All procedures in this study were approved by Tufts Medical Center Human Investigation Review Board (IRB). Young (22–35 years) and elderly (65–69 years) healthy human volunteers were recruited through the HNRCA Volunteer Services Department and the Department of Molecular Biology and Microbiology, Tufts University School of Medicine. All enrolled subjects signed IRB approved consent forms. Individuals that were pregnant, taking medication or reporting symptoms indicative of infection within the prior 2 weeks were excluded from the study. Blood was drawn between 8 am and 10 am and study subjects were asked not to have any food intake after midnight on the day of blood draw.

Streptococcus pneumoniae TIGR4 strain (serotype 4), were grown at 37°C with 5% CO₂ in Todd-Hewitt broth (BD Biosciences) supplemented with 0.5% yeast extract and Oxyrase (Oxyrase) till mid-exponential phase. Aliquots were then frozen at −80°C in the growth media with 25% (v/v) glycerol. Before use, bacterial aliquots were thawed on ice, washed once and diluted in PBS to the required concentrations. Bacterial titers were confirmed by plating on Tryptic Soy Agar plates supplemented with 5% sheep blood agar (Northeast Laboratory Services).

Whole blood was drawn using acid citrate/dextrose as an anti-coagulant. PMNs were then purified using a 2% gelatin sedimentation as previously described (Bou Ghanem et al., 2014). This method allows for isolation of active PMNs with ~90% purity.

A vitamin E stock solution (ADM) of 30 mg/ml of d-α-tocopherol in ethanol was prepared. To increase cellular uptake (Marko et al., 2007) the stock solution was diluted in FBS to a final concentration of 2 mg/ml and incubated for 30 min at 37°C with gentle vortexing at 10 min intervals. PMNs (2 × 10⁷ cells/ml) were incubated with the α-tocopherol form of vitamin E (α-Toc) at a final concentration of 25 μg/ml or with 0.06% ethanol as vehicle control in HBSS lacking calcium and magnesium for 1 h, washed and then added to the Transwells for the migration assay or used in the opsonophagocytic assay (detailed below).

Transmigration assay was performed as previously described (Bou Ghanem et al., 2014). Briefly, Human pulmonary mucoepidermoid carcinoma-derived NCI-H292 (H292) (ATCC) cells were seeded on inverted Transwell filters collagen-coated Transwell filters (0.33-cm², Corning Life Sciences) and then allowed to grow and polarize for 1 week in RPMI 1640 medium (ATCC) with 2 mM L-glutamine, 10% FBS (Invitrogen) and 100 U penicillin/streptomycin. On the day of the migration assay, the epithelial monolayers were washed out of the antibiotic-containing media and equilibrated in HBSS for 30 min. The apical side of monolayers was infected with S. pneumoniae at a multiplicity of infection (MOI) of 20 for 2.5 h. Uninfected wells were treated with HBSS. The monolayers were washed and placed into 24-well plates. 600 μl of HBSS was added to the lower (apical) chamber and 100 μl of PMNs (1 × 10⁶) was added to the top (basolateral) chamber. PMNs that transmigrated into the apical chamber were collected and lysed in 10% Triton-X 100. Migrated neutrophils were quantitated using myeloperoxidase ELISA following a well-established assay (McCormick et al., 1995) where serial dilutions of known numbers of neutrophils were used to establish a standard curve. For each donor the average migration from triplicate wells per condition was assessed and plotted.

The ability of human PMNs to kill pneumococci was assessed ex vivo as described previously (Dalia et al., 2010) with some modifications. Briefly, 5 × 10⁵ PMNs were incubated with 1 × 10³ bacteria grown to mid log phase and pre-opsonized with 10 μl rabbit complement (pel-freez) in 200 μl reactions in Hank's buffer/0.1% gelatin. The reactions were incubated rotating for 45 min at 37°C. Percent killing in comparison to incubations with no PMNs was determined by plating serial dilutions on blood agar plates. For each donor six total replicates were performed and n = 3 were plated to calculate efficiency of killing and the other three used for quantification of serine proteases (described below). To measure if bacterial uptake by PMNs decreased with age, we followed a previously published assay where bacteria are pre-labeled with a Fluorescein isothiocyanate (FITC) dye and phagocytosis is assessed by flow cytometry following addition of trypan blue that quenches any extracellular signal allowing detection of internalized bacteria (Dalia et al., 2010). We saw that age had no effect on the percentage of PMNs that were FITC-positive, however, although FITC-labeling of bacteria did not have any significant effect on bacterial viability directly, it did interfere with the ability of PMNs to kill pneumococci (data not shown).

For each donor, pooled PMN pellets from three opsonophagocytic reactions (1.5 × 10⁶ PMNs) were lysed with 300 μl of the Cathepsin G Activity Assay Kit (abcam) lysis buffer. PMN elastase levels (abcam PMN Elastase Human ELISA Kit) and activity (abcam PMN Elastase Fluorometric Activity Assay Kit) as well as Cathepsin G levels (Aviva Systems Biology CTSG Human ELISA Kit) and activity (abcam Cathepsin G Activity Assay Kit) in the cell pellets were then determined following manufacturer's instructions.

All statistical analysis was performed using Prism5 for Macintosh (Graph Pad). D'Agostino & Pearson omnibus normality test was used to determine if the data were normally distributed. Paired student t-test, unpaired student t-test or Mann-Whitney test were used for comparison between groups as indicated. Pearson or Spearman tests were used to determine correlation. One sample t-test was used to measure whether α-Toc -indices were different from 1.0. All comparisons with p < 0.05 were considered significantly different. Individual points representing data from a single donor were plotted. Lines and error bars represent the mean values ± SEM.

Prior studies demonstrated that aging was associated with an increase in PMN recruitment in to the lungs following S. pneumoniae infection (Bou Ghanem et al., 2014; Menter et al., 2014). To test whether PMNs from elderly individuals possess an enhanced intrinsic ability to migrate in response to pneumococcal infection, we measured PMN migration across lung epithelial cells in vitro. Polarized monolayers of H292 human epithelial cells on Transwell filters were mock-infected or apically infected with S. pneumoniae TIGR4 at an MOI of 20. PMNs from young (22–35) or elderly (65–69) healthy human volunteers were then added to the basolateral side and cumulative PMN migration was measured after 120 min. No detectable migration was observed in the absence of infection in either group (data not shown). As previously observed (Bou Ghanem et al., 2014), S. pneumoniae infection elicited robust trans-epithelial movement of PMNs from all young donors, with some variation in the ability of PMNs from different donors to migrate (2.7 ± 1.9 × 10⁵; Figure 1A). Transepithelial migration by PMNs isolated from elderly donors (2.6 ± 1.4 × 10⁵; Figure 1A) was indistinguishable from that of young controls. PMN migration across the epithelium in response to the chemoattractant peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP) was also indistinguishable between the age groups (Figure 1B).

We previously found that vitamin E mitigates the harmful inflammatory response during pneumococcal infection of aged mice partially due to its direct effect on PMN migration (Bou Ghanem et al., 2014). To test the effect of vitamin E on the transmigration response of PMNs from elderly individuals, PMNs were pretreated with 25 μg/ml of α-tocopherol form of vitamin E (α-Toc), a concentration that was previously found to blunt migration of PMNs from young donors and is within the range of levels measured in plasma of humans taking a daily supplement of 200 International Units of vitamin E (Meydani et al., 1997). This concentration of α-Toc had no effect on either PMN or bacterial viability (data not shown) (Bou Ghanem et al., 2014). α-Toc treatment significantly reduced PMN migration in 5 out of 8 elderly donors and 4 out of 6 young donors tested (p < 0.05 by paired t-test). To more thoroughly analyze the magnitude of the effect of α-Toc, for PMNs from each individual tested for transepithelial migration in the presence or absence of α-Toc, we calculated a “α-Toc PMN migration index,” which is the ratio of migration in the presence of α-Toc to migration in its absence (Figure 2). As previously observed, α-Toc treatment had no effect on fMLP-induced migration of PMNs from young donors (Bou Ghanem et al., 2014), and here we found that α-Toc also had no effect on migration of PMNs from aged donors, with α-Toc migration indices of close to 1.0 (Figure 2A). In contrast, PMN migration in response to pneumococcal infection from young and elderly donors was diminished by α-Toc treatment (Figure 2B), with average α-Toc migration indices of 0.66 and 0.83, respectively. Although the α-Toc migration indices were not significantly different between the age groups (p = 0.39 as measured by Student's t-test), α-Toc treatment significantly reduced average migration in PMNs isolated from young (p = 0.043), but not elderly (p = 0.249) donors as measured by one sample t-test. Finally, although α-Toc treatment blunted PMN transepithelial migration in response to pneumococcal infection in the majority of individuals, the migration of PMN of some individuals was either not affected or slightly increased by α-Toc (Figure 2B), consistent with previous findings that the effect of vitamin E can vary between individuals (Belisle et al., 2010).

The absence of age-specific transepithelial migration responses left open the question whether intrinsic PMN anti-pneumococcal function declines with age. Throughout their lifetimes, the majority of individuals are intermittently nasopharyngeally colonized by S. pneumoniae strains (Simell et al., 2012), a process that can generate a humoral immune response (Ferreira et al., 2013). Therefore, rather than opsonizing bacteria with (potentially antibody-containing) donor sera, which could confound our interpretation, we opsonized bacteria with purified complement prior to incubation with PMNs isolated from young and elderly donors. Surprisingly, PMNs isolated from elderly donors killed complement-opsonized bacteria significantly more efficiently than their younger counterparts (Figure 3A), and analysis of bacteriocidal activity after stratification by age revealed a significant positive correlation between bacterial killing and age (Figure 3B).

Killing of engulfed pneumococci by human PMNs was previously shown to be dependent on serine proteases, such as cathepsin G (CG) and neutrophil elastase (NE) (Standish and Weiser, 2009), so we compared the amounts and activities of these proteases in young or aged S. pneumoniae-infected PMNs (see Materials and Methods). First, irrespective of donor age, we detected very low amounts and activities of both cathepsin G and neutrophil elastase in the supernatants following pneumococcal infection of PMNs (data not shown), suggesting that S. pneumoniae infection does not trigger large scale exocytosis of serine protease-containing granules in our assay. Second, ELISAs of cell pellets revealed no significant age-associated difference in cathepsin G amounts (Figure 4A); activity measurements showed that PMNs isolated from elderly donors had on average 3-fold higher cathepsin G activity compared to young PMNs, but this difference did not reach statistical significance (p = 0.16; Figure 4B). Neutrophil elastase levels did not significantly differ between groups (Figure 4C), but PMNs from elderly donors displayed a significant (p = 0.0158), 15-fold higher level of neutrophil elastase activity (Figure 4D) compared to young PMNs. Finally, F-test of variances indicated significantly (p < 0.05) more individual-to-individual variation in activity levels of both cathepsin G and neutrophil elastase in PMNs from elderly donors compared to young controls. These data suggest that, in the majority of subjects, aging is accompanied by increased activity of serine proteases, which could contribute to the heightened intrinsic anti-microbial activity of PMNs from elderly donors.

We next tested the effect of α-Toc on the serine protease and bacteriocidal activities of PMNs isolated from young and elderly donors following exposure to S. pneumoniae ex vivo. For both activities, we determined the respective α-Toc indices, i.e., the ratio of activity in the presence of α-Toc to the activity in its absence. The baseline activity of cathepsin G and neutrophil elastase was elevated in PMNs from aged donors compared to young donors (Figure 4), and the α-Toc treatment did not increase these activities in PMNs from aged donors any further, resulting in α-Toc activity index of 1.1 and 0.96 for cathepsin G and neutrophil elastase, respectively (Figure 5A). The α-Toc bacteriocidal index of PMNs from elderly donors also was 1.0, indicating that α-Toc had no effect on the ability of these PMNs to kill pneumococci in our assay (Figure 5B). In contrast, the average α-Toc activity indices for cathepsin G and neutrophil elastase from PMNs from young donors were 1.85 and 2.1, respectively. These α-Toc activity indices were not significantly higher than 1.0 or than the activity indices of PMNs from elderly donors. The average α-Toc bacteriocidal index in young donors, at 2.0, was elevated and was both significantly higher than 1.0 (p = 0.027) and significantly higher than that of elderly donors (p = 0.0054; Figure 5B). In fact, while α-Toc treatment significantly (p < 0.05) boosted PMN killing in only 1 out of the 8 elderly donors by paired t-test, PMNs from 4 out of 6 young donors tested showed significantly increased bactericidal activity upon exposure to α-Toc. Furthermore, statistical analyses upon numeric stratification by age revealed that the ability of α-Toc to boost neutrophil elastase activity and bacterial killing both negatively correlated with age (Figures 5C,D), suggesting that the effect of α-Toc on PMN function is age-dependent. These findings suggest that α-Toc boosts the ability of PMNs from young donors to kill S. pneumoniae, in part by increasing the activity of anti-microbial serine proteases, in particular neutrophil elastase.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.