| Crohn's disease (CD) is an idiopathic chronic disease in colon. It occurs with increasing frequency in developed and industrial countries, but remains sporadically in tropical third world countries with poor sanitation. Hygiene hypothesis may explain this phenomenon. It stated that pathogen infections may protect the host from immunological disease. Although the cause of CD is complicated, it is proved that CD is not only the consequence of inappropriate stimulation of effector responses but also due to a dysregulation of the normal immunosuppressive mechanisms. The development of CD is related to a polarized type-1 immune response and TNBS-induced colitis shares features with CD. Trinitrobenzene sulfonic acid (TNBS)-induced colitis is a murine model of human CD. In TNBS-induced murine model, ethanol was used to break the mucosa barrier of intestine, TNBS served as a hapten reagent and combined with the protein on the damaged intestinal mucosa to form the complete antigen. The similar immune lesion to CD happens in the intestine of murine. It is known that helminth infections might regulate a polarized type-1 or type-2 immune response and the immune regulatory process is associated with Tregs (CD4~+ CD25~+ regulatory T cells). Shistosome infection switches immunologically from an early Th1 response to a Th2-dominated response after the onset of parasite egg-laying. During schistosome infection,Tregs are also involved in its immunomodulation in its host.Tregs are important components of the homeostasis of the immune system, they prevent the development of autoimmunity and control virtually all forms of immune response, including inflammation. Foxp3 (forkhead box P3), a member of the forkhead/winged-helix family of transcriptional regulators, is a master regulatory gene for the development of Tregs and specifically expressed in CD4~+CD25~+ Tregs. In mice, the lack of foxp3-expressing Tregs is sufficient to break down self-tolerance and induce autoimmune disease. CD4~+ CD25~+ Foxp3~+ T cells can suppress and modulate the excessive immune response. The role of CD4~+ CD25~+ Foxp3~+ regulatory T cells in murine colitis with schistosome eggs treatment has not been reported.Elliott showed that exposure to Schistosoma mansoni eggs protects mice from TNBS-induced colitis. In the present paper, we demonstrate that exposure to Schistosoma japonicum eggs protects mice from TNBS-induced colitis and Tregs may be involved in the protection.Specific pathogen-free 6-8 week-old female BALB/c mice were used. Eggs of S. japonicum were collected from the livers of schistosome-infected rabbits, which had been infected with 1,500 cercariae 46 days previously. The eggs were collected and washed in phosphate buffered saline (PBS). Mice were intraperitoneally injected with 10,000 eggs in 100μl PBS at day 0. At days 7, 14 and 21, the animals were boosted in the same way and with the same dosage. Control mice received sterile PBS with the same volume and timing. In each group 5 mice were used and all experiments were duplicated. Mice were lightly anesthetized with metofane (methoxyflurane) at day 6 after the last immunization. A 3.5F catheter, fitted onto a l-ml syringe, was carefully inserted into the colon such that the tip was 4 cm proximal to the anus and 0.5 mg of TNBS in 50% ethanol was slowly administered into the lumen of the colon. In control experiments, mice were given 50% ethanol alone using the same technique described above. The total injection volume was 100μl for each mouse allowing TNBS or ethanol to reach the entire colon, including the caecum and appendix. Animals were then kept in a vertical position for 30 s and returned to their cages. Colons of mice were removed on day 6 after the induction of colitis and embedded in paraffin. Sections were stained with hematoxylin and eosin. The degree of inflammation of the colon was graded semiquantitatively from 0 to 4 (0, no evidence of inflammation; 1, low level of lymphocyte infiltration with infiltration seen in10% high-power field (hpf), no structural changes observed; 2, moderate lymphocyte infiltration with infiltration seen in 10%–25% hpf, crypt elongation, bowel wall thickening which does not extend beyond the mucosal layer, no evidence of ulceration; 3, high level of lymphocyte infiltration with infiltration seen in 25%– 50% hpf, high vascular density, thickening of bowel wall which extends beyond mucosal layer; and 4, marked degree of lymphocyte infiltration with infiltration seen in 50% hpf, high vascular density, crypt elongation with distortion, transmural bowel wall thickening with ulceration). Grading the inflammation in colons of mice was done in a blinded fashion by the same pathologist. Spleen cells were isolated from the experimental mice on day 6 after TNBS treatment. Cells were cultured in RPMI 1640 supplemented with 2 mM L-glutamine, 10 mM HEPES buffer, 10μg/ml gentamicin, 10% FCS, 100 U/ml penicillin and 100μg/ml streptomycin. Cells were cultured for 72 h at 3×106 cells/well in 1 ml microtiter wells and stimulated with 5μg/ml Con A. The concentrations of IFN-γ, IL-4, IL-5 and IL-10 in cell culture supernatants were determined by ELISA Kits. In brief, mouse IFN-γ, IL-4, IL-5 and IL-10 were detected by biotinylated monoclonal antibodies, which were evidenced by avidin-conjugated horseradish peroxidase followed by incubation with TMB substrate. The reactions were measured at 450 nm in an ELISA reader. Single-cell suspensions of spleen cells were stained and analyzed on FACS Calibur using CellQuest software. For determination of the phenotype of lymphocyte populations,mouse regulatory T cell staining Kit was used. The following conjugated antibodies were incubated with lymphocyte populations: fluorescein isothiocyanate (FITC)-conjugated anti-mouse CD4, allophycocyanin (APC)-conjugated anti-mouse CD25 and phycoerythrin (PE)-conjugated anti-mouse Foxp3 FJK-16s (a master regulatory gene for the development of Tregs). Before incubation with PE anti-mouse Foxp3 FJK-16s,cells were fixed with permeabilization buffer and Fc receptors were blocked with anti-CD16/32 antibody. PE -conjugated rat IgG2a served as the isotype control.The colons of mice were removed on day 6 after administration of TNBS or ethanol. TNBS-treated mice showed ulcerations of the colons surrounded by hyperemia and inflammation, whereas the colons of control mice treated with ethanol alone or S. japonicum eggs followed by TNBS showed neither macroscopic signs of inflammation nor obvious hyperemia and edema. Histologically, transmural inflammation affecting the entire colon was observed in TNBS-treated mice, lymphocyte infiltrates were associated with thickening of the colon wall, ulcerations, loss of goblet cells, and the presence of granulomas in TNBS-treated mice. The inflammatory responses were significantly milder in the colitis mice exposed to egg prior to TNBS-treatment. Histological score from egg-unexposed and -exposed mice was 3.6±0.49 and 1.6±0.70, respectively (P< 0.0005), thus confirming the prevention of TNBS-induced inflammation by previous injection of eggs. Spleen cells from TNBS-treated mice generated in vitro large amounts of IFN-γ, which were about twice the level of IFN-γafter administration of schistosome eggs prior to TNBS treatment (P<0.01). The secretion of IL-4, IL-5 and IL-10 in vitro was significantly (P<0.01) higher in colitis mice previously exposured to schistosome eggs compared to the animals treated with TNBS alone. The percentage of CD4~+ CD25~+ Foxp3~+ T cells in spleens of egg-treated mice was not significantly different from untreated control mice. These cells increased, however significantly (P<0.05) after TNBS-treatment, egg-exposed mice, but not after TNBS treatment alone.In the research, the results indicated that CD4~+ CD25~+ Foxp3~+ regulatory T cells may be involved in the modulation during TNBS-induced colitis with schistosome eggs exposure and the excessive Th1 response was converted. As a result, the lesion of TNBS-induced colitis in mice was attenuated.
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