An ex-vivo Human Intestinal Model to Study Entamoeba histolytica Pathogenesis

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

The parasite Entamoeba histolytica exploits the activities of human matrix metalloproteinases to invade colonic tissue

International audienceIntestinal invasion by the protozoan parasite Entamoeba histolytica is characterized by remodelling of the extracellular matrix (ECM). The parasite cysteine proteinase A5 (CP-A5) is thought to cooperate with human matrix metalloproteinases (MMPs) involved in ECM degradation. Here, we investigate the role CP-A5 plays in the regulation of MMPs upon mucosal invasion. We use human colon explants to determine whether CP-A5 activates human MMPs. Inhibition of the MMPs' proteolytic activities abolishes remodelling of the fibrillar collagen structure and prevents trophozoite invasion of the mucosa. In the presence of trophozoites, MMPs-1 and-3 are overexpressed and are associated with fibrillar collagen remodelling. In vitro, CP-A5 performs the catalytic cleavage needed to activate pro-MMP-3, which in turn activates pro-MMP-1. Ex vivo, incubation with recombinant CP-A5 was enough to rescue CP-A5-defective trophozoites. Our results suggest that MMP-3 and/or CP-A5 inhibitors may be of value in further studies aiming to treat intestinal amoebiasis

Newly visualized fibrillar collagen scaffolds dictate Entamoeba histolytica invasion route in the human colon

International audienceThe extracellular matrix (ECM) and its role in the outcome of infectious diseases have been poorly investigated. In this study, we determined the impact of the collagen fibres architecture on the invasive process of the enteric parasite Entamoeba histolytica. The behaviour of E. histolytica wild-type and silenced for the cysteine protease A5 (CP-A5) were compared on a three-dimensional collagen matrix and within human colon fragments for fibrillar collagen cleavage and migration. The interstitial collagen fibres within the connective tissue of the human colon, visualized by multiphoton and second harmonic generation signals imaging, presented a dense scaffold at the subepithelial level and a loose meshwork within the chorion. To penetrate the tissue, E. histolytica migrated on the dense scaffold that remained intact, reached the crypt of Lieberkhün, migrated along and then disorganized the loose scaffold to escape into the mucosa. Interestingly, in vitro, CP-A5 was not required for collagenase activity and migration through the matrix but was necessary within the tissue environment for collagen meshwork remodelling and subsequent invasion. The data point out that further step of invasion relay with ECM destruction that requires human components induced or activated in the presence of CP-A5

Cell cytotoxicity during interaction between human colonic explants and <i>E. histolytica</i> or <i>E. dispar</i>.

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

<i>E. histolytica</i>-induced secretion of pro-inflammatory cytokines.

<p>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, <i>E. dispar</i> 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 (<b>*</b><i>p</i><0.05) and 7 hours (<b># </b><i>p</i><0.03) in the explants incubated with WT, in comparison with both those secreted by explants incubated with <i>E. dispar</i> and the amoeba-free control.</p

Cell cytotoxicity during interplay between <i>E. histolytica</i> trophozoites affected for virulent factors and human colonic explants.

<p>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). <b>*</b> indicates a significant difference between HGL2 and control (<i>P</i><0.01), NEO and control (<i>P</i><0.03) and WT and control (<i>P</i><0.05). <b>#</b> indicates a significant difference between HGL2 and control (<i>p</i><0.008), NEO and control (<i>p</i><0.01), RBV and control (<i>p</i><0.01), G3 and control (<i>p</i><0.009), WT and control (<i>p</i><0.007), RBV and RB8 (<i>p</i><0.03), G3 and RB8 (<i>p</i><0.01) and WT and RB8 (<i>p</i><0.01)</p

Pro-inflammatory cytokines secretion induced in the <i>ex-vivo</i> human colonic model by sub-strains of <i>E. histolytica</i>.

<p>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 (<i>p</i><0.05) and 7 hours (<i>p</i><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 (<i>p</i><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.</p

Migration through the lamina propria of <i>E. histolytica</i> sub-strains impaired in virulent functions.

<p>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.</p