Concurrent infection with Schistosoma mansoni attenuates inflammation induced changes in colonic morphology, cytokine levels, and smooth muscle contractility of trinitrobenzene sulphonic acid induced colitis in rats

Abstract

Background and aims: Crohn's disease, characterised by chronic T helper 1 (Th1) inflammation and dysmotility of the gut, is most prevalent in developed countries. Parasitic infections are most prevalent in developing countries and induce a T helper 2 (Th2) immune response. We hypothesised that this Th2 immune response protects against Th1 gut inflammation.

Methods: The parasite Schistosoma mansoni induces a transient Th2 immune response in the semipermissive rat host. 2,4,6-Trinitrobenzene sulphonic acid (TNBS) induced colitis is an experimental model of Th1-like gut inflammation. The effect of concurrent infection with S mansoni on the course of TNBS induced colitis was assessed using macroscopic and microscopic damage scores, histology, myeloperoxidase (MPO) activity assay, cytokine production assay, and by studying in vitro contractility of longitudinal and circular colonic muscle strips.

Results: TNBS induced colitis that spontaneously healed after four weeks. Concurrent infection with S mansoni significantly reduced the duration of TNBS induced colitis to two weeks, as shown by macroscopic and microscopic damage scores and by a faster decrease in colonic MPO activity. TNBS increased colonic interleukin 2 (IL-2) production whereas S mansoni increased splenic IL-4 and IL-2 levels. Contractility of longitudinal and circular muscle strips was maximally inhibited one week after TNBS and normalised after three weeks. After four weeks, longitudinal muscle strip contractility was significantly increased. Concurrent infection with S mansoni normalised longitudinal muscle contractility after one week whereas circular muscle contractility remained inhibited.

Conclusions: Concurrent infection with S mansoni significantly attenuates TNBS induced colitis in the rat. Inflammation induced disturbances in contractility of longitudinal and circular colonic muscle strips may outlast the inflammatory reaction.

Figure 1

Experimental chronology of the study. Experiments were performed one day, four days, one week, two weeks, three weeks, and four weeks after intracolonic injection. Each experiment at any time interval for any experimental group was repeated at least four to six times.

Figure 2

Macroscopic damage score of the colon in the four groups of rats: control rats; rats infected with Schistosoma mansoni alone; rats given an intracolonic injection of 2,4,6-trinitrobenzene sulphonic acid (TNBS) alone; and rats with concurrent infection with S mansoni plus intracolonic injection of TNBS. Results are expressed as boxplots of the median. *p<0.05 compared with the TNBS group (Kruskal-Wallis test followed by a post hoc Mann-Whitney test, n = 4–6).

Figure 3

Cross section of the distal colon of a control rat (A), a rat four days after intracolonic injection of 2,4,6-trinitrobenzene sulphonic acid (TNBS) (B), and a Schistosoma mansoni infected rat four days after intracolonic injection of TNBS (C). The images are printed at the same final magnification. Note disruption of the epithelial barrier, distortion of the mucosal villi, and transmural inflammatory infiltrate after intracolonic injection of TNBS. Concurrent infection with S mansoni markedly attenuated these histological signs of inflammation. Bar represents 200 µm (Sirius red staining, ×100).

Figure 4

Microscopic damage score validated on histological sections of the colon in the four groups of rats: control rats; rats infected with Schistosoma mansoni alone; rats given an intracolonic injection of 2,4,6-trinitrobenzene sulphonic acid (TNBS) alone; and rats with concurrent infection with S mansoni plus intracolonic injection of TNBS. Results are expressed as boxplots of the median. *p<0.05 compared with the TNBS group (Kruskal-Wallis test followed by a post hoc Mann-Whitney test, n = 4–6).

Figure 5

Myeloperoxidase (MPO) activity of the colonic mucosa (A) and spleen (B) in the four groups of rats: control rats; rats infected with Schistosoma mansoni alone; rats given an intracolonic injection of 2,4,6-trinitrobenzene sulphonic acid (TNBS) alone; and rats with concurrent infection with S mansoni plus intracolonic injection of TNBS. *p<0.05 compared with controls and S mansoni infected rats; †p<0.05 compared with controls and TNBS alone (one way ANOVA followed by Student-Newman-Keuls test, n = 4–6). Results are expressed as units per gram tissue colonic mucosa or spleen. Splenic MPO activity was significantly increased in all S mansoni infected rats, independent of intracolonic injection of TNBS or saline. Differences were independent of the time course of infection.

Figure 6

Interferon γ (IFN-γ) (A), interleukin (IL)-2 (B), and IL-4 (C) production in the spleen in the four groups of rats: control rats; rats infected with Schistosoma mansoni alone; rats given an intracolonic injection of 2,4,6-trinitrobenzene sulphonic acid (TNBS) alone; and rats with concurrent infection with S mansoni plus intracolonic injection of TNBS. *p<0.05 compared with controls and TNBS alone; †p<0.05 compared with rats infected with S mansoni alone (one way ANOVA followed by Student-Newman-Keuls test, n = 4–6).

Figure 7

Interferon γ (IFN-γ) (A), interleukin (IL)-2 (B), and IL-4 (C) production in the colonic mucosa in the four groups of rats: control rats; rats infected with Schistosoma mansoni alone; rats given an intracolonic injection of 2,4,6-trinitrobenzene sulphonic acid (TNBS) alone; and rats with concurrent infection with S mansoni plus intracolonic injection of TNBS. No significant differences were found in colonic IL-4 production (one way ANOVA followed by Student-Newman-Keuls test, n = 4–6). *p<0.05 compared with controls, S mansoni infected rats, and S mansoni with intracolonic injection of TNBS rats (one way ANOVA followed by Student-Newman-Keuls test, n = 4–6).

Figure 8

Cumulative concentration-response curves to acetylcholine (ACh) in longitudinal and circular muscle strips of the colon of control rats. Contractions are expressed as g contraction per cross sectional area (CSA) of the longitudinal and circular muscle layers. E_max represents the maximal contractile response. pD₂ represents the negative logarithm of the molar concentration inducing 50% of the maximal contractile response.

Figure 9

Time course of the maximal contractile response (E_max) values for acetylcholine in longitudinal (A) and circular (B) muscle strips in the four groups of rats: control rats; rats infected with Schistosoma mansoni alone; rats given an intracolonic injection of 2,4,6-trinitrobenzene sulphonic acid (TNBS) alone; and rats with concurrent infection with S mansoni plus intracolonic injection of TNBS. *p<0.05 compared with the other groups; †p<0.05 compared with the other groups (one way ANOVA followed by the Student-Newman-Keuls test, n = 4–6).