involved (Inv) colon tissue from UC patients. 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 Amyloid b-peptide (42-1) (human) mice with oxazolone-induced distal colitis, treatment with LAP-depleting antibody was associated with the development of considerable colitis. Conclusions: Our findings suggest that CD3+CD4+LAP+Foxp3-Tregs limit the extension of inflammatory lesions in UC patients. 0.05. Human study All participants provided written informed consent prior to inclusion in the study. Ethical approval was provided by the Ethical Committee of the Istituto Superiore di Sanit (Reference: Pre-C-871/14, 25/11/2014). Animal studies This study was carried out in accordance with the recommendations of Decreto Legislativo 4 marzo 2014, n. 26 according with 2010/63/UE(14G00036) direction. The protocol was approved by the Italian Ministry of Health (Research: 16/2014-PR [DGSAF 12073-A, 05/06/2014], 03/10/2014). Results The frequency of LP CD3+CD4+LAP+ cells is usually higher in uninvolved vs. involved colon tissue from ulcerative colitis patients Preliminary analyses of the percentage of CD3+CD4+LAP+ and CD3+CD4+Foxp3+ Tregs isolated from biopsies from different portions of control colons revealed no differences between colon regions (Supplementary Physique 1). We next evaluated the frequency of LP CD3+CD4+LAP+ and CD3+CD4+Foxp3+ Tregs in LPMCs isolated from biopsies from controls and from patients with endoscopically and histologically active UC with varying degrees of disease extension. In patients with either proctitis or left-sided colitis, the percentage of CD4+Foxp3+ cells was significantly higher in involved tissue than in uninvolved tissue (Physique ?(Figure1A).1A). As reverse % of CD4+LAP+ T regs was significantly higher in uninvolved vs. involved tissue (Physique ?(Physique1B)1B) In agreement with previous observations (14, 24, 25), the percentage of Foxp3+ Tregs in the LP CD3+CD4+ T-cell population was significantly higher in involved tissue, regardless of disease extension, than in controls (Physique ?(Figure1A).1A). Similarly, in line with previous reports (14), the percentage of CD4+LAP+ Tregs was significantly higher in involved tissue from patients with considerable colitis and left-sided colitis than in controls (Physique ?(Figure1B).1B). In uninvolved tissue, the Amyloid b-peptide (42-1) (human) percentage of CD4+Foxp3+ cells was comparable to that of controls, while the percentage of CD4+LAP+ Tregs tissue was significantly higher. As previously reported (14), the majority of LAP+ cells detected were Foxp3- (Physique ?(Physique1C).1C). Some biopsy specimens sections were also immunofluorescence stained for tissue assessment of CD4+LAP+ cells by confocal microscopy. As illustrated in Figures 1D,E, uninvolved tissue showed significantly more CD4+LAP+ double-fluorescent cells when compared with involved and control tissue. Open in a separate window Physique 1 The frequency of LP CD3+CD4+LAP+ cells is usually higher in uninvolved (Uninv) vs. involved (Inv) colon tissue from UC patients. (A) Frequency of LP Foxp3+ cells in the CD3+CD4+-gated LPMC populace: * 0.05 (Mann-Whitney (Wilcoxon signed-rank test) for uninvolved vs. involved tissue in proctitis and left-sided colitis. Data symbolize the imply SE of ERCC3 79 UC patients and 29 controls. (B) Frequency of LP LAP+ cells in the CD3+CD4+-gated LPMC populace: *for Ctrl vs. proctitis (Uninv + Inv) and left-sided colitis (Inv); *(Mann-Whitney (Wilcoxon signed-rank test) for uninvolved vs. involved tissue in proctitis and left-sided colitis. Data symbolize the imply SE of 79 UC patients and 29 controls. (C) Representative density plots of Foxp3 and LAP expression Amyloid b-peptide (42-1) (human) in LP CD3+CD4+-gated cells. (D) Representative CD4+LAP+ T cells in colonic mucosa.
involved (Inv) colon tissue from UC patients