (Mm00441242_ m1), CCL3 (Mm01302427_m1), CD11b (Mm00434455_m1), MHCII (Mm00482914_m1) CD68 (Mm03047343_m1). Real-time PCR was performed using an ABI Prism 7300 instrument (Applied Biosystems, UK), βactin was the endogenous control and relative gene expression was calculated with reference to untreated cells using the ΔΔCT method with Applied Biosystems RQ software (Applied Biosystems, UK).
Supernatant samples from control- and IFNγ-treated macrophages were assessed for nitrite using the Greiss assay. Samples or standards (serial dilutions of sodium nitrite) were added to a 96-well plate and incubated (10 min, room temperature) in the presence of Greiss Reagent I (25 μl; 1% sulphanamide, orthophosphoric acid, H2O2). Greiss Reagent II (25 μl; 0.1% N-1-naphthyl-ethlenediamine dihydrochloride, distilled water) was added, incubation continued (10 min, room temperature), and absorbance was read at and nitrite concentrations were calculated with reference to the standard curve. 2.9. Statistical analysis
2.7. Analysis of TNFα concentration by ELISA
Data were analysed using a Student's t-test for independent means and are expressed as means + SEM.
Supernatant concentration of TNFα (R&D Systems, UK) obtained from macrophages was measured using ELISA. Briefly, standards or samples (100 μl) were added to antibody-coated 96-well plates and incubated for 2 h at room temperature, plates were washed and samples were incubated in detection antibody for 2 h. Plates were washed and incubated in horseradish peroxidase-conjugated streptavidin (1:200 in PBS containing 1% BSA) for 20 min at room temperature. Substrate solution (tetramethylbenzidine, Sigma-Aldrich, UK) was added, incubation continued at room temperature in the dark for 30 min and the reaction was stopped using 1 M h2SO4. Absorbance measurements were read at 450 nm using a microplate reader (Bio Tek Instruments, US). Concentrations were calculated relative to the appropriate standard curve.
3. Results Analysis of plasma from aged, compared with young mice revealed marked increases in the inflammatory cytokines, IL-1β, TNFα and IL-6, but no change in IL-12p70 (Fig. 1A–D; *p < 0.05; Student's t-test for independent means; n = 4 or 5). Significant increases in the chemokine, CXCL1 and the anti-inflammatory cytokine, IL-10, were also observed (*p < 0.05). It has been suggested that peripheral cells can infiltrate the brain with age (Barrett et al., 2015a; Minogue et al., 2014) and here we show that there was a small but significant increase in the number of CD11b+ Ly6G+ neutrophils (Fig. 2A,B) and a greater infiltration of CD11b+ 86
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Fig. 2. Macrophages and neutrophils infiltrate the brain with age. A single cell suspension, prepared from perfused brain tissue of 3 and 18 month-old euthatal-anaesthetized mice, was assessed by flow cytometry, as described in the Methods. A,B. Neutrophils, identified as CD11b+Ly6G+ cells, were increased in the brains of aged mice compared with their younger counterparts as indicated by the mean data and sample plot (*p < 0.05; Student’s t-test for independent means). D,E. Macrophages, identified CD11b+CD45high cells, were also increased in the brains of aged, compared with their young, mice (*p < 0.05; Student’s t-test for independent means). C,F. Hippocampal expression of CXCL1 mRNA and CCL2 mRNA was increased in aged, compared with young, mice (*p < 0.05; **p < 0.01; Student’s t-test for independent means). Data are expressed as the mean, expressed as a % of the total cell number ± SEM (n = 4 (young) and 5 (aged)).
CD45high macrophages (Fig. 2D,E) in the brain of aged, compared with young mice (*p < 0.05; ***p < 0.001; n = 4 or 5). These changes were associated with increased expression of CXCL1 (**p < 0.01; Fig. 2C) and CCL2 (*p < 0.05; Fig. 2F) in the hippocampus, which may provide the chemotactic signals for neutrophils and macrophages respectively. The data indicated that there was an increase in CD11b+ CD45low CXCR2+ and CD11b+ CD45low CCR1+ microglia (*p < 0.05; Fig. 3A–D; n = 4 or 5). CXCR2 is one of the receptors that is activated by CXCL1 which is increased in hippocampus of aged mice (Fig. 2C) while CCR1 binds many chemokines including CCL3, expression of which is increased in hippocampus from aged, compared with young, mice (Fig. 3E). These changes were associated with evidence of increased microglial activation including increased expression of CD11b, MHCII, CD68, TNFα and IL-6 (*p < 0.05; **p < 0.01; Fig. 4A–E; n = 4 or 6). To assess the possibility that infiltrating macrophages are responsible for driving the increase in microglial activation, an in vitro model was used. We stimulated BMDMs with IFNγ for 4 h and in IFNγfree medium for 20 h and show that this increased expression of TNFα and iNOS (*p < 0.05; **p < 0.01; Fig. 5A,B; n = 4). The CM from control-stimulated and IFNγ-stimulated macrophages was collected, microglia were incubated in this CM and the data show that mRNA expression of TNFα and iNOS was significantly increased in microglia that were incubated with CM prepared from IFNγ-stimulated, compared with control-stimulated, macrophages (***p < 0.001; Fig. 5C,D; n = 5). This suggests that inflammatory macrophages that infiltrate the brain may contribute to microglial activation. As shown here and in other studies, microglia in the brain of aged animals are inflammatory, contributing to the neuroinflammation that characterizes the aged brain. Therefore infiltrating macrophages
encounter this microenvironment. We used an in vitro approach to model the impact of inflammatory microglia on macrophages. Microglia were incubated with or without IFNγ (50 ng/ml) for 6 h and in IFNγ-free DMEM for 18 h. Cells were harvested for analysis of TNFα and iNOS mRNA; IFNγ significantly increased TNFα mRNA from 0.82 ± 0.09 (mean ± SEM expressed in RQ relative to β-actin) to 7.68 ± 1.08 (p < 0.001; student’s t-test for independent means; n = 5). The equivalent changes in iNOS mRNA were 0.88 ± 0.18 (control-treated) and 19.41 ± 4.92 (IFNγ-treated; p < 0.001; student’s t-test for independent means; n = 5). The supernatant was removed and incubated with macrophages for 24 h. The data show that mRNA expression of TNFα and iNOS and TNFα, as well as supernatant concentrations of TNFα and nitrite were increased in macrophages exposed to CM from IFNγ-treated microglia (*p < 0.05; Fig. 6A–D; n = 3–6), suggesting that there is a positive feedback loop operating between macrophages and microglia such that when stimulated with an inflammatory stimulus, a self-perpetuating loop of inflammation is initiated. 4. Discussion Inflammation is a recognized age-related change and here we show that this is manifest by increased circulating concentrations of inflammatory cytokines as well as microglial activation and increased expression of inflammatory markers in the hippocampus. The evidence presented suggests that one possible contributor to microglial activation is the infiltration of macrophages. The age-related increases in circulating concentrations of IL-1β, TNFα and IL-6 observed here are consistent with the concept of inflammaging and are similar to the findings of recent reports that show 87
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Fig. 3. Expression of CXCR2 and CCR1 is increased on microglia from aged, compared with young, mice. A single cell suspension, prepared from perfused brain tissue of 3 and 18 month-old euthatal-anaesthetized mice, was assessed by flow cytometry, as described in the Methods. The expression of CXCR2 (A,B) and CCR1 (C,D) on CD11b+CD45low microglia was significantly greater in samples from aged, compared with young, mice (*p < 0.05; ***p < 0.001; Student’s t-test for independent means). Representative FACS plots are shown. Data are expressed as the mean, expressed as a % of the total cell number ± SEM (n = 4–5). E. Hippocampal expression of CCL3 mRNA was increased in aged, compared with young, mice (*p < 0.05; Student’s t-test for independent means). Data are expressed as the mean (RQ) ± SEM (n = 4 (young) and 5 (aged)). Fig. 4. Microglial activation is increased with age. Hippocampal tissue from 3 and 18 month-old euthatal-anaesthetized mice was prepared for analysis of markers of microglial activation by RT-PCR as described in the Methods. A–F. mRNA expression of CD11b (A) MHCII (B) and CD68 (C), as well as TNFα (D) and IL-6 (E) was increased in hippocampal tissue from aged, compared with young, mice (*p < 0.05; **p < 0.01; Student’s t-test for independent means). Data are expressed as the mean (RQ) ± SEM (n = 4 (young) and 6 (aged)).
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Fig. 5. Conditioned medium from IFNγ-stimulated macrophages increases microglial activation. A,B. BMDMs were incubated in the presence or absence of IFNγ (50 ng/ml) for 4 h and in IFNγ-free medium for 20 h which significantly increased TNFα mRNA and iNOS mRNA (*p < 0.05; **p < 0.01; n = 4). C,D. CM from IFNγ-stimulated macrophages significantly increased TNFα mRNA and iNOS mRNA in microglia, compared with CM from control-stimulated macrophages (***p < 0.001; Student’s t-test for independent means). Data are expressed as the mean (RQ) ± SEM (n = 5).
Fig. 6. Conditioned medium from IFNγ-stimulated microglia increases TNFα and iNOS in macrophages. Microglia were incubated in the presence or absence of IFNγ (50 ng/ml) for 6 h and in IFNγ-free medium for 18 h (*p < 0.05; **p < 0.01). BMDMs were incubated with CM from control- or IFNγ-treated microglia for 24 h. CM from IFNγ-treated microglia significantly increased TNFα mRNA (A) and protein (B) as well as iNOS mRNA (C) and nitrite (D; *p < 0.05; ***p < 0.001; Student’s t-test for independent means). Data are expressed as the mean ± SEM (n = 3–6).
similar age-related increases in inflammatory cytokines in mice (Puchta et al., 2016; Scott et al., 2017). In contrast to these findings of agerelated increases in inflammatory cytokines, it has been reported that the concentrations of TNFα and IL-12p70 were decreased in plasma from 24 month-old, compared with 3 month-old, rats (Katharesan et al., 2016). Interestingly, age-related increased circulating TNFα and IL-6 is also a feature in humans (Scott et al., 2017). We show that neutrophils infiltrated the brains of older mice, albeit to a lesser extent than macrophages. Infiltration of neutrophils has also been reported in APP/PS1 mice, where it is associated with increased amyloid pathology and neuroinflammation (Minogue et al., 2014). More recently, infiltration was observed in 2 other models of AD and the cells were shown to release the inflammatory cytokine, IL-17, and neutrophil extracellular traps (Zenaro et al., 2015). When neutrophil infiltration was inhibited by blocking LFA-1 integrin, microgliosis and amyloid pathology were reduced and improvements in cognition were observed in the mice (Zenaro et al., 2015). The neutrophil infiltration described here was associated with increased expression of CXCL1, which is a major neutrophil chemotactic factor (De Filippo et al., 2008), in the hippocampus. Indeed increased CXCL1, which is evident in several areas of the brain including hippocampus 3 h after seizure, directly precedes and positively correlates with neutrophil infiltration (Johnson et al., 2011). The data indicate that macrophages infiltrate the brain in 18 monthold mice, confirming our earlier findings in rats (Barrett et al., 2015a). Infiltration of macrophages has also been observed in APP/PS1 compared with WT mice (Barrett et al., 2015b; Kelly et al., 2013; Minogue et al., 2014) and also in the ArcAβ model of amyloidosis (Ferretti et al., 2016). The infiltration of cells has been shown to be exacerbated with infection (McManus et al., 2014).
We have consistently found that microglial activation is evident in circumstances of macrophage infiltration, for example in CD200-deficient mice (Denieffe et al., 2013), and APP/PS1 mice (Minogue et al., 2014), particularly following Bordetella pertussis infection (McManus et al., 2014). Here we provide further data indicating a correlation between infiltrating macrophages and microglial activation perhaps implying a causal relationship between the changes. Expression of several markers of microglial activation was increased in aged, compared with young, mice including cell surface markers CD11b, CD40 and MHCII as well as TNFα and IL-6 and these changes mirror those that have been previously reported (Minogue et al., 2014; Murphy et al., 2012). There are number of factors that act as chemotactic signals for macrophages; one of these is CCL2 (Deshmane et al., 2009) which, in the present study, was increased in hippocampus of aged, compared with young, mice and which is produced by several cells including microglia. The data indicate that microglia expressed CXCR2 and CCR1, receptors for several chemokines including CXCL1 and CCL3, both of which are increased with age in the hippocampus and increase microglial activation (Skuljec et al., 2011). These chemokines may therefore contribute to the age-related changes in microglia, although it is also possible that infiltrating macrophages may be a contributory factor. At present there is no consensus about the impact of infiltrating macrophages with evidence suggesting beneficial effects in some circumstances but not others (Herz et al., 2017). Here we used an in vitro approach to model the impact of activated macrophages on 89
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microglia incubating microglia in the presence of CM from control-treated or IFNγ-treated macrophages. The data show that CM from IFNγ-stimulated macrophages markedly increased TNFα and iNOS indicative of increased microglial activation and suggesting that inflammatory macrophages may contribute to the neuroinflammation that is observed with age. It has been proposed that ageing affects macrophages and microglia differently; microglia become sensitized to inflammatory stimuli perhaps as a result of priming (Perry and Holmes, 2014) but it appears that macrophages become de-sensitized and production of inflammatory mediators is reduced (Rawji et al., 2016; Shaw et al., 2013). However exactly when this desensitization becomes evident during the ageing process remains to determined and, at least in our hands, BMDMs from 16 month-old rats responded more to IFNγ in terms of TNFα release than BMDMs from 3 to 4 month-old rats (Barrett et al., 2015a). With respect to microglia, there is a great deal of data that indicate they are sensitized to inflammatory stimuli at least in middle-age; specifically, microglia from 14 to 16 month-old mice were more responsive to LPS and PamCSK3 than microglia from 1–2 month-old mice (Njie et al., 2012) and a similar LPS-induced responsiveness was observed in 18 month-old compared with 2 month-old mice (Sierra et al., 2007), while IL-6 expression was constitutively higher in hippocampal tissue and glia from 24 month-old mice compared with adult or neonatal mice (Ye and Johnson, 1999). It is important to note that the selection of mouse strain impacts on readouts (Koks et al., 2016) and, for example, genetic background results in distinct inflammatory signatures in models of inflammatory angiogenesis (Marques et al., 2011) and fat/ethanol-induced liver inflammation (Bavia et al., 2015), while it also affects neuroinflammatory changes resulting from infection with Toxoplasma gondii (Brandao et al., 2011). These differences in mouse strain serve to emphasise the difficulty in extrapolating from animal studies into the human context. The evidence presented indicates that the age-related inflammatory markers in plasma are reflected by neuroinflammatory changes in the brain and that infiltrating macrophages may play a role in driving the neuroinflammatory changes. It is likely that macrophage infiltration is facilitated by chemotactic signals including increased CCL2 in hippocampus, an additional factor may be the increased permeability of the blood brain barrier which occurs with age (Minogue et al., 2014) and is negatively impacted by inflammatory cytokines (Varatharaj and Galea, 2017).
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