Age-Related Oxidative Stress and Mitochondrial Dysfunction in Lymph Node Stromal Cells Limit the Peripheral T Cell Homeostatic Maintenance and Function

Abstract

Lymph nodes (LN) are the key organs in charge of long-term maintenance of naïve lymphocytes and their initial, primary activation upon infection. Accumulating evidence indicates that LN stromal cells undergo degenerative changes with aging that critically impair LN function, including the generation of protective primary immune responses. The nature of these defects remains incompletely understood. We here demonstrate that age-related LN stromal changes manifest themselves in mitochondrial dysfunction and oxidative stress. Ex vivo, all three major stromal cell subsets, fibroblastic reticular cells (FRC), lymphatic endothelial cells (LEC), and blood endothelial cells (BEC) exhibit elevated mitochondrial reactive oxygen species (ROS) stress, reduced mitochondrial potential, and elevated mitochondrial mass with aging. Old FRC also exhibited elevated cytoplasmic ROS production. This was accompanied by the reduced ability of old LN stromal cells to support Tn survival in vitro, a defect alleviated by pretreating old LN stroma with the general antioxidant N-acetyl cysteine (NAC) as well as by mitochondrial ROS-reducing (mitoquinone) and mitophagy-inducing (urolithin A) compounds. Mitochondrial dysfunction and, in particular, reduced mitochondrial potential in old FRC were also seen upon vaccination or infection in vivo. Consistent with these results, in vivo antioxidant treatment of old mice with NAC restored to adult levels the numbers of antigen-specific CD8⁺ effector T cells and their production of granzyme B in response to antigenic challenge.

Keywords: T cell homeostasis; aging; lymph node stromal cells; mitochondrial dysfunction; oxidative stress.

FIGURE 1

Aging of lymph node stromal cells limits peripheral T cell survival and maintenance. Purified adult and old CD8⁺CD62L^hiCD44^lo naïve T cells were co‐cultured with LN stromal cells from adult and old naïve C57BL/6 mice for 4 days, and survival and maintenance of naive CD8⁺ T cells in culture was analyzed by flow cytometry. (a) Data show geometric mean fluorescence intensity (MFI) of cell surface molecules on adult naïve CD8⁺ T cells. The percentage of (b) live (Annexin‐V⁻Live/Dead‐Zombie Aqua⁻), (c) early apoptotic (Annexin‐V⁺Live/Dead‐Zombie Aqua⁻), and (d) late apoptotic (Annexin‐V⁺Live/Dead‐Zombie Aqua⁺) adult (left) and old (right) CD8⁺ T cells were shown. The geometric MFI (gMFI) of intracellular (e) BCL2 and (f) Bax protein in co‐cultured adult (left) and old (right) CD8⁺ Tn cells was shown. (g) The ratio of intracellular BAX to BCL2 proteins in co‐cultured CD8⁺ Tn cells was shown. (h) A representative histogram (left) and gMFI (right) of intracellular cleaved caspase 3 expression in adult or old CD8⁺ Tn cells co‐cultured with adult and old lymph node stromal cells were shown. Data represent measurements performed on CD8⁺ T cells co‐cultured with adult stroma (open circle) or old stroma (filled circle). Data represent 3–4 independent experiments. Two‐way ANOVA followed by Tukey's multiple comparison correction test (a), Mann–Whitney U test (b–h). ns, nonsignificant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001.

FIGURE 2

Old LN stromal cells experience oxidative stress and exhibit mitochondrial dysfunction. Peripheral LN (pooled inguinal, axillary and brachial LN) were digested with Liberase‐TL and DNase‐I and single cells suspension were treated with (a) 2′,7′‐dichlorofluorescin‐diacetate (DCF‐DA; 1 μM) for 15 min, (b) MitoSOX‐Red (5 μM) for 30 min, (c, d) MitoTracker Green‐FM (200 nM) and MitoTracker Deep Red‐FM (200 nM) for 30 min at 37°C in 5% CO₂ incubator. Cells were washed and stained for surface markers to identify stromal cell populations and analyzed by flow cytometry. Data show level of (a) cellular (cytoplasmic) ROS, (b) mitochondrial ROS, (c) mitochondrial mass, and (d) mitochondrial membrane potential in LN stromal cell subsets, FRC (CD45⁻Pdpn⁺CD31⁻), LEC (CD45⁻Pdpn⁺CD31⁺), BEC (CD45⁻Pdpn⁻CD31⁺), and double negative (CD45⁻Pdpn⁻CD31⁻) cells from young adult (2–3 months), mid‐age (9 months), and old (18–19 months) mice. Data represent normalized MFI of the indicated parameters relative to 2–3 months of the corresponding cell population. Each dot represents an individual mouse. (e, f) Adult and old lymph node stromal cells were treated with the indicated concentrations of (e) Mitoquinone (Mito‐Q) or N‐acetyl cysteine (NAC; 1 mM) and (f) urolithin‐A (Uro‐A) for 24 h, cultures were washed, and purified adult CD8⁺ Tn cells were co‐cultured with treated and untreated stromal cells for 4 days. (e) Data show Annexin‐V⁻Live/Dead⁻ live (left) and Annexin‐V⁺Live/Dead⁺ late apoptotic (right) CD8⁺ Tn cells co‐cultured with Mito‐Q or NAC treated stromal cells. (f) Data show Annexin‐V⁻Live/Dead⁻ live (left) and Annexin‐V⁺Live/Dead⁺ late apoptotic (right) CD8⁺ Tn cells co‐cultured with Uro‐A treated stromal cells. Data are representative of four (a–d) or two (e, f) independent experiments and expressed as mean ± SEM. ANOVA followed by Tukey's multiple comparison correction test (a–d), Unpaired t‐test (e, f). ns, nonsignificant; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001.

FIGURE 3

Mitigating lymph node stromal cell oxidative stress improves the survival of naïve T cells. Purified pLN stromal cells from adult and old naïve C57BL/6 mice were cultured overnight and treated with N‐acetyl cysteine (NAC, 1 mM) or anti‐LTβR (2 μg/mL) for 24 h. The next day, purified CD8⁺CD62L^hiCD44^lo naïve T cells from adult and old C57BL/6 mice were co‐cultured with stromal cells for 4 days, and survival of CD8⁺ T cells in culture was analyzed by flow cytometry. (a) Representative flow cytometry plots (gated on CD8⁺ T cells) show staining of Annexin‐V and Live/Dead‐Zombie Aqua. Numbers in the quadrant indicate the percentage of cells. (b) Percentage of live (Annexin‐V⁻Live/Dead‐Zombie Aqua⁻) and (c) apoptotic (Annexin‐V⁺Live/Dead‐Zombie Aqua⁺) adult (left) and old (right) CD8⁺ T cells co‐cultured with either adult stroma (open bars) or old stroma (filled bars) were shown. (d) Data show the percentage of intracellular Bcl2⁺ cells within the CD8⁺ T cell population. Data pooled from 3 to 4 independent experiments and expressed as mean ± SEM. Each dot represents the mean of duplicate measurements performed in each experiment. Two‐way ANOVA followed by Tukey's multiple comparison correction test. ns, nonsignificant; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

FIGURE 4

Oxidative stress and mitochondrial dysfunction in old lymph node stromal cells limit their response to West Nile virus infection. Adult (2–4 months) and old (18–20 months) C57BL/6 mice were infected with WNV via s.c. in hind foot‐pads. At day 7, draining LN were harvested and digested to isolated stromal cells. As in Figure 2, mitochondrial parameters were analyzed in LN stromal cells. Representative flow cytometry histograms show staining profile for (a) MitoTracker Green indicating mitochondrial mass, (c) MitoSOX Red indicating mitochondrial ROS, and (e) MitoTracker Deep Red indicating membrane potential‐dependent mitochondrial mass within FRC (CD45⁻Pdpn⁺CD31⁻) population at 0 and 7 dpi. The corresponding group level comparison of (b) mitochondrial mass, (d) mitochondrial ROS, and (f) membrane potential in FRC (CD45⁻Pdpn⁺CD31⁻), LEC (CD45⁻Pdpn⁺CD31⁺), BEC (CD45⁻Pdpn⁻CD31⁺), and DN (CD45⁻Pdpn⁻CD31⁺) population were depicted. Each dot represents an individual mouse. Data are representative of three independent experiments and expressed as mean ± SEM. Each dot represents an individual mouse. Two‐way ANOVA followed by Tukey's multiple comparison correction test (b, d, f). ns, nonsignificant; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001.

FIGURE 5

Targeting oxidative stress improved functional T cell immunity to West Nile virus infection in old mice. Old (18–20 months) C57BL/6 male mice treated with N‐acetyl cysteine (NAC) every alternate day by oral gavage for four continuous weeks followed by s.c. infection with WNV in both hind foot‐pads. At 7 dpi, WNV‐specific T cell response in blood and dLN was analyzed. (a) Representative flow cytometry plots (gated on live, CD3⁺CD4⁻CD8⁺ T cells) show staining of CD44 and H‐2D^b‐NS4b‐PE tetramer in blood from untreated adult and old, and NAC‐treated old mice. (b) Absolute numbers of H‐2D^b‐NS4b‐tetramer⁺CD8⁺ T cells in the dLN (left) and blood (right) were shown. (c) Absolute numbers of granzyme B⁺H‐2D^b‐NS4b‐tetramer⁺CD8⁺ T cells in the dLN (left) and blood (right) were shown. Data are pooled from two independent experiments and expressed as mean ± SEM. Each dot represents an individual mouse. Numbers next to the box indicate the percentage of positive cell population (a). One‐way ANOVA followed by Tukey's multiple comparison test (b, c). ns, nonsignificant; *p ≤ 0.05, and **p ≤ 0.01(b, c).