Oral Tolerance Induced by OVA Intake Ameliorates TNBS-Induced Colitis in Mice

Abstract

Introduction: Literature data have shown that the consumption of dietary proteins may cause modulatory effects on the host immune system, process denominated oral tolerance by bystander suppression. It has been shown that the bystander suppression induced by dietary proteins can improve inflammatory diseases such as experimental arthritis. Here, we evaluated the effects of oral tolerance induced by ingestion of ovalbumin (OVA) on TNBS-induced colitis in mice, an experimental model for human Crohn's disease.

Methods and results: Colitis was induced in BALB/c mice by instilling a single dose of TNBS (100 mg/kg) in ethanol into the colon. Tolerized mice received OVA (4mg/mL) dissolved in the drinking water for seven consecutive days, prior to or concomitantly with the intrarectal instillation. Control groups received protein-free water and ethanol by intrarectal route. We observed that either the prior or concomitant induction of oral tolerance were able to reduce the severity of colitis as noted by recovery of body weight gain, improvement of clinical signs and reduction of histological abnormalities. The in vitro proliferation of spleen cells from tolerant colitic mice was lower than that of control mice, the same as the frequencies of CD4+ T cells secreting IL-17 and IFN-γ. The frequencies of regulatory T cells and T cells secreting IL-10 have increased significantly in mice orally treated with OVA. The levels of inflammatory cytokines (IL-17A, TNF-α, IL-6 and IFN-γ) were lower in supernatants of cells from tolerant colitic mice, whereas IL-10 levels were higher.

Conclusion: Our data show that the modulation of immune response induced by oral tolerance reduces the severity of experimental colitis. Such modulation may be partially attributed to the increase of Treg cells and reduction of pro-inflammatory cytokines in peripheral lymphoid organs of tolerant mice by bystander suppression.

Fig 1. Flowchart of the TNBS-induced colitis and oral tolerance design.

Panel A: Previous oral tolerance. Panel B: Concomitant oral tolerance.

Fig 2. OVA intake improves the severity of TNBS-induced colitis in mice.

For induction of tolerance an OVA solution (4mg / ml) was provided in drinking water to BALB/c mice (n = 5) for seven consecutive days, either 3 or 10 days before colitis induction. The experimental colitis was induced in BALB/c mice by intrarectal instillation of TNBS in 50% ethanol (1 mg / mL, 100 μL). Two control groups were included: mice that received protein-free water and were instilled with either ethanol (white bar) or TNBS (light gray bar). Panel A: Temporal changes in body weight, in percentage. All mice were weight daily and the weight alteration was represented by percentage in comparison with the mean initial value found on day 1 and represented as 100%. Body weights in orally treated mice were significantly higher than in non-tolerant mice. Panel B: Clinical signs were evaluated for the presence of diarrhea, rectal prolapse, bleeding and cachexia, assigning a score ranging from 0 to 2, with 0: no change, 1: slight change, and 2: severe change. Panel C: Oral tolerance to OVA modulates antibody production. Oral tolerance and experimental colitis was induced as illustrated in Fig 1. Five days after TNBS instillation, mice of all groups were immunized with OVA (10 mg) in aluminum hydroxide (1 mg) by intraperitoneal route, and 14 days later challenged i.p. with 10mg OVA in saline solution. On day 21, sera were collected, diluted 1: 100 to 1: 12,800 and tested by ELISA for anti-OVA antibodies (A) and anti-TNP (B). Values are means ± SEM sum of the optical densities (O.D.) Data are representative of three independent experiments (n = 5). ANOVA followed by Bonferroni post-test were used to determine statistical significance (p <0.05).

Fig 3. The oral tolerance to OVA changes histopathological features in the large intestine of mice with TNBS-induced colitis.

BALB / c mice were exposed to the treatments described in Fig 2 and sacrificed 5 days after induction of colitis. Distal part of large intestine was collected and fixed in paraffin for histological processing. Panel A: Histological sections of 5μm were stained with hematoxylin and eosin and examined by light microscopy (200X). Panel B: Sections were assessed for the presence of folds, hemorrhage, and infiltration of leukocytes at two distal portions of the intestines (1 to 2 cm and 2 to 3 cm from the rectum), assigning a score ranging from 0 to 20. Panel C: Thickening of the colon wall, measured in micrometers in two distal portions was analyzed with Infinity Analyze Nikon H600L program (100X). Two independent analyzes were performed.

Fig 4. The oral tolerance to OVA alters the proliferative response and cytokine producing cells from mice with TNBS-induced colitis.

The induction of tolerance and colitis was performed as described in Fig 2. Mice were sacrificed five days after TNBS instillation. Spleens were aseptically removed; cells were labeled with CFSE and cultured at a concentration of 2x10⁶ cells / ml in the presence of Concanavalin A (ConA; 2,5μg / ml) for 72 hours at 37°C and 5% CO2. Panel A: Spleen cell proliferation. Cells were fixed in 1% formaldehyde and the readings performed in flow cytometer (FACSCalibur, BD). Proliferation was calculated using the software FCS Express and represents the inverse of the ratio of the fluorescence exhibited by the cells after 72 hours of culture and those immediately after labeling with CFSE. The cell frequency (Panel B) and the mean fluorescence intensity (MFI) of T regulatory CD25⁺Foxp3⁺ cells (Panel C), and the frequency of cytokine producing cells (Panels D-I) were monitored within the CD4⁺ T cell gate. The values correspond to the mean ± S.E.M. of two independent experiments (n = 5). ANOVA followed by Bonferroni a posteriori test was used to determine statistical significance.

Fig 5. The oral tolerance to OVA alters cytokine levels in supernatants of ConA-stimulated spleen cells from mice with TNBS-induced colitis.

Cultures of spleen cells were carried out as described in Fig 4. Cytokine levels were evaluated in the culture supernatants by using the CBA Multiplex kit (Cytometric Bead Array Th1 / Th2 / Th17, BD) and readings were performed in a flow cytometer (FACSCalibur, BD). Cytokine concentrations were determined using the array FCAP TM Version 3.0 Software (BD). Results were expressed as means ± S.E.M. obtained from two independent experiments (n = 5).ANOVA followed by Bonferroni a posteriori test was used to determine statistical significance.