Tbet Expression in Regulatory T Cells Is Required to Initiate Th1-Mediated Colitis

Abstract

In normal conditions gut homeostasis is maintained by the suppressive activity of regulatory T cells (Tregs), characterized by the expression of the transcription factor FoxP3. In human inflammatory bowel disease, which is believed to be the consequence of the loss of tolerance toward antigens normally contained in the gut lumen, Tregs have been found to be increased and functionally active, thus pointing against their possible role in the pathogenesis of this immune-mediated disease. Though, in inflammatory conditions, Tregs have been shown to upregulate the T helper (Th) type 1-related transcription factor Tbet and to express the pro-inflammatory cytokine IFNγ, thus suggesting that at a certain point of the inflammatory process, Tregs might contribute to inflammation rather than suppress it. Starting from the observation that Tregs isolated from the lamina propria of active but not inactive IBD patients or uninflamed controls express Tbet and IFNγ, we investigated the functional role of Th1-like Tregs in the dextran sulfate model of colitis. As observed in human IBD, Th1-like Tregs were upregulated in the inflamed lamina propria of treated mice and the expression of Tbet and IFNγ in Tregs preceded the accumulation of conventional Th1 cells. By using a Treg-specific Tbet conditional knockout, we demonstrated that Tbet expression in Tregs is required for the development of colitis. Indeed, Tbet knockout mice developed milder colitis and showed an impaired Th1 immune response. In these mice not only the Tbet deficient Tregs but also the Tbet proficient conventional T cells showed reduced IFNγ expression. However, Tbet deficiency did not affect the Tregs suppressive capacity in vitro and in vivo in the adoptive transfer model of colitis. In conclusion here we show that Tbet expression by Tregs sustains the early phase of the Th1-mediated inflammatory response in the gut.

Keywords: Tbet; Th1-like Tregs; Treg cells; inflammation; inflammatory bowel disease.

Figure 1

Lamina propria (LP) FoxP3+CD4+ T cells from IBD patients express IFNγ. (A) Flow cytometric analysis of IFNγ expression in CD4+FoxP3+ regulatory T cells (Tregs) and CD4+FoxP3- conventional T cells (ConvT) cells from LP cells of IBD patients and controls: gating strategy. (B) Flow cytometric analysis of IFNγ expression in Tregs (upper panel) and ConvT (lower panel) from ileal Crohn's disease (CD; n = 8), control ileum (n = 7), colonic CD (n = 5), active ulcerative colitis (UC; n = 7), inactive UC (n = 4), and control colon (n = 5). (C,D) Representative dot plot representing FoxP3 and Tbet expression in LP CD4+ T cells (C) and Tbet and IFNγ expression in LP FoxP3+CD4+ T cells (D) from IBD patients. (E) Frequency of IFNγ+ cells among Tbet+ and Tbet- Tregs in CD (n = 4) and UC (n = 3) patients. Numbers in the dot plot quadrants and histograms represent the relative frequency of cell subpopulations. Horizontal bars in (B,D) represent the mean value ±SD. *p < 0.05; **p < 0.01.

Figure 2

(A) Representative dot plots showing FoxP3^eGFP and Tbet expression in LP CD3+CD4+ T cells isolated from untreated and DSS-treated mice. Frequency (B) and absolute numbers (C) of Tbet+ cells among total CD4+, Treg and ConvT cells as indicated. (D) Representative FoxP3^eGFP and IFNγ expression of the same cells as in (A). Frequency (E) and absolute (F) numbers of IFNγ+ cells among total CD4+, Treg, and ConvT cells as indicated. Numbers in the dot plot quadrants represent the relative frequency of cell subpopulations. Horizontal bars indicate the mean, symbols indicate each analyzed mouse. Vertical bars indicate mean ±SEM. **p < 0.01, ns, not significant.

Figure 3

(A) Representative histological sections of colons from DSS-treated mice (upper panel 20x magnification, lower panel 40x magnification) at different time points as indicated. Bars in the pictures indicate the scale. (B) Cumulative histologic score of DSS-treated mice pooled from three independent experiments at different time points, bars indicate the mean ±SD. (C) Percent of Tbet+ cells among LP Tregs and ConvT cells from DSS-treated mice at different time points, symbols indicate each analyzed mouse, bars indicate the mean. (D) Tbet+ cells fold increase relative to baseline (day 0) in Tregs and ConvT cells. Numbers in the dot plot quadrants represent the cell relative frequency. *p < 0.05; **p < 0.01; ns, not significant.

Figure 4

Total CD3+CD4+ T cells from the spleen of reporter mice were polyclonally activated in vitro. (A) Representative dot plots representing Tbet and IFNγ expression gating on Tregs and ConvT after 48 h of stimulation. Tbet and IFNγ isotype control stainings gating on CD4+ cells are shown (upper right plot). Percent of Tbet+ (B) and IFNγ+ (C) cells among Treg and ConvT cells at different time points. Each point represents the mean ±SD of three independent experiment performed.

Figure 5

Total CD3+CD4+ T cells from the spleen of reporter mice were polyclonally activated in the presence of neutralizing anti-IFNγ antibody or control IgG. (A) Representative dot plots showing Tbet and IFNγ expression on gated Treg and ConvT cells after 24 h of stimulation. Tbet and IFNγ isotype control stainings gating on CD4+ cells are shown (upper right plot). (B,C) Percent of Tbet+ cells among Treg (B) and ConvT (C) cells in the presence of anti-IFNγ or control IgG at different time points. Each point represents the mean ±SD of three experiment performed. (D) Representative dot plots showing FoxP3 and Tbet expression in LP CD3+CD4+ T cells of DSS-treated IFNγ^ko and IFNγ^het mice. Numbers in the dot plot quadrants represent the relative frequency of cell subpopulations.

Figure 6

(A) Body weight variation relative to the baseline of Treg-specific FoxP3 conditional knockout (FoxP3^CreTbx21^fl/fl) and control mice (FoxP3^CreTbx21^wt/wt) upon DSS treatment. Results from one representative experiment out of four performed is shown (mean percentage ±SEM). (B) Endoscopic pictures and endoscopic severity grading of knockout and control mice at the end of one representative experiment (Day 10). (C) Representative histological section from the colons of FoxP3 knockout and control mice at the end of the experiment and cumulative histologic severity scoring of the experiment shown in (A). lcn-2 (D) and cytokine (E,G) mRNA expression in the colon tissue of DSS-treated FoxP3 knockout and control mice. Vertical bars indicate the average ±SD analyzed in the pool of mice from four independent experiments. (F) Fold change of cytokine mRNA expression, as indicated, in FoxP3 knockout mice relative to control mice. *p < 0.05; **p < 0.01.

Figure 7

(A) Representative dot plots showing FoxP3^eGFP and Tbet expression in LP CD3+CD4+ cells isolated from the colons of Treg-specific FoxP3 conditional knockout (FoxP3^CreTbx21^fl/fl) and control mice (FoxP3^CreTbx21^wt/wt). (B) Frequency of Tbet+ cells among LP Treg and ConvT cells from Tbet knock out and control mice. (C) Representative dot plots showing IFNγ and Tbet expression in LP ConvT cells isolated from Tbet knockout and control mice. (D) Frequency of Tbet+IFNγ cells among LP Treg and ConvT cells from Tbet knockout and control mice. Numbers in the dot plot quadrants represent the cell subpopulation relative frequency. Horizontal bars in (B,D) indicate the mean value and symbols represent each animal analyzed from three independent experiments. *p < 0.05; **p < 0.01.

Figure 8

(A) Representative dot plots showing the expression of FoxP3^eGFP and Tbet in CD3+CD4+ T cells from the spleen of Treg-specific Tbet conditional knockout (Foxp3^CreTbx21^fl/fl) or control mice (Foxp3^CreTbx21^wt/wt) polyclonally activated in vitro for 12 h. (B) Frequency of Tbet+ cells gated on ConvT cells of CD3+CD4+ T cells from Tbet knockout or control mice polyclonally activated in vitro for 12 h or left unstimulated. (C) Frequency of Tbet+IFNγ+ cells gated on Treg and ConvT cells of CD3+CD4+ Tcells knockout and control mice as in (B). Numbers in the dot plot quadrants represent the relative frequency of cell subpopulations. Horizontal bars in (B,C) indicate the mean value of results obtained from three independent experiments. **p < 0.01.