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* = Presenting author

P035 Lamina propria CD4+LAP+ Foxp3- regulatory cells are involved in limiting the disease extension in Ulcerative Colitis

A. Butera1, A. Amendola1, M. Sanchez2, S. Piroli3, A. Pronio3, D. De Nitto4, R. Pica4, M. Boirivant*1

1Istituto Superiore di Sanità, Dept Infectious, Parasitic and Immune-mediated Diseases, Roma, Italy, 2Istituto Superiore di Sanità, Dept Cell Biology and Neurosciences, Rome, Italy, 3University ‘Sapienza’, Dept General Surgery, ‘P. Stefanini’, Roma, Italy, 4Sandro Pertini Hospital, IBD, GE Unit, Roma, Italy

Background

In ulcerative colitis (UC), disease extension influences disease prognosis. Biological factors involved in the disease extent and proximal extension are presently unknown. Aim: we evaluated the % of CD4+ Foxp3+ and CD4+LAP+ Foxp3- regulatory cells according to disease extension in UC active patients undergoing colonoscopy and in control patients undergoing colonoscopy for non-inflammatory conditions and in oxazolone (OXZ)–induced colitis, an animal model for UC.

Methods

Patients: clinical and endoscopic activity was evaluated according to Montreal classification and to Mayo endoscopic score, respectively. Extension of disease at the time of endoscopy was classified according to Montreal classification. Time of unvaried disease extension from the diagnosis was evaluated. Multiple biopsies were taken from involved and uninvolved tissue when available. OXZ colitis: colitis was induced by intrarectal administration of OXZ 6 mg or 2 mg in 150 ul of ethanol 50%. Mice were sacrificed after 
48 hours, and colons were analysed for macroscopic extension of the disease. LPMC from human biopsies and colonic murine tissue were isolated using DTT-EDTA-Collagenase sequence and % of CD4+ Foxp3+ and CD4+LAP+Foxp3- cells was evaluated by immunofluorescence. IL-10 mRNA tissue content was evaluated by real-time polymerase chain reaction (PCR).

Results

UC patients: patients with proctitis and left -sided colitis with a median time of unvaried disease extension from the diagnosis of 6 months (range: 0–48 mo) showed a significantly increased % of CD4+ LAP+ Foxp3- cells, both in involved and uninvolved tissue when compared with controls, whereas patients with extensive colitis showed values comparable with those of controls. Patients with proctitis with a median time of unvaried disease extension from the diagnosis of 13 years (range: 10–23 yr) showed a significantly increased proportion of CD4+ LAP+Foxp3- cells and IL-10 mRNA tissue content in uninvolved tissue when compared with involved tissue. Percentage of CD4+ Foxp3+ cells was significantly increased in involved tissue irrespective from disease extension and time from diagnosis, whereas uninvolved tissue showed values comparable with those of controls. OXZ colitis: administration of OXZ 6 mg was associated with a distal colitis with increased % of LP CD4+LAP+Foxp3- cells and CD4+Foxp3+ cells, whereas OXZ 2 mg administration was associated with extensive colitis with % of CD4+LAP+Foxp3- comparable to controls. Blockade of LAP by anti-LAP antibody i.p. administration before induction of OXZ 6 mg colitis was associated with extensive colitis with no variation in LP CD4+Foxp3+ cells.

Conclusion

CD4+LAP+Foxp3- regulatory cells are involved in limiting the extension of inflammatory lesions in UC.