Aging leads to dysfunctional innate immune responses to TLR2 and TLR4 agonists

Abstract

Background: Sepsis is more common in the elderly. TNF⍺ is recognized as an important mediator in sepsis and Toll-like receptors (TLRs) play an important role in initiating signaling cascades to produce TNF⍺. Little is known about how innate immunity is altered in healthy human aging that predisposes to sepsis.

Aims and methods: We tested the hypothesis that aging dysregulates the innate immune response to TLR 2 and 4 ligands. We performed whole blood assays on 554 healthy subjects aged 40-80 years. TNFα production was measured at baseline and after stimulation with the TLR2 agonists: peptidoglycan, lipoteichoic acid, Pam3CysK, Zymosan A and the TLR4 agonist lipopolysaccharide (LPS). In a subset of subjects (n = 250), we measured Toll-like receptor (TLR) 2, 4 and MyD88 expression using real-time PCR.

Results and discussion: We measured a 2.5% increase per year in basal secretion of TNFα with aging (n = 554 p = 0.02). Likewise, TNFα secretion was increased with aging after stimulation with peptidoglycan (1.3% increase/year; p = 0.0005) and zymosan A (1.1% increase/year p = 0.03). We also examined the difference between baseline and stimulated TNFα for each individual. We found that the increase was driven by the elevated baseline levels. In fact, there was a diminished stimulated response to LPS (1.9% decrease/year; p = 0.05), lipoteichoic acid (2.1% decrease/year p = 0.03), and Pam3CysK (2.6% decrease/year p = 0.0007). There were no differences in TLR or MyD88 mRNA expression with aging, however, there was an inverse relationship between TLR expression and stimulated TNFα production.

Conclusions: With aging, circulating leukocytes produce high levels of TNFα at baseline and have inadequate responses to TLR2 and TLR4 agonists. These defects likely contribute to the increased susceptibility to sepsis in older adults.

Keywords: Aging; Immunosenescence; Inflammaging; Innate immunity; Sepsis; Shock; TLR signaling; TNFα.

Figure 1:. TLR2 mRNA expression inversely correlates with stimulated TNFα production.

TLR2 mRNA was measured from unstimulated whole blood. TLR2 was normalized to the housekeeping gene 18s RNA using the 2^-ΔCT method. TNFα is measured after stimulation with Lipotechoic acid (20 μg/ml), Peptidoglycan (10 μg/ml), Zymosan (10 μg/ml), and Pam3CysK (10 μg/ml) for 24 hours. TNFα (Logged) is reported as pg/mL/WBC. The center line is the line of best fit of a simple linear regression and the dashed lines are the 95% confidence intervals. The reported p-value is whether the slope of the line is significantly non-zero.

Figure 2:. TLR4 mRNA expression inversely correlates with TNFα production.

TLR4 mRNA was measured from unstimulated whole blood. TLR4 was normalized to the housekeeping gene 18s RNA using the 2^-ΔCT method. TNFα is measured after stimulation with lipopolysaccharide (LPS) (1 ng/ml) for 24 hours. TNFα (Logged) is reported as pg/mL/WBC. The center line is the line of best fit of a simple linear regression and the dashed lines are the 95% confidence intervals. The reported p-value is whether the slope of the line is significantly non-zero.