Myeloid-derived suppressor cells are involved in lysosomal acid lipase deficiency-induced endothelial cell dysfunctions

Abstract

The underlying mechanisms that lysosomal acid lipase (LAL) deficiency causes infiltration of myeloid-derived suppressor cells (MDSCs) in multiple organs and subsequent inflammation remain incompletely understood. Endothelial cells (ECs), lining the inner layer of blood vessels, constitute barriers regulating leukocytes transmigration to the site of inflammation. Therefore, we hypothesized that ECs are dysfunctional in LAL-deficient (lal(-/-)) mice. We found that Ly6G(+) cells transmigrated more efficiently across lal(-/-) ECs than wild-type (lal(+/+)) ECs, which were associated with increased levels of PECAM-1 and MCP-1 in lal(-/-) ECs. In addition, lal(-/-) ECs showed enhanced migration and proliferation, decreased apoptosis, but impaired tube formation and angiogenesis. lal(-/-) ECs also suppressed T cell proliferation in vitro. Interestingly, lal(-/-) Ly6G(+) cells promoted in vivo angiogenesis (including a tumor model), EC tube formation, and proliferation. Finally, the mammalian target of rapamycin (mTOR) pathway was activated in lal(-/-) ECs, and inhibition of mTOR reversed EC dysfunctions, including decreasing Ly6G(+) cell transmigration, delaying migration, and relieving suppression of T cell proliferation, which was mediated by decreasing production of reactive oxygen species. Our results indicate that LAL regulates EC functions through interaction with MDSCs and modulation of the mTOR pathway, which may provide a mechanistic basis for targeting MDSCs or mTOR to rejuvenate EC functions in LAL deficiency-related diseases.

Figure 1. LAL deficiency in ECs leads to increased transendothelial migration of Ly6G⁺ cells

(A) Transwell assay was performed to determine Ly6G⁺ cells transmigration across the endothelial monolayer formed by lal^+/+ or lal^−/− ECs. Six hours after seeding Ly6G⁺ cells on the EC monolayer, the number of Ly6G⁺ cells that have migrated to the lower chamber was counted. Bars represent 250 μm. (B) CD4⁺ T cell transmigration across the endothelial monolayer formed by lal^+/+ or lal^−/− ECs was examined. Six hours later, the number of transmigrating CD4⁺ T cells was counted. (C) Expressions of PECAM-1 and ICAM-2 in ECs were determined by Western blot analysis. β-actin was used as control. Representative blots of 4 individual experiments were shown. (D) Transwell assay was performed to determine Ly6G⁺ cells transmigration across the ECs that were transfected with PECAM-1 siRNA; (E) Transwell assay was performed to determine Ly6G⁺ cells transmigration across the ECs that were pre-treated with anti-PECAM-1 neutralizing antibodies or control IgG; (F) Real-time PCR analysis of mRNA expression levels of MCP-1, IL-6 and TNFα in lal^+/+ vs. lal^−/− ECs. The relative gene expression was normalized to GAPDH mRNA, and analysis was performed by the 2^−ΔΔCT method. (G) Real-time PCR analysis of mRNA expression level of CCR2 in lal^+/+ vs. lal^−/− Ly6G⁺ cells. The relative gene expression was normalized to GAPDH mRNA, and analysis was performed by the 2^−ΔΔCT method. (H) To block chemokines and cytokines, ECs were pre-treated with 10 μg/mL neutralizing antibody against MCP-1, IL-6, TNF-α individually or in combination, or control IgG for 1 h. Six hours after seeding Ly6G⁺ cells on the EC monolayer, the number of migrating Ly6G⁺ cells was counted. In all above experiments, data were expressed as mean ± SD; n = 4. *P < 0.05, **P < 0.01.

Figure 2. LAL deficiency influences EC angiogenic functions

(A) In vitro matrigel tube formation was performed to compare the tube-forming capability between lal^+/+ and lal^−/− ECs. Top: representative micrographs of matrigel tube formation in ECs from lal^+/+ and lal^−/− mice at different time interval. Bottom: statistical analysis of cumulative tube lengths at 6 h. Data were normalized to lal^+/+ ECs and expressed as mean ±SD; n = 4. *P < 0.05. (B) In vivo angiogenesis was assessed by matrigel plug assay. Matrigel plugs containing ECs isolated from lungs of lal^+/+ or lal^−/− mice were implanted into lal^+/+ mice. Plugs were harvested for H&E and immunohistochemical staining 10 d after implanting in vivo. Representative microphotographs of matrigel plug sections stained with H&E and CD31 antibody were shown. Original magnification ×400. (C) Perfusion of matrigel plugs was determined by measuring the hemoglobin content. Data were normalized to lal^+/+ ECs and expressed as mean ±SD; n = 4, **P < 0.01; (D) The in vitro wound healing assay was conducted to determine EC migration in the presence of mitomycin C. Left: Representative pictures of wound healing assay of ECs from lal^+/+ or lal^−/− mice at the beginning and end of incubation (0 and 15h, respectively). The dotted lines define the areas lacking cells. Right: Quantification of distance from one end to the other end of the wound area. Data were normalized to lal^+/+ ECs at 0 h and expressed as mean ±SD; n = 4. *P < 0.05, **P < 0.01. Bars represent 500 μm.

Figure 3. LAL deficiency facilitates EC proliferation

(A) Comparison of the number of CD31⁺ cells in the lungs of lal^+/+ or lal^−/− mice. Lung cells from lal^+/+ or lal^−/− mice were purified by anti-CD31 microbeads and counted. (B) ECs after 3 days’ culture were harvested, and the number was compared between lal^+/+ and lal^−/− mice. (C) The percentage of BrdU incorporation into lal^+/+ or lal^−/− ECs were analyzed by flow cytometry. (D) The percentage of Annexin V positive cells in lung CD31⁺ cells from lal^+/+ or lal^−/− mice. (E) ECs were cultured in medium containing 20% plasma from lal^+/+ or lal^−/− mice for 72 h, and the cell number was counted afterwards. (F) Flow cytometry analysis of VEGFR2 expression in lal^+/+ vs. lal^−/− ECs. Data were normalized to lal^+/+ ECs. (G) ECs transfected with VEGFR2 or control siRNA were cultured in medium containing 20% plasma from lal^+/+ or lal^−/− mice for 72 h, and the cell number was counted afterwards. In all above experiments, data were expressed as mean ± SD; n = 3-4. *P < 0.05, **P < 0.01.

Figure 4. ECs from lal^−/− mice suppress T cell proliferation and function

(A) CFSE-labeled lal^+/+ CD4⁺ T cells were stimulated with anti-CD3 mAb plus anti-CD28 mAb for 4 days in the presence or absence of ECs from the lungs of lal^+/+ or lal^−/− mice at 10:1 ratio between CD4⁺ T cells: ECs. The proliferation of labeled CD4⁺ T cells was analyzed by flow cytometry. Peaks represent cell division cycles. PBS was used as a negative control. (B) The secretions of IL-4, IFN-γ, IL-10, and IL-17 of CD4⁺ T cells in the culture medium were measured by ELISA analysis. Data were expressed as mean ± SD; n = 3~4. **P < 0.01.

Figure 5. Ly6G⁺ cells from lal^−/− mice influence EC functions

(A) The effect of Ly6G⁺ cells on EC tube-forming capability was determined by matrigel tube formation assay. Left: representative micrographs of tube formation in ECs co-cultured with lal^+/+ or lal^−/− Ly6G⁺ cells. Right: statistical analysis of cumulative tube lengths. Data were normalized to lal^+/+ ECs only. Bars represent 500 μm. (B) The effects of macrophages (F4/80⁺ and CD11b⁺) and CD4⁺ T cells on EC tube-forming capability were determined by matrigel tube formation assay. (C) The effect of Ly6G⁺ cells on angiogenesis in the in vivo matrigel plug assay. Matrigel plugs containing Ly6G⁺ cells isolated from bone marrow of lal^+/+ or lal^−/− mice were implanted into lal^+/+ mice. Plugs were harvested 14 d after implantation and analyzed by H&E and immunohistochemical staining. Representative microphotographs of matrigel plug sections stained with H&E and CD31 antibody were shown. Original magnification ×200. (D) The effect of Ly6G⁺ cells on angiogenesis in the B16 melanoma tumor model. Matrigel mixed with B16 melanoma cells (1× 10⁵) and lal^+/+ or lal^−/− Ly6G⁺ cells (1× 10⁶) was implanted subcutaneously into lal^+/+ mice for 10 days. Representative microphotographs of matrigel plug sections stained with CD31 antibody were shown. Original magnification ×200. n=10. (E) Real-time PCR analysis of the mRNA expression level of VEGF in lal^+/+ vs. lal^−/− Ly6G⁺ cells. The relative gene expression was normalized to GAPDH mRNA, and determined by the 2^−ΔΔCT. (F) ECs were transfected with VEGFR2 or control siRNA, and then the effect of Ly6G⁺ cells on EC tube-forming capability was determined by matrigel tube formation assay. Statistical analysis of cumulative tube lengths was shown. Data were normalized to lal^+/+ ECs only. (G) ECs after 3 days’ co-culture with lal^+/+ or lal^−/− Ly6G⁺ cells were harvested, and the number was counted. (H) The percentage of BrdU incorporation into lal^+/+ or lal^−/− ECs co-cultured with Ly6G⁺ cells was analyzed by flow cytometry. In above experiments, data were expressed as mean ± SD; n = 3-4. *P < 0.05, **P < 0.01.

Figure 6. Activation of the mTOR pathway is involved in EC dysfunctions

(A) Expressions of phosphorylated-S6 and S6 in lal^+/+ or lal^−/− ECs were determined by Western blot analysis. Representative blots of 4 individual experiments were shown. (B) After inhibition of mTOR in ECs by siRNA transfection, the expressions of phosphorylated-S6 and S6 were examined afterwards. Representative blots of 3 individual experiments were shown. (C) Ly6G⁺ cells transmigration was determined after mTOR knockdown by siRNA transfection in ECs. Data were normalized to lal^+/+ Ly6G⁺ cells transmigrating across lal^+/+ ECs with control siRNA (C siRNA) transfection and expressed as mean ± SD; n = 4-5. *P < 0.05, **P < 0.01. (D) EC migration after mTOR knockdown was assessed by in vitro wound healing assay in the presence of mitomycin C. Data were normalized to lal^+/+ ECs with control siRNA transfection at 0 h and expressed as mean ± SD; n = 3. *P < 0.05, **P < 0.01. Bars represent 250 μm (C) and 500 μm (D). (E) Proliferation of CFSE-labeled lal^+/+ CD4⁺ T cells in the presence or absence of lal^+/+ or lal^−/− ECs with mTOR or control siRNA transfection was analyzed by flow cytometry. (F) The secretion of IL-4, IL-10 and IFN-γ of CD4⁺ T cells in the culture medium was measured by ELISA analysis. Data were expressed as mean ± SD; n = 4. *P < 0.05, **P < 0.01.

Figure 7. ROS over-production causes EC dysfunctions

(A) ROS production was increased in lal^−/− ECs, which was reversed by mTOR inhibitor rapamycin. Statistical analysis of mean fluorescent intensity (MFI) of the ROS level by flow cytometry is shown. (B) Ly6G⁺ cell transmigration was determined after antioxidant NAC pre-treatment of ECs. (C) Tube formation of ECs after NAC pre-treatment. Data were normalized to lal^+/+ ECs. (D) EC migration after NAC treatment by in vitro wound healing assay at 15h in the presence of mitomycin C. Data were normalized to lal^+/+ ECs at 0 h. (E) EC proliferation after NAC treatment. (F) The proliferation of lal^+/+ CD4⁺ T cells in the presence of lal^+/+ or lal^−/− ECs with or without NAC pre-treatment was analyzed by flow cytometry. In all above experiments, data were expressed as mean ± SD; n = 4. *P < 0.05, **P < 0.01.