Age-related differences in neuroinflammatory responses associated with a distinct profile of regulatory markers on neonatal microglia

All animal research was carried out in adherence with protocols approved by the National Institutes of Health Rocky Mountain Laboratories Animal Care and Use Committee with animal protocols 2008-46 and 2009-70.

At the indicated time points, all animals were anaesthetized by inhalation of isofluorane and euthanized using axillary incision. Brain tissue was snap frozen in liquid nitrogen and stored at −80°C until use.

TLR4 agonist ultra-pure LPS (catalog number tlrl-3pelps) and TLR9 agonist phosphorothioated CpG-ODN type B [5′-tcc atg acg ttc ctg acg tt-3′] (catalog number tlrl-1826) were purchased from InvivoGen (San Diego, CA, USA). Agonist stocks were suspended in endotoxin-free water, aliquoted and stored at −20°C. Immediately prior to use, agonists were diluted in endotoxin-free, PBS-buffered solution.

For protein quantification, brain tissue was weighed and homogenized in Bio-Plex cell lysis buffer (Bio-Rad, Hercules, CA, USA; catalog number 171-304012) containing PMSF (Sigma, St. Louis, MO, USA; catalog number P-7626) and Complete Mini Protease Inhibitor Cocktail (Roche, Basel, CH; catalog number 11836153001) as previously described [24]. All samples were diluted to a final concentration of 300 mg/ml of brain tissue in lysis buffer. Samples were then centrifuged at 4,500 × g for 15 minutes at 4°C to remove debris. Cytokine and chemokine protein levels in brain homogenate supernatants were analyzed using the Invitrogen Mouse Cytokine Twenty-Plex Antibody Bead kit (Carlsbad, CA, USA; catalog number LMC006) on a Bio-Rad Bio-Plex 200 system (Hercules, CA, USA). Individual protein concentrations were calculated using standard curves generated from standards provided with the Twenty-Plex kit.

Total RNA was extracted from brain tissue using the Qiagen RNeasy Mini Kit (Valencia, CA, USA; catalog number 74106) per the manufacturer’s instructions. RNA was then treated with DNase (Ambion, Foster City, CA, USA; catalog number AM2224) for 30 minutes at 37°C, followed by a final purification and concentration using the Zymo Research RNA Clean-up kit (catalog number R1018). Complimentary DNA (cDNA) was generated from the isolated RNA using the iScript cDNA Synthesis kit (Bio-Rad, Hercules, CA, USA; catalog number 170-8891). All primers were designed using Primer3 and were gene-specific in blast searches performed using the National Center for Biotechnology Information database as previously described [24]. Real-time PCR was performed using iTAQ SYBR Green Supermix with ROX (Bio-Rad, Hercules, CA, USA, catalog number 1725852) on an Applied Biosystems (Grand Island, NY, USA) PRISM 7900HT instrument. All samples were run in triplicate. The baseline was automatically set and the C_T was manually set to intersect the mid-log phase of PCR curves at 0.19. Dissociation curves were used to verify that only a single gene product was amplified in each sample. Data analysis was completed following the published criteria for real-time quantitative PCR method using the comparative C_T method [25, 26]. Gene expression was calculated relative to the average expression of three housekeeping genes (Actb, Gusb, Rpl32) for each sample within an experiment. The same housekeeping genes were used for each experiment. Data are shown as the percent of the average of the housekeeping genes (% of housekeeping genes). RNA that was not reverse transcribed and water were used as negative controls.

Animals were anaesthetized by inhalation of isofluorane, followed by perfusion through the left ventricle of the heart with ice cold 1× Hank’s balanced salt solution (HBSS) without calcium and magnesium (Gibco, Grand Island, NY, USA; catalog number 14185). Whole brains were removed and sliced into several pieces prior to homogenization with a Dounce homogenizer and/or trituration using a 5 ml pipet. Samples were further triturated using a 20-gauge needle. For some experiments, brain homogenates were further dissociated by incubation in 0.05% Collagenase D (Roche, Basel, CH; catalog number 11 088 882 001), 0.09 U/ml Dispase I (Sigma, St. Louis, MO, USA; catalog number D4818) and 0.025 U/ml DNase I (Sigma, catalog number D4527) in 1× HBSS at room temperature for 30 minutes with continuous rocking. To isolate the myeloid and immune cells, cells were resuspended in 70% Percoll/HBSS and fractionated using a Percoll gradient as previously described [27]. Cells were harvested from the 30/70% interface of the Percoll gradient, washed in HBSS and analyzed by flow cytometry or used for quantitative real-time PCR.

Following isolation by Percoll gradient, samples were plated onto 96-well plates and analyzed for cell surface markers as previously described [28]. In short, cells were fixed in 2% paraformaldehyde, then permeabilized in 0.1% saponin/2% BSA/1× PBS. Samples were incubated in an F_C blocking solution containing rat anti-mouse CD16/CD32 Fcγ III/II antibody (BD Pharmingen, San Diego, CA, USA; catalog number 553142) in 2% donkey serum/0.1% saponin/2% BSA/1× PBS. Cells were incubated with fluorochrome-conjugated antibodies at room temperature. After washing twice, cells were resuspended in PBS and analyzed on a FACSAria flow cytometer (BD Biosciences, San Jose, CA, USA) using FACSDiva software (BD Biosciences, San Jose, CA, USA). Cells were gated as described in the figure legends.

To determine if the neuroinflammatory response changes in the first weeks of life, we inoculated neonatal (two day-old) and weanling (21 day-old) mice ic with either the TLR4 agonist, LPS, or the TLR9 agonist, CpG-ODNs. Control mice were inoculated with vehicle controls.

Cytokine protein levels were examined at 12 hours post inoculation (hpi), a time point previously shown to be the peak of cytokine production following TLR ligand inoculation in the CNS [29, 30]. Twelve cytokines were detectable at significant levels following TLR stimulation of the CNS. Several of these cytokines, including IL-1α, IL-1β, IL-2, IL-5, IL-6, TNF, CC chemokine ligand (CCL)2, CCL3 and CXCL9 were significantly higher in neonatal brain tissue than in weanling brain tissue in response to LPS (Figure 1). CpG-ODN also induced higher responses in neonates than in weanling mice for IL-2, IL-5, TNF, CCL2 and CXCL9. No significant age-dependent differences were observed for IL-12 or CXCL1. The cytokines tested were undetectable at later time points of 24, 48 or 72 hours for both neonates and weanling mice. Overall, the levels of several cytokines induced by TLR4 and/or TLR9 ligand induction were significantly higher in neonates than those observed in weanlings.

Since a difference in the expression of several cytokines was observed between weanling and neonates, we further examined neuroinflammatory responses using real-time PCR analysis. RNA was analyzed at 6 hpi, a timepoint that correlates with peak mRNA expression of cytokines following TLR stimulation both in vitro and in vivo [28, 30]. Analysis of mRNA expression also indicated similarly increased levels of several of these cytokines in neonatal mice as well as increased expression of inducible nitric oxide (Nos2, iNos) mRNA and adhesion molecule Icam1 mRNA (Figure 2, data not shown). In contrast, expression of Csf2 (GM-CSF) mRNA, a growth factor for monocytes, was elevated in brain tissue from weanling mice compared with neonatal mice (Figure 2). Thus, LPS inoculation induced different neuroinflammatory responses in neonates compared with weanlings with increased production of several cytokines and decreased expression of Csf2 mRNA.

To examine the time course of these neuroinflammatory responses, we measured mRNA expression levels from 2 to 48 hpi after treatment with either LPS or vehicle control. Modest increases in gene expression for Ccl2 and Ccl3 mRNA were observed in vehicle control-treated animals for both neonatal and weanling brains at 2 hpi but not at other time points. At 2 hpi after stimulation with LPS, Ccl2 and Il6 mRNA levels were comparable between the two ages, while Ccl3 mRNA was higher in neonates (Figure 3). However, by 6 hpi, these LPS responses in weanling mice had dropped to nearly basal levels while neonatal Il6 and Ccl2 levels remained elevated (Figure 3). Thus, expression of certain cytokine mRNAs were generally higher in neonates and peak expression occurred later than that observed in weanlings. This difference in kinetics may contribute to the increased protein levels of these cytokines observed at 12 hpi (Figure 1).

One possible explanation for the increased neuroinflammatory responses in neonatal mice would be higher expression of TLRs in the brains of neonates than adult mice. However, analysis of mRNA demonstrated lower levels of Tlr4 and Tlr9 mRNA in neonatal brain compared with other ages (Figure 4). Thus, the increased neuroinflammatory response is not likely to be due to increased expression of TLRs in the neonatal brain.

To investigate which cell types might be contributing to the heightened neonatal inflammatory response, we compared mRNA expression levels of inflammatory cells and glial cells in brain tissue from neonatal and weanling mice. Analysis of gene expression for inflammatory cells including T cells (Cd3ϵ, Cd8α) and neutrophils (Ela2) showed that these genes were only induced at 12 to 48 hpi (Figure 5A-C), after the peak cytokine response (Figure 3). Thus, an influx of T cells or neutrophils would probably be responding to, rather than producing, early increases in cytokine levels. The level of mRNA for the astrocyte activation marker Gfap was lower in neonatal brain than weanling brains (Figure 5D), suggesting that more activated astrocytes were also not likely to be the source of heightened cytokine responses in neonates. However, mRNAs for activation markers of myeloid cells (for example, microglia and macrophages), namely Itgax (Cd11c) and Cd80, were elevated at 6 to 12 hpi in neonates compared with weanlings (Figure 5E-F), in concert with the increases in the various cytokine mRNAs. Interestingly, at these time points we also observed elevated mRNA levels for Slamf7 (Figure 5G), whose expression has been reported on natural killer (NK) cells and other immune cells [31, 32]. Thus, the only cell markers that correlated with the early neuroinflammatory response in neonates were those thought to be primarily for myeloid cells and/or NK cells.

Since resident myeloid cells and/or inflammatory cells such as NK cells could be contributing to the cytokine response, we isolated these cells from the brains of neonatal and weanling mice using a Percoll gradient. The 6 hpi time point was used to allow for RNA analysis of isolated cell fractions. We analyzed cells from the 30/70% interface, which are predominantly microglia, macrophages and infiltrating immune cells [33]. Analysis of this cell fraction for CD4+ T cells, CD8+ T cells, NK cells and B cells indicated no significant populations of these cells in the brain for either neonatal or weanling mice (data not shown). Instead, the primary cells in this fraction for both neonates and weanling mice were CD45+ CD11b + myeloid cells; primarily microglia, monocytes and resident macrophages (data not shown).

RNA analysis of the myeloid cell fraction for genes analyzed in whole brain fractions demonstrated greater upregulation of Ccl2, Nos2 and Slamf7 mRNA following LPS stimulation in neonates compared with weanlings (Figure 6A-C). However, other cytokines, such as Il6, that were upregulated in neonates at 6 hpi in whole brain tissue (Figure 3) were not significantly different in the myeloid cell fractions at this time point (Figure 6D). Thus, myeloid cells isolated from the brain tissue of neonatal mice had increased upregulation of some of the same proinflammatory genes that were upregulated in the whole brain, indicating that these cells were likely contributing to the increased neuroinflammatory response in neonatal mice.

Interestingly, analyses of Tlr4 and Tlr9 mRNA expression in the myeloid cell fractions indicated no significant difference in Tlr4 mRNA expression in the myeloid fraction from neonatal and weanling mice, while a slight increase in Tlr9 mRNA was observed in weanlings (Figure 6E-F). Thus, the difference in the cytokine response between neonatal and weanling myeloid cells did not correlate with differences in TLR mRNA expression.

Since RNA analysis of myeloid cells from neonatal and weanling mice indicated differences in their neuroinflammatory responses, we further analyzed these cells by flow cytometry at 6 hpi, the time point that mRNA differences were observed. Analysis of cells expressing leukocyte common antigen CD45 and the myeloid-specific marker CD11b identified four distinct cell populations. These included CD45^lo, CD11b^lo microglia (mic, dark blue gate), and three populations of CD45^hi cells that varied in their expression of CD11b (light blue, green and fuchsia gates) (Figure 7). Further analysis using the inflammatory monocyte marker Ly6C indicated that these three cell populations were either Ly6C + monocytes (m1 green and m2 fuchsia gates) or Ly6C⁻ resident macrophages (mac, light blue gate). The monocyte populations have two distinct profiles in the weanling mice which differed in the expression of CD11b and F4/80, being either CD11b^int, F4/80^hi (m1, green gate) or CD11b^hi and F4/80^lo (m2, fuschia gate). Lower numbers of all cell populations were observed in neonates compared with weanlings; most notably, the microglia population (mic, dark blue gate) had five-fold fewer cells in neonates than weanlings (Figure 7). In addition, microglia had higher levels of F4/80 (middle panel, X axis) compared with weanlings indicating that these cells change phenotypically between these two ages. Of the CD45^hi populations, the resident macrophage (mac, light blue gate) was also reduced in neonates and was barely detectable.

To examine the functional state of these cell types, we examined cell surface markers related to cell activation: CD11a (integrin α L) and CD86; as well as those associated with inhibition of activation: CD200R (OX2R) and CD172a (SIRPα) [34‐37]. Cells were analyzed at 6 hpi, that is, at the peak of cytokine mRNA expression. A significant difference was observed between neonatal and weanling mice in the mean fluorescent intensity (MFI) for these molecules (Figure 8), although the relative surface expression of these molecules was not altered by LPS stimulation (data not shown). Neonatal microglia had higher levels of CD11a and CD172a, but lower levels of CD86 (Figure 8). Neonatal monocytes had lower levels of CD11a than weanling monocytes, with varying levels of CD86, CD200R and CD172a depending on the subpopulation (Figure 8). Thus, there were distinct differences in the expression of cell surface regulatory markers between neonates and weanlings, but these varied substantially between the cell populations.

Slamf7 was one of the markers whose mRNA expression was upregulated in whole brain tissue and in the myeloid/immune cells fraction from neonatal mice (Figure 5G, Figure 6C). SLAMF7 is often used as an activation marker for NK cells [31, 32, 38], however, we were unable to detect NK cells in the CNS by flow cytometry (data not shown). Although SLAMF7 was recently detected on activated human monocytes [39], expression in the CNS has not been reported. Analysis of SLAMF7 on the myeloid cells in the CNS demonstrated clear expression of SLAMF7 on neonatal microglia and lower levels of SLAMF7 on weanling microglia (Figure 9A), but was not upregulated following LPS stimulation (Figure 9B). SLAMF7 was not expressed at levels above background on the monocyte or macrophage population (Figure 9C, data not shown) for either PBS or LPS inoculated mice. Thus, SLAMF7 appears to be primarily expressed by neonatal microglia and may be a useful marker for identifying this population. The higher expression of SLAMF7, CD11a, F4/80 and CD172a on neonatal microglia compared with weanling microglia indicates a clear phenotype on these cells that correlates with the heightened inflammatory response in the CNS.