Increased susceptibility to Trichuris muris infection and exacerbation of colitis in Mdr1a-/- mice

Abstract

Aim: To investigate the influence of Trichuris muris (T. muris) infection in a mouse model of genetic susceptibility to inflammatory bowel disease, Mdr1a-/-.

Methods: Mdr1a-/- mice were housed under specific pathogen free conditions to slow the development of colitis and compared to congenic FVB controls. Mice were infected with approximately 200 embryonated ova from T. muris and assessed for worm burden and histological and functional markers of gut inflammation on day 19 post infection.

Results: Mdr1a-/- mice exhibited a marked increase in susceptibility to T. muris infection with a 10-fold increase in colonic worm count by day 19 pi compared to FVB controls. Prior to infection, Mdr1a-/- exhibited low-level mucosal inflammation with evidence of an enhanced Th1 environment. T. muris infection accelerated the progression of colitis in Mdr1a-/- as evidenced by marked increases in several indicators including histological damage score, mucosal CD⁺ T-cell and DC infiltration and dramatically increased production of pro-inflammatory cytokines.

Conclusion: These data provide further evidence of the complex interaction between T. muris and an inflammatory bowel disease (IBD)-susceptible host which may have relevance to the application of helminth therapy in the treatment of human IBD.

Keywords: Colitis; Helminth; Inflammatory bowel disease; Mdr1a; P-glycoprotein.

Figure 1

Mdr1a-/- (KO) mice show increased susceptibility to Trichuris muris infection compared to FVB controls (WT). Data shows large intestinal worm burden measured on day 19 following infection with approximately 200 embryonated Trichuris muris eggs. Each data point represents a single mouse (n = 6 in each group); the horizontal bar denotes the mean for each group. ^bP < 0.01.

Figure 2

Parasite-specific IgG1 and IgG2a antibody responses in the serum of naïve and Trichuris muris infected FVB (WT) and mdr1a-/- (KO) mice. Serum was taken from mice at day 19 post infection. Data shows mean ± SD for n = 5-7 animals in each group. OD: Optical density.

Figure 3

Cytokine production by mesenteric lymph nodes from naïve and Trichuris muris infected FVB (WT) and mdr1a-/- (PGP-KO) mice. Levels of Th1 [interferon (IFN)γ, A; interleukin (IL)-12p40, C] and Th2 (IL-13, D; IL-5, E) and the pro-inflammatory cytokine [tumor necrosis factor (TNF)α, B] were measured in mesenteric lymph nodes (MLN) following in vitro stimulation with Trichuris muris (T. muris) E/S antigen. MLN were isolated on day 19 post infection in T. muris infected groups. Data is shown as mean + SD for n = 5 animals in each group ^aP < 0.05; ^bP < 0.01.

Figure 4

Expression of interferon γ-dependent genes IDO and CXCL-10 in colonocytes from naïve Mdr1a-/- and FVB mice. mRNA expression was measured relative to β-actin in each case. Data is expressed as fold change in expression in Mdr1a-/- colonocytes relative to FVB controls and is shown as mean ± SD for n = 4 preparations for each group. ^aP < 0.05, ^bP < 0.01 vs FVB groups.

Figure 5

Histological analysis of proximal colon from FVB (WT) and mdr1a-/- (PGP-KO) mice. A-E: H and E stained sections from proximal colon of WT-N (A), WT-INF (B), KO-N (C) and KO-INF (D-E). Tissues were isolated on day 19 post-infection in Trichuris muris (T. muris) infected mice; F: Histological disease scores for colon from naïve (WT-N; KO-N) and T. muris infected (WT-INF; KO-INF) mice based on the grading system described by Collett et al[11], 2008. Bar shows mean values for each group ^aP < 0.05, ^bP < 0.01, n = 5 for each group.

Figure 6

CD4⁺ T-cell infiltration associated with Trichuris muris infection in the proximal colon of FVB (WT) and mdr1a-/- (PGP-KO) mice. A: Immunohistochemical staining for CD4⁺ T cells in naïve (WT-N; KO-N) and Trichuris muris (T. muris) infected (WT-INF; KO-INF) WT and PGP-KO mice; B: Mean data (± SD) from 5 animals in each group showing CD4⁺ T cells present per 20 crypts units. Tissues were isolated on day 19 post-infection in T. muris infected mice, ^bP < 0.01.

Figure 7

Dendritic cell infiltration associated with Trichuris muris infection in the proximal colon of FVB (WT) and mdr1a-/- (PGP-KO) mice. Fluorescence staining (pink) for dendritic cells in frozen sections of proximal colon from naïve (WT-N; KO-N) and Trichuris muris (T. muris) infected (WT-INF; KO-INF) mice. Tissues were removed on day 19 post infection in T. muris infected mice. Original magnification × 20. Images are representative of tissues from 5 mice in each group.