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

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

Age-Dependent Cell Trafficking Defects in Draining Lymph Nodes Impair Adaptive Immunity and Control of West Nile Virus Infection

Impaired immune responses in the elderly lead to reduced vaccine efficacy and increased susceptibility to viral infections. Although several groups have documented age-dependent defects in adaptive immune priming, the deficits that occur prior to antigen encounter remain largely unexplored. Herein, we identify novel mechanisms for compromised adaptive immunity that occurs with aging in the context of infection with West Nile virus (WNV), an encephalitic flavivirus that preferentially causes disease in the elderly. An impaired IgM and IgG response and enhanced vulnerability to WNV infection during aging was linked to delayed germinal center formation in the draining lymph node (DLN). Adoptive transfer studies and two-photon intravital microscopy revealed a decreased trafficking capacity of donor naïve CD4+ T cells from old mice, which manifested as impaired T cell diapedesis at high endothelial venules and reduced cell motility within DLN prior to antigen encounter. Furthermore, leukocyte accumulation in the DLN within the first few days of WNV infection or antigen-adjuvant administration was diminished more generally in old mice and associated with a second aging-related defect in local cytokine and chemokine production. Thus, age-dependent cell-intrinsic and environmental defects in the DLN result in delayed immune cell recruitment and antigen recognition. These deficits compromise priming of early adaptive immune responses and likely contribute to the susceptibility of old animals to acute WNV infection

Decreased accumulation of CD4⁺ T cells and CD19⁺ B cells in DLN of old mice after immunization with ovalbumin.

<p><b>A-C</b>. Adult and old mice were immunized with ovalbumin (OVA) complexed with complete Freund’s adjuvant in the footpad. At the indicated days post infection, the draining popliteal LN was harvested, and cells were counted (<b>A</b>). Antibody staining detected specific lymphocyte populations including CD4⁺ T cells (<b>B</b>) and CD19⁺ B cells (<b>C</b>). The results were pooled from two independent experiments with a total of 5 mice per group and data is expressed as the mean ± the standard error of the mean (SEM). <b>D</b>. Numbers of IL-2 secreting CD4⁺ T cells as judged by ELISPOT assay following OVA_323–337 peptide stimulation in the DLN of adult and old mice at day 7 after immunization. <b>E-F.</b> Draining popliteal LN cells were analyzed by flow cytometry at 6 days after infection, and the numbers of GC B cells (CD19⁺ Fas⁺ GL7⁺) (<b>E</b>) and T_FH cells (CD4⁺ PD1⁺ CXCR5⁺) (<b>F</b>) were quantified. Each data point represents and individual mice and the results are pooled from two independent experiments. Asterisks indicate statistical significance (*, <i>P</i> < 0.05; **, <i>P</i> < 0.01; ***, <i>P</i> < 0.001; unpaired t test).</p

Humoral immune response of donor bone marrow into irradiated recipient mice.

<p><b>A.</b> Scheme of reconstitution studies with bone marrow from adult or old mice. 10⁷ bone marrow cells from adult or old mice were mixed with 10⁷ bone marrow cells from μMT (B cell deficient) mice and transferred into irradiated recipient CD45.1 mice. After 12 weeks, mice were infected with WNV (New York strain) and serum was harvested 5, 8, and 15 dpi. <b>B.</b> After 12 weeks post transfer, blood was sampled and tested for reconstitution efficiency. The vast majority (>95%) of circulating CD19⁺ B cells was derived from the CD45.2 donor mice. <b>C-D</b>. IgM (<b>C</b>) and IgG (<b>D</b>) levels were measured by ELISA for reactivity with WNV E protein. Data is plotted as the reciprocal log₁₀ titer and represents data from 5 mice per group.</p

Transfer of naïve CD4⁺ T cells from old mice results in reduced GC responses.

<p><b>A.</b> TCR β/δ^-/- recipient mice were infected with WNV (New York strain). Two days after infection, 3 x 10⁶ naïve sorted CD4⁺ T cells from adult or old mice were transferred adoptively into the recipient mice and 6 days post transfer (at 8 days post infection) spleens were analyzed for GC responses. Percentages of CD4⁺ T cells (<b>B</b>), T_FH cells (CD4⁺ PD1⁺ CXCR5⁺ cells) (<b>C</b>), and GC B cells (Fas⁺ GL7⁺ of CD19⁺ cells) (<b>D</b>) were quantified. The results are pooled from three independent experiments with a total of 11 to 12 mice per group, and each data symbol represents a single mouse. Asterisks indicate statistical significance (*, <i>P</i> < 0.05; ***, <i>P</i> < 0.001; unpaired t-test).</p

Defects in migration of naïve CD4⁺ T cells from old mice into inflamed LN.

<p><b>A.</b> Scheme of adoptive transfer studies. 2 x 10⁶ FACS-sorted naïve (CD44^- CD62L⁺) CD4⁺ T cells from adult or old mice were differentially labeled with fluorescent dyes, mixed in a 1:1 ratio, and transferred to recipient adult or old mice that had been infected with WNV-KUN 48 hours earlier via a subcutaneous route. <b>B-C</b>. One hour later, draining popliteal LN (<b>B</b>) and spleen (<b>C</b>) were harvested. Cells were counted and the frequency of adult and old donor cells was determined by flow cytometry. In each tissue, the data was normalized to the levels of adult donor cells in the adult recipient organ and is a composite of four independent experiments. Asterisks indicate statistical significance (*, <i>P</i> < 0.05; Mann-Whitney test). <b>D-E</b>. Time-lapse two-photon intravital imaging of diapedesis of labeled donor naïve CD4⁺ T cells (adult = green; old = blue) in the HEV from the DLN of a recipient WNV-KUN-infected mouse. Images are individual frames from a continuous time-lapse movie (see <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005027#ppat.1005027.s007" target="_blank">S1</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005027#ppat.1005027.s008" target="_blank">S2</a> Movies</b>). Relative time is displayed in min:sec. Panel <b>D</b> shows a picture of a Q-dot labeled vessel in a LN with multiple old (blue) and adult (green) CD4⁺ T cells adhering to the endothelium. The white dotted line is drawn to highlight the edge of the vessel lumen. The white box shows a zoomed in view of an extravasation "hot spot" that is then presented as a 4 time-lapse images in panel <b>E</b>. Two T cells (cells 1 and 2) from adult mice are shown undergoing diapedesis, whereas two representative T cells (cells 3 and 4) from old mice remain in the lumen over the same time frame. <b>F</b>. Cellular deformation of donor naïve CD4⁺ T cells in the HEV from the DLN of a recipient WNV-KUN-infected mouse as determined by analysis of two-photon microscopic images. <b>G.</b> Time-lapse two-photon imaging of movement of labeled donor naïve CD4⁺ T cells (adult = green; old = blue) in explanted DLN of a recipient WNV-KUN-infected mouse (see <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005027#ppat.1005027.s009" target="_blank">S3 Movie</a></b>). The figure presents representative cell tracking for adult and old CD4⁺ T cells followed by evaluation of displacement. White opaque dots represent cells with tracked paths (green or blue). Red arrow indicates cell displacement. Scale bar (white): 40 μm. <b>H-L.</b> Analysis of movement parameters of adult and old donor naïve CD4⁺ T cells in explanted LN 6 to 8 hours post-transfer to recipient mice infected with WNV-KUN 48 hours earlier. Individual cells were tracked and (<b>H</b>) speed (μM/min), (<b>I</b>) cell displacement factor (μm/min^1/2), (<b>J</b>) mean square cell displacement over time (μm²), (<b>K</b>) randomness of migration (Hotelling’s test of directionality) and (<b>L</b>) the predicted time of search efficiency for antigen. For panel <b>K,</b> the migration directionality was of a small magnitude, and no significant difference was observed between the cells from adult and old mice. For panel <b>L</b>, to judge the impact that the observed motility defects of old T cells would have on their ability to search antigen in DLN, we used a mathematical model [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005027#ppat.1005027.ref030" target="_blank">30</a>] to predict the time needed by adult and old T cells to first reach increasingly distant locations (first passage time). The first passage time is proportional to the motility coefficient, which we estimated from the mean square displacement data. The predicted time to reach a location 500 μm away is ~20 hours for adult T cells, but ~50 hours for old T cells. The data in <b>F</b>, <b>H and I</b> are shown as a scatter plot and reflects three independent experiments. Asterisks indicate statistical significance (***, <i>P</i> < 0.001, ****, <i>P</i> < 0.0001; Mann-Whitney test).</p

Survival rate, viral burden, and B cell responses after WNV infection.

<p><b>A</b>. Adult or old mice were infected with 10² PFU of WNV (New York strain) via subcutaneous injection in the footpad. Survival was monitored for 30 days: <i>n</i> = 20 adult (16 weeks) and <i>n</i> = 34 old (18 months) mice from at least three independent experiments. The difference was statistically different (<i>P</i> < 0.01; log rank test). <b>B-D.</b> Viral burden after WNV infection of adult and old mice was measured by qRT-PCR from serum (<b>B</b>) and plaque assay from spleen and brain (<b>C-D</b>). The data is shown as the mean titer (log₁₀ PFU/ml) ± standard deviation (SD) and reflects 8 to 9 mice per time point from at least two independent experiments. The dashed lines represent the limit of sensitivity of the assay. Asterisks indicate statistical significance (*, <i>P</i> < 0.05; **, <i>P</i> < 0.01; ***, <i>P</i> < 0.001; Mann-Whitney test). <b>E-H</b>. Serum was obtained from adult and old mice at days 5, 8, 15, and 90 after WNV infection. <b>E and H</b>. Neutralizing antibody titers were determined by a focus-reduction assay. Results are shown as the mean of the reciprocal log₁₀ titer ± SD that yields 50% inhibition of infection and reflects data from 5 to 11 mice per group from three independent experiments. <b>F-G</b>. Anti-WNV IgM (<b>F</b>) and IgG (<b>G</b>) levels were measured by ELISA. Data is plotted as the reciprocal log₁₀ titer, defined as three times greater than background. <b>I-J.</b> Ninety days after infection, WNV-specific antibody secreting long-lived plasma cells in the bone marrow (LLPC) were quantified. Shown is the LLPC frequency per 10⁶ bone marrow cells ± SD (<b>I</b>), as well as representative ELISPOT images from three adult and three old mice (<b>J</b>). Data is pooled from 6 to 9 mice per group from three independent experiments.</p

Delayed germinal center formation and decreased accumulation of CD4⁺ T cells and CD19⁺ B cells in DLN after WNV infection of old mice.

<p>Mice were infected with 10² PFU of WNV (New York strain) in the footpad. <b>A-D</b>. Draining popliteal LN cells were analyzed by flow cytometry at the indicated days after infection. Numbers of GC B cells (CD19⁺ Fas⁺ GL7⁺ cells) (<b>A</b>) and T_FH cells (CD4⁺ PD1⁺ CXCR5⁺ cells) were quantified (<b>C</b>). <b>B and D.</b> Representative flow cytometry plots of GC B cells (<b>B</b>, on gated CD19⁺ cells) and T_FH cells (<b>D</b>, on gated CD4⁺ cells) from adult and old mice at 8 days post infection. <b>E</b>. Immunofluorescence staining of germinal centers in DLN from 6 days post WNV-infected adult and old mice. Slides were stained with GL7-PE, and IgD-FITC. Arrows point to sites of active GCs indicated by GL7 staining in the red channel. In the graph, the number of GCs per section is recorded with each point representing a section taken from the center of draining popliteal lymph nodes from adult or old mice from two independent experiments. <b>F.</b> Hematoxylin and eosin stained sections of the draining popliteal LN from adult and old mice 4 days post infection. <b>G-M</b>. At the indicated days post infection, the DLN was harvested, and total cells were counted (<b>G</b>). Antibody staining detected specific lymphocyte subsets including CD4⁺ T cells (<b>H</b>) and CD19⁺ B cells (<b>K</b>). CD4⁺ T cells (<b>I, J</b>) and CD19⁺ B cells (<b>L, M</b>) were co-stained with antibodies to CD69 (<b>I, L</b>) or Ki-67 (<b>J, M</b>) to establish their state of activation or proliferation. The results are pooled from at least three independent experiments with a total of 6 to 14 mice per group. Data is expressed as the mean ± the standard error of the mean (SEM). Asterisks indicate statistical significance (*, <i>P</i> < 0.05; **, <i>P</i> < 0.01; ***, <i>P</i> < 0.001; Mann-Whitney test).</p

Leukocyte infiltration in the DLN of WNV-infected adult and old mice.

<p>Adult and old mice were infected with 10² PFU of WNV (New York strain) in the footpad. One or two days later, the draining popliteal LN was harvested, cells were stained with antibodies to detect specific cell populations including (<b>A</b>) NK1.1⁺ NK cells, (<b>B</b>) CD11b⁺ F4/80⁺ macrophages, (<b>C</b>) CD11c⁺ dendritic cells, and (<b>D</b>) γδ T cells. The results are depicted as a scatter plot and are pooled from two independent experiments with a total of 6 mice per group (with the exception of γδ T cells, which reflects 3 mice per group). Asterisks indicate statistical significance (*, <i>P</i> < 0.05; **, <i>P</i> < 0.01; ***, <i>P</i> < 0.001; Mann-Whitney test).</p