Age-related decline of the acute local inflammation response: a mitigating role for the adenosine A_2A receptor

Aging has a negative impact on the accumulation and viability of neutrophils in the air pouch

Injection of LPS into a dorsal air pouch (500 ng/pouch) elicits the recruitment of neutrophils [8]. Fractions of Ly6G⁺ neutrophils remained relatively constant throughout age groups, ranging from 70% to 82% in WT mice (Fig. 1B, left panel). In 3-month-old WT mice, approximately 1.5 x 10⁶ neutrophils had accumulated 4 h after injection (Fig. 1B, right panel). Neutrophil infiltration was 52% greater in KO mice. However, by the age of 15 months, the numbers had dropped by more than half for both genotypes. No such decrease was observed in the bloodstream of WT mice: counts of leukocytes, including lymphocytes, monocytes, and neutrophils, were all even higher at 15 months (Suppl. Figure S1). The LPS injection caused bloodstream neutrophil counts to double in young and old mice, while lymphocyte and monocyte counts were unchanged. The viability of Ly6G⁺ neutrophils harvested from the air pouches dropped as the mice aged (Fig. 1C, top panel). This occurred faster in KO mice, in which the final counts were approximately 30% lower than in WT, and is reflected in the proportions of early apoptotic neutrophils and late apoptotic/dead cells. Increased neutrophil apoptosis in A_2AR-KO mice is consistent with earlier reports indicating that activation of A_2AR delays apoptosis in human neutrophils [19,20]. The viability of non-neutrophil leukocytes (Ly6G^-) similarly decreased with aging (fig 1C, bottom panel).

Local tissue permeability and neutrophil degranulation decrease with aging

The permeability of local interstitial tissues decreased markedly as the animals aged, based on the total protein content of the air pouch fluid, both in saline-injected (basal condition) and LPS-injected animals (Fig. 1D). In WT mice injected with LPS, total protein decreased by 83% between the ages of 3 months and 15 months. The corresponding decrease in KO mice was more pronounced. Measured as an indication of neutrophil azurophilic degranulation [21], elastase activity also decreased as the mice aged (Fig. 1E), noticeably more in KO mice.

Aging affects the profile of cytokine accumulation at the inflammatory site

Levels of each cytokine and chemokine measured in air pouch exudates of WT and KO mice at three months of age were for the most part comparable, whether the mice were injected with LPS or not (Fig. 2A). However, as the animals aged, levels of TNF, IL-6, IL-10, CXCL1, and CCL2-4 and G-CSF decreased markedly while CXCL2-3 remained elevated. Figure 2B illustrates the changes observed respectively in LPS-injected WT and KO mice, relative to cytokine status at 3 months. The decrease was faster and greater in KO mice than in WT mice, with levels of CXCL 1, 2 and 3 dropping more slowly. An age-matched comparison between the two genotypes shows that the effect of A_2AR knockout was most apparent at the age of 6 months and that CXCL 1, 2 and 3 were the least affected (Fig. 2C). Additional cytokines and growth factors were measured, including IL-1α, IL-1β, IL-2 to IL-5, IL-7, IL-9, IL-12, IL-13, IL-15, IL-17, LIF, M-CSF, GM-CSF, IFN-γ, and VEGF, but their concentrations were stable and remained below 50 pg/ml consistently under all conditions tested (data not shown).

Figure 2. Cytokine/chemokine levels in dorsal air pouches. (A) Concentrations of cytokine, chemokine or growth factor in cell-free exudate recovered from WT and A_2AR KO mice after injecting air-pouches with phosphate buffered saline (-) or with buffer + LPS. Mice were aged 3, 6 or 15 months (m). Concentrations were measured using a multiplex immunoassay as described in Methods and are expressed as mean ± SEM for n = 12 mice. *Significantly different from the concentration measured at 3m. ^#Significantly different from the age-matched wild-type group. (B) Changes (%) in concentrations measured at 6m and 15m relative to 3m, in LPS-injected air pouches. (C) Variations between wild-type and A_2AR-knockout mice concerning cytokine concentrations measured in LPS-injected dorsal air pouches. Values are % changes relative to age-matched WT. (D) The effect of neutrophils influx on cytokine/chemokine levels in dorsal air pouches. WT mice aged 3m were injected with anti (α)-Ly6G before injection of LPS into air pouches. Cytokine concentrations and neutrophil counts (insert) in the pouches are expressed as the change (%) relative to control mice (not injected with the antibody). Values are expressed as mean ± SEM (n = 5).

Such clear age-related decreases in cytokine concentrations were not observed in the bloodstream. Basal circulating levels remained quite constant over the 1-year period of study (Suppl. Figure S2). Only IL-15 and to a lesser extent CXCL 1, 2 and 3 and CCL 2 and 3 tended to decrease with age, but not to a significant extent. Injection of LPS into the pouch increased the levels of several cytokines in the bloodstream. The highest levels measured were by far for G-CSF, in young and older mice. Levels of IL-6, CXCL9 (MIG), CXCL10 (IP-10), CCL2 and to a lesser extent CCL4 also increased. For most analytes, blood levels in LPS-injected mice were comparable at 3 and 15 months. Exceptions included IL-6, which increased significantly in older mice, while levels of G-CSF and CXCL10 decreased. Levels of CCL3, CCL4, and IL-15 tended to increase in older mice but did not reach significant levels. IFN-γ, IL-2, IL-3, IL-4, IL-5, IL-7, IL-9, IL-10, IL-12, IL-13, IL-17, LIF, M-CSF, and VEGF were stable and consistently below 100 pg/ml under all tested conditions (data not shown).

Incoming neutrophils are required for normal cytokine production at the site of inflammation

To hinder neutrophil migration to the inflammation sites, 3-month-old WT mice that were to be treated with LPS received an intravenous injection of anti-Ly6G, which works via a β2-integrin-dependent mechanism [22]. Neutrophil counts in LPS-injected pouches were thus decreased by as much as 80% (Fig. 2D, insert). Subsequent accumulation of TNF, IL-6, IL-10, CCL2-4, CXCL9-10 and G-CSF was also decreased, often by more than 75% (Fig. 2D), while levels of CXCL 1, 2 and 3 increased by 2–4 fold. Preventing neutrophil migration thus appears to replicate the effects of aging on the local accumulation of cytokines.

Aging affects gene expression profiles in migrated leukocytes

Increased expression of genes encoding transcription factors, cytokines, enzymes, regulatory elements and receptors is an additional indication of leukocyte activation [7,8]. We calculated the stimulation indexes of various genes in neutrophils as a function of age under basal and inflamed (LPS) conditions, WT and KO mice (Suppl. Figures S3, S4). Consistent with our previous observations [8], mRNA encoding the cytokines IL-1β, CXCL2, CCL 3 & 4 and phosphatase DUSP1 were among the most abundant, under basal and LPS-inflamed conditions. Messenger RNA encoding IL-1α, IL-1β, CCL 3 & 4, CXCL 2 & 3, MAPK signaling factor GADD45B and suppressor signals SOCS3 and TNFAIP3 had the highest stimulation indexes. CCL3, CCL4, CXCL1, and CXCL3 mRNA levels increased in leukocytes recovered from air-pouches of WT mice aged 6 and 15 months, under basal and LPS conditions, by more than 10-fold in the case of CCL4 (Fig. 3A & B, top and middle panels). In contrast, TNF mRNA diminished consistently. IL-1α mRNA faded under conditions of LPS stimulation. The CCL4 stimulation index increased at six months, while indexes for CXCL1 and IL-1α decreased (Fig. 3A & B, bottom panels). Increases in CXCL2 and CXCL3 and decreases in TNF under basal and LPS conditions were greater in KO than in WT mice (Figure 4A & B). Stimulation indexes of IL-1α, IL-1β, CCL3, CCL4, CXCL2, CXCL3, EDN1, and TNF were affected negatively as the animals aged. Age-matched comparisons between KO and WT revealed CCL4 expression up to 16 times higher in leukocytes recovered from dorsal air pouches of KO compared to WT mice (Fig. 5), while CXCL2 and IL-α decreased. Stimulation indexes for CCL3, CCL4 were higher. In 15-month-old mice, the vast majority of the affected genes were expressed more weakly in KO than in WT mice. A compilation of the most salient gene expression trends is presented in Table 1.

Figure 3. Effect of age on gene expression in leukocytes recovered from dorsal air pouches raised on WT mice. Gene mRNA transcripts were quantitated as described in Methods and categorized as transcription factors, receptors, cytokines or enzymes. In each category, genes are ranked from the largest increase to the largest decrease under basal conditions (saline injection only). (A) 6-month to 3-month age comparison. (B) 15-month to 3-month comparison. “Stimulation index” is the change due to stimulation by LPS injection, relative to the basal condition. Dotted lines indicate one doubling or halving of gene expression. Values are expressed as the base-2 logarithm.

Figure 4. Effect of age on gene expression in leukocytes recovered from dorsal air pouches raised on A_2AR KO mice. Please refer to the legend for Figure 3.

Figure 5. Age-matched comparisons of gene expression in leukocytes recovered from dorsal air pouches raised on wild-type (WT) and A_2AR-knockout (KO) mice. Ratios were determined for the basal condition (injection of PBS only, top row), the LPS-stimulated condition (middle) and the stimulation indexes (bottom).

Experimental design

A_2AR heterozygotes (A_2AR^+/-) CD1 mice were paired. Offsprings were genotyped to select A_2AR^-/- (KO) and A_2AR^+/+ (wild-type) animals, as described previously [6]. Mice were kept for up to 15 months in groups of 4 per cage at 20 °C and 60% relative humidity with a light-dark cycle of 12 h. Access to food and water was ad libitum. Age groups (3, 6, 15 months old), genotypes and age-matched genotypes were compared. The dependent variables were: counts and viability of granulocytes recovered from the dorsal air pouch, expression (mRNA) of inflammatory genes in leukocytes and in the tissue lining the air pouch, accumulation of cytokines/chemokines and elastase activity in the pouch exudates.

Dorsal air pouches

The Université Laval animal protection committee approved all air-pouch experiments. These were conducted as described previously [8]. Briefly, dorsal pouches (one per mouse) were raised by subcutaneous injection of 5 mL of sterile air on day 0 and 3 mL on day 3. On day 6, individual air pouches were injected with 500 µL of pre-warmed, endotoxin-free phosphate-buffered saline (PBS), or PBS containing 500 ng of lipopolysaccharide from Escherichia coli (LPS, E. coli 0111:B4, Sigma-Aldrich Canada Co., Oakville, ON, Canada). Mice were sacrificed 4 h after LPS injection, and air pouches were washed twice with ice-cold PBS (total of 2 mL). Leukocyte suspensions were assayed for cell enumeration with a Cellometer Auto T4 Plus cell counter (Nexcelom Bioscience LLC, Lawrence, MA, USA). Blood was collected by cardiac puncture. The experimental groups contained an average of 8 mice and equal numbers of females and males. The entire experiment was repeated five times.

Cell subtyping

Identification of leukocyte and subtypes were performed using V450-conjugated rat anti-mouse CD45 IgG2b, k (leukocyte marker), APC-conjugated rat anti-mouse Ly6G (1A8) IgG2a, k (granulocyte marker), FITC-conjugated rat anti-mouse CD3 IgG2b, k (BD Biosciences, Mississauga, ON, Canada; lymphocyte marker), and PE-Cyanine7-conjugated rat anti-mouse CD115 IgG2a, k (eBioscience, San Diego, CA, USA; monocyte marker). Briefly, 100 μL of cell suspension was incubated with 0.2 μg of anti-CD45, 0.2 μg of anti-Ly6G, 0.5 μg of anti-CD3 and 0.2 μg of anti-CD115 for 30 min in the dark. PBS was added (400 μL) and samples were analyzed using a FACS Canto II flow cytometer with FACSDiva software, version 6.1.3 (BD Biosciences).

Neutrophil viability

Cell viability was assessed using V450-conjugated rat anti-mouse CD45, APC-conjugated anti-Ly6G, and a FITC Annexin V Apoptosis Detection Kit (BD Biosciences). Briefly, 100 μL of cell suspension was incubated with 0.2 μg each of anti-CD45 (leukocyte marker) and anti-Ly6G for 30 min in the dark. After centrifugation, cell pellets were suspended in 100 μL of the binding buffer 1X provided with the apoptosis detection kit. Annexin V (5 μL) and 5 μL of propidium iodide were added to each sample. After 15 min, 400 μL of binding buffer was added, and samples were analyzed using a FACS Canto II flow cytometer with FACSDiva software, version 6.1.3 (BD Biosciences). Gating was determined using control samples labeled individually with either Annexin V or propidium iodide.

Elastase, and total proteins

Mouse elastase and total proteins in air pouch exudates were measured using respectively an ELISA method (R&D Systems, Minneapolis, MN, USA) and a protein assay (Bio-Rad, Mississauga, ON, Canada) per the manufacturers' instructions.

Metabolites

Cytokine/chemokine levels in cell-free supernatants recovered from dorsal pouch exudates were measured using a multiplexed bead-based immunoassay (BD^™ Cytometric Bead Array) according to the manufacturer's protocol. TNF (C8), IL-6 (B4), IL-10 (C4), IL-1β (E5), GM-CSF (B9), CCL2/MCP-1 (B7), CCL3/MIP-1α (C7), CCL4/MIP-1β (C9) and CXCL1/KC (A9) levels were determined using a FACS Canto II flow cytometer with FCAP Array software, version 3.0 (BD Biosciences). CXCL2-3/MIP-2 measurements were performed using a commercially available ELISA kit (R&D systems Inc., Minneapolis, MN, USA) according to the manufacturer's instructions. Plasma cytokines and G-CSF were measured using Eve Technologies (Calgary, AB, Canada).

RNA isolation

Total RNA was isolated from approximately 5x10⁶ leukocytes (pellet of centrifuged dorsal pouch cell suspension) using Ribozol™ (Amresco, Solon, OH, USA) according to the manufacturer's protocol, with modifications [8,9]. Briefly, the sample was homogenized in 1 mL of Ribozol™, and 200 µL of chloroform were added. Samples were mixed and then centrifuged at 12,000 x g for 15 min (4 °C), and the aqueous phase (450 µL, on top) was transferred to a tube containing an equal volume of isopropanol, mixed thoroughly using a vortex device and centrifuged at 12,000 x g for 10 min (4 °C). The supernatant was discarded, and the precipitated RNA pellet was washed twice in 500 µL of 75% ethanol with centrifugation at 12,000 x g for 5 min (4 °C). The final pellet was allowed to air-dry for 5-10 min and then re-suspended in RNase-free water. RNA was quantitated using a Qubit^® Fluorometer (Life Technologies Inc., Burlington, ON, Canada).

Comparative real-time PCR

Gene expression (mRNA transcript abundance) was monitored in leukocytes using real-time PCR. Reverse transcription was performed using 1 µg of total RNA with a Transcriptor First Strand cDNA Synthesis Kit (Roche Applied Science, Laval, QC, Canada) following the manufacturer's instructions. Real-time PCR was performed as described previously [40]. Briefly, cDNA amplification was carried out in a Rotor-Gene Q operated with Q-series software version 2.0.2 (Qiagen Inc, Mississauga, ON, Canada) using 35 cycles of 95 °C for 17 seconds, 58 °C for 25 seconds and 72 °C for 25 seconds. Each reaction mixture contained 40 ng of cDNA, 2 μL of 10X buffer (100 mM Tris, 500 mM KCl, 30 mM MgCl₂, 1.5% Triton X-100), 100 μM dNTP, 500 nM of primers, 0.1 unit of Taq DNA polymerase (Roche Applied Science) and SYBR^® Green I dye (Life Technologies Inc.) diluted 1:30 000 in a volume of 20 µL. Reaction specificity was ascertained by performing the Melt^® procedure (58–99 °C, 1 °C per 5 s) at the end of the amplification protocol, according to the manufacturer's instructions. For each gene of interest, specific primers were designed as described previously [8]. Briefly, primers were selected systematically within the coding region, with a theoretical melting point of 58 °C, GC content of 50% (± 10%) and 18–24 base pair length, for an average product length of 200 base pairs. Primers thus designed were all tested with gradient PCR before use in real-time PCR [8]. Internal control genes were ranked using RefFinder, a web-based tool (http://150.216.56.64/referencegene.php?type=reference) developed for reference gene screening and evaluation based on published datasets and four ranking methods [8]. The RefFinder overall final ranking of each gene is a weighting calculated as the geometric mean of the rankings obtained from each method. Six genes were selected as candidate control genes: GAPDH, GUSB, H2AFZ, PPIA, TUBB4A, and UBC. The gene with the best ranking was used as the normalizing factor for presenting relative mRNA expression. In this study, GAPDH consistently ranked best.

Statistical analysis

Where applicable, values are expressed as mean ± SEM. Unless stated otherwise, statistical analyses were performed using Student's two-tailed unpaired t-test. The criterion for declaring a difference to be significant was p < 0.05.