Aged mice are unable to mount an effective myeloid response to sepsis

Abstract

The elderly have increased morbidity and mortality following sepsis; however, the cause(s) remains unclear. We hypothesized that these poor outcomes are due in part to defects in innate immunity, rather than to an exaggerated early inflammatory response. Young (6-12 wk) or aged (20-24 mo) mice underwent polymicrobial sepsis, and subsequently, the aged mice had increased mortality and defective peritoneal bacterial clearance compared with young mice. No differences were found in the magnitude of the plasma cytokine responses. Although septic aged mice displayed equivalent or increased numbers of circulating, splenic, and bone marrow myeloid cells, some of these cells exhibited decreased phagocytosis, reactive oxygen species production, and chemotaxis. Blood leukocyte gene expression was less altered in aged versus young mice 1 d after sepsis. Aged mice had a relative inability to upregulate gene expression of pathways related to neutrophil-mediated protective immunity, chemokine/chemokine receptor binding, and responses to exogenous molecules. Expression of most MHC genes remained more downregulated in aged mice at day 3. Despite their increased myeloid response to sepsis, the increased susceptibility of aged mice to sepsis appears not to be due to an exaggerated inflammatory response, but rather, a failure to mount an effective innate immune response.

Figure 1. Aged mice have increased mortality and defective bacterial clearance after septic insult

(A) Young (6–10 weeks; n=10) and old (20–24 months; n=10) B6 mice underwent CLP using 25-gauge needle and survival was monitored for seven days. Differences in survival were calculated using Log-rank (Mantel-Cox) test (*p=0.02). (B) Young (n=9) and old (n=9) mice underwent CLP and were sacrificed one day later. Peritoneal lavage was performed under aseptic technique. Bacterial colonies were determined from serial dilutions of peritoneal lavage fluid (**p=0.004, Mann Whitney test). Data shown were from two or more independent experiments.

Figure 2. Aged mice do not have significantly increased plasma cytokine and chemokine concentrations after sepsis

Blood was collected from young and old mice one and three days after CLP using heparinized syringe by intra-cardiac puncture. Blood from naïve mice served as controls. Plasma cytokine levels were measured using multiplex Luminex kit (***p<0.001 by 2-way ANOVA). Data shown were from two or more independent experiments.

Figure 3. After sepsis, aged mice have relative and absolute increased numbers of myeloid cells

Blood, spleen, and BM were collected from young and old mice one day and three days after CLP. Myeloid cells were analyzed by flow cytometry using anti-CD11b, anti-Ly6G, anti-CD11c, and anti-Gr-1 (*p<0.05, **p<0.01, ***p<0.001 by two-way ANOVA). Data shown were from two or more independent experiments.

Figure 4. Myeloid cells from aged mice have decreased functional capacity

Blood, spleen, BM, and peritoneal cells were collected from young and old B6 mice one day after CLP. (A) ROS production was measured by the mean fluorescence intensity (MFI) of DHR 123 after PMA stimulation from PMNs (CD11b⁺Ly6G⁺) and MOs (CD11b⁺Ly6G⁻) cells. (B) Spleen and BM cells were incubated with FITC latex beads and stained for PMNs and MOs. FITC⁺ cells were considered phagocytic. (C) Migration of blood, BM, and peritoneal PMNs to KC (30 ng/ml) and MOs to MCP-1 (30 ng/ml) were determined after two hours incubation. Migration index was calculated by dividing the number of cells that migrated toward MCP-1 or KC by the number of cells that migrated to medium alone. (*p<0.05, **p<0.01 by t-test or two way ANOVA). Data shown were from two or more independent experiments.

Figure 5. Murine hematopoietic cell numbers and function from the elderly after sepsis are different as compared to younger mice

One day after CLP, (A) BM from young and aged mice were analyzed for LSK (lin⁻sca-1⁺ckit⁺), LT-HSCs (CD150⁺CD135⁻LSK), and ST-HSCs (CD150⁻CD135⁺LSK) (B) BM LSKs from young and aged mice were sorted and cultured in methylcellulose media with indicated cytokines. Colonies were counted 10–14 days later. (*p<0.05, **p<0.01, ***p<0.001 by paired t-test or two way ANOVA). The data shown were obtained from 3–6 mice per group from at least three independent experiments.

Figure 6. The genomic response of aged leukocytes to sepsis is inadequate as compared to young mice

(A) Blood from young and aged mice were collected one day after CLP and analyzed for comprehensive CBC. Percentage of leukocyte subsets is shown. (B)The genomic response of total circulating leukocytes of young and aged mice that were sacrificed at two hours, one and three days after CLP. A distance from reference (DFR) calculated for 28,464 significant probe sets (p<0.001) that differentiated the genomic expression of the various groups. DFR calculations illustrate that the genomic response of old mice to CLP at day one is significantly greater to that of young mice (p<0.05) indicating leukocytes from the elderly are incapable of mounting an appropriate response to polymicrobial sepsis. Subsequently, aged mice continue to increase their genomic abnormalities while young mice trend toward returning to baseline at day three. Data shown were from three or more independent experiments.

Figure 7. Gene Ontology heat maps of neutrophil chemotaxis, chemokine binding, and response to exogenous dsRNA, with concordant DFRs at 24hours

Aged mice significantly differ in their upregulation of these pathways from young mice. Pathways important to innate immunity have significantly less genomic upregulation 24 hours after CLP in aged mice as compared to juvenile rodents.