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Development of a primary mouse intestinal epithelial cell monolayer culture system to evaluate factors that modulate IgA transcytosis.

ABSTRACT: There is significant interest in the use of primary intestinal epithelial cells in monolayer culture to model intestinal biology. However, it has proven to be challenging to create functional, differentiated monolayers using current culture methods, likely due to the difficulty in expanding these cells. Here, we adapted our recently developed method for the culture of intestinal epithelial spheroids to establish primary epithelial cell monolayers from the colon of multiple genetic mouse strains. These monolayers contained differentiated epithelial cells that displayed robust transepithelial electrical resistance. We then functionally tested them by examining immunoglobulin A (IgA) transcytosis across Transwells. IgA transcytosis required induction of polymeric Ig receptor (pIgR) expression, which could be stimulated by a combination of lipopolysaccharide and inhibition of ?-secretase. In agreement with previous studies using immortalized cell lines, we found that tumor necrosis factor-?, interleukin (IL)-1?, IL-17, and heat-killed microbes also stimulated pIgR expression and IgA transcytosis. We used wild-type and knockout cells to establish that among these cytokines, IL-17 was the most potent inducer of pIgR expression/IgA transcytosis. Interferon-?, however, did not induce pIgR expression, and instead led to cell death. This new method will allow the use of primary cells for studies of intestinal physiology.

SUBMITTER: Moon C

PROVIDER: S-EPMC4019725 | biostudies-literature | 2014 Jul

REPOSITORIES: biostudies-literature

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Development of a primary mouse intestinal epithelial cell monolayer culture system to evaluate factors that modulate IgA transcytosis.

Moon C C VanDussen K L KL Miyoshi H H Stappenbeck T S TS

Mucosal immunology 20131113 4

There is significant interest in the use of primary intestinal epithelial cells in monolayer culture to model intestinal biology. However, it has proven to be challenging to create functional, differentiated monolayers using current culture methods, likely due to the difficulty in expanding these cells. Here, we adapted our recently developed method for the culture of intestinal epithelial spheroids to establish primary epithelial cell monolayers from the colon of multiple genetic mouse strains. ...[more]

PMID: 24220295

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Project description:BackgroundProtozoan parasite secretions can be triggered by various modified media and diverse physicochemical stressors. Equally, host-parasite interactions are known to co-opt the exchange and secretion of soluble biochemical components. Analysis of Eimeria falciformis sporozoite secretions in response to interaction with mouse intestinal epithelial cells (MIECs) may reveal parasite secretory motifs, protein composition and inflammatory activities of E. falciformis extracellular vesicles (EVs).MethodsEimeria falciformis sporozoites were allowed to interact with inactivated MIECs. Parasite secretions were separated into EV and vesicle-free (VF) fractions by discontinuous centrifugation and ultracentrifugation. Secreted EVs were purified in an iodixanol density gradient medium and the protein composition of both EV and VF fractions were analyzed by liquid chromatoraphy-tandem mass spectroscopy. The inflammatory activities of E. falciformis sporozoite EV on MIECs were then investigated.ResultsDuring the interaction of E. falciformis sporozoites with inactivated MIECs, the parasite secreted VF and vesicle-bound molecules. Eimeria falciformis vesicles are typical pathogenic protozoan EVs with a mean diameter of 264 ± 2 nm, and enclosed heat shock protein (Hsp) 70 as classical EV marker. Refractile body-associated aspartyl proteinase (or eimepsin), GAP45 and aminopeptidase were the main components of E. falciformis sporozoite EVs, while VF proteins include Hsp90, actin, Vps54 and kinases, among others. Proteomic data revealed that E. falciformis EV and VF proteins are aggregates of bioactive, antigenic and immunogenic molecules which act in concert for E. falciformis sporozoite motility, pathogenesis and survival. Moreover, in MIECs, E. falciformis EVs induced upregulation of gene expression and secretion of IL-1β, IL-6, IL-17, IL-18, MCP1 as well as pyroptosis-dependent caspase 11 and NLRP6 inflammasomes with the concomitant secretion of lactate dehydrogenase.ConclusionsEimeria falciformis sporozoite interaction with MIECs triggered the secretion of immunogenic and antigenic proteins. In addition, E. falciformis sporozoite EVs constitute parasite-associated molecular pattern that induced inflammatory response and cell death. This study offers additional insight in the secretion and protein composition of E. falciformis secretomes as well as the proinflammatory functions of E. falciformis sporozoite EVs.

Dataset Information

Development of a primary mouse intestinal epithelial cell monolayer culture system to evaluate factors that modulate IgA transcytosis.

Publications

Development of a primary mouse intestinal epithelial cell monolayer culture system to evaluate factors that modulate IgA transcytosis.

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