Age-associated decrease in TLR function in primary human dendritic cells predicts influenza vaccine response

Abstract

We evaluated TLR function in primary human dendritic cells (DCs) from 104 young (age 21-30 y) and older (> or =65 y) individuals. We used multicolor flow cytometry and intracellular cytokine staining of myeloid DCs (mDCs) and plasmacytoid DCs (pDCs) and found substantial decreases in older compared with young individuals in TNF-alpha, IL-6, and/or IL-12 (p40) production in mDCs and in TNF-alpha and IFN-alpha production in pDCs in response to TLR1/2, TLR2/6, TLR3, TLR5, and TLR8 engagement in mDCs and TLR7 and TLR9 in pDCs. These differences were highly significant after adjustment for heterogeneity between young and older groups (e.g., gender, race, body mass index, number of comorbid medical conditions) using mixed-effect statistical modeling. Studies of surface and intracellular expression of TLR proteins and of TLR gene expression in purified mDCs and pDCs revealed potential contributions for both transcriptional and posttranscriptional mechanisms in these age-associated effects. Moreover, intracellular cytokine production in the absence of TLR ligand stimulation was elevated in cells from older compared with young individuals, suggesting a dysregulation of cytokine production that may limit further activation by TLR engagement. Our results provide evidence for immunosenescence in DCs; notably, defects in cytokine production were strongly associated with poor Ab response to influenza immunization, a functional consequence of impaired TLR function in the aging innate immune response.

Figure 1

Representative example of identification of mDC and pDC populations from peripheral blood. Lin⁻ HLADR⁺ cells were further fractionated into CD11c⁺ (mDC) or CD123⁺ (pDC) populations. In this example, following treatment with R848, production of TNF-α and IL-6 are evaluated using intracellular cytokine staining, gating in this case on mDCs.

Figure 2

Age-associated alteration in Toll-like receptor (TLR)–induced cytokine production in mDCs and pDCs in older, compared to young adults. A. Age-associated defects (n=50 young, n=54 older) in IL-12p40, IL-6 and TNF-α production in mDCs (as measured by intracellular cytokine staining) following stimulation with indicated TLR ligands B. Age-associated defects in IFN-α and TNF-α production in pDCs (n=50 young, n=54 older) following stimulation with R848 and CpG. Stable defects in TLR8-induced cytokine production in mDCs (C) and in TLR7-induced cytokine production in pDCs (D), assessed both with R848 and TLR7- or TLR8-specific agonists 4–6 weeks after studies in A and B (n=36 young, n=34 older). Each plot depicts the mean ± SEM percent positive difference in intracellular cytokine production between samples stimulated with the indicated TLR ligand and unstimulated samples.; P values after adjustment for covariates are indicated: *< 0.0001, ^#<0.01, ^$<0.05, ^&<NS).

Figure 3

Boolean gating of DC subpopulations. (A) the frequency of mDCs expressing each of the seven possible combinations of IL-6, TNF-α, and IL-12 (p40) after stimulation with the TLR7/8 ligand R848; (B) the frequency of pDCs expressing each of the three possible combinations of IFN-α and TNF-α after stimulation with R848.

Figure 4

Basal cytokine levels in older, compared to young adults. A. Baseline IL-12 (p40,) IL-6 and TNF-α production in mDCs (as measured by intracellular cytokine staining) and IFN-α and TNF-α production in pDCs (n=50 young, n=54 older). The mean ± SEM percent positive mDCs/pDCs for cyokine production in the absence of TLR stimulation are depicted (in some cases because of low variability in the samples, error bars are too narrow to be distinguished). P values after adjustment for covariates are indicated: *< 0.0001. B and C. Representative examples of baseline cytokine production, depicted here baseline TNF-α and IL-6 production in mDCs from 6 young and 6 older adults following R848 treatment.

Figure 5

Age-associated alteration in Toll-like receptor (TLR)–induced cytokine production in purified DCs isolated using magnetic bead sorting in 6 older, compared to 7 young adults two years after the original study. A. Age-associated decrease (n=13) in IL-12 (p40,) IL-6 and TNF-α production in mDCs (as measured by intracellular cytokine staining) following stimulation with indicated TLR ligands. B. Age-associated decrease in IFN-α and TNF-α production in pDCs (n=13) following stimulation with R848 and CpG. The mean ± SEM percent positive difference in intracellular cytokine production between samples stimulated with the indicated TLR ligand and unstimulated samples are depicted; P values after adjustment for covariates are indicated: #<0.01, $<0.05. C and D. Representative examples of TLR ligand induced cytokine production, depicted here TNF-α and IL-6 production in 6 young (Panel A) and 6 older adults (Panel B) after stimulation with TLR7/8 ligand R848.

Figure 6

Age-associated decrease in TLR protein expression. Shown are baseline percent positive mDCs for TLR 1, TLR 2, TLR3 and TLR8 expression and pDCs for TLR9 expression (compared with isotype control). Values indicate the mean ± SEM (because of the low amount of variability in these samples, in some cases error bars are too narrow to be distinguished) of the young adults (n = 24) and older adults (n = 24). P values after adjustment for covariates: *< 0.0001, ^$<0.05, ^&<NS.

Figure 7

Effect of aging on expression of TLRs in human mDC and pDC. The data display mRNA expression levels of indicated TLRs in purified mDCs (A), and pDCs (B) from 40 individuals (Young, N=20; Older, N=20). mRNA levels were quantified by Q-PCR and normalized to β-actin. The median values and interquartile range are indicated . $ indicates statistical significance between young and older cohort (Mann Whitney, p < 0.05).

Figure 8

Responses to influenza vaccination in older adults and young adults. A. Pre-and postvaccine titers showing a ≥ 4-fold increase to each of the influenza strains in the 2007–2008 vaccine (A/Solomon Islands/3/2006-like [H1N1], A/Wisconsin/67/2005-like [H3N2], and B/Malaysia/2506/2004-like [B]) are shown. B. The proportion in each age group with a ≥ 4-fold increase in titer to none, 1, 2, or all of the vaccine strains. C. Pre-and postvaccine hemagglutination inhibition (HI) titers ≥ 1:64. D. The proportion in each age group with a postvaccination titer ≥ 1:64 to none, 1, 2, or all of the vaccine strains. For seroprotection, we excluded members of the cohort who had pre-vaccine titers of ≥ 1:64, who therefore already met criteria for protection prior to vaccination. Consequently, the analysis of seroprotection included n=32 young and n=49 older for the H1N1 vaccine strain; n=47 young and n=30 older for H3N2; and n=44 young and n=34 older for the B strain. P values after adjustment for covariates: *< 0.0001, #<0.01 for difference in distribution in panels A and C.

Figure 9

Prediction of antibody responses to the 2007–2008 influenza vaccine by TLR-induced cytokine production in mDCs and pDCs. Data depict the least squares mean of TLR–induced cytokine production. A. Association of seroconversion to vaccine strains indicated with TLR-induced production of p40 subunit of IL12, IL6, and TNF-α in mDCs. P values after adjustment for covariates. B. Relation of seroconversion to indicated vaccine strains with TLR-induced IFN-α and TNF-α production in pDCs. P values after adjustment for covariates: C. Relation of seroprotection to indicated vaccine strains TLR-induced production of p40 subunit of IL12, IL6, and TNF-α in mDCs. D. Relation of seroprotection to indicated vaccine strains with TLR-induced IFN-α and TNF-α production in pDCs. *< 0.0001, ^#<0.001, ^&<NS.