Figure 1.
Helminth co-infection exacerbates C. rodentium-induced colitis and mortality in MyD88 KO mice.
MyD88 knockout and wild-type C57BL/6 mice were infected with H. polygyrus (200 L3) and inoculated with C. rodentium (5×108 CFU) 7 days later. A & C: Body weight changes of wild-type (A) and MyD88 knockout (C) mice that were infected with C. rodentium, H. polygyrus, both H. polygyrus and C. rodentium, and normal control mice during the course of the experiment (8 days) are shown. Data shown are pooled from three independent experiments and are expressed as the body weight change as a percentage of the individual mouse initial body weight ± SE (n = 10–15) at each time point. B &D: Survival curve in wild-type (B) and MyD88 knockout (D) mice. H. polygyrus co-infection results in a significantly increased mortality in MyD88 KO mice.
Figure 2.
Helminth co-infection exacerbates C. rodentium-induced colitis and intestinal injury in MyD88 knockout mice.
A. Macroscopic examination of colon tissues of MyD88 knockout mice from different treatment groups. Arrows indicate intestinal bleeding. B. Colon tissues were removed from uninfected mice or from mice infected with H. polygyrus, C. rodentium, or both two weeks after bacterial infection, frozen in Tissue Tek OCT compound, and the sections were stained with hematoxylin and eosin. B: Magnification, ×40 and C: Magnification, ×100. Duplicate samples are presented from co-infected and C. rodentium-infected MyD88 knockout mice. Co-inf (chronic): C57BL/6 and MyD88 knockout mice were pre-infected with H. polygyrus for 3 weeks and then co-infected with C. rodentium. Arrows indicate cellular infiltration and loss of intestinal architecture. D. Histopathological score of colonic inflammation in mice infected with C. rodentium or both. The scores were assessed by determination of infiltration of inflammatory cells (score range, 0 to 4), together with the evaluation of cecal tissue damage (score range, 0 to 4). The data shown are pooled from three independent experiments with total (n = 9 to 12 per group). *** p<0.001, **p<0.005.
Figure 3.
Helminth infection results in enhanced C. rodentium translocation in colonic tissue of MyD88 KO mice.
A. Immunofluorescence microscopic analysis of bacterial distribution and localization in the colonic tissues at day 5 post C. rodentium infection. Most of the bacteria were detected in the intestinal epithelial surface in MyD88 knockout mice with bacterial infection alone. An increased bacterial translocation was detected in MyD88 knockout mice with helminth-coinfection. Green: GFP-C. rodentium, Blue: DAPI. Red: CD11c in MLN. B. Numbers of bacteria recovered from fecal samples of C. rodentium-infected and co-infected MyD88 knockout mice at 7 days post-infection. The data shown are represented as the mean ± the SEM (n = 6 to 7 mice). *p<0.05.
Figure 4.
Helminth co-infection results in decreased number of CD11b+ cells and antimicrobial peptide expression in colonic tissue.
A–C, MyD88 knockout mice were infected with H. polygyrus and inoculated with C. rodentium orally 7 days later. Mice infected with C. rodentium (C.r) (A), H. polygyrus (Hp) (B) and both (Co-inf) (C) were sacrificed. Histological sections of the colon were stained with anti-CD11b-FITC (green) and DAPI (blue), and analyzed by immunofluorescence microscopy. Magnification, ×100. D, Colonic tissue MPO level was determined by ELISA. E–H. Colon tissues were collected from C. rodentium-infected (E: MyD88 knockout; F: C57BL/B6) and co-infected (G: MyD88 knockout and H: C57BL/B6) mice and stained with anti-GR1-FITC (green), anti-F4/80 (red) and DAPI (blue), and analyzed by immunofluorescence microscopy. All images were digitized and cropped in Adobe Photoshop LE 5.0 (Adobe Systems). I–L:Colon tissues were collected from normal control, C. rodentium-infected, H. polygyrus infected, and co-infected wild-type C57BL/6 and MyD88 knockout mice. The expression of Arginase 1, a marker for alternatively active macrophages, was determined in MyD88 knockout mice (I) and B6 mice (J) using quantitative RT-PCR. iNOS expression was determined in MyD88 knockout mice (K) and B6 mice (L). Values are the fold increase compared with baseline obtained from uninfected mice. The data shown are the mean ± the SEM (n = 3–5 mice/group) from one of three experiments performed showing similar results. *p<0.05.
Figure 5.
Co-infection with H. polygyrus results in dysregulation of Citrobacter-induced colonic cytokines.
Colon tissues were collected from normal control, C. rodentium-infected, H. polygyrus infected, and co-infected wild-type C57BL/6 and MyD88 knockout mice. Total RNA was isolated. The expression of KC, TNF-α, IL-10 and IL1β was determined using quantitative RT-PCR. Values are the fold increase compared with baseline obtained from uninfected mice. The data shown are the mean ± the SEM (n = 3–5 mice/group) from one of three experiments performed showing similar results. *p<0.05.
Figure 6.
H. polygyrus-infection induces the inhibition of LPS-, but not IL-1β-, induced pro-inflammatory cytokine production.
The peritoneal macrophages were collected from normal and helminth-infected mice and re-stimulated the cells in vitro with LPS (100 ng/ml) or IL-1β (1 ng/ml) for 1 or 6 h. Culture supernatants were collected (at 6 h). TNF-α (A) and IL-6 (B) production was measured by Cytokine ELISA. KC expression (C) (at 1 h) was determined by Real time RT-PCR. *p<0.05, n = 5–10 mice per group.
Figure 7.
Co-infection with H. polygyrus results in down-regulation of C. rodentium-induced colonic anti-microbial peptide expression.
Colon tissues were collected from control, C. rodentium-infected, H. polygyrus infected, and co-infected MyD88 knockout and wild-type mice. Total RNA was isolated. IL-22, Reg3γ and Reg3β expression was determined using quantitative RT-PCR. Values are the fold increase compared with baseline obtained from uninfected mice. The data shown are the mean ± the SEM (n = 3–5 mice/group) from one of three experiments performed showing similar results. *p<0.05.