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The Complement System Is Essential for the Phagocytosis of Mesenchymal Stromal Cells by Monocytes - PubMed

  • ️Tue Jan 01 2019

The Complement System Is Essential for the Phagocytosis of Mesenchymal Stromal Cells by Monocytes

Caroline Gavin et al. Front Immunol. 2019.

Abstract

Mesenchymal stromal cell (MSC) therapy is a promising tool in the treatment of chronic inflammatory diseases. This has been ascribed to the capacity of MSC to release a large variety of immune-modulatory factors. However, all aspects of the mode of therapeutic MSC action in different diseases remain unresolved, mainly because most of the infused MSC are undetectable in the circulation within hours after infusion. The aim of this study was to elucidate the fate of MSC after contact with plasma. We found that upon contact with blood, complement proteins including C3b/iC3b are deposited on MSC. Importantly, we also found that complement bound to MSC enhanced their phagocytosis by classical and intermediate monocytes via a mechanism that involves C3 but not C5. Thus, we describe for the first time a mechanism which might explain, at least partly, why MSC are not found in the blood circulation after infusion. Our results indicate that MSC immune-modulatory effects could be mediated by monocytes that have phagocytosed them.

Keywords: MSC; complement; fate; live; monocytes; phagocytosis; plasma.

Copyright © 2019 Gavin, Meinke, Heldring, Heck, Achour, Iacobaeus, Höglund, Le Blanc and Kadri.

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Figures

Figure 1
Figure 1

Survival and function of MSC after their contact with plasma. (A) Contour plots from flow cytometry analysis of MSC stained with anti-C3c FITC or specific isotope control to detect C3b/iC3b deposition after exposure to plasma, heat inactivated (HI) plasma or untreated MSC. (B) FlowSight images of MSC incubated with blood compounds in the presence or absence of 10 mM EDTA for 30 min. MSC were stained with C3c-FITC and CD73-PE. Brightfield image of MSC is shown in the left part of the image. (C) Percentage of C3c-FITC binding to MSC after exposure to different conditions (media, EDTA-plasma, HI-plasma, plasma, purified C3 protein) for 1 h. (D) Representative contour plot showing FSC and SSC of MSC after their exposure to plasma. Bars represent percentage of live (aqua live dead negative) MSC after 1 h incubation in control vs. plasma from two different experiments. Data shown are representative of two experiments of five different MSC. (E) Inhibition of proliferation of 5 days activated T cells (n = 3) in the presence of MSC (n = 7) at the indicated ratios. Data shown are means and SD of two independent experiments. (F) Intracellular IDO or IL-6 expression was measured by flow cytometry in MSC exposed to control media, inactive complement plasma (+ EDTA, 10 mM) or active complement plasma for 1 h, thereafter washed and treated with TNF-α and IFN-γ for 72 h. Data shown are representative from four MSC. (G) Expression of complement regulatory proteins CD46, CD55, and CD59 on MSC cultured in complete medium, HI plasma or plasma was analyzed by flow cytometry. Mean fluorescent intensities of MSC from five MSC in two different experiments are displayed with mean and SD.

Figure 2
Figure 2

Effects of blocking complement regulators on MSC pre-treated with plasma. (A) Representative contour plots of calcein RO-stained MSC exposed to plasma, HI plasma or medium for 1 h in the presence or absence of blocking antibodies against complement inhibitors CD46, CD55, or CD59. (B) Percentage of calcein leakage, and (C) percentage of C3b/iC3b binding to MSC pre-treated with anti-CD46, anti-CD55, and anti-CD59 blocking antibodies in the presence of active plasma. Data shown are means and SD (n = 4). (D) FlowSight images showing C3b/iC3b binding and 7-AAD (death marker) on MSC in the presence or absence of anti-CD59 blocking antibody in the blood chamber experiments. (E) Percentage of live cells (aqua live dead negative) of MSC pre-treated with plasma in the presence or absence of anti-CD59 blocking antibodies. Data shown are means and SD (n = 4). Data are representative of two independent experiments. Statistical significance was determined using paired t-test *p = 0.05 and ***p = 0.0001.

Figure 3
Figure 3

Complement enhances phagocytosis of MSC by monocytes. MSC were labeled with pHrodo and incubated with or without active plasma for 1 h, washed and co-cultured with THP-1 cells (A,B) or PBMC (D,E). Phagocytosis was analyzed after 2 h co-culture. Phagocytosis inhibitor Cytochalasin D (CytoD) was added as a negative control. (A) Representative plots of flow-cytometric analysis of phagocytosis by THP-1 cells. pHrodo bright fluorescence indicates cells that have phagocytosed labeled MSC. (B) Pooled data of THP-1 cell phagocytosis, bars represent means with SD (n = 4) from two independent experiments. (C) Gating strategy of CD14+ monocytes in freshly isolated PBMC. (D) Representative plots of flow-cytometry analysis of phagocytosis by monocytes. pHrodo bright fluorescence indicates cells that have phagocytosed labeled MSC. (E) Pooled data from MSC phagocytosis by monocytes, bars represent means with SD (n = 4) of two independent experiments. Statistical significance was determined using ANOVA followed by Holm-Sidak's multiple comparisons test *p = 0.05, **p = 0.001.

Figure 4
Figure 4

Phagocytosis of MSC is mediated by classical and intermediate monocytes. (A) Gating strategy on different subsets of monocytes in freshly isolated PBMC. Classical monocytes (CD14+ CD16−), intermediate monocytes (CD14+ CD16+), and non-classical monocytes (CD14− CD16+). (B) Representative flow cytometry plots showing distribution of different subsets of monocytes on gated pHrodo Bright and pHrodo Low. (C) Distribution of the three monocyte subsets within the pHrodo Bright population. Bars represent mean values with SD (n = 4 PBMC) of pooled data from two different experiments.

Figure 5
Figure 5

Mechanism of MSC phagocytosis by monocytes. MSC were labeled with pHrodo and incubated with or without active plasma for 1 h in the presence or absence of the C3 inhibitor compstatin or the C5 inhibitor soliris. Cells were washed and co-cultured with PBMC for 2 h. (A) Representative plots of flow-cytometric analysis of phagocytosis by monocytes. pHrodo bright fluorescence indicates cells that have phagocytosed labeled MSC. (B) Pooled data from two independent experiments using a total of four different MSC. Bars represent mean values with SD (n = 4). Statistical significance was determined using ANOVA followed by Holm-Sidak's multiple comparisons test **p = 0.001. (C) Percentage of reduction in phagocytosis in the presence of C3 or C5 inhibitor calculated from the data shown in (B) and the percentage of phagocytosis observed in plasma free medium was subtracted. (D) Correlation between percentage of C3b/iC3b binding to MSC and percentage of pHrodo bright monocytes from HI plasma and plasma in the phagocytosis experiments.

Figure 6
Figure 6

Model of MSC fate after interaction with blood. (A) Increase in C3 binding and decrease in the survival of plasma pre-treated MSC in the presence of anti-CD59. (B) C3 binds to the surface of MSC, probably through the alternative complement pathway. CD59 blocks the membrane attack complex from forming. MSC are phagocytosed by classical and intermediate monocytes, mainly mediated by the presence of C3 on the MSC surface. The receptor binding to C3 and inducing the phagocytosis remains unknown.

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