Human Oral Epithelial Cells Impair Bacteria-Mediated Maturation of Dendritic Cells and Render T Cells Unresponsive to Stimulation

Abstract

The oral mucosa is a first line of defense against pathogenic organisms and yet tolerates food antigens and resident bacteria. Mucosal epithelial cells are emerging as important regulators of innate and adaptive immune responses. However, the contribution of oral epithelial cells (OECs) determining oral immunity is understudied. Here, we evaluated the ability of H413 and TR146 cells, two OEC lines derived from human oral squamous cell carcinomas, and primary OECs to modulate immune responses to a cocktail of Gram+ and Gram- bacteria known as MV130. OECs expressed CD40 constitutively and class II major histocompatibility complex (MHC II) molecules when stimulated with IFNγ, but not CD80 or CD86. Dendritic cells (DCs) treated with bacteria in co-culture with OECs did not fully mature, as judged by the expression of MHC II, CD80 and CD86, and barely released IL-12 and TNFα, compared to control DCs. Furthermore, in the presence of OECs, DCs were unable to stimulate allogenic naive CD4 T cells to produce IFNγ and TNFα. Similarly, OECs in culture with total CD4 T cells or Th1 cells stimulated with anti-CD3 and anti-CD28 antibodies abrogated CD25 and CD69 expression, T cell proliferation and the release of IFNγ and TNFα. The inhibition on T cell activation by OECs was cell-contact dependent, TGFβ independent and largely irreversible. Overall, this behavior of OECs is likely key to avoid immune system over-reaction against resident bacteria.

Keywords: T cells; bacteria; dendritic cells; immunomodulation; oral epithelial cells.

Figures

Figure 1

Cytokine secretion and surface markers expression in OECs. (A) Figure depicts the levels of various cytokines determined by ELISA on cell culture supernatants obtained from H413 and TR146 OEC lines and primary OECs incubated with media (gray bars) or the bacterial cocktail MV130 (black bars). Statistically significant differences (p < 0.05) between media and MV130 conditions are represented by “*”. Data were obtained from a total of seven independent experiments and we plotted mean values with error bars corresponding to standard errors of the mean (SEM). (B) Figure shows surface expression of CD40 and HLA-DR in H413, TR146 and primary OECs cultured with media, MV130 or IFNγ for 48 h, as determined by flow cytometry (black profiles). Isotype specific antibodies were used as negative control (gray profiles). Result shows MFI of a representative experiment out of three independent assays.

Figure 2

Effect of OECs on DC maturation and cytokine expression. (A) Expression of CD86, CD80, CD40 and HLA-DR in monocyte-derived DCs cultured alone (DC Ctrl) or in co-culture with OECs during 48 h (DC:H413; DC:TR146; DC: primary OECs). Black bars represent cells incubated with MV130 and gray bars represent cells in media. DCs are gated based on CD11c staining. (B) Cytokine secretion by DCs cultured with OECs. Figure shows the levels of TNFα, IL-12, IL-6, IL-8, IL-10 and TGFβ (pg/ml) determined by ELISA in supernatants of DCs cultured alone (DC Ctrl) or in co-culture with OECs during 48 h (DC:H413; DC:TR146; DC: primary OECs). Black bars represent cells incubated with MV130 and gray bars represent cells with media. Statistically significant differences (p < 0.05) between the different DC conditions and DC Ctrl were noted as “#” for media or as “*” for MV130. Data were obtained from a total of four independent experiments and we plotted mean values with error bars corresponding to SEM.

Figure 3

Analysis of cytokines in DC:T cell cultures under OEC conditioning. Figure depicts the levels of IFNγ, TNFα, IL-6, IL-4, TGFβ and IL-10 detected by ELISA in cultures of naive CD4 T cells activated by allogenic DCs (DC:CD4) alone (controls) or in co-culture with H413, TR146, or primary OECs (DC:OEC) with media (gray bars) or MV130 (black bars). Statistically significant differences (p < 0.05) between the different DC:CD4 conditions and DC:CD4 Ctrl were noted as “#” for media or as “*” for MV130. Data were obtained from a total of three independent experiments and we plotted mean values with error bars corresponding to SEM.

Figure 4

OECs alter the cytokine levels produced by CD4 T cells. Levels of IFNγ, TNFα and TGFβ determined by ELISA on cultures consisting of CD3/CD28 activated total CD4 T cells (A) or Th1 cells (B) alone (CD4 Ctrl), in the presence of OEC (CD4:OEC) or their conditioned media (CD4:CM). The experiments were carried out using H413, TR146 cell lines and primary OECs. Media and MV130 conditions are represented by gray and black bars, respectively. Statistically significant differences (p < 0.05) between the different CD4 T cells conditions and CD4 T cells Ctrl were noted as “#” for media or as “*” for MV130. Data were obtained from a total of three independent experiments and we plotted mean values with error bars corresponding to SEM.

Figure 5

OECs suppressed the activation and proliferation of activated CD4 T cells. (A) Expression of CD25 and CD69 activation markers on CD3/CD28 stimulated CD4 T cells cultured alone or with H413 cells. (B) CFSE staining and (C) annexin V and 7-AAD staining of activated CD4 T cells alone (CD4) or in culture with OEC (CD4:H413). (D) IFNγ, TNFα, and TFGβ on supernatants of CD3/CD28 stimulated CD4 T cell cultures consisting of: control CD4 T cells (CD4 Ctrl); CD4 T cells cultured with H413 cells (CD4:H413) and the same CD4 T cells isolated from H413 co-cultures and activated again with anti-CD3/CD28 antibodies (CD4 React). Statistically significant differences (p < 0.05) between the different CD4 T cells conditions and CD4 T cells Ctrl were noted with “*”. Data were obtained from a total of three independent experiments and we plotted mean values with error bars corresponding to SEM.

Figure 6

OEC-conditioning is not determined by TGFβ or cytokine consumption. (A) IFNγ and TNFα levels measured in co-cultures of H413 and CD3/CD28 stimulated CD4 T cells containing different concentrations of anti-TGFβ blocking antibody (ng/ml). Activated CD4 T cells alone were used as control. (B) Supernatants of CD3/CD28 activated CD4 T cells (CD4 SN) were assayed for IFNγ and TNFα prior to and after 24 and 48 h (gray and black bars, respectively) of being added to H413 cell cultures (H413: CD4 SN). Statistically significant differences (p < 0.05) shown in panel A between different CD4 T cell cultures and control CD4 T cells (left column) are noted as “*”. Data were obtained from a total of three independent experiments and we plotted mean values with error bars corresponding to SEM.

Figure 7

OEC-mediated immunomodulation under bacterial stimulation. Figure illustrates the experimental settings used in this work and the most relevant findings. (A) OECs response to MV130 stimulation. H413 and TR146 cells but not primary OECs constitutively released IL-6 and IL-8. These cytokines level increased with MV130 stimulation. (B) DCs did not fully mature in the presence of OECs. DC stimulated with MV130 expressed CD80, CD86 and HLA-DR, and released IL-12, IL-10 and TNFα (left), while in co-culture with OECs (right) showed decreased expression of HLA-DR, CD80 and CD86, and decreased release of IL-12, IL-10, and TNFα. (C) DCs failed to stimulate allogenic CD4 T cells in the presence of OECs. CD4 T cells stimulated with allogenic DCs previously treated with MV130 (left) released IFNγ and TNFα but did not when in co-culture with OEC (right). (D) CD4 T cell activation was compromised in the presence of OECs. CD3/CD28-activated CD4 T cells expressed CD25 and CD69 and released IFNγ and TNFα (left) unlike their counterparts in co-culture with OECs (right).