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. 2019 Apr 18:2019:7029726.
doi: 10.1155/2019/7029726. eCollection 2019.

The Effects of Immunosuppressive Factors on Primary Dendritic Cells from C57BL/6 and CBA Mice

Vasiliy V Kurilin  1 , Julia N Khantakova  1   2 , Valeriy P Tereschenko  1 , Julia A Lopatnikova  1 , Irina A Obleukhova  1 , Sergey V Sennikov  1   2
Affiliations

The Effects of Immunosuppressive Factors on Primary Dendritic Cells from C57BL/6 and CBA Mice

Vasiliy V Kurilin et al. J Immunol Res. .

Abstract

Introduction: Dendritic cells (DCs) control immune responses by modulating T and B cells towards effector or tolerogenic responses. In this study, we evaluated the effects of different immunosuppressive molecules on the phenotypic and functional characteristics of primary dendritic cells from C57BL/6 and CBA mice.

Methods: DCs were derived from bone marrow cells in the presence of rmGM-CSF and rmIL-4. DCs were then treated with different types of immunosuppressive molecules (rmIL-10, rmTGF-β, and BAY 11-7082) and cocultured with syngeneic splenocytes. The amount of CD4+CD25hiFoxP3+ Tregs, IL-10 expression, and proliferation were evaluated.

Results: Tolerogenic factors were found to have different effects on DCs C57Bl/6 mice. In C57Bl/6 mice, BAY 11-7082 alone had no effect on the expression of DC maturation molecules (CD80, CD86). Transforming growth factor beta (TGF-β), alone and in combination with BAY 11-7082, reduced the expression of these molecules. Cocultivation of DCs with splenocytes in the presence of TGF-β and BAY 11-7082 favored regulatory T cell (CD4+CD25hiFoxP3+) differentiation and disfavored differentiation of CD4+ T cells producing IL-10. In CBA mice, we found that rmIL-10 and rmTGF-β have a weak effect on maturation of DCs and their functional properties to induce Treg cells and IL-10 production.

Conclusion: These results indicate that TGF-β and IL-10 have different effects on the phenotypic and functional characteristics of DCs and that the NF-κB inhibitor, BAY 11-7082, has no synergistic effect on these treatments. In mice with an opposite nature of the immune response, the effects of immunoregulatory cytokines (IL-10 and TGF-b) differ on maturation of dendritic cells.

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Figures

Figure 1
Figure 1
Expression of costimulatory molecules on bone marrow-derived DCs obtained from C57BL/6 mice. (a) Expression of H-2b on CD11c+ DCs. (b) Expression of CD86 on CD11c+H-2b+ DCs. (c) Expression of CD80 on CD11c+H-2b+DCs. (d) Expression of CD80/86 on CD11c+H-2b+ DCs. N = 18 mice per group. At least three independent experiments were performed. The arrows show significant intergroup differences (p < 0.05). Notes: GM-CSF+IL-4—BMCs cultured in the presence of GM-CSF and IL-4 alone; Bay—BMCs cultured in the presence of GM-CSF, IL-4, and BAY11-7082 added on day 2 of culturing; IL-10—BMCs cultured in the presence of GM-CSF, IL-4, and IL-10 added on day 3 of culturing; TGF-β—BMCs cultured in the presence of GM-CSF, IL-4, and TGF-β added on day 3 of culturing. Bay+IL-10—BMCs cultured in the presence of GM-CSF, IL-4, and BAY 11-7082 added on day 2 of culturing and IL-10 added on day 3 of culturing; Bay+TGFβ—BMCs cultured in the presence of GM-CSF, IL-4, and BAY 11-7082 added on day 2 of culturing and TGF-β added on day 3 of culturing.
Figure 2
Figure 2
Expression of costimulatory molecules on bone marrow-derived DCs from C57BL/6 mice. (a) Expression of H-2b on CD11c+ DCs. (b) Expression of CD80/86 on CD11c+H-2b+ DCs.
Figure 3
Figure 3
Expression of costimulatory molecules on bone marrow-derived DCs from CBA. (a) Expression of H-2K on CD11c+ DCs. (b) Expression of CD86 on CD11c+H-2k+ DCs. (c) Expression of CD80 on CD11c+H-2k+ DCs. (d) Expression of CD80/86 on CD11c+H-2k+ DCs. N = 18 mice per group. At least three independent experiments were performed. The arrows show significant intergroup differences (p < 0.05). Notes: GM-CSF+IL-4—BMCs cultured in the presence of GM-CSF and IL-4 alone; Bay—BMCs cultured in the presence of GM-CSF, IL-4, and BAY11-7082 added on day 2 of culturing; IL-10—BMCs cultured in the presence of GM-CSF, IL-4, and IL-10 added on day 3 of culturing; TGF-β—BMCs cultured in the presence of GM-CSF, IL-4, and TGF-β added on day 3 of culturing.
Figure 4
Figure 4
Expression of Treg markers in the cocultures of splenocytes and DCs from C57BL/6 and CBA mice. (a) The relative frequency of CD4+CD25+ cells in C57Bl/6 mice. (b) The relative frequency of FoxP3+ cells within CD4+CD25+ cells in C57Bl/6 mice. (c) The relative frequency of CD4+CD25+ cells in CBA mice. (d) The relative frequency of FoxP3+ cells in the CD4+CD25+ population in CBA mice. N = 18 mice per group. At least three independent experiments were performed. The arrows show significant intergroup differences (p < 0.05). Note: DC+Spl—coculture of immature DCs and splenocytes; DC-Bay+Spl—coculture of DCs treated with BAY 11-7082 and splenocytes; DC-IL-10+Spl—coculture of DCs treated with IL-10 and splenocytes; DC-TGF-β+Spl—coculture of DCs treated TGF-β and splenocytes; DC (Bay+IL-10)+Spl—coculture of DCs treated with BAY 11-7082 and IL-10 and splenocytes; DC (Bay+TGFβ)+Spl—cocultures of DCs treated with a combination of BAY 11-7082 and TGF-β and splenocytes.
Figure 5
Figure 5
The frequency of IL-10-producing cells among CD4+ lymphocytes in the coculture of splenocytes and DCs in (a) C57Bl/6 mice and (b) CBA mice. N = 18 mice per group. At least three independent experiments were performed. The arrows show significant intergroup differences (p < 0.05, N = 18). Note: DC+Spl—coculture of immature DCs and splenocytes; DC-Bay+Spl—coculture of DCs treated with BAY 11-7082 and splenocytes; DC-IL-10+Spl—coculture of DCs treated with IL-10 and splenocytes; DC-TGF-β+Spl—coculture of DCs treated with TGF-β and splenocytes; DC (Bay+IL-10)+Spl—coculture of DCs treated with BAY 11-7082 and IL-10 and splenocytes; DC (Bay+TGFβ)+Spl—coculture of DCs treated with BAY 11-7082 and TGF-β and splenocytes.
Figure 6
Figure 6
IL-10 level in the conditioned media of cocultures of splenocytes and induced tolerogenic DCs in (a) C57Bl/6 mice and (b) CBA mice. N = 18 mice per group. At least three independent experiments were performed. The arrows indicate significant intergroup differences (p < 0.05, N = 18). Note: DC+Spl—coculture of immature DCs and splenocytes; DC-Bay+Spl—coculture of DCs treated with BAY 11-7082 and splenocytes; DC-IL-10+Spl—coculture of DCs treated with IL-10 and splenocytes; DC-TGF-β+Spl—coculture of DCs treated with TGF-β and splenocytes; DC (Bay+IL-10)+Spl—coculture of DCs treated with BAY 11-7082 and IL-10 and splenocytes; DC (Bay+TGFβ)+Spl—coculture of DCs treated with BAY 11-7082 and TGF-β and splenocytes.
Figure 7
Figure 7
The proliferation index of the cocultures of splenocytes and DCs. The arrows indicate significant intergroup differences (p < 0.05, N = 18). Note: DC+Spl—coculture of immature DCs and splenocytes; DC-Bay+Spl—coculture of DCs treated with BAY 11-7082 and splenocytes; DC-IL-10+Spl—coculture of DCs treated with IL-10 and splenocytes; DC-TGF-β+Spl—coculture of DCs treated with TGF-β and splenocytes; DC (Bay+IL-10)+Spl—coculture of DCs treated with BAY 11-7082 and IL-10 and splenocytes; DC (Bay+TGFβ)+Spl—coculture of DCs treated with BAY 11-7082 and TGF-β and splenocytes.
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