No abstract

The protozoan parasite Giardia duodenalis is responsible for more than 280 million cases of gastrointestinal complaints (“giardiasis”) every year, worldwide. Infections are acquired orally, mostly via uptake of cysts in contaminated drinking water. After transformation into the trophozoite stage, parasites start to colonize the duodenum and upper jejunum where they attach to the intestinal epithelium and replicate vegetatively. Outcome of Giardia infections vary between individuals, from self-limiting to chronic, and asymptomatic to severely symptomatic infection, with unspecific gastrointestinal complaints. One proposed mechanism for pathogenesis is the breakdown of intestinal barrier function. This has been studied by analyzing trans-epithelial electric resistances (TEER) or by indicators of epithelial permeability using labeled sugar compounds in in vitro cell culture systems, mouse models or human biopsies and epidemiological studies. Here, we discuss the results obtained mainly with epithelial cell models to highlight contradictory findings. We relate published studies to our own findings that suggest a lack of barrier compromising activities of recent G. duodenalis isolates of assemblage A, B, and E in a Caco-2 model system. We propose that this epithelial cell model be viewed as mimicking asymptomatic infection. This view will likely lead to a more informative use of the model if emphasis is shifted from aiming to identify Giardia virulence factors to defining non-parasite factors that arguably appear to be more decisive for disease.

Background and aimsThe protozoa Giardia duodenalis is a major cause of gastrointestinal illness worldwide, but underlying pathophysiological mechanisms remain obscure, partly due to the absence of adequate cellular models. We aimed to overcome these limitations and to recapitulate the authentic series of events in the primary human duodenal tissue by using the human organoid system.MethodsWe established a compartmentalized cellular transwell system with electrophysiological and barrier properties akin to duodenal mucosa and dissected the events leading to G. duodenalis-induced barrier breakdown by functional analysis of transcriptional, electrophysiological and tight junction components.ResultsOrganoid-derived cell layers of different donors showed a time- and parasite load-dependent leak flux indicated by collapse of epithelial barrier upon G. duodenalis infection. Transcriptomic analysis suggested major expression changes in genes contributing to ion transport and tight junction structure. SLC12A2/NKCC1- and CFTR-dependent chloride secretion was reduced early after infection, while changes in the tight junction composition, localization and structural organization occurred later as revealed by immunofluorescence analysis and freeze fracture electron microscopy.ConclusionData suggest a previously unknown sequence of events culminating in intestinal barrier dysfunction upon G. duodenalis infection ignited by alterations of cellular ion transport followed by breakdown of the tight junctional complex and loss of epithelial integrity. The newly established organoid-derived model to study G. duodenalis infection will help enable further molecular dissection of the disease mechanism and, thus, can help to find new options treating disease and infection, in particular relevant for chronic cases of giardiasis.

Usually, duodenal barriers are investigated using intestinal cell lines like Caco-2, which in contrast to native tissue are limited in cell-type representation. Organoids can consist of all intestinal cell types and are supposed to better reflect the in vivo situation.Growing three-dimensionally, with the apical side facing the lumen, application of typical physiological techniques to analyze the barrier is difficult. Organoid-derived monolayers (ODMs) were developed to overcome this. After optimizing culturing conditions, ODMs were characterized and compared to Caco-2 and duodenal tissue. Tight junction composition and appearance were analyzed, and electrophysiological barrier properties, like paracellular and transcellular barrier function and macromolecule permeability, were evaluated. Furthermore, transcriptomic data were analyzed. ODMs had tight junction protein expression and paracellular barrier properties much more resembling the originating tissue than Caco-2. Transcellular barrier was similar between ODMs and native tissue but was increased in Caco-2. Transcriptomic data showed that Caco-2 expressed fewer solute carriers than ODMs and native tissue. In conclusion, while Caco-2 cells differ mostly in transcellular properties, ODMs reflect trans-and paracellular properties of the originating tissue. If cultured under optimized conditions, ODMs possess reproducible functionality, and the variety of different cell types makes them a suitable model for human tissue-specific investigations.