Cornea-Derived Mesenchymal Stromal Cells Therapeutically Modulate Macrophage Immunophenotype and Angiogenic Function

Abstract

Macrophages are crucial drivers of inflammatory corneal neovascularization and thus are potential targets for immunomodulatory therapies. We hypothesized that therapeutic use of cornea-derived mesenchymal stromal cells (cMSCs) may alter the function of macrophages. We found that cMSCs can modulate the phenotype and angiogenic function of macrophages. In vitro, cMSCs induce apoptosis of macrophages while preferentially promoting a distinct CD14^hi CD16^hi CD163^hi CD206^hi immunophenotype that has significantly reduced angiogenic effects based on in vitro angiogenesis assays. In vivo, application of cMSCs to murine corneas after injury leads to reduced macrophage infiltration and higher expression of CD206 in macrophages. Macrophages cocultured ("educated") by cMSCs express significantly higher levels of anti-angiogenic and anti-inflammatory factors compared with control macrophages. In vivo, injured corneas treated with cMSC-educated macrophages demonstrate significantly less neovascularization compared with corneas treated with control macrophages. Knocking down the expression of pigment epithelial derived factor (PEDF) in cMSCs significantly abrogates its modulating effects on macrophages, as shown by the reduced rate of apoptosis, decreased expression of sFLT-1/PEDF, and increased expression of vascular endothelial growth factor-A in the cocultured macrophages. Similarly, cMSCs isolated from PEDF knockout mice are less effective compared with wild-type cMSCs at inhibiting macrophage infiltration when applied to wild-type corneas after injury. Overall, these results demonstrate that cMSCs therapeutically suppress the angiogenic capacity of macrophages and highlight the role of cMSC secreted PEDF in the modulation of macrophage phenotype and function. Stem Cells 2018;36:775-784.

Keywords: Angiogenesis; Cornea; Inflammation; Macrophages; Mesenchymal stromal cells; Pigment epithelial derived factor.

Figure 1. cMSCs modulate macrophage immunophenotype

A: Control Mq and cMEMqs were expanded as described in the methods. On day +14, human control Mq or cMEMqs were analyzed by flow cytometry. Cell surface expression of CD86, HLA-DR, CD206 (Mannose receptor), CD163 (scavenger receptor), CD14 and CD16 were determined in control Mq and cMEMqs. Histograms represent MFI (Mean fluorescent intensity) in unstained Mq, control Mq and cMEMqs. B: The mean from independent biological replicated are presented as the bar charts. Educating macrophages with cMSCs reduced the expression of CD86 and HLA-DR, and increased the expression of CD163, CD206, CD14 and CD16 (n=4, 2-sided t-test * P <0.001). The values shown are mean ± SD (error bars). C: Conditioned media from human cMSCs induced more apoptosis in macrophages compared to unconditioned media (n=5, Mann-Whitney U-test: P = 0.0079) Boxes show the interquartile (25%–75%) range, whiskers encompass the range (minimum–maximum), and horizontal lines represent the mean. MFI: median fluorescein intensity, Cas: Caspase, ^CM: conditioned media, Ctrl: control, Mq: macrophage, cMEMq: cMSC-educated macrophage.

Figure 2. cMSCs modulate macrophage phenotype and recruitment into the corneal stroma after injury

A: Representative immunofluorescence staining after topical application of murine cMSCs to the injured murine cornea, demonstrating significantly reduced infiltration of F4/80+ macrophages, and increased CD206+ cells in the stroma compared to control treated corneas. Scale bar, 50 μm. B: To quantify, corneas were digested and subjected to flow cytometry which demonstrated significantly reduced the percentage of F4/80+ cells to total cells in murine cMSCs treated corneas compared to the control. Among the F4/80+ gated cells, there was the higher expression of CD206 in the cMSCs treated corneas compared to control treated corneas (n=5, 2-sided t-test * P <0.001).

Figure 3. cMSCs modulate the angiogenic properties of macrophages

A: CD14+CD163+ population of human cMEMqs were isolated by fluorescence-activated cell sorting and subjected to quantitative RT-PCR demonstrating lower expression of VEGF-A and higher expression of PEDF, sFLT-1, and TIMP-1 compared to CD14+CD163+ sorted control macrophages (n=5, P <0.001 for all comparison). B: The conditioned media from cMEMqs contains more sFLT-1 (1296 ± 715.3 vs. 276.6 ± 168.0 pg/ml), PEDF (17741 ± 8546 vs. 4956 ± 3851 pg/ml) and TSG-6 (10357 ± 2190 vs. 2757 ± 823.7 pg/ml) compared to the conditioned media from control untreated macrophages (n=5). The values shown are mean ± SD (error bars). 2-sided t-test: * P < 0.0001. Mq: macrophage, cMEMq: cMSC-educated macrophage.

Figure 4. cMSC-educated macrophages demonstrate reduced angiogenic properties

A: Murine macrophages were co-cultured with murine cMSCs as described in methods (cMEmMq). Applying cMEmMqs in fibrin gels to the cornea of wild-type mice after injury resulted in significantly less neovascularization compared to control macrophages on day ten (n=5, 2-sided t-test: * P < 0.0001). Scale bar, 500 μm. B: Conditioned media from cMEMqs inhibited tubule formation by human umbilical vein endothelial cells (HUVEC cells) compared to conditioned media from control untreated macrophages (n=5, 2-sided t-test: * P < 0.0001). Mq: macrophage, cMEmMq: cMSC-educated mouse macrophage, NV: neovascularization, CM: conditioned media.

Figure 5. cMSC derived PEDF is a key mediator of macrophage modulation

A: Injured wild-type corneas treated either with cMSCs obtained from the PEDF^−/− mice (placed in fibrin gels) or from wild-type PEDF ^+/+ mice were digested and subjected to flow cytometry. Injured wild-type corneas treated with cMSC^PEDF−/− had more infiltration of F4/80+ macrophages with lower expression of CD206 compared to the those treated with cMSC^PEDF+/+ (n=5; *P = 0.03). B: knocking down PEDF by siRNA decreased PEDF protein by almost 90% compared to scrambled siRNA (n=9, 2-sided t-test: *P<0.0001). Conditioned media from PEDF knockdown cMSCs induced less apoptosis in macrophages compared to conditioned media from control cMSC (n=5, Mann-Whitney U-test: P = 0.007). The values shown in bar graphs are mean ± SD (error bars). Boxes show the interquartile (25%–75%) range, whiskers encompass the range (minimum–maximum), and horizontal lines represent the mean. 2-sided t-test used for all comparison. MFI: median fluorescein intensity, SC: scrambled, Mq: macrophage, Cas: Caspase.

Figure 6. cMSC derived PEDF is a key mediator of macrophage angiogenic function

A: RT-qPCR shows mouse macrophages educated with wild type PEDF^+/+ cMSCs, expressed more arginase-1, CD206, IL-10, PEDF, and sFLT-1; and less iNOS mRNA compared to control untreated macrophages. Conversely, macrophages educated with PEDF ^−/− cMSCs, expressed less arginase-1, CD206, IL-10, PEDF, and sFLT-1 with more iNOS (n=5; P <0.001 for all comparison). B: Macrophages educated with PEDF knockdown cMSCs secreted more VEGF-A (448.3 ± 104.1 vs. 229.4 ± 125.1 pg/ml; P = 0.03) and less PEDF (7162.1 ± 2918.0 vs. 22373 ± 9683.0 pg/ml; P = 0.02) and sFLT-1 (249.1 ± 98.54 vs. 1172 ± 473.4 pg/ml; P = 0.008) compared to those trained with control cMSCs. The mean TSG-6 expression although decreased was not statistically significant (1160 ± 1309.0 vs. 3035 ± 3032.0 pg/ml) (P = 0.32). The values shown in bar graphs are mean ± SD (error bars). 2-sided t-test used for all comparison. M^PEDF EmMq: cMSC^PEDF-educated mouse macrophage, M^{Sc siRNA} EMq: cMSC^{Scrambled siRNA} educated macrophage, M^{PEDF siRNA} EMq: cMSC^{PEDF siRNA} educated macrophage.