CD3+CD4+LAP+Foxp3-Regulatory Cells of the Colonic Lamina Propria Limit Disease Extension in Ulcerative Colitis

Abstract

Background and Aims: In ulcerative colitis (UC), inflammation begins in the rectum and can extend proximally throughout the entire colon. The extension of inflammation is an important determinant of disease course, and may be limited by the action of regulatory T cells (Tregs). In this cross-sectional study, we evaluated the relationship between UC extension and the proportions of CD3+CD4+Foxp3+ and CD3+CD4+LAP+Foxp3-Tregs in the colonic lamina propria (LP) of 79 UC patients and 29 controls. The role of these cells in UC extension was also investigated in the murine oxazolone-induced colitis model. Methods: Patients: Disease extension was classified according to the Montreal classification. Where possible, endoscopic biopsies of involved and uninvolved tissue were obtained from UC patients. Mouse model: Colitis was induced by intrarectal oxazolone administration. Lamina propria mononuclear cells were isolated from patient biopsies and mouse colon tissue using enzymatic method and the percentage of CD3+CD4+Foxp3+ and CD3+CD4+LAP+Foxp3-cells evaluated by immunofluorescence. Confocal microscopy was applied for the visualization and quantification of CD4+LAP+ cells on tissue histological sections. Results: In UC patients with distal colitis the proportion of LP CD3+CD4+Foxp3+ Tregs was significantly higher in inflamed tissue than uninvolved tissue. As opposite, the proportion of LP CD3+CD4+LAP+ Tregs was significantly higher in uninvolved tissue than involved tissue. Both LP CD3+CD4+Foxp3+ and LP CD3+CD4+LAP+ Tregs proportion in involved tissue was significantly higher than in controls irrespective of the extension of inflammation. In mice with oxazolone-induced distal colitis, treatment with LAP-depleting antibody was associated with the development of extensive colitis. Conclusions: Our findings suggest that CD3+CD4+LAP+Foxp3-Tregs limit the extension of inflammatory lesions in UC patients.

Keywords: CD3+CD4+LAP+Foxp3-regulatory cells; immunity; inflammation; oxazolone colitis; regulatory T cells; ulcerative colitis.

Figure 1

The frequency of LP CD3+CD4+LAP+ cells is higher in uninvolved (Uninv) vs. involved (Inv) colon tissue from UC patients. (A) Frequency of LP Foxp3+ cells in the CD3+CD4+-gated LPMC population: *P < 0.05 (Mann-Whitney U-test) for controls (Ctrl) vs. proctitis (Uninv + Inv), left-sided colitis (Inv), and extensive colitis; **P<0.05 (Wilcoxon signed-rank test) for uninvolved vs. involved tissue in proctitis and left-sided colitis. Data represent the mean ± SE of 79 UC patients and 29 controls. (B) Frequency of LP LAP+ cells in the CD3+CD4+-gated LPMC population: *P<0.05 for Ctrl vs. proctitis (Uninv + Inv) and left-sided colitis (Inv); *P<0.05 (Mann-Whitney U-test) for controls vs. left-sided colitis (Uninv + Inv) and extensive colitis; **P<0.05 (Wilcoxon signed-rank test) for uninvolved vs. involved tissue in proctitis and left-sided colitis. Data represent the mean ± SE of 79 UC patients and 29 controls. (C) Representative density plots of Foxp3 and LAP expression in LP CD3+CD4+-gated cells. (D) Representative CD4+LAP+ T cells in colonic mucosa tissue of UC patients and controls. Confocal microscopy images of CD4 (green), LAP (red) and nuclei (blue) of matched involved and uninvolved colonic mucosa of a patient with UC, and a control subject (original magnification 630x). CD4+LAP+ double positive T cells are indicated by white arrows. For UC, 1 representative staining of 4 patients is shown. For control subjects, 1 representative staining of 2 subjects is shown. H&E stained sections of the corresponding subjects are also illustrated (original magnification 200x). (E) % of CD4+LAP+ cells in UC patients (involved and uninvolved tissue) and controls. For quantification at least 3 images for each patient (n = 4) or control subjects (n = 2) were assessed. Data represent mean ± SE, *P<0.05 (Mann-Whitney U-test) for controls vs. uninvolved tissue; **P < 0.05 (Wilcoxon signed-rank test) for uninvolved vs. involved tissue.

Figure 2

Ratio between IL-10 and IL-17 expressing CD3+CD8-(CD4)LAP+ cells is significantly higher in uninvolved (Uninv) vs. involved (Inv) colon tissue from ulcerative colitis patients. (A) Frequency of LP LAP+ cells expressing intracellular IL-17 *P < 0.05 (Mann-Whitney U-test) for controls vs. Involved (Inv) tissue in proctitis, left sided colitis and extensive colitis. **P < 0.05 (Wilcoxon signed-rank test) for uninvolved vs. involved tissue in both proctitis and left-sided colitis. (B) Frequency of LP LAP+ cells expressing intracellular IL-10: *P < 0.05 (Mann-Whitney U-test) for controls vs. proctitis (Uninv), left-sided colitis (Uninv + Inv), and extensive colitis; **P < 0.05 (Wilcoxon signed-rank test) for uninvolved vs. involved tissue in both proctitis and left-sided colitis. (C) % LP LAP+ cells expressing intracellular IL-10 / % LP LAP+ cells expressing intracellular IL-17 (ratio). **P < 0.05 (Wilcoxon signed-rank test) for uninvolved vs. involved tissue in proctitis, and left sided colitis. *P < 0.05 (Mann-Whitney U-test) proctitis and left-sided colitis univolved tissue vs. controls *P < 0.05 (Mann-Whitney U-test) Extensive colitis vs. proctitis and vs. left-sided colitis involved tissue. Data represent mean ± SE of 43 UC patients (proctitis, 16; left-sided colitis, 17; extensive colitis, 10) and 26 controls.

Figure 3

Reduced disease extension is associated with a higher percentage of CD3+CD8-(CD4) LAP+ cells in uninvolved vs. involved colon tissue from UC patients. (A) Frequency of LP LAP+ cells in CD3+CD8- (CD4)-gated cells: *P < 0.05 (Mann-Whitney U-test) for controls vs. uninvolved tissue in proctitis + left-sided colitis; **P < 0.05 (Wilcoxon signed-rank test) for uninvolved vs. involved tissue in proctitis + left-sided colitis. (B) Frequency of LP LAP+ cells expressing intracellular IL-10: **P < 0.05 (Wilcoxon signed-rank test) for uninvolved vs. involved tissue in proctitis + left-sided colitis. (C) Frequency of LP Foxp3+ cells in CD3+CD8- (CD4+)-gated cells: **P < 0.05 (Wilcoxon signed-rank test) for uninvolved vs. involved tissue in proctitis + left-sided colitis. In all cases, data represent the mean ± SE of 26 controls and of 13 UC patients with a history of extensive colitis in whom inflammatory lesions were limited to the rectum or left colon at the moment of endoscopy. Patient clinicopathological variables of this UC subgroup are not different from the whole UC patients population [Age: 53 ± 3–53 (29–73) (years) mean ± SE-median (range); Sex: 7/6 M/F; Disease duration since diagnosis:14 ± 3.2–12 (1–32); (years) mean ± SE-median (range). Mayo endoscopic score: 2 ± 0.2–2 (1–3) mean ± SE-median (range)].

Figure 4

Oxazolone-induced colitis reproduces the observations made in UC patients. (A) Weight changes in mice after intrarectal administration of oxazolone (6 mg/ethanol 50%). Each point represents the cumulative mean (± SE) weight from 3 separate experiments in which 5 mice per group were studied. *P < 0.05 (Student's t-test) for EtOH 50% vs. OXZ 6 mg/EtOH 50%. (B) Representative macroscopic (on the left) and corresponding microscopic (on the right) images of the colons from untreated (a), EtOH 50%-treated (b), and OXZ 6 mg /EtOH 50%-treated (c) mice, all of which were sacrificed 2 days post-treatment. H&E staining of distal (untreated), distal and medial colonic tract of EtOH 50%-treated and oxazolone-treated miceat 40 × magnification. HS, histopathologic score (see methods). (C) Percentage of LAP+ cells among CD3+CD4+-gated LMPCs isolated from the colon of untreated, ethanol-treated and oxazolone/ethanol-treated mice: *P < 0.05 for untreated mice vs. OXZ-treated mice (uninvolved tissue); **P < 0.05 for uninvolved vs. involved tissue in OXZ-treated mice. Each point represents mean ± SE of pooled values derived from 3 experiments in which 5 mice per group were evaluated. (D) Percentage of Foxp3+ cells among CD3+CD4+-gated LMPCs isolated from the colon of untreated, ethanol-treated, and oxazolone/ethanol-treated mice: *P < 0.05 (Mann-Whitney U-test) for untreated mice vs. OXZ-treated mice (involved tissue); **P < 0.05 (Wilcoxon signed-rank test) for uninvolved vs. involved tissue in OXZ-treated mice. Each point represents the mean ± SE of pooled values derived from 3 experiments in which 5 mice/group were evaluated. (E) Representative CD4+LAP+ T cells in colonic mucosa of involved and uninvolved tissue of oxazolone-treated mice. Confocal microscopy images of CD4 (green), LAP (red) and nuclei (blue) of matched involved and uninvolved colonic mucosa of a oxazolone-treated mouse (original magnification 630x). CD4+LAP+ double positive T cells are indicated by white arrows. 1 representative staining of 3 mice is shown. H&E stained sections of the corresponding confocal images are also illustrated (original magnification 40x). The rectangles highlight the area shown in the confocal microscopy images. (F) % of CD4+LAP+ cells in colons of oxazolone-treated mice (involved and uninvolved tissue). For quantification at least 3 images for each colon (n = 3) were assessed. Data represent mean ± SE, **P < 0.05 (Wilcoxon signed-rank test) for uninvolved vs. involved tissue.

Figure 5

Depletion of LAP+ cells is associated with proximal extension of inflammatory lesions in the mouse oxazolone-induced colitis model. (A) Weight changes in oxazolone-treated mice (6 mg/ethanol 50%) pretreated with either Isotype Ab or anti-LAP Ab. Each point represents the cumulative mean (± SE) weight from 3 separate experiments in which 5 mice per group were studied. (B) Representative macroscopic images of colons of oxazolone-treated mice pretreated with (a) Isotype Ab or (b) anti-LAP Ab. (C) H&E stained colonic specimens (40 × magnification) of distal, medial and proximal colonic portions of oxazolone-treated mice pretreated with (a) Isotype Ab (a1-a4) or (b) anti-LAP Ab Isotype Ab (b1-b4). HS, histopathologic score (see methods).