Reduced bioenergetics and toll-like receptor 1 function in human polymorphonuclear leukocytes in aging

Abstract

Aging is associated with a progressive decline in immune function (immunosenescence) resulting in an increased susceptibility to viral and bacterial infections. Here we show reduced expression of Toll-like receptor 1 (TLR1) in polymorphonuclear leukocytes (PMN) and an underlying age-dependent deficiency in PMN bioenergetics. In older (>65 years) adults, stimulation through TLR1 led to lower activation of integrins (CD11b and CD18), lower production of the chemokine IL-8, and lower levels of the phosphorylated signaling intermediate p38 MAP kinase than in PMN from younger donors (21-30 years). In addition, loss of CD62L, a marker of PMN activation, was reduced in PMN of older adults stimulated through multiple pathways. Rescue of PMN from apoptosis by stimulation with TLR1 was reduced in PMN from older adults. In seeking an explanation for effects of aging across multiple pathways, we examined PMN energy utilization and found that glucose uptake after stimulation through TLR1 was dramatically lower in PMN of older adults. Our results demonstrate a reduction in TLR1 expression and TLR1-mediated responses in PMN with aging, and reduced efficiency of bioenergetics in PMN. These changes likely contribute to reduced PMN efficiency in aging through multiple aspects of PMN function and suggest potential therapeutic opportunities.

Figure 1. Effect of aging on expression of TLRs in human PMN

Whole blood of younger (n=31) and older (n =22) adults was labeled for flow cytometry with lineage markers and TLRs at 4°C for 30 min following RBC lysis. Labeling was detected by LSR II. Data shown are % positive neutrophils for TLR surface expression. Values indicate the means ± SEM in young and older adults. Asterisks indicate statistical significance between younger and older cohort (Unadjusted t-test accounting for unequal variances, p < 0.02).

Figure 2. Age-associated alterations in PMN surface markers

Whole blood of younger (n=32) and older (n =35) adults was stimulated with indicated ligands for 0, 15 min at 37 °C. Following RBC lysis, PMN were labeled for PMN markers at 4°C for 30 min. Flow cytometric labeling was detected by LSR II. Data shown are normalized MFI (Mean Fluorescence Intensity) of CD11b, CD18 and CD62L surface expression on PMN. Values indicate the means ± SEM in younger and older adults; asterisks indicate statistical significance between younger and older cohort (*P<0.05, **P < 0.01). For normalized CD11b and CD18 all stimulated PMN increased over time (P<0.001) and for CD62L all stimulated PMN decreased over time (P<0.001).

Figure 3. Effect of aging on TLR-stimulated production of cytokines by PMN

In unstimulated vs TLR ligand-stimulated cells, the difference in the production of IL-8 in supernatants of PMN from younger (N=36) and older (N=34) adults. IL-8 was detected by ELISA after stimulation as shown. Values indicate the mean ± SEM concentration of IL-8 produced by PMN. Asterisks indicate statistical significance between younger and older cohorts (p < 0.05).

Figure 4. Pam3CSK4 stimulated neutrophils from the young show increased phosphorylation of p38 MAPK

(A) immunoblot of total p38 MAPK and active phosphorylated p38 in a representative result of neutrophils from younger and older adults after stimulation with the TLR1/2 ligand Pam3CSK4 for 15 min. (B) Densitometry of immunoblot of p38 and phospho-p38 in neutrophils from 21 pairs of younger and older subjects after stimulation with Pam3CSK4 for 15min. Densitometry shows the means ± SEM of the ratio of total p38 and phospho-p38 to β-actin, and of phospho-p38 to total p38. Asterisks indicate statistical significance between younger and older cohort (** p < 0.01).

Figure 5. Age-related alterations in TLR1-mediated stimulation of PMN

PMN from younger and older adults (10⁶/ml, n=10/group) were stimulated with TNF-α (20ng/ml), LPS (0.5μg/ml), or Pam3CSK4 (5μg/ml) in RPMI/10% human serum. Cells were incubated for 18 hr and labeled with Annexin V and PI for detection of apoptosis by flow cytometry (A). The level of fluorescent glucose (MFI of 2-NBDG) was quantified from aliquots of the same PMN by flow cytometry after 2 hr (B). Values indicate the mean ± SEM of apoptotic cells (A., ** P<0.04) and glucose level (B., * p< 0.04, ** p< 0.02) and asterisks indicate statistical significance between younger and older cohort.