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. 2015 Jun;14(3):421-32.
doi: 10.1111/acel.12320. Epub 2015 Feb 27.

Global analyses revealed age-related alterations in innate immune responses after stimulation of pathogen recognition receptors

Talibah U Metcalf  1 , Rafael A Cubas, Khader Ghneim, Michael J Cartwright, Julien Van Grevenynghe, Justin M Richner, David P Olagnier, Peter A Wilkinson, Mark J Cameron, Byung S Park, John B Hiscott, Michael S Diamond, Anne M Wertheimer, Janko Nikolich-Zugich, Elias K Haddad
Affiliations

Global analyses revealed age-related alterations in innate immune responses after stimulation of pathogen recognition receptors

Talibah U Metcalf et al. Aging Cell. 2015 Jun.

Abstract

Aging leads to dysregulation of multiple components of the immune system that results in increased susceptibility to infections and poor response to vaccines in the aging population. The dysfunctions of adaptive B and T cells are well documented, but the effect of aging on innate immunity remains incompletely understood. Using a heterogeneous population of peripheral blood mononuclear cells (PBMCs), we first undertook transcriptional profiling and found that PBMCs isolated from old individuals (≥ 65 years) exhibited a delayed and altered response to stimulation with TLR4, TLR7/8, and RIG-I agonists compared to cells obtained from adults (≤ 40 years). This delayed response to innate immune agonists resulted in the reduced production of pro-inflammatory and antiviral cytokines and chemokines including TNFα, IL-6, IL-1β, IFNα, IFNγ, CCL2, and CCL7. While the major monocyte and dendritic cell subsets did not change numerically with aging, activation of specific cell types was altered. PBMCs from old subjects also had a lower frequency of CD40+ monocytes, impaired up-regulation of PD-L1 on monocytes and T cells, and increased expression of PD-L2 and B7-H4 on B cells. The defective immune response to innate agonists adversely affected adaptive immunity as TLR-stimulated PBMCs (minus CD3 T cells) from old subjects elicited significantly lower levels of adult T-cell proliferation than those from adult subjects in an allogeneic mixed lymphocyte reaction (MLR). Collectively, these age-associated changes in cytokine, chemokine and interferon production, as well as co-stimulatory protein expression could contribute to the blunted memory B- and T-cell immune responses to vaccines and infections.

Keywords: immunosenescence; innate immune agonists; innate immunity; interferon signaling; pattern recognition receptors; peripheral blood mononuclear cells.

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Figures

Fig 1
Fig 1
Single-gene analysis shows unique age-related differences in immune responses to PRR agonists. Agonist-treated cells (LPS, CLO97, poly I:C/Lyovec, or 5′-pppRNA/Lyovec) from adult and old subjects (n = 8/group) were normalized to their respective negative controls (untreated or Lyovec only treated). Gene array analysis was performed using an Illumina platform, and differentially expressed genes (DEGs) were selected to have a FDR 5% with FC ≥ 1.3 or ≤ −1.3. (A) Histograms depict the kinetics of expression and number of DEGs for agonist-treated adult and old subjects for 6 and 24 h. (B) Venn diagram analysis of agonist-treated adult (A) compared to treated old (O) subjects at 6 (top row) and 24 h (bottom row). Diagrams show the number of unique and common statistically significant genes based on the above criteria. Listed above and below each diagram are cytokines and chemokines that are uniquely elicited by each agonist at 6 and 24 h, respectively. Upregulated and downregulated genes in respect to negative controls are depicted by up- and downward arrows, respectively.
Fig 2
Fig 2
Adults show a faster and greater enrichment of functional pathways relative to old subjects. (A) Transcriptional profiles for adult and old individuals (n = 8/group) generated by comparing agonist-treated PBMCs (LPS, CLO97, poly I:C/Lyovec, or 5′-pppRNA/Lyovec) to negative controls (untreated or Lyovec only treated) were analyzed by IPA canonical pathway database. Genes undergoing pathway analysis passed nominal P < 0.05 and FC ≥ 1.3 or ≤ −1.3 statistical cutoffs. Heat map depicts enrichment of a custom selected list of canonical pathways (y-axis) at different contrasts (x-axis). Pathways are considered statistically significant (P < 0.05) if they pass a threshold score > 0.15 (indicating 15% gene enrichment) based on a Fisher's test. Scale ranges from blue (pathway not significantly enriched, score < 0.15) to orange (significantly enriched pathway). (B) Table shows enrichment scores and P values of selected pathways elicited by LPS, CLO97, and 5′-pppRNA/Lyovec compared to negative controls, which shows the delay response at 6 h in old subjects.
Fig 3
Fig 3
Agonist stimulation induced a monocyte cell signature in adults compared to old counterparts. Transcriptional profiles generated from comparing adult stimulated PBMCs to old stimulated PBMCs (n = 8/group) were analyzed using cell type-specific modules. The radial plots illustrate the enrichment of genes associated with T and B cells, natural killer (NK), plasmacytoid (p), and myeloid (m) DCs, and monocyte subsets for a given treatment (LPS, CLO97, poly I:C/Lyovec, or 5′-pppRNA/Lyovec) at 6 (A) and 24 h (B), by plotting the Normalized Enrichment Score (NES) calculated by GSEA that takes into account the FDR using the Benjamini and Hochberg method (Subramanian et al., 2005). Gene sets induced in a specific cell type that was significantly enriched in adults or old subjects are denoted by asterisks: * for P value < 0.05. A positive enrichment score (+NES; radially outward from 0) represents upregulation in adults, whereas a negative score (−NES; inward from 0 to center) represents upregulation in old subjects.
Fig 4
Fig 4
Age-related changes in surface expression of co-stimulatory molecules. (A) PBMCs were stained for CD3+ T cells, CD19+ B cells, CD14+ monocytes, and either CTLA, PD-L2, or CD40. CTLA and PD-L2 (n = 21/group) and CD40 (n = 12/group). Individual values along with mean ± SEM are shown. P values were determined by comparing adult to old subjects using the Mann–Whitney U-test. (B) PBMCs isolated from adult and old subjects (n = 8/group) were stimulated with CLO97 for 24 h. PBMCs were stained for CD3+ (T cells), CD19+ (B cells), CD14+ (monocytes), and either PD-L1, PD-L2, or B7-H4. MFI values along with mean ± SEM are shown. Statistical significance between adult (A) and old (O) subjects was determined by two-way ANOVA followed by Sidak correction for multiple comparisons. For untreated vs. treated, P values are not shown for significant upregulation: PD-L1 on monocytes and B cells from both age groups (P < 0.001) and on T cells from A (P < 0.001) and O (P < 0.01); PD-L2 on monocytes from A (P < 0.001) and O (P < 0.01) and B cells from A (P < 0.01) and O (P < 0.001); B7-H4 on B cells from O (P < 0.05).
Fig 5
Fig 5
PBMCs from adults produced higher levels of inflammatory cytokines, IFNs, and chemokines in response to PRR agonists. (A) Graphs depict the means ± SEM of IFNα, TNFα, and CCL2 concentrations in response to stimulation with CLO97 and LPS for 6 and 24 h for adult and old subjects. TNFα and CCL2 were measured by CBA, whereas IFNα was measured by ELISA. (B-D) Table depicts the means ± SEM for cytokines and chemokines measured by Milliplex that showed age-related alternations after stimulation with (B) CLO97, (C) LPS, and (D) 5′-pppRNA/Lyovec for 24 h. Asterisks indicate statistical significance (***) P < 0.001, (**) P < 0.01, and (*) P < 0.05) between treated PBMCs vs. negative controls and adult vs. old subjects using two-way ANOVA followed by Sidak correction for multiple comparisons. n = 8/group.
Fig 6
Fig 6
PBMCs from adults induced higher levels of proliferation of allogeneic T cells. Allogeneic mixed lymphocyte reaction (MLR) using LPS- or CLO97-stimulated PBMCs (minus CD3 T cells) from adults and old donors (n = 10/group) cultured with pooled CFSE-labeled CD3+ T cells from adults (n = 2). Graphs depict the frequency of dividing CD3 T cells (CFSE low) after day 3 (D3) (A) and day 5 (D5) (B) of culturing. Individual values along with mean are shown. Frequencies > 1 (baseline) were considered significant, indicating a gating of > 20 events. For D3, the percentage of donors above baseline (dash line) is listed in table below graph. Statistical significance between adults and old subjects was determined by Mann–Whitney U-test.
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References

    1. Abb J, Abb H, Deinhardt F. Age-related decline of human interferon alpha and interferon gamma production. Blut. 1984;48:285–289. - PubMed
    1. Alberti S, Cevenini E, Ostan R, Capri M, Salvioli S, Bucci L, Ginaldi L, De Martinis M, Franceschi C, Monti D. Age-dependent modifications of Type 1 and Type 2 cytokines within virgin and memory CD4+ T cells in humans. Mech. Ageing Dev. 2006;127:560–566. - PubMed
    1. Alvarez-Rodriguez L, Lopez-Hoyos M, Munoz-Cacho P, Martinez-Taboada VM. Aging is associated with circulating cytokine dysregulation. Cell. Immunol. 2012;273:124–132. - PubMed
    1. Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, Davis AP, Dolinski K, Dwight SS, Eppig JT, Harris MA, Hill DP, Issel-Tarver L, Kasarskis A, Lewis S, Matese JC, Richardson JE, Ringwald M, Rubin GM, Sherlock G. Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat. Genet. 2000;25:25–29. - PMC - PubMed
    1. Benjamini Y, Hochberg Y. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J. R. Stat. Soc. Series B Stat. Methodol. 1995;57:289–300.

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