Ly6C^high monocytes are increased with age, express more CCR2 and less F4/80.

<p>(A) Total numbers of Ly6C^high and Ly6C^low monocytes were quantitated in the blood of old (18–22 mo) WT C57Bl6/J mice and compared to that from young (10–14 wk) mice. The data represent the mean (± SEM) of 6 mice. (B) Analysis of the Ly6C^high monocytes as a percentage of CD45⁺ cells in the blood and bone marrow of young and old mice (± SEM; <i>n</i> = 6). (C) CCR2 expression on Ly6C^high monocytes in the bone marrow and blood of old mice is higher than young controls as determined by flow cytometry (<i>n</i> = 6–8). (D) The mean expression of the macrophage maturity marker, F4/80, on Ly6C^high monocytes in the bone marrow and blood of young and old mice (<i>n</i> = 6–8). (E) Cells recruited to the peritoneum were quantitated 4 hours after administration of 100 nM CCL2. The recruitment of Ly6C^high and Ly6C^low monocytes was greater in old mice (± SEM; <i>n</i> = 5). Statistical significance was determined by two-tailed Mann-Whitney-Wilcoxon test or two-way ANOVA with Fisher's LSD post-test where appropriate. * indicates <i>p</i> < .05, ** indicates <i>p</i> < 0.005, *** indicates <i>p</i> < 0.0005 and **** indicates p < 0.00005. (A-D) is representative of 4 independent experiments; (E) is representative of 2 independent experiments.</p

Ly6C^high monocytes contribute to elevated levels of serum IL6 and TNF in aged mice.

<p>Young and old mice were injected with 500 nm negatively-charged polystyrene microparticles (PS-MPs) previously shown to reduce numbers of circulating Ly6C^high monocytes. Circulating monocyte populations (A) and IL6 levels in whole blood (B) were quantitated after 24 hours. Statistical significance was determined by two-tailed Mann-Whitney-Wilcoxon test. * indicates <i>p</i> < .05, ** indicates <i>p</i> < 0.005, *** indicates <i>p</i> < 0.0005 and **** indicates p < 0.00005. (A-B) is representative of ± SEM of 5 mice from 2 independent experiments.</p

Reducing TNF-regulated recruitment of Ly6C^high monocytes during <i>S</i>. <i>pneumoniae</i> colonization in old mice reduced nasopharyngeal bacterial loads.

<p>(A-B) TNF in the (A) nasopharnyx and (B) serum of young and old mice during <i>S</i>. <i>pneumoniae</i> colonization as measured by qPCR and ELISA, respectively (± SEM; <i>n</i> = 3–5). (C) CFUs in nasal lavages of old WT and old TNF mice on day 4 after colonization with <i>S</i>. <i>pneumoniae</i> (± SEM; <i>n</i> = 6–8, one independent experiment of two shown). (D) Ly6C^high monocytes as a percent of circulating CD45+ cells in old WT and TNF KO mice on day 4 of <i>S</i>. <i>pneumoniae</i> colonization (± SEM; <i>n</i> = 3–4, one independent experiment of two shown). Statistical significance was determined by two-tailed Mann-Whitney-Wilcoxon test, one-way ANOVA or two-way ANOVA with Fisher's LSD post-test where appropriate. * indicates <i>p</i> < .05, ** indicates <i>p</i> < 0.005, *** indicates <i>p</i> < 0.0005 and **** indicates p < 0.00005.</p

Depletion of inflammatory monocytes improves outcome to S. pneumoniae infection in old mice.

<p>Mice (n = 7-10/group) were injected with PS-MP day -1, 0, +1, +3 and +5 during colonization <i>with S</i>. <i>pneumoniae</i>. A) The percentage of Ly6C^high monocytes was significantly reduced in old mice treated with PS-MP (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005368#ppat.1005368.s003" target="_blank">S3 Fig</a>). B) Survival was significantly improved in old mice treated with PS-MP (p = 0.005, Mantel-Cox log-rank test). C) Both young and old mice treated with PS-MP lost less weight than their control counterparts (*,p<0.05, one-way ANOVA with uncorrected Fisher's LSD). Levels of <i>S</i>. <i>pneumoniae</i> in the D) nasal wash, E) lungs and F) spleen were lower in old mice treated with PS-MP. Fewer young mice had bacteria in their lungs and spleens when they were treated with PS-MP. (*,p<0.05, **,p<0.005 one-way ANOVA with uncorrected Fisher's LSD). CFU count for mice that reached endpoint before day 7 are not included.</p

Human CD14⁺⁺CD16⁺HLA-DR⁺ (intermediate) monocytes produce more inflammatory cytokines with age.

<p>Intracellular production of TNF (A) and IL-6 (B) in classical (CD14⁺⁺), intermediate (CD14⁺⁺CD16⁺) and non-classical (CD14⁺CD16⁺) monocytes from young and elderly donors in response to LPS (50 ng/ml) and <i>S</i>. <i>pneumoniae (5 x10</i>^<i>6</i><i>CFU)</i>. C) The secretion of TNF and D) IL-6 for isolated CD14+ monocytes in response to LPS for young and older donors. E) The frequency of intermediate monocytes were found to have a significant, positive correlation with the levels of serum TNF (β = 2.78, p<0.006). (A-D) is representative of ± SEM of n = 7 young donors (26–52 yrs) and n = 6 older donors (63–70 yrs) *indicates p<0.05, and ** indicates p< 0.05. Intermediate monocyte (CD14++CD16+HLA-DR+) count (cells per microlitre of whole blood) increases relative to serum levels of TNF in older donors (n = 94, 61-100yrs).</p

Old mice have increased numbers of circulating and recruited Ly6C^high monocytes during the course of <i>S</i>. <i>pneumoniae</i> colonization.

<p>(A) Colony forming units (CFUs) in nasal lavages from young and old WT mice were quantified on days 3, 7, 14 and 21 following intranasal colonization with <i>S</i>. <i>pneumoniae</i> (± SEM; <i>n</i> = 5–22). (B) CFUs of <i>S</i>. <i>pneumoniae</i> in the lungs at day 3 following intranasal colonization (± SEM; <i>n</i> = 9–22). (C) Survival of young and old mice after intranasal <i>S</i>. <i>pneumoniae</i> colonization (± SEM; <i>n</i> = 12). (D) Total serum CCL2 in young and old mice following intranasal <i>S</i>. <i>pneumoniae</i> colonization was measured by a high sensitivity ELISA. The data represent the mean (± SEM) of 3 mice per time point. (E) Ly6C^high monocytes as a percent of CD45⁺ cells in the blood of young and old WT mice during nasopharyngeal <i>S</i>. <i>pneumoniae</i> colonization (± SEM; <i>n</i> = 5–8) was measured by flow cytometry. (F) Mean expression of F4/80 on Ly6C^high monocytes in the blood of old mice during <i>S</i>. <i>pneumoniae</i> colonization is decreased as compared to young mice. (G) Levels of CCL2 transcript in the nasopharynx during the course of <i>S</i>. <i>pneumoniae</i> colonization were measured by quantitative PCR. (± SEM; <i>n</i> = 3). (H-I) Total numbers of (H) Ly6C^high monocytes and (I) macrophages detected by flow cytometry in the nasopharnyx of young and old mice during <i>S</i>. <i>pneumoniae</i> colonization (± SEM; <i>n</i> = 3–8). (J) Mean F4/80 expression on nasopharyngeal macrophages is lower in old mice during <i>S</i>. <i>pneumoniae</i> colonization (± SEM; <i>n</i> = 3–8). Statistical significance was determined by two-tailed Mann-Whitney-Wilcoxon test, one-way or two-way ANOVA with Fisher's LSD post-test. (K) Circulating blood monocytes from old mice bind fewer TRITC-labelled <i>S</i>. <i>pneumoniae</i> (4°C) but there is no difference in internalization of the bacteria (37°C). Survival in (C) was determined by the Mantel-Cox Log-rank test. * indicates <i>p</i> < .05, ** indicates <i>p</i> < 0.005, *** indicates <i>p</i> < 0.0005 and **** indicates p < 0.00005. (A-J) is representative of 3 independent experiments.</p

Anti-TNF therapy can reverse the age-associated increase in circulating Ly6C^high monocytes.

<p>(A-B) Young mice were give 200 ng/ml of TNF intraperitoneally every other day for 3 weeks. Numbers of circulating Ly6C^high and Ly6C^low monocytes (A) and serum IL6 (B) were quantitated. The data represent the mean (± SEM) of 5 mice. (C) Young and old WT mice were treated for 3 weeks with a neutralizing TNF antibody or IgG control and total numbers of circulating Ly6C^high monocytes were quantitated by flow cytometry. The data represent the mean (± SEM) of 4 mice. (D) The mean CCR2 expression on circulating Ly6C^high monocytes in young and old mice treated with either anti-TNF or IgG was quantitated and found to be reduced with anti-TNF treatment (<i>n</i> = 4). (E) Intracellular staining of IL6 and TNF on blood monocytes after a 4 hour stimulation with LPS from young and old WT mice treated with either anti-TNF or IgG demonstrates that the number of monocytes that stain positive for IL6 or TNF are decreased with anti-TNF therapy(± SEM; <i>n</i> = of 4). (F) Serum IL6 is reduced in old mice treated with anti-TNF but not the IgG control. (G) IL6 production in whole blood following stimulation with LPS or a vehicle control after 24 hours from young and old WT mice given either anti-TNF or IgG (± SEM; <i>n</i> = 4). Statistical significance was determined by two-tailed Mann-Whitney-Wilcoxon test, one-way or two-way ANOVA with Fisher's LSD post-test where appropriate. * indicates <i>p</i> < .05, ** indicates <i>p</i> < 0.005, *** indicates <i>p</i> < 0.0005 and **** indicates <i>p</i> < 0.00005. (A-G) are representative of 1 experiment with n = 4 mice.</p