Suppression of Neutrophil-Mediated Tissue Damage-A Novel Skill of Mesenchymal Stem Cells

Abstract

Mesenchymal stem cells (MSCs) are crucial for tissue homeostasis and regeneration. Though of prime interest, their potentially protective role on neutrophil-induced tissue damage, associated with high morbidity and mortality, has not been explored in sufficient detail. Here we report the therapeutic skill of MSCs to suppress unrestrained neutrophil activation and to attenuate severe tissue damage in a murine immune-complex mediated vasculitis model of unbalanced neutrophil activation. MSC-mediated neutrophil suppression was due to intercellular adhesion molecule 1-dependent engulfment of neutrophils by MSCs, decreasing overall neutrophil numbers. Similar to MSCs in their endogenous niche of murine and human vasculitis, therapeutically injected MSCs via upregulation of the extracellular superoxide dismutase (SOD3), reduced superoxide anion concentrations and consequently prevented neutrophil death, neutrophil extracellular trap formation and spillage of matrix degrading neutrophil elastase, gelatinase and myeloperoxidase. SOD3-silenced MSCs did not exert tissue protective effects. Thus, MSCs hold substantial therapeutic promise to counteract tissue damage in conditions with unrestrained neutrophil activation. Stem Cells 2016;34:2393-2406.

Keywords: Mesenchymal stem cells; Neutrophil extracellular traps; Neutrophils; Superoxide dismutase; Vasculitis.

Figure 1

MSCs suppress oxidative burst, death, peroxidase and proteases release. 2 × 10⁶ mNeu per well were cultured alone or cocultured with AT-MSCs at mNeu:MSC ratios of 10:1, 100:1, and 1000:1. (A): 4 hours after coculture, mNeu were activated by 100 ng/ml PMA at 37°C for 10 minutes. mNeu treated with DMSO served as nonactivated control. Cocultures were labeled with 5 µM dihydrorhodamine 123 (DHR) and APC-conjugated Gr-1 antibody at 37°C for 30 minutes, and subjected to flow cytometric analysis. DHR intensity was analyzed within the Gr-1⁺ gate. The depicted histograms are representative of three independent experiments. (B): Cocultures were harvested 24 hours later, and labeled with Annexin V-FITC, PI, and Gr-1-APC, and analyzed by flow cytometry. The depicted dot plots and histograms were gated on Gr-1⁺ population, and are representative of three independent experiments. (C): Depicted are percentages of Annexin V⁺ cells expressed as mean ± SEM of three independent experiments. ***, p < .001. (D–H): Four hours after coculture, mNeu were activated by 100 ng/ml PMA for 30 minutes. Supernatants from cocultures were harvested. (D) MPO concentrations were measured by ELISA. (E) The MPO activity was assessed using the TMB substrate based activity assay in a 10 minute interval. (F–G) Total and active neutrophil gelatinase (MMP-9) was evaluated based on its proteolytic activity of converting a pro-detecting enzyme to an active-detecting enzyme, which converts peptide substrate into colored product. The proMMP-9 in the supernatants was activated by p-aminophenyl mercuric acetate. (H) NE concentrations were calculated from its proteolytic activity of converting a nonfluorescent substrate to a fluorescent product. *, p < .05; **, p < .01; ***, p < .001 by Student’s t tests. These experiments were independently repeated three times with similar results. Abbreviations: MPO, myeloperoxidase; mNeu, murine bone marrow neutrophils; MSCs, mesenchymal stem cells; NE, neutrophil elastase, TMB, 3,3′,5,5′-Tetramethylbenzidine.

Figure 2

MSCs reduce NETs formation and engulf neutrophils. (A): 2 × 10⁵ hNeu were cultured alone or cocultured with 2 × 10⁴ AT-MSCs or HDF in wells of glass chamber slides and activated by 100 ng/ml PMA for 3 hours. Slides were fixed and labeled with anti-DNA/histone-1 followed by AF555-conjugated anti-mouse IgG (red) and counterstained with DAPI (blue). Scale bar: 200 µm. (B): 1 × 10⁵ hNeu were cultured alone or with AT-MSCs at hNeu:MSC ratios of 10:1, 100:1 and 1000:1 and subsequently activated with 100 ng/ml PMA for 3 hours. Cocultures were labeled with 0.25 µM Sytox orange for 5 minutes at room temperature and fluorescence intensities were measured by microplate reader. Culture medium containing Sytox orange served as background fluorescent control. Fluorescence intensities were normalized on the intensity of non-activated controls. Results are expressed as mean ±SD of five-plicate measurements. This experiment was independently repeated three times with similar results. ***, p < .001. (C–D): 1×10⁶ mNeu were labeled with 2 µM PKH26 and cultured at 37°C overnight to induce apoptosis. Thereafter, mNeu were cocultured with 1×10⁵ of 2.5 µM CFSE labeled AT-MSCs or murine bone marrow derived macrophages for 24 hours. (C) Cocultured cells were fixed on chamber slides and photographed under confocal microscope with 1 µm light section. White scale bar: 50 µm; yellow scale bar: 10 µm. The squares in the “Merge” panel indicate the areas enlarged in the “Enlarge” panel. (D) Cocultures were analyzed by flow cytometry. AT-MSCs or macrophages cocultured with PKH26 labeled mNeu are depicted in filled grey histograms, and the counterparts cocultured with nonlabeled mNeu are shown in empty histograms. The depicted photographs and histograms are representative of three independent experiments. Mϕ, macrophages. (E): AT-MSCs were cultured alone or with PMA activated mNeu at a mNeu:MSCs ratio of 10:1. 24 hours after culture, cells were labeled with CD73-APC and ICAM-1-PE or VCAM-1-PE antibodies and subjected to flow cytometric analysis. The ICAM-1 and VCAM-1 expression were analyzed in CD73⁺ MSC gate. (F–G): CFSE labeled MSCs (green) were cocultured with PKH26 labeled mNeu (red) from wild-type (CD18^+/+) or CD18^−/− mice at a mNeu:MSC ratio of 10:1 for 24 hours. The engulfment of neutrophils by MSCs were examined by (F) confocal microscopy, white scale bar: 50 µm; yellow scale bar: 10 µm or (G) flow cytometry. Abbreviations: AU, arbitrary unit. HDF, human dermal fibroblasts; ICAM-1, intercellular adhesion molecule 1; hNeu, human peripheral neutrophils; MSCs, mesenchymal stem cells; mNeu, murine bone marrow neutrophils; NETs, neutrophil extracellular traps; RFI, relative fluorescence intensity.

Figure 2

MSCs reduce NETs formation and engulf neutrophils. (A): 2 × 10⁵ hNeu were cultured alone or cocultured with 2 × 10⁴ AT-MSCs or HDF in wells of glass chamber slides and activated by 100 ng/ml PMA for 3 hours. Slides were fixed and labeled with anti-DNA/histone-1 followed by AF555-conjugated anti-mouse IgG (red) and counterstained with DAPI (blue). Scale bar: 200 µm. (B): 1 × 10⁵ hNeu were cultured alone or with AT-MSCs at hNeu:MSC ratios of 10:1, 100:1 and 1000:1 and subsequently activated with 100 ng/ml PMA for 3 hours. Cocultures were labeled with 0.25 µM Sytox orange for 5 minutes at room temperature and fluorescence intensities were measured by microplate reader. Culture medium containing Sytox orange served as background fluorescent control. Fluorescence intensities were normalized on the intensity of non-activated controls. Results are expressed as mean ±SD of five-plicate measurements. This experiment was independently repeated three times with similar results. ***, p < .001. (C–D): 1×10⁶ mNeu were labeled with 2 µM PKH26 and cultured at 37°C overnight to induce apoptosis. Thereafter, mNeu were cocultured with 1×10⁵ of 2.5 µM CFSE labeled AT-MSCs or murine bone marrow derived macrophages for 24 hours. (C) Cocultured cells were fixed on chamber slides and photographed under confocal microscope with 1 µm light section. White scale bar: 50 µm; yellow scale bar: 10 µm. The squares in the “Merge” panel indicate the areas enlarged in the “Enlarge” panel. (D) Cocultures were analyzed by flow cytometry. AT-MSCs or macrophages cocultured with PKH26 labeled mNeu are depicted in filled grey histograms, and the counterparts cocultured with nonlabeled mNeu are shown in empty histograms. The depicted photographs and histograms are representative of three independent experiments. Mϕ, macrophages. (E): AT-MSCs were cultured alone or with PMA activated mNeu at a mNeu:MSCs ratio of 10:1. 24 hours after culture, cells were labeled with CD73-APC and ICAM-1-PE or VCAM-1-PE antibodies and subjected to flow cytometric analysis. The ICAM-1 and VCAM-1 expression were analyzed in CD73⁺ MSC gate. (F–G): CFSE labeled MSCs (green) were cocultured with PKH26 labeled mNeu (red) from wild-type (CD18^+/+) or CD18^−/− mice at a mNeu:MSC ratio of 10:1 for 24 hours. The engulfment of neutrophils by MSCs were examined by (F) confocal microscopy, white scale bar: 50 µm; yellow scale bar: 10 µm or (G) flow cytometry. Abbreviations: AU, arbitrary unit. HDF, human dermal fibroblasts; ICAM-1, intercellular adhesion molecule 1; hNeu, human peripheral neutrophils; MSCs, mesenchymal stem cells; mNeu, murine bone marrow neutrophils; NETs, neutrophil extracellular traps; RFI, relative fluorescence intensity.

Figure 3

Activated neutrophils mediated tissue damage in vasculitis patients. (A–D): Representative microphotographs of paraffin-embedded skin sections from six healthy donor (left panel) and six vasculitis patients (right panel) with H&E staining (A–B), immunostaining of human neutrophil marker CD15 (green) and neutrophil extracellular traps marker DNA/Histone-1 (red) (C), and immunostaining of NE (green) and MPO (red) (D). In immunostainings nuclei were counterstained with DAPI (blue). The squares in (A) indicate the enlarged areas in (B). White arrows indicate blood vessels, black arrows indicate neutrophils in (B). Scale bar: 50 µm. (E–F): The percentage of CD15⁺ neutrophils and NE⁺ MPO⁺ neutrophils was calculated from each skin section of 6 different healthy donors and 6 patients with vasculitis and expressed as mean ± SEM of CD15⁺ neutrophils (E) and NE⁺ MPO⁺ neutrophils (F). n = 6; **, p< .01 by Mann–Whitney tests. Abbreviations: MPO, myeloperoxidase; NE, neutrophil elastase.

Figure 4

MSCs reduce vessel destruction and hemorrhage in immune complex-mediated vasculitis. (A): 100 µl PBS or 2.5 × 10⁵ AT-MSCs were intradermally injected to both sides of shaved dorsal skin of mice. The Arthus reaction was elicited by i.v. injection of 100 µl PBS solution containing 2% BSA and 1% Evans blue. Afterward anti-BSA antibody was intradermally injected to the area that had been injected with AT-MSCs or PBS. Four hours later, mice were sacrificed and skin specimen were harvested and digitally photographed. (B– C): The areas of Evans blue spots on skin were analyzed by Image J. n = 10; **, p < .01 by Mann-Whitney tests. (D–T): Mice were intra-dermally injected with PBS or nontransfected MSCs or SOD3-siRNA transfected MSCs or control-siRNA transfected MSCs and subjected to Arthus reaction. Mice received rabbit IgG served as healthy controls. Representative pictures of paraffin-embedded skin sections from 4 mice of each group with H&E staining (D, G, J, M, P), immunostaining of murine neutrophil marker Ly6G (green) and NETs marker DNA/Histone-1 (red) (E, H, K, N, Q), and immunostaining of NE (green) and MPO (red) (F, I, L, O, R) are shown. Nuclei were counterstained with DAPI (blue) in immunostainings. White arrows in H&E staining indicate blood vessels. Scale bar: 50 µm. (S–T) The depicted are mean ± SEM of Ly6G⁺ cells (S) and NE⁺ MPO⁺ cells (T) counted from each section of 4 mice. n = 4; *, p < .05; **, p < .01 by Mann-Whitney tests. Abbreviations: AU, arbitrary unit; MSCs, mesenchymal stem cells; MPO, myeloperoxidase; NE, neutrophil elastase.

Figure 5

Induced SOD3 from MSCs is responsible for neutrophil suppressive effects. (A–B): 2 × 10⁵ AT-MSCs were cultured alone or with 2 × 10⁶ mNeu activated by 100 ng/ml PMA. mNeu treated with DMSO served as nonactivated controls. AT-MSCs treated with 100 ng/ml PMA served as control for PMA effects. Supernatants were harvested 2 hours (A) and 24 hours (B) after onset of coculture, and SOD3 concentrations in supernatants were measured by ELISA. This experiment was independently performed three times with similar results. (C): 2.5 × 10⁵ AT-MSCs were intradermally injected to shaved dorsal skin of mice. The Arthus reaction was elicited by i.v. injection of 100 µl of 2% BSA in PBS and intradermal injection of anti-BSA antibody to the area that had been injected with AT-MSCs. 4 hours later, mice were sacrificed and skin specimen were processed for cryosections. Sections were immunostained with human specific beta-2 microglobulin (β2M, green, indicative of MSCs) and SOD3 (red), and nuclei were counterstained with DAPI (blue). The square in the left panel indicates the area enlarged in the right panel. White scale bar: 100 µm; yellow scale bar: 20 µm. (D–F): Endogenous ABCB5⁺ dermal MSCs express SOD3 in situ. Depicted in (D) are representative microphotographs of paraffin-embedded skin sections from human vasculitis patients with immunostaining of human ABCB5 (green) and human SOD3 (red) (left panel), and mice subjected to IC-mediated vasculitis with immunostaining of ABCB5 (green) and murine SOD3 (red) (right panel), respectively. Scale bar: 10 µm. The percentage of ABCB5⁺ SOD3⁺ cells in total dermal cells in each section are expressed as mean ± SEM in human (E) and murine (F) samples, respectively. n = 8; **, p < .01 by Student’s t tests. (G–H): 2 × 10⁶ mNeu were cultured alone or with non-transfected MSCs or control-siRNA transfected MSCs, or SOD3-silenced MSCs at a mNeu:MSC ratio of 10:1, and subjected to PMA-induced oxidative burst assay with reactive oxygen species indicator dihydrorhodamine 123. The depicted histograms in (G) are representative of three independent experiments. The mean fluorescence intensity (MFI) of each sample in (G) was normalized to MFI of PMA activated neutrophils as percentages in the same experiment. The chart depicted in (H) is mean ± SEM of percentage of MFI derived from three experiments. **, p < .01; ***, p < .001 by Student’s t tests. (I–J): 100 µl PBS or 2.5 × 10⁵ control-siRNA transfected MSCs or SOD3-silenced MSCs were intradermally injected into dorsal skin of mice. The Arthus reaction was elicited at the areas that had been injected with MSCs or PBS. Four hours later, mice were sacrificed and skin specimen were harvested and digitally photographed. The areas of Evans blue on skin specimen with MSCs treatment were normalized to Evans blue stained areas after PBS injection on the same mouse and expressed as percentages. n = 8; **, p < .01; ***, p < .001 by Mann–Whitney tests. Abbreviations: MSCs, mesenchymal stem cells; mNeu, murine bone marrow neutrophils; SOD3, superoxide dismutase 3.

Figure 5

Induced SOD3 from MSCs is responsible for neutrophil suppressive effects. (A–B): 2 × 10⁵ AT-MSCs were cultured alone or with 2 × 10⁶ mNeu activated by 100 ng/ml PMA. mNeu treated with DMSO served as nonactivated controls. AT-MSCs treated with 100 ng/ml PMA served as control for PMA effects. Supernatants were harvested 2 hours (A) and 24 hours (B) after onset of coculture, and SOD3 concentrations in supernatants were measured by ELISA. This experiment was independently performed three times with similar results. (C): 2.5 × 10⁵ AT-MSCs were intradermally injected to shaved dorsal skin of mice. The Arthus reaction was elicited by i.v. injection of 100 µl of 2% BSA in PBS and intradermal injection of anti-BSA antibody to the area that had been injected with AT-MSCs. 4 hours later, mice were sacrificed and skin specimen were processed for cryosections. Sections were immunostained with human specific beta-2 microglobulin (β2M, green, indicative of MSCs) and SOD3 (red), and nuclei were counterstained with DAPI (blue). The square in the left panel indicates the area enlarged in the right panel. White scale bar: 100 µm; yellow scale bar: 20 µm. (D–F): Endogenous ABCB5⁺ dermal MSCs express SOD3 in situ. Depicted in (D) are representative microphotographs of paraffin-embedded skin sections from human vasculitis patients with immunostaining of human ABCB5 (green) and human SOD3 (red) (left panel), and mice subjected to IC-mediated vasculitis with immunostaining of ABCB5 (green) and murine SOD3 (red) (right panel), respectively. Scale bar: 10 µm. The percentage of ABCB5⁺ SOD3⁺ cells in total dermal cells in each section are expressed as mean ± SEM in human (E) and murine (F) samples, respectively. n = 8; **, p < .01 by Student’s t tests. (G–H): 2 × 10⁶ mNeu were cultured alone or with non-transfected MSCs or control-siRNA transfected MSCs, or SOD3-silenced MSCs at a mNeu:MSC ratio of 10:1, and subjected to PMA-induced oxidative burst assay with reactive oxygen species indicator dihydrorhodamine 123. The depicted histograms in (G) are representative of three independent experiments. The mean fluorescence intensity (MFI) of each sample in (G) was normalized to MFI of PMA activated neutrophils as percentages in the same experiment. The chart depicted in (H) is mean ± SEM of percentage of MFI derived from three experiments. **, p < .01; ***, p < .001 by Student’s t tests. (I–J): 100 µl PBS or 2.5 × 10⁵ control-siRNA transfected MSCs or SOD3-silenced MSCs were intradermally injected into dorsal skin of mice. The Arthus reaction was elicited at the areas that had been injected with MSCs or PBS. Four hours later, mice were sacrificed and skin specimen were harvested and digitally photographed. The areas of Evans blue on skin specimen with MSCs treatment were normalized to Evans blue stained areas after PBS injection on the same mouse and expressed as percentages. n = 8; **, p < .01; ***, p < .001 by Mann–Whitney tests. Abbreviations: MSCs, mesenchymal stem cells; mNeu, murine bone marrow neutrophils; SOD3, superoxide dismutase 3.

Figure 6

Scheme of adaptive MSC responses to unrestrained neutrophil activation. Immune complexes inside and outside the vessel lead to unrestrained neutrophil activation with enhanced generation and release of $O_2^{-.}$ which can stimulate NET formation with expulsion of chromatin decorated with granules and highly concentrated MPO, NE and MMP-9, neutrophil death with spillage of their toxic cargo leading to tissue damage and tissue break down. Further damage to macromolecules occur by $O_2^{-.}$ and derivatives thereof like H₂O₂, ONNO. and hydroxyl radicals (HO^·). MSCs are able to mount an adaptive response to unrestrained neutrophil activation, with the release of $O_2^{-.}$ detoxifying extracellular SOD3 and ICAM-1/CD18 dependent engulfment of dying neutrophils thus preventing the spillage of their toxic cargo. By employing independent strategies MSCs effectively protect from tissue damage due to unrestrained neutrophil activation. Abbreviations: ICAM-1, intercellular adhesion molecule 1; H₂O₂, hydrogen peroxide; MMP-9, matrix metalloproteinase-9; MPO, myeloperoxidase; MSCs, mesenchymal stem cells; NE, neutrophil elastase; NET, neutrophil extracellular trap; ONNO., peroxynitrate; SOD3, superoxide dismutase 3.