Senescence regulates macrophage activation and angiogenic fate at sites of tissue injury in mice (original) (raw)
Aging is associated with enhanced angiogenesis. We have previously demonstrated that IL-10–/– mice have significantly reduced ocular angiogenesis following laser injury (1). IL-10 is a critical regulator of macrophage migration and influx into the site of tissue injury and of the subsequent development of CNV (1). Regulation of ocular angiogenesis is particularly important in the elderly who develop CNV in the eye as a function of unbridled angiogenesis that eventually leads to blindness. In order to directly test the effect of tissue injury on angiogenesis in aged animals, we lasered the eyes of old mice (>18 months of age) and compared the CNV response on day 7 after laser injury with that of young mice (<3 months of age). Old mice had significantly more CNV than did young mice (Figure 1), a finding similar to previous reports that tested mice over 16 months of age (14). These findings suggest that antiangiogenic responses in old mice are impaired.
Quantification of CNV in young (<3 months of age) and old (>18 months of age) mice on day 7 following laser injury. Mice were perfused intracardially with FITC-dextran (green) prior to harvesting eyeballs and sclerochoroidal flat mounts were made in order to quantify CNV volumes by confocal microscopy. (A) Old mice demonstrated significantly more CNV than did young mice. Error bars represent SEM. *P < 0.05 vs. old. (B and C) Representative CNV from a young (B) and an old (C) mouse.
Macrophages from senescent mice fail to inhibit postdevelopmental angiogenesis. Because macrophages have been shown to inhibit angiogenesis after laser-induced injury to the eye, we isolated F4/80+ splenic macrophages from C57BL/6 young or old mice by positive selection. In order to determine whether there is a functional difference between these macrophages in their ability to regulate angiogenesis, 1 × 105 macrophages from young and old mice were injected in to the eyes of old C57BL/6 host mice on the day of laser, and CNV response was measured on day 7 following laser injury. Macrophages from young mice significantly inhibited the CNV response, but macrophages from old mice failed to demonstrate any inhibition compared with the sham control mice injected with PBS (Figure 2). We have previously demonstrated that bone marrow–derived macrophages infiltrate into the laser lesions in the choroid after tissue injury (1). Here we isolated and dissected laser lesions as described in Methods, and F4/80 immunostaining demonstrated no significant differences in macrophage numbers within the laser lesions of young and old mice (data not shown). Enriched macrophage cytokine gene expression signature within these lesions was assessed by quantitative real-time PCR. Uninjured choroidal tissue was used as a control. We analyzed the cytokine profile of ocular macrophages isolated from young and old mice by measuring their cytokine gene expression 6 hours after tissue injury by real-time quantitative PCR. Macrophage-rich CNV lesions from old mice demonstrated significant downregulation of IL-12, _TNF-_α, and Fas ligand (FasL) 6 hours after the laser-induced injury, while these genes were highly upregulated or relatively unaltered in young mice (Figure 3, A–C). In addition, IL-6 and IL-23 were upregulated after tissue injury in young mice at levels exponentially higher than those seen in old mice (Figure 3, D and E). IL-10 expression was upregulated in both young and old mice (Figure 3F).
Assessment of CNV inhibition following intraocular injection of macrophages (1 × 105 cells/eye). Old mice were injected with PBS (sham) or macrophages from either old or young mice on the day of laser, and CNV was measured at day 7 following laser injury. Macrophages isolated from young mice demonstrated significant inhibition of CNV following intraocular injection in to old eyes, while macrophages isolated from old mice fail to inhibit CNV. *P < 0.02, young vs. old.
Quantitative real-time PCR gene expression analysis of macrophage-rich lesions from young and old mouse eyes after laser injury. Matching unlasered tissue was used as the baseline control. Macrophages from old mice demonstrated significant (asterisks) downregulation of IL-12 (A, P = 0.0068627), _TNF-_α (B, P = 0.0370), and FasL (C, P = 0.0302) compared with young mice. IL-6 (D, P = 0.00017) and IL-23 (E, P = 0.1024) were upregulated at levels exponentially higher in young mice than in old mice. (F) IL-10 was upregulated in both young and old mice, although baseline levels of IL-10 (P = 0.0701) were significantly higher in old mice than young mice. RQ, relative quantification.
IL-10 regulates macrophage function and promotes angiogenesis. These results are especially interesting given our previously published observations that IL-10–/– mice, which lack native IL-10, have reduced angiogenesis after laser injury to the eye (1). IL-10 is a key regulator of macrophage polarization and regulation of angiogenesis (1, 13). We tested the ability of IL-10–/– macrophages to inhibit CNV after intraocular injection to determine whether macrophage polarization affects angiogenic fate, i.e., CNV. Host C57BL/6 mice were injected intraocularly with 1 × 105 F4/80+ splenic macrophages from either syngeneic IL-10–/– mice or WT mice on the day of laser. Control mice were injected with PBS. Macrophages from IL-10–/– mice inhibited CNV even more efficiently than did those from young syngeneic WT mice, suggesting that the presence of native IL-10 causes a drift of macrophage function to a proangiogenic phenotype, in contrast to the effect of senescence on macrophage function (Figure 4). Given that IL-10 is such a critical cytokine in regulating abnormal angiogenesis in the eye, we wanted to test the gene expression of IL-10 in the posterior compartment of the eye at baseline in young mice and compare the levels to those of old mice using IL-10–/– eyes as a negative control. As shown in Figure 5, dissected sclerochoroidal tissue from old C57BL/6 mice at baseline had exponentially higher levels of IL-10 than did young WT or IL-10–/– mice. This result suggests that the micromilieu in old mice is rich in IL-10 and might create an environment that promotes polarization of infiltrating macrophages to a proangiogenic M2 phenotype. In addition, high baseline levels of IL-10 in old mice might explain the modest upregulation of IL-10 in macrophage-rich lesions of old mice compared with young mice after injury (Figure 3F). We then compared the effect of tissue injury on cytokine gene expression of macrophages within the laser lesions from eyes of IL-10–/– mice with that in C57BL/6 syngeneic WT young mice. Macrophage-enriched CNV lesions from IL-10–/– mice demonstrated marked upregulation of IL-6, _TNF-_α, IL-12, and FasL after laser injury in contrast to WT mice (Figure 6, A–D). As expected, IL-10–/– mice had a paucity of IL-10 gene expression, but WT mice had significant upregulation of IL-10 after injury (Figure 6E). IL-23 was upregulated in both young and IL-10–/– mice (data not shown). This signature defines the IL-10–/– macrophage as a TNFhi, IL-6hi, IL-12hi, IL-23hi, FasLhi, antiangiogenic macrophage and is consistent with our results that (a) IL-10–/– mice had reduced CNV and (b) IL-10–/– mouse–derived macrophages inhibited CNV after injection into the eye of a WT mouse. Our results also demonstrated that as mice age, senescent macrophages had elevated IL-10 at baseline and after injury. Old macrophages have a TNFlo, IL-6lo, IL-12lo, IL-23lo, FasLlo, and IL-10 hi phenotype after tissue injury to the eye, a phenotype that impairs their ability to regulate angiogenesis.
Assessment of CNV inhibition following intraocular injection of macrophages (1 × 105 cells/eye). Syngeneic old mice were injected with PBS (sham) or macrophages from young WT or IL-10–/– mice on the day of laser, and CNV was measured at day 7 following laser injury. (A) Macrophages isolated from IL-10–/– mice demonstrated significant inhibition of CNV following intraocular injection into old eyes, at levels beyond those seen following intraocular injection of macrophages from young WT mice. *P < 0.008 vs. sham. (B–D) Representative CNV from mice injected with IL-10–/– macrophages (B), young WT macrophages (C), and sham-injected mice (D).
Relative gene expression of IL-10 at baseline in dissected sclerochoroidal tissue from IL-10–/– mice, young WT mice, and old WT mice. IL-10 gene expression was performed using real-time PCR; data are represented using the IL-10–/– eye as the baseline. *P < 0.001 vs. old.
Quantitative real-time PCR gene expression analysis of macrophage-rich lesions from young WT and IL-10–/– mouse eyes after laser injury. Matching unlasered tissue was used as the baseline control. (A–D) Macrophages from IL-10–/– mice demonstrated upregulation of IL-6 (A, P = 0.0558), _TNF-_α (B, P = 0.3579), IL-12 (C, P = 0.1824), and FasL (D, P = 0.4569) compared with young mice. (E) IL-10 was upregulated in young mice and deficient in IL-10–/– mice. *P = 0.0513.
Effect of IL-10 and IFN-γ on macrophage polarization and angiogenesis. The presence of IL-10, as seen in the tissue micromilieu in old mice, was associated with a proangiogenic phenotype, as seen in Figure 1 and in previous studies (1). In order to directly assess the effect of polarizing cytokines such as IL-10 or IFN-γ on macrophages and their regulation of angiogenesis, we isolated F4/80+ macrophages from the spleens of WT mice and incubated them with 100 ng/ml of IL-10 or IFN-γ for 24 hours prior to injection. Host WT mice were lasered on day 0, and their eyes were injected with 1 × 105 F4/80+ splenic macrophages. IL-10–treated macrophages failed to inhibit CNV, while IFN-γ–treated macrophages demonstrated significant inhibition of CNV (Figure 7). These data clearly highlight the important role of IL-10 as a proangiogenic cytokine and demonstrate that in presence of IL-10, macrophages polarize to an M2 phenotype and fail to demonstrate antiangiogenic properties.
Ability of macrophages to inhibit CNV after treatment with polarizing cytokines. F4/80+ macrophages were isolated from the spleens of C57BL/6 young WT mice, treated with 100 ng/ml IL-10 or IFN-γ, and then injected in the eyes of host WT mice on the day of laser (1 × 105 cells/eye). Macrophages treated with IFN-γ significantly inhibited CNV, while macrophages treated with IL-10 failed to do so. *P < 0.000003 vs. sham.
Aging impairs the ability of macrophages to regulate vascular endothelial cell proliferation. The results thus far suggest that as mice age, their macrophages lose antiangiogenic function and that IL-10 is a key proangiogenic regulator of macrophage fate and function. We have previously demonstrated that macrophages can directly inhibit vascular endothelial cell proliferation and angiogenesis in vivo (1). We wanted to test directly whether senescence-associated alterations in macrophage gene expression impair the ability of macrophages to inhibit the growth and proliferation of vascular endothelial cells. Thioglycollate-induced peritoneal macrophages (effectors) were harvested from WT young, IL-10–/–, gld, or old mice and incubated with human dermal microvascular endothelial cells (HMVECs; targets) at a 25:1 ratio in order to assess the effect of macrophages on proliferation of HMVECs. As shown in Figure 8, while macrophages from young and IL-10–/– mice significantly inhibited the proliferation of HMVECs, macrophages from old and gld mice failed to do so. The effect was contact dependent: macrophages from gld or old mice placed in transwells above the target HMVECs did not inhibit or stimulate proliferation when compared with macrophages from young or IL-10–/– mice placed in transwells (data not shown). This confirms that the loss of antiangiogenic function of senescent macrophages is likely caused by downregulation of FasL and suggests that old macrophages might actually promote abnormal angiogenesis as seen in diseases of aging such as AMD, atheromatous heart disease, and certain cancers.
Effect of senescence on macrophage ability to regulate proliferation of vascular endothelial cells. HMVECs were cultured alone, unlabeled [H(UNL)] or labeled [H(L)], or were cultured with macrophages from young WT [M/H(25:1)B6], old WT [M/H(25:1)Old], gld [M/H(25:1)_Gld_], and IL-10–/– [M/H(25:1)_IL-10–/–_] mice for 12 hours prior to measuring the proliferation of HMVEC with [3H] thymidine (TdR). Young WT and IL-10–/– macrophages inhibited the proliferation of HMVECs, while old and gld macrophages failed to do so. *P = 0.009; **P < 0.00086.