Fig 1.
Gross pathology of colonic loops in Muc2+/+, Muc2-/- and germ-free (GF) mice inoculated with Entamoeba histolytica (Eh).
Eh-colonic loops were performed in antibiotic treated (Abx) and untreated Muc2+/+ and Muc2-/- littermates and in GF mice. Gross pathology was measured and scored among treatments as indicated in Material and Methods. (A) Eh inoculated in Muc2+/+ colons elicited extensive mucoid secretions with bloating. (B) Eh in Muc2-/- mice showed a similar ballooning effect with abundant watery secretion under intense pressure. (C) Colonic loops in GF mice evoked a less robust watery/mucoid secretions with less ballooning as Muc2 genotypes. (D) Gross pathology score of colons. Note no differences in gross pathology were observed among Eh-inoculated groups compared to homologous controls. n = 6, *** P < 0.001.
Fig 2.
Entamoeba histolytica (Eh) infection in antibiotic treated mice exacerbates pro-inflammatory responses in Muc2+/+ and Muc2-/- littermates.
(A-C) Muc2+/+ and Muc2-/- littermates pro-inflammatory cytokines mRNA expression in colonic tissue were measured by qPCR and compared with their corresponding untreated control (line). (D-F) Colonic luminal pro-inflammatory cytokines and chemokines (G-I) protein levels were measured using a mouse cytokine/chemokine array. n = 6. *P < 0.05, **P < 0.01.
Fig 3.
Entamoeba histolytica (Eh) in antibiotic-treated mice triggers robust mucus and Muc2 mRNA expression.
(A) Colonic loops from Muc2+/+ mice were fixed in Carnoy’s solution to preserve the mucus layers and stained with Periodic acid Schiff (PAS) reagent to visualize secreted and goblet cell mucus. Top left untreated control proximal colon with densely packed pear-shaped collection of goblet cells in the crypts filled with PAS+ material (arrow). Top right is the Abx- treated group showing similar morphology to the controls. Bottom left is Eh-inoculated colonic loops with secreted mucus is a watery exudate and less PAS+ cells in the crypts. Bottom right is the Abx treated mice inoculated with Eh showing fewer filled PAS+ cells in the crypts and with a dense mucus plugs on the surface of the mucosa (inset). (B) Quantification on the number of filled goblet cells per crypt. (C) Gene expression for Muc2, (D) Math1 and (E) Spdef. (F) Confocal microscopy using Muc2 specific antibody (red) and counterstained with DAPI (blue) to visualize Muc2 filled goblet cells in sections for A above. (G) Fluorescence quantification of Muc2 signal per crypt from images in F was analyzed using ImageJ software. Scale bar = 50 μm. n = 6. *P < 0.05, **P < 0.01, ***P < 0.001.
Fig 4.
Reduction in bacterial load with antibiotics increases Eh-induced colonic mucus and non-mucus secretions.
To quantify the release of newly synthesized mucus, mucin was metabolically labeled with 3H-glucosamine that incorporates into mucin galactose and N-acetylgalactosamine glycan residues. (A, D, F) In response to Eh in closed colonic loops, secreted 3H-labeled glycoproteins were fractionated on a Sepharose 4B column to separate high molecular weight mucin (Vo; fractions 15–20) from non-mucin glycoproteins (fractions 25–50). (B, E) Total 3H- counts (CPM) released constitutively in control, Abx-treated and fecal microbial transplant (FMT) animals in response to Eh. (C) Fold change in 3H-mucin V0 mucin and non-mucin release among the treated groups as shown in the legend. The column was equilibrated with BD: blue dextran (2,000 kilodaltons), TG: thyroglobulin (660 kilodaltons), Ova: chicken ovalbumin (42.7 kilodaltons), B12: Vitamin B12 (1.4 kilodaltons). n = 8, *P < 0.05, **P < 0.01.
Fig 5.
Entamoeba histolytica (Eh) supresses Math1 mRNA expression in colonic goblet cells ex vivo and in vitro.
(A) Math1 expression heatmap of Math1GFP mice colons, the dotted line indicates the colonic loop ligation. (B) Each colon was divided into proximal and distal regions and GFP signal quantified. (C) Monolayers of LS 174T human goblet cells where either exposed to Eh for 30 min (m), 1 h, fixed Eh with glutaraldehyde 2.5% (Glut), or pre-treated with IL-1β. mRNA expression levels of transcription factor MATH1 were measured by qPCR and normalized with GAPDH levels. GFP: green fluorescent protein, AU: arbitrary units. n = 6, *P < 0.05, ***P< 0.001.
Fig 6.
E. histolytica promotes bacterial translocation.
(A) Representative heat map image of the small intestine, cecum and colon of wild type mice inoculated with bioluminescent E. coli XEN 14 and infected with Eh in colonic loops (arrows). The mean bioluminescent signal was quantified and plotted on a histogram. (B) Representative heatmap of Math1 expression in the gastrointestinal tract of Math1GFP mice inoculated with a sub lethal dose of LPS (5mg/kg BW) showing increase Math1 GFP activity in the cecum and distal colon (arrows). Mean GFP signal was quantified and plotted on a histogram. (C) In colonic loops inoculated with Eh, intestinal permeability was measured by serum concentration of FITC-dextran (ng/mL) and (D) myeloperoxidase activity in the ileum (absorbance 450nm). (E) Visualization of bacterial localization by florescence in situ hybridization (FISH) in the ileum (a, d), proximal colon (b, e), and distal colon (c, f) of Eh inoculated loops (d, e, f) and PBS inoculated control loops (a, b, c). Translocated bacteria in response to Eh in the ileum, proximal colon and distal colon are show by the arrows. Red: bacteria, blue: enterocytes nuclei, green: mucus. The dotted line delimits area where commensal bacteria were present. Scale bar = 50 μm. (F) Quantification of the distance between bacteria and the epithelium from images in E was analyzed using ImageJ software. (G) Quantification of colony forming units (CFU) per gram present in the spleen and mesenteric lymph nodes (MLN) following Eh inoculation in colonic loops. GFP: green fluorescent protein, AU: arbitrary units, Ctrl: control, MPO: myeloperoxidase. n = 6, *P < 0.05.
Fig 7.
Germ-free mice have a deficient immune response towards E. histolytica.
Amebic colonic loops were performed in germ-free (GF) mice and pro-inflammatory response towards Eh was measured and compared with specific pathogen free (SPF) animals. (A) Relative expression of pro-inflammatory cytokines TNF-α and IL-1β in the proximal colon. (B) Relative expression of the transcription factor Math1, and (C) Muc2 in the proximal colon. (D) Myeloperoxidase activity in the proximal and distal colons of SPF and GF mice inoculated with Eh; fold change was compared with untreated SPF mice. (E) Number of filled goblet cells/crypt was blindly counted from the distal and proximal colons in GF and SPF mice. Abx: antibiotics, MPO: myeloperoxidase. n = 6, *P < 0.05, **P < 0.01.
Fig 8.
E. histolytica disrupts the colonic epithelium in germ-free mice.
Colonic loops inoculated with Eh in specific pathogen-free (SPF) and germ-free (GF) mice were fixed in Carnoy’s solution and stained with periodic acid-Schiff reagent. (A) Representative images of SPF animals inoculated with Eh showing thick adherent inner mucus (IM) layer and a loose outer mucus (OM) layer, with a cavitated goblet cell (GC; arrow) due to mucus depletion shown. (B) Intense mucus secretion in response to Eh in GF (arrows) mice; note the lower number of goblet cells in the surface epithelium and crypts. (C) Eh attaching to GF colonic epithelial cells (EC) (arrows), the epithelial cell on the right appear to be plucked out (inset) by Eh. (D) The arrows point at disruption of the single layer of epithelial cells by Eh in close proximity to the erosion.
Table 1.
Primer sequences used for quantitative real-time PCR.