Age-dependent changes in innate immune phenotype and function in rhesus macaques (Macaca mulatta)

Abstract

Aged individuals are more susceptible to infections due to a general decline in immune function broadly referred to as immune senescence. While age-related changes in the adaptive immune system are well documented, aging of the innate immune system remains less well understood, particularly in nonhuman primates. A more robust understanding of age-related changes in innate immune function would provide mechanistic insight into the increased susceptibility of the elderly to infection. Rhesus macaques have proved a critical translational model for aging research, and present a unique opportunity to dissect age-dependent modulation of the innate immune system. We examined age-related changes in: (i) innate immune cell frequencies; (ii) expression of pattern recognition receptors (PRRs) and innate signaling molecules; (iii) cytokine responses of monocytes and dendritic cells (DC) following stimulation with PRR agonists; and (iv) plasma cytokine levels in this model. We found marked changes in both the phenotype and function of innate immune cells. This included an age-associated increased frequency of myeloid DC (mDC). Moreover, we found toll-like receptor (TLR) agonists lipopolysaccharide (TLR4), fibroblast stimulating ligand-1 (TLR2/6), and ODN2006 (TLR7/9) induced reduced cytokine responses in aged mDC. Interestingly, with the exception of the monocyte-derived TNFα response to LPS, which increased with age, TNFα, IL-6, and IFNα responses declined with age. We also found that TLR4, TLR5, and innate negative regulator, sterile alpha and TIR motif containing protein (SARM), were all expressed at lower levels in young animals. By contrast, absent in melanoma 2 and retinoic acid-inducible gene I expression was lowest in aged animals. Together, these observations indicate that several parameters of innate immunity are significantly modulated by age and contribute to differential immune function in aged macaques.

Keywords: immune senscence; innate immunity; myeloid; nonhuman primate; pattern recognition receptor.

Fig. 1

T cell frequencies in young, adult, and aged macaque. Young (≤4 yrs, n=69), adult (5–18 yrs, n=163), and aged (≥19 yrs, n=85) PBMC were stained to determine the frequency of naïve (A,B), central memory (C,D), and effector memory (E,F) CD4 or CD8 T cells. The total frequency of CD4 (G) and CD8 (H) T cells is also shown, as well as CD/CD8 ratios (I). Correlations between age and cell frequencies/ratios were determined by Spearman's rank correlation.

Fig. 2

Innate immune cell frequencies in young, adult, and aged macaque. (A) Flow cytometric gating strategy to identify monocytes and dendritic cell (DC) populations. (B–D) The frequency of nonlymph (CD3^-CD20^-), monocytes and DCs (as frequencies of nonlymph cells) was determined in young (≤4 yrs, n=47), adult (5–18 yrs, n=119), and aged (≥19 yrs, n=43) animals. The frequency of myeloid DC (E), plasmacytoid DC (F), and ‘other DC’ (G) amongst total DC (CD14^-HLA-DR⁺ cells) is also shown. *p≤0.05 (as compared to adults).

Fig. 3

Toll-Like Receptor expression in young, adult, and aged macaque PBMC. Expression levels of TLR1-9 relative to housekeeping gene MGSS were determined by qRT-PCR using primers and probes designed for Macaca mulatta. Young (1–4 yrs), adult (8–14yrs), and aged (≥19 yrs) cohorts are shown. Bars graphs represent group mean±SEM. n=17–28/age group. *p≤0.05.

Fig. 4

NLR/RLR/signaling adaptor expression in young, adult, and aged macaque PBMC. Expression levels of RIG-I, MDA5, DAI, IFI16, AIM2, NLRP3, MyD88, MAVS, and STING relative to housekeeping gene MGSS were determined by qRT-PCR using primers and probes designed for Macaca mulatta. Young (1–4 yrs), adult (8–14 yrs), and aged (≥19 yrs) cohorts are shown. Bars graphs represent group mean±SEM. n=12–18/age group. *p≤0.05.

Fig. 5

Innate negative regulator expression in macaque PBMC Expression levels of innate negative regulators Tollip, IRAK-M, NLRX1, ATF3, SARM1, and RBCK relative to housekeeping gene MGSS were determined by qRT-PCR using primers and probes designed for Macaca mulatta. Young (1–4 yrs), adult (8–14 yrs), and aged (≥19 yrs) cohorts are shown. Bars graphs represent group mean±SEM. n=12–18/age group. *p≤0.05; ***p≤0.001.

Fig. 6

Age-dependent innate TLR responses in macaque monocytes and myeloid DC. Two million PBMC were stimulated for 6 h in the presence of brefeldin A with the following TLR agonists: TLR2/6 (FSL-1 – 1 µg/ml); TLR4 (LPS – 20 ng/ml); and TLR9 (ODN2006 – 10 µM). Cells were stained to distinguish monocytes and mDC and stained intracellularly for cytokines. Representative flow cytometry is shown for unstimulated and LPS-stimulated monocytes (A). Frequency of TNF+ or IL-6+ responding monocytes (B,C) and mDC (D,E) is shown for each agonist. Bar graphs represent mean frequency of cytokine producing cells ±SEM (adjusted for background using unstimulated controls). n=12–15 animals/age group. *p≤0.05; **p≤0.01; ***p≤0.001.

Fig. 7

Age-dependent innate TLR responses in macaque DC subpopulations. Two million PBMC were stimulated for 6 h in the presence of brefeldin A with agonists for the following TLR: TLR2/6 (FSL-1 – 1 µg/ml); TLR4 (LPS – 20 ng/ml); TLR7/8 (Imiquimod − 10 µg/ml); and TLR9 (ODN2006 − 10 µM). Cells were stained to distinguish CD11c-CD123-DC (‘other’ DC) and pDC and stained intracellularly for cytokines. Frequency of TNF+ or IL-6+ responding ‘other DC’ (A,B) and pDC (C,D) is shown for the agonists described. Bar graphs represent mean frequency of cytokine producing cells ±SEM (adjusted for background using unstimulated controls). n=12–15 animals/age group. *p≤0.05; ***p≤0.001.

Fig. 8

IFNα responses in young, adult, and aged macaque plamacytoid DC. Two million PBMC were stimulated with Imiquimod (TLR7 agonist; 10 µg/ml) for 6 h in the presence of brefeldin A. Frequency of IFNα responding plasmacytoid DC was subsequently measured by FCS (A), with representative flow cytometry also shown (B). Bar graphs represent mean frequency of cytokine producing cells ±SEM (adjusted for background using unstimulated controls). n=12–15 animals/age group.

Fig. 9

Age-dependent circulating cytokine levels in rhesus macaque. Serum cytokine levels were determine by cytometric bead assay (luminex) in young (n=34), adult (n=35), and aged (n=37) rhesus macaques. Graphs represent mean±SEM. *p<0.05. nd, Not detected.