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Membrane particles generated from mesenchymal stromal cells modulate immune responses by selective targeting of pro-inflammatory monocytes - PubMed

  • ️Sun Jan 01 2017

Membrane particles generated from mesenchymal stromal cells modulate immune responses by selective targeting of pro-inflammatory monocytes

Fabiany da C Gonçalves et al. Sci Rep. 2017.

Abstract

Mesenchymal stromal cells (MSC) are a promising therapy for immunological disorders. However, culture expanded MSC are large and get trapped in the capillary networks of the lungs after intravenous infusion, where they have a short survival time. Hypothetically, living cells are a risk for tumor formation. To reduce risks associated with MSC infusion and improve the distribution in the body, we generated membrane particles (MP) of MSC and MSC stimulated with IFN-γ (MPγ). Tracking analysis and electron microscopy indicated that the average size of MP was 120 nm, and they showed a round shape. MP exhibited ATPase, nucleotidase and esterase activity, indicating they are enzymatically active. MP and MPγ did not physically interact with T cells and had no effect on CD4+ and CD8+ T cells proliferation. However, MP and MPγ selectively bound to monocytes and decreased the frequency of pro-inflammatory CD14+CD16+ monocytes by induction of selective apoptosis. MP and MPγ increased the percentage of CD90 positive monocytes, and MPγ but not MP increased the percentage of anti-inflammatory PD-L1 monocytes. MPγ increased mRNA expression of PD-L1 in monocytes. These data demonstrate that MP have immunomodulatory properties and have potential as a novel cell-free therapy for treatment of immunological disorders.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1

Immunophenotype of unstimulated and IFN-γ stimulated AT-MSC. (a) Representative flow cytometry analysis of the commonly used markers for MSC (CD45 and CD31, both negative, and CD105, CD13, CD73, CD90), and the immune-markers HLA-I, HLA-II, and PD-L1. Isotype (white histograms), unstimulated AT-MSC (grey histograms) and IFN-γ AT-MSC (black histograms). (b) Percentage positive cells and (c) Mean fluorescence intensities (MFI) of the markers on unstimulated and IFN-γ stimulated AT-MSC. Data are presented as mean ± SD from 5 independent experiments. P values refer to the condition without IFN-γ. Unpaired t-test was used for statistical analysis.

Figure 2
Figure 2

Characterization of Membrane Particles generated from unstimulated and IFN-γ stimulated AT-MSC (MP and MPγ, respectively). (a) Nanoparticle tracking analysis (NTA) profiles of MP and MPγ. The NTA software generates a distribution graph on a particle-by-particle basis, a count (in terms of absolute number and concentration), and (b) size distribution of MP and MPγ. (c) The average number of particles generated per MSC. Data are presented as mean ± SD from 10 independent preparations of MP. There was no statistical difference with respect to concentration and size between MP and MPγ. The statistic test used was unpaired t-test. (d) Transmission electron microscopy analysis of MP. White arrows point to areas zoomed in on at the images on the right side. Most of the MP showed a round shape and a size below 200 nm.

Figure 3
Figure 3

Enzymatic activity of Membrane Particles. (a) ATPase activity was measured at four different concentrations of MP (1 × 1012, 1 × 1011, 1 × 1010 and 1 × 109 particles/ml). MP and MPγ were able to catalyze the breakdown of ATP and the detection of free phosphate was dependent on the concentration of MP. (b) The nucleotidase activity of the MSC marker CD73 was measured for three concentrations of MP (1 × 1012, 1 × 1011 and 1 × 1010 particles/ml). MP and MPγ were able to produce free phosphates after adding AMP substrate in a dose-dependent fashion. CD73 enzyme (2 and 1 ng) was used to calculate the concentration of CD73 in the MP. There was no statistical difference in enzyme activity between MP and MPγ. (c) Esterase activity of three concentrations of MP (1 × 109, 1 × 108 and 1 × 107 particles/ml) was measured by the conversion of CFDA-SE to CFSE by flow cytometry. Fluorescent events were observed in MP labeled with CFSE (CFSE-MP), and the number of CFSE-MP detected was dependent on the concentration of MP. There was no statistical difference between MP and MPγ in esterase activity. Controls (PBS + CFSE and non-labeled MP) were negative. Data are presented as mean ± SD. Enzyme activities were detected in MP generated from 5 different MSC donors.

Figure 4
Figure 4

Effect of Membrane Particles on lymphocyte proliferation. CFSE loaded PBMC stimulated with anti-CD3/antiCD28 antibody were cultured with different ratios of MP for 4 days (1:5,000, 1:10,000, 1:40,000 and 1:80,000). CFSE dilution in CD4+ and CD8+ T cells was measured. (a and b) Addition of MP or MPγ did not affect the proliferation of CD4+ and CD8+ T cells. (n = 8; mean ± SD). Two-way ANOVA was used for statistical analysis.

Figure 5
Figure 5

Effect of MP on CD14+ cells. Monocytes were cultured with different ratios of MP for 24 h (1:10,000, 1:40,000 and 1:80,000) to determine the effect of MP on monocyte immunophenotype. (a) Expression of CD16 on monocytes cultured in the presence of MP or MPγ (n = 6; mean ± SD). (b and c) Monocyte cell surface levels of CD90 and PD-L1 in the presence of MP or MPγ (n = 7; mean ± SD). (d) mRNA expression of monocytes after culture with MP. After 24 h of culture with MP or MPγ, monocytes were separated from MP and assessed by real-time RT-PCR for CD90, IDO, PD-L1, IL-6, TNF-α and IL-10 expression (n = 6; mean ± SD). Multiple comparison test (two-way ANOVA) was used for statistical analysis, *p < 0.05, **p < 0.01 and ***p < 0.001 vs control; # p < 0.05 and ## p < 0.01 vs MP group.

Figure 6
Figure 6

Effect of MP on apoptosis of monocyte subsets measured by Annexin V staining. Monocytes were cultured overnight with 3 ratios of MP or MPγ (1:10,000, 1:40,000 and 1:80,000). (a) Percentage of Annexin V positive CD14+CD16− classical monocytes, and (b) percentage of Annexin V positive CD14+CD16+ pro-inflammatory monocytes. Data represent mean ± SD of 5 experiments using MP from 3 different donors. Two-way ANOVA was used for statistical analysis. P values (*p < 0.05) refer to the control without MP.

Figure 7
Figure 7

Uptake of MP by monocytes. MSC were labeled with PKH-26 before generation of MP (PKH-MP). PKH-MP were added to PBMC (ratio 1:40,000) and incubated for 1 h and 24 h at 37 °C. As a control the experiment was incubated at 4 °C. (a and b) Representative flow cytometry analysis of PKH-MP uptake by lymphocytes (CD3) and monocytes (CD14) at time points 1 h and 24 h at 4 °C and at 37 °C. (c) Percentage of CD3+ T cells positive for PKH-MP, and (d) Percentage of CD14+ monocytes positive for PKH-MP. Data are presented as mean ± SD from 6 experiments. Two-way ANOVA was used for statistical analysis. P values (*p < 0.05) refer to the 4 °C control at the 1 h time point.

Figure 8
Figure 8

Confocal microscopy analysis of MP uptake by monocytes at 24 h. Z-stack images were collected at 1.2 μm intervals ranging from 0 to 17.6 μm. Staining for monocyte membrane (green), MP (red), and nucleus (blue) shows that MP are localized on the membrane of the monocytes (white arrows) and are not internalized. Scale bars: 5 μm.

Figure 9
Figure 9

Schematic overview of the interaction of MP with monocytes. MP generated from MSC bind to monocyte plasma membranes. As an effect of the MP-monocyte interaction, MP modulate monocyte function by affecting gene expression and inducing apoptosis of pro-inflammatory monocytes.

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References

    1. Bernardo, M. E. & Fibbe, W. E. Mesenchymal stromal cells: sensors and switchers of inflammation. Cell Stem Cell 13, 392–402, 10.1016/j.stem.2013.09.006 (2013). - PubMed
    1. Eggenhofer E, Luk F, Dahlke MH, Hoogduijn MJ. The life and fate of mesenchymal stem cells. Front Immunol. 2014;5:148. doi: 10.3389/fimmu.2014.00148. - DOI - PMC - PubMed
    1. Hao L, et al. Mesenchymal stromal cells for cell therapy: besides supporting hematopoiesis. Int J Hematol. 2012;95:34–46. doi: 10.1007/s12185-011-0991-8. - DOI - PubMed
    1. Le Blanc K, et al. Mesenchymal stem cells for treatment of steroid-resistant, severe, acute graft-versus-host disease: a phase II study. Lancet. 2008;371:1579–1586. doi: 10.1016/S0140-6736(08)60690-X. - DOI - PubMed
    1. Reinders ME, et al. Autologous bone marrow-derived mesenchymal stromal cells for the treatment of allograft rejection after renal transplantation: results of a phase I study. Stem Cells Transl Med. 2013;2:107–111. doi: 10.5966/sctm.2012-0114. - DOI - PMC - PubMed

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