Aging mesenchymal stem cells fail to protect because of impaired migration and antiinflammatory response

Abstract

Rationale: Aging is characterized by functional impairment and reduced capacity to respond appropriately to environmental stimuli and injury. With age, there is an increase in the incidence and severity of chronic and acute lung diseases. However, the relationship between age and the lung's reduced ability to repair is far from established and necessitates further research in the field.

Objectives: Little is currently known about age-related phenomena in mesenchymal stem cells (MSCs). On account of their ability to protect the endothelium and the alveolar epithelium through multiple paracrine mechanisms, we looked for adverse effects that aging might cause in MSC biology. Such age-related changes might partly account for the increased susceptibility of the aging lung to injury.

Measurements and main results: We demonstrated that old mice have more inflammation in response to acute lung injury. To investigate the causes, we compared the global gene expression of aged and young bone marrow-derived MSCs (B-MSCs). Our results revealed that the expression levels of inflammatory response genes depended on the age of the B-MSCs. We demonstrated that the age-dependent decrease in expression of several cytokine and chemokine receptors is important for the migration and activation of B-MSCs. Finally, we showed by adoptive transfer of aged B-MSCs to young endotoxemic mice that aged cells lacked the antiinflammatory protective effect of their young counterparts.

Conclusions: Taken together, the decreased expression of cytokine and chemokine receptors in aged B-MSCs compromises their protective role by perturbing the potential of B-MSCs to become activated and mobilize to the site of injury.

Figure 1.

Lung inflammation increases with age in a murine model of LPS-induced lung injury. Several parameters of lung inflammation were assessed 48 hours after LPS-induced acute respiratory distress syndrome (ARDS) in young and old mice. (A) Wet-to-dry ratio indicates significantly increased water content in the lungs of old mice after injury. (B) Cytokine levels are increased in the bronchoalveolar lavage (BAL) of old mice (n_o = 4, n_y = 6, analyzed in triplicate by Luminex, Austin, TX). (C) Total counts of inflammatory cells are higher in the BAL of old mice (n_o = 4, n_y = 6). (D) Macrophages, lymphocytes, and neutrophils are elevated in the BAL of old mice (n_o = 4, n_y = 6). (B and D) Open bars, young mice; solid bars, old mice. (E) Representative histological sections of lung obtained from young mice (panel 1) and old mice (panel 2) after endotoxin treatment. (F) Lung inflammation score based on multiple histological sections from all animals. Data are represented as means ± SEM. Lym = lymphocytes; MAC = macrophages; n_o = number of old mice analyzed per experiment; n_y = number of young mice analyzed per experiment; Neu = neutrophils; TNF = tumor necrosis factor. Scale bars: 100 μm. *P < 0.05, **P < 0.005, ***P < 0.0005.

Figure 2.

Differential gene expression in aged versus young bone marrow–derived mesenchymal stem cells (B-MSCs). (A) Microarray map of sorted B-MSCs from young and old mice, showing 927 genes that are differentially expressed. (B–E) Real-time PCR detection of genes that are differentially expressed in microarrays including (B) cytokine receptors, (C) chemokines, (D) Marcks, Mmp9, and Timp2, and (E) markers of cell senescence. Open bars, young mice; solid bars, old mice. Values are expressed as means ± SEM; n = 3 samples per group, done in triplicate. *P < 0.05, **P < 0.005, ***P < 0.0005.

Figure 3.

Down-regulation of cytokine receptors in aged bone marrow–derived mesenchymal stem cells (B-MSCs) affects their activation. (A) The expression (RT-PCR) of antiinflammatory factors is significantly lower in aged B-MSCs after 0.5% LPS-serum cell activation. Open bars, young mice; solid bars, old mice. (B) Western blot showing that phosphorylated c-Jun N-terminal kinase (p-JNK) is lower in aged activated B-MSCs (column O) compared with young activated B-MSCs (column Y). Blocking of TNFR2 alone (column YT) or together with IFNGR1 (column YTI) in young activated B-MSCs inhibits the phosphorylation of JNK. β-Actin was used as a loading control. (C) Bar graph depicting the results of two TNFR2 blocking experiments. (D–H) Real-time PCR detection of (D) Il1rn, (E) Nos2, (F) Tgfb3, (G) Mmp9, and (H) Il10 after blocking of TNFR2 and/or IFNGR1 in young activated B-MSCs. The values are expressed as means ± SEM; n = 3 samples per group, done in triplicate. *P < 0.05, **P < 0.005, ***P < 0.0005. IFNGR1 = IFN-γ receptor-1; inh = inhibitor; TNFR2 = tumor necrosis factor receptor-2.

Figure 4.

Aged bone marrow–derived mesenchymal stem cells (B-MSCs) are not as protective as their young counterparts in LPS-induced lung injury. Several parameters of lung inflammation were assessed 48 hours after LPS-induced acute respiratory distress syndrome (ARDS) in young mice and concomitant administration of either young or aged B-MSCs (5 × 10⁵). (A) Wet-to-dry ratio indicates increased water content in the lungs of mice that received aged B-MSCs (n_o = 6, n_y = 7). (B) Cytokine levels are increased in the bronchoalveolar lavage (BAL) of mice that received aged B-MSCs (n_o = 6, n_y = 6, analyzed in triplicate by multiplexed bead-based immunoassays). (C) Total counts of inflammatory cells are higher in the BAL of mice that received aged B-MSCs (n_o = 6, n_y = 6). (D) Macrophages, lymphocytes, and neutrophils are elevated in the BAL of mice that received aged B-MSCs (n_o = 6, n_y = 6). (B and D) Open bars, young mice; solid bars, old mice. (E) Representative histological sections of lung obtained from LPS-treated mice without B-MSC treatment (panel 1), mice treated with young B-MSCs (panel 2), and mice treated with aged B-MSCs (panel 3). (F) Lung inflammation score based on multiple histological sections of all animals. To evaluate the protective effect of young B-MSCs in old mice, 5 × 10⁵ young B-MSCs were administered intravenously to old (24-mo-old) mice. After 48 hours mice were killed and (G) BAL cell counts and (H) histological sections stained with hematoxylin and eosin were analyzed. Data are presented as means ± SEM. Lym = lymphocytes; Mac = macrophages; n_o = old mice analyzed per experiment; n_y = young mice analyzed per experiment; Neu = neutrophils; TNF-α = tumor necrosis factor-α. Scale bars: 100 μm. *P < 0.05, **P < 0.005, ***P < 0.0005.

Figure 5.

Aged bone marrow–derived mesenchymal stem cells (B-MSCs) migrate less than young MSCs. (A) Wound-healing assays of young and aged B-MSCs incubated with serum from endotoxemic mice (LPS-serum): aged B-MSCs at (panel 1) time 0 and (panel 2) 48 hours, and young B-MSCs at (panel 3) time 0 and (panel 4) 48 hours. (B) Western blot showing that phosphorylated JNK (p-JNK) is lower in aged activated B-MSCs (Old) compared with young activated B-MSCs (Young). Antibody blocking of CCR7 in young activated B-MSCs inhibits the phosphorylation of JNK (Young + CCR7 inhib). β-Actin was used as a loading control. (C) Bar graph depicting the results of CCR7-blocking experiments (n = 2). (D) Migration of B-MSCs in Transwell chambers after 48 hours of stimulation with LPS-serum: (panel 1) aged B-MSCs, (panel 2) young B-MSCs, and (panel 3) young B-MSCs subjected to CCR7 blocking. (E) Bar graph depicting the number of migrating cells (n = 3). Solid bars, old B-MSCs; open bars, young B-MSCs. Values represent means ± SEM; n = number of experiments performed. Scale bars: 100 μm. *P < 0.05, **P < 0.005, ***P < 0.0005.

Figure 6.

Migration of aged bone marrow–derived mesenchymal stem cells (B-MSCs) in parabiotic mice. Young wild-type acute respiratory distress syndrome (ARDS) mice were coupled with either young or old green fluorescent protein–positive (GFP⁺) mice to create parabiotic pairs. (A) The migration of GFP⁺ B-MSCs after 48 hours was tracked by flow cytometric analysis of total lung cell suspension in the wild-type mice. (B and C) The frequency of GFP⁺ B-MSCs derived from either young or old donors in (B) the lung and (C) the blood of wild-type mice. (D) Bronchoalveolar lavage (BAL) cytokine levels, (E) water content in the lung as measured by wet-to-dry ratio, (F) total count of cells in BAL, and (G) BAL macrophage, lymphocyte, and neutrophil ratios were elevated in the parabionts coupled with old GFP⁺ mice. (H) Histology of lungs from wild-type parabionts with (panel 1) young and (panel 2) old GFP⁺ donors. (I) Lung inflammation score based on histological sections. Open bars, young mice; solid bars, old mice. Values represent means ± SEM; n = 6 mice per group. Scale bars: 100 μm. *P < 0.05, **P < 0.005, ***P < 0.0005. Lym = lymphocytes; Mac = macrophages; Neu = neutrophils; TNF-α = tumor necrosis factor-α.

Figure 7.

Aged human bone marrow–derived mesenchymal stem cells (hB-MSCs) are not as protective as their young counterparts in lung injury. (A) Real-time PCR of cytokine receptors expressed in aged and young hB-MSCs (n = 3, in triplicate). (B) Migration of hB-MSCs in Transwell chambers after 48 hours of stimulation with serum from patients with acute respiratory distress syndrome (ARDS) (n = 4). (C) Wound-healing assays of hB-MSCs incubated with serum from patients with ARDS: aged hB-MSCs at (panel 1) time 0 and (panel 2) 48 hours; young hB-MSCs at (panel 3) time 0 and (panel 4) 48 hours. Several parameters of lung inflammation were assessed 48 hours after LPS-induced ARDS in young mice and concomitant administration of saline or either young or aged hB-MSCs (5 × 10⁵): (D) cytokine levels, (E) wet-to-dry ratio, (F) total counts of inflammatory cells, and (G) macrophage, lymphocyte, and neutrophil content in mice treated with saline (gray bars), young hB-MSCs (open bars), or aged hB-MSCs (black bars) (n = 6). (H) Representative histological sections of lung obtained from injured mice treated with saline (panel 1), mice treated with young hB-MSCs (panel 2), and mice treated with aged hB-MSCs (panel 3). (I) Lung inflammation score based on histological sections (n = 6). Values are expressed as means ± SEM. Scale bars: 100 μm. *P < 0.05, **P < 0.005, ***P < 0.0005. BAL = bronchoalveolar lavage; Lym = lymphocytes; Mac = macrophages; Neu = neutrophils; TNF-α = tumor necrosis factor-α.