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Interleukin-10 production and T cell-suppressive capacity in B cell subsets from atherosclerotic apoE −/− mice

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Abstract

The evidence regarding the role of regulatory B cells (Breg) in atherosclerosis are scarce, and there are contradictory data about their atheroprotective properties. Due to the demonstrated protective function of Breg in different inflammatory diseases mainly through interleukin-10 (IL-10) production, the knowledge of their participation in atherosclerosis immunopathology would be very valuable. To further study which B cell subsets participate in IL-10 production and their regulatory role, splenocytes from apolipoprotein-E-deficient mice were evaluated by ex vivo and in vitro cultures. Atherosclerotic mice had increased frequency of IL-10+ B cells, which presented high CD1d, CD19, and IgM, but variable CD5, CD21, and CD23 expression. IL-10+ B cells were not enriched in B cell subsets previously reported as Breg. Increased frequency of IL-10+ B cells with transitional 1-like (T1-like) and follicular (FO) and reduced CD5+ and marginal zone (MZ) phenotypes were observed ex vivo. Increased frequency of IL-10+ B cells with T1-like and MZ, and decreased IL-10+ FO and T2 phenotypes were also observed in vitro. To determine regulatory capacity of B cells in the atherosclerotic model, each subset were co-cultured with CD4+CD25− T cells. CD5+, FO, MZ, and T1-like cells from atherosclerotic mice exhibited regulation in an IL-10-dependent manner. However, only FO cells decreased both frequency of interferon gamma (IFN-γ)+ and tumor necrosis factor alpha (TNF-α)+ and proliferation of T cells. Finally, splenocytes showed increased frequency of IFN-γ+ and TNF-α+ cells only when FO-depleted B cells were evaluated. These results suggest that mainly FO B cells can modulate in some level the inflammatory responses observed in atherosclerosis.

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Acknowledgments

The authors thank Professor Andres Baena (Grupo de Inmunología Celular e Inmunogenética, Universidad de Antioquia, Colombia) for providing us with some of the reagents used in this study. We are also grateful to Julio Jaramillo for their support in animal breeding and care. This work received financial support from Colciencias (project number 111554431390), Sostenibilidad de la Universidad de Antioquia, and the program Jóvenes Investigadores e Innovadores de Colciencias. Professor Lina Yassin was supported by Universidad CES (Facultad de Medicina-Dirección de Gestión del conocimiento).

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Author notes

  1. Diana Castaño and Lina M. Yassin contributed equally to the present work.

Authors and Affiliations

  1. Grupo de Inmunología Celular e Inmunogenética, Instituto de Investigaciones Médicas, Facultad de Medicina, Universidad de Antioquia UdeA, Calle 70 No 52-21, Medellín, Colombia

    Héctor Rincón-Arévalo, Mauricio Rojas, Gloria Vásquez & Diana Castaño

  2. Grupo Inmunomodulación, Instituto de Investigaciones Médicas, Facultad de Medicina, Universidad de Antioquia UdeA, Calle 70 No 52-21, Medellín, Colombia

    Janny Villa-Pulgarín, Jorge Tabares & José R. Ramírez-Pineda

  3. Unidad de Citometría, Facultad de Medicina, Sede de Investigación Universitaria, Universidad de Antioquia UdeA, Calle 70 No 52-21, Medellín, Colombia

    Mauricio Rojas

  4. Grupo de Investigaciones Biomédicas Uniremington, Corporación Universitaria Remington, Medellín, Colombia

    Lina M. Yassin

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  1. Héctor Rincón-Arévalo
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  2. Janny Villa-Pulgarín
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  3. Jorge Tabares
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All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. All procedures performed in studies involving animals were in accordance with the ethical standards of the institution or practice at which the studies were conducted.

Electronic supplementary material

ESM S1

Frequency of B cells secreting IL-10 in atherosclerotic mice. Percentage of IL-10 secreting splenic B cells from WT-HFD and apoE −/−HFD mice after A. ex vivo and B. in vitro cultures. Briefly, isolated B cells were cultured as previously described, incubated for the last 45 min with an anti-IL-10 conjugated to an anti-CD45 antibody to capture secreted IL-10 by each B cell, and finally extracellularly labeled with anti-IL-10. Median and interquartile range of IL-10+ B cells from three to seven mice per group are shown from two independent experiments. Mann-Whitney test. *p < 0.05. (GIF 9 kb)

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ESM S2

Frequency of aortic IL-10+ B cells after ex vivo culture. Aortic B cells were cultured ex vivo as explained in the “Materials and methods.” Median and interquartile range from nine to ten mice per group are shown from two independent experiments. Mann-Whitney test. (GIF 3 kb)

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ESM S3

Frequency of Foxp3+ cells in IL-10+ B cells region. A. Representative pseudocolor plots of Foxp3 expression in splenic IL-10+ (left) and IL-10− B cells (right) after ex vivo culture. B-C. Frequency of Foxp3+ in IL-10+ and IL-10-splenic B cells after B. ex vivo and C. in vitro cultures. Median and interquartile range from nine mice (WT-HFD and apoE −/−) are shown. D-E. Frequency of Foxp3+ cells in splenic IL-10+ B cells from WT-HFD and apoE −/−HFD mice after D. ex vivo and E. in vitro cultures. Median and interquartile range from four to five mice per group are shown. Mann-Whitney test. ∞ p < 0.0001; Φ p < 0.001; *p < 0.05. (GIF 27 kb)

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ESM S4

B cell subsets analysis. Representative pseudocolor plots of CD5+, follicular (FO, CD5−CD21lowCD23+CD24low), transitional 1-like (T1-like, CD5−CD21lowCD23−CD24low), transitional 2 (T2, CD5−CD21hiCD23+CD24hi), and marginal zone (MZ, CD5−CD21hiCD23−CD24hi) B cell subsets from WT-HFD mouse after A ex vivo and B in vitro cultures. (GIF 18 kb)

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ESM S5

TNF-α+ and IFN-γ+ T cells analysis. A. Representative pseudocolor plot of gating strategy used in co-cultures and depleted cultures. After exclusion of aggregates (left), cells were selected by negative live/dead expression (center). Only in co-cultures CD4+ were gated (right). B. Representative pseudocolor plot for TNF-α+ and IFN-γ+ cells in CD4+ T cells (from A.) in splenic-enriched T cells from a young WT mice cultured without B cells from apoE −/−HFD mice, as is mentioned in “Materials and methods.” C. Representative analysis of CD4+ T cells proliferation in the co-cultures. Orange non-proliferating CD4+ T cells. Pink proliferating CD4+ T cells. (GIF 68 kb)

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ESM S6

Representative pseudocolor plots of enriched B cell subsets from a WT-HFD mouse, according to CD5, CD21, and CD23 markers for the sorting of B cells subsets. A. B cells before sorting. B-F. B cell subsets after sorting. B. CD5+, C. follicular (FO, CD5−CD21lowCD23+CD24low), D. transitional 1-like (T1-like, CD5−CD21lowCD23−CD24low), E. transitional 2 (T2, CD5−CD21hiCD23+CD24hi), and F. marginal zone (MZ, CD5−CD21hiCD23−CD24hi) B cell subsets. (GIF 22 kb)

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ESM S7

Proliferation of CD4+CD25− T cells co-cultured with each B cell subsets from atherosclerotic mice. A. Fold change in the proliferation index (total number of divisions per the number of cells that went into division) of CD4+CD25− T cells co-cultured with each B cell subset (CD5+, follicular (FO), marginal zone (MZ), transitional 1-like (T1-like), and transitional 2 (T2)) from apoE −/−HFD mice. B. Fold change in the division index (average number of cell divisions that a cell in the original population has undergone) of T cells co-cultured with B cell subsets from apoE −/−HFD mice. Median and interquartile range from 8 apoE −/−HFD mice for each co-culture are shown from two independent experiments. Two-way ANOVA test with Šídák post-test. Differences between co-cultures and cultures without B cells were indicated as follows: ∞ p < 0.0001; Φ p < 0.001; # p < 0.01; *p < 0.05. Symbol on the bracket indicates difference between co-cultures at a ratio of 1:1 and 2:1. The threshold on the y-axis was stablished based on T cells cultured without B cells. (GIF 27 kb)

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ESM S8

Frequencies of B cell subsets after culture. A. Percentage of B cell subsets after A. ex vivo and B. in vitro cultures. Median and interquartile range from 13 to 14 mice per group are shown from two independent experiments. Kruskal-Wallis test with Dunn’s post-test. ∞ p < 0.0001; Φ p < 0.001; # p < 0.01; *p < 0.05. (GIF 48 kb)

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Rincón-Arévalo, H., Villa-Pulgarín, J., Tabares, J. et al. Interleukin-10 production and T cell-suppressive capacity in B cell subsets from atherosclerotic apoE −/− mice. Immunol Res 65, 995–1008 (2017). https://doi.org/10.1007/s12026-017-8939-6

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