An ex-vivo human intestinal model to study Entamoeba histolytica pathogenesis

Abstract

Amoebiasis (a human intestinal infection affecting 50 million people every year) is caused by the protozoan parasite Entamoeba histolytica. To study the molecular mechanisms underlying human colon invasion by E. histolytica, we have set up an ex vivo human colon model to study the early steps in amoebiasis. Using scanning electron microscopy and histological analyses, we have established that E. histolytica caused the removal of the protective mucus coat during the first two hours of incubation, detached the enterocytes, and then penetrated into the lamina propria by following the crypts of Lieberkühn. Significant cell lysis (determined by the release of lactodehydrogenase) and inflammation (marked by the secretion of pro-inflammatory molecules such as interleukin 1 beta, interferon gamma, interleukin 6, interleukin 8 and tumour necrosis factor) were detected after four hours of incubation. Entamoeba dispar (a closely related non-pathogenic amoeba that also colonizes the human colon) was unable to invade colonic mucosa, lyse cells or induce an inflammatory response. We also examined the behaviour of trophozoites in which genes coding for known virulent factors (such as amoebapores, the Gal/GalNAc lectin and the cysteine protease 5 (CP-A5), which have major roles in cell death, adhesion (to target cells or mucus) and mucus degradation, respectively) were silenced, together with the corresponding tissue responses. Our data revealed that the signalling via the heavy chain Hgl2 or via the light chain Lgl1 of the Gal/GalNAc lectin is not essential to penetrate the human colonic mucosa. In addition, our study demonstrates that E. histolytica silenced for CP-A5 does not penetrate the colonic lamina propria and does not induce the host's pro-inflammatory cytokine secretion.

Figure 1. Cell cytotoxicity during interaction between human colonic explants and E. histolytica or E. dispar.

Mean LDH concentrations (IU/L) released into the supernatant of the organotypic culture after incubation with E. histolytica WT or E. dispar or in the absence of amoeba (control) from 1 to 7 hours. Data are from 8 individual experiments. * indicates a significant difference between WT and control (p<0.03) and between WT and E. dispar (p<0.05). # indicates a significant difference between WT and control (p<0.001) and between WT and E. dispar (p<0.02)

Figure 2. Interaction between Entamoeba and the lumen surface of the human colonic explants.

A. Analyse by histology of the mucus layer at the surface of Human colonic fragments incubated for seven hours without Entamoeba (left panel) and with E. histolytica (right panel). The mucus layer covering the epithelium at the surface was observable after seven hours of organotypic culture but not in the presence of E. histolytica. B. Scanning electron micrographs of the luminal surface of the human colonic explants incubated with E. histolytica or E. dispar. Representative images from three individual experiments are shown. (a) E. histolytica trophozoites adhering to the mucus layer at time 0; (b) 2 hours after incubation, the mucus layer had been degraded by E. histolytica and the regular mucosal architecture of the colonic epithelium was visible. Holes corresponded to the crypts of Lieberkühn and abundant aggregates were seen in the interglandular regions. (c) The aggregates were composed of human cells and trophozoites, as seen in an enlargement of this region (d) After 4 hours, the epithelium was damaged and (e) E. histolytica trophozoites began to penetrate into the tissue (f) After 4 hours, E. dispar trophozoites were still adhering to the mucus but had not degraded it and (g) had not evoked the recruitment of cells to the interglandular region, as shown after manually scraping the mucus after SEM fixation procedure of the sample.

Figure 3. Structure of the mucosa during E. histolytica's invasion of human colonic explants.

Time lapse histological studies of the colonic mucosa. Representative images are shown from eight individual experiments; (a) After 2 hours of incubation, E. histolytica trophozoites adhered to the epithelium and had detached enterocytes in the interglandular region (b) The trophozoites then migrated along the basal side of the epithelium lining the crypts (c) The trophozoites penetrated deeper into the mucosa after four and (d) seven hours of incubation, leading to the disruption of the mucosal normal architecture. (e) In the absence of amoeba, no alteration of the mucosa architecture was visible, even after 7 hrs of incubation (f) Massive destruction of the mucosal architecture was observed in the presence of E. histolytica (g) The non-virulent E. dispar was unable to cross the mucus barrier or degrade the epithelium.

Figure 4. E. histolytica-induced secretion of pro-inflammatory cytokines.

Histogram showing mean ± SD concentrations (pg/ml) of individual analytes (IL-1β, IL-6, IL-8, IFN-γ and TNF) secreted from 8 human colonic explants incubated with WT, E. dispar or without amoeba (control) after 4 and 7 hours of incubation, as measured on a Luminex100 system. Levels of secreted pro-inflammatory cytokines (IL-1β, IL-6, IL-8, IFN-γ and TNF) were significantly higher at 4 hours (* p<0.05) and 7 hours (# p<0.03) in the explants incubated with WT, in comparison with both those secreted by explants incubated with E. dispar and the amoeba-free control.

Figure 5. Cell cytotoxicity during interplay between E. histolytica trophozoites affected for virulent factors and human colonic explants.

Mean LDH concentrations (IU/L, from 3 individual experiments) released after incubation of human colonic explants with HGL2, NEO, RBV, G3, WT and RB8 trophozoites and in the absence of amoeba (control) for 4 hours (grey bars) and 7 hours (black bars). * indicates a significant difference between HGL2 and control (P<0.01), NEO and control (P<0.03) and WT and control (P<0.05). # indicates a significant difference between HGL2 and control (p<0.008), NEO and control (p<0.01), RBV and control (p<0.01), G3 and control (p<0.009), WT and control (p<0.007), RBV and RB8 (p<0.03), G3 and RB8 (p<0.01) and WT and RB8 (p<0.01)

Figure 6. Pro-inflammatory cytokines secretion induced in the ex-vivo human colonic model by sub-strains of E. histolytica.

Mean concentrations (pg/ml) of IL-1β, IL-8, IFN-γ and TNF secreted after 4 hours (grey bars) and 7 hours (black bars) of incubation of HGL2, NEO, RBV, G3, WT and RB8 trophozoites with 3 individual human colonic explants. NEO and HGL2 parasites induced significantly higher levels of pro-inflammatory cytokines IL-1β, IL-6, IL-8, IFN-γ and TNF in the explants incubated for 4 hours (p<0.05) and 7 hours (p<0.02), compared with the control in the absence of amoeba. RBV, G3 and WT strains induced significantly higher levels of pro-inflammatory cytokines [IL-1β (p<0.05,<0.01 and <0.01 respectively) IL-8 (<0.04,<0.01 and <0.01 respectively), IFN-γ(<0.009,<0.003 and <0,01 respectively) and TNF (<0.02,<0.02 and <0.02 respectively)] in the explants incubated for 4 hours and IL-8 (<0.009, <0.001 and <0.01 respectively), IFN-γ (<0.002,<0.0004 and <0.01 respectively) and TNF (<0.008,<0.008 and <0.02 respectively)] at 7 hours, compared with the control in the absence of amoeba. WT secreted IL1β (0.04/0.03), IL-8 (0.001/0.001), IFN-γ (0.001/0.002) and TNF (0.008/0.01) at 4 and 7 hours respectively, compared with RB8.

Figure 7. Migration through the lamina propria of E. histolytica sub-strains impaired in virulent functions.

Representative images from three individual experiments are shown. Histological examination of colonic tissue sections after seven hours of incubation, with HGL2, G3, RBV and RB8. Transversal tissue slices were stained with haematoxylin-eosin. Trophozoites were immunostained with antibodies against the Gal/GalNAc lectin. Experiments with HGL2, G3 and RBV revealed that trophozoites were able to invade the mucosa, as described for the WT. In contrast, RB8 parasites were unable to penetrate deeper into the lamina propria and were blocked at the surface of the mucosa, although they were still able to disorganize and detach cells from the upper side of the mucosa.