Increased Activity of Epithelial Cdc42 Rho GTPase and Tight Junction Permeability in the Cftr Knockout Intestine.

Increased intestinal permeability is a manifestation of cystic fibrosis (CF) in people with CF (pwCF) and in CF mouse models. CF transmembrane conductance regulator knockout (Cftr KO) mouse intestine exhibits increased proliferation and Wnt/ß-catenin signaling relative to wild-type mice (WT). Since the Rho GTPase Cdc42 plays a central role in intestinal epithelial proliferation and tight junction remodeling, we hypothesized that Cdc42 may be altered in the Cftr KO crypts. Immunofluorescence showed distinct tight junction localization of Cdc42 in Cftr KO fresh crypts and enteroids, the latter indicating an epithelial-autonomous feature. Quantitative PCR and immunoblots revealed similar expression of Cdc42 in the Cftr KO crypts/enteroids relative to WT, whereas pull-down assays showed increased GTP-bound (active) Cdc42 in proportion to total Cdc42 in Cftr KO enteroids. Cdc42 activity in the Cftr KO and WT enteroids could be reduced by inhibition of the Wnt transducer Disheveled 2. Using a dye permeability assay, Cftr KO enteroids exhibited increased paracellular permeability to 3kD dextran relative to WT. In Cftr KO relative to WT enteroids, leak permeability and Cdc42 tight junction localization were reduced to a greater extent by inhibition of Wnt/ß-catenin signaling with Endo-IWR1. Increased proliferation or inhibition of Cdc42 activity with ML141 had no effect on WT enteroid permeability. In contrast, inhibition of Cdc42 with ML141 increased permeability to both 3kD dextran and tight-junction impermeant 500 kD dextran in Cftr KO enteroids. These data suggest that increased constitutive Cdc42 activity may alter the stability of paracellular permeability in Cftr KO crypt epithelium.

Equine enteroid-derived monolayers recapitulate key features of parasitic intestinal nematode infection.

Stem cell-derived organoid cultures have emerged as attractive experimental models for infection biology research regarding various types of gastro-intestinal pathogens and host species. However, the large size of infectious nematode larvae and the closed structure of 3-dimensional organoids often hinder studies of the natural route of infection. To enable easy administration to the apical surface of the epithelium, organoids from the equine small intestine, i.e. enteroids, were used in the present study to establish epithelial monolayer cultures. These monolayers were functionally tested by stimulation with IL-4 and IL-13, and/or exposure to infectious stage larvae of the equine nematodes Parascaris univalens, cyathostominae and/or Strongylus vulgaris. Effects were recorded using transcriptional analysis combined with histochemistry, immunofluorescence-, live-cell- and scanning electron microscopy. These analyses revealed heterogeneous monolayers containing both immature and differentiated cells including tuft cells and mucus-producing goblet cells. Stimulation with IL-4/IL-13 increased tuft- and goblet cell differentiation as demonstrated by the expression of DCLK1 and MUC2. In these cytokine-primed monolayers, the expression of MUC2 was further promoted by co-culture with P. univalens. Moreover, live-cell imaging revealed morphological alterations of the epithelial cells following exposure to larvae even in the absence of cytokine stimulation. Thus, the present work describes the design, characterization and usability of an experimental model representing the equine nematode-infected small intestinal epithelium. The presence of tuft cells and goblet cells whose mucus production is affected by Th2 cytokines and/or the presence of larvae opens up for mechanistic studies of the physical interactions between nematodes and the equine intestinal mucosa.

Expression and functional characterization of bovine receptor activator of NF-κB ligand (RANKL).

Receptor activator of nuclear factor Kappa-B Ligand (RANKL) is a member of the tumor necrosis factor ligand (TNF) family involved in immune responses and immunomodulation. Expressed in various cells types around the body, RANKL plays a crucial role in bone remodeling and development of the thymus, lymph nodes and mammary glands. Research in other species demonstrates that RANKL is required for the development of microfold cells (M cells) in the gut, however limited information specific to cattle is available. Cloning and expression of bovine RANKL (BoRANKL) was carried out and bioactivity of the protein was demonstrated in the induction of osteoclast differentiation from both bovine and ovine bone marrow cells. The effects of BoRANKL on particle uptake in bovine enteroids was also assessed. The production of cross-reactive bovine RANKL protein will enable further investigations into cell differentiation using the available ruminant organoid systems, and their role in investigating host-pathogen interactions in cattle and sheep.

A novel in vitro model of the small intestinal epithelium in co-culture with 'gut-like' dendritic cells.

Cross-talk between dendritic cells (DCs) and the intestinal epithelium is important in the decision to mount a protective immune response to a pathogen or to regulate potentially damaging responses to food antigens and the microbiota. Failures in this decision-making process contribute to the development of intestinal inflammation, making the molecular signals that pass between DCs and intestinal epithelial cells potential therapeutic targets. Until now, in vitro models with sufficient complexity to understand these interactions have been lacking. Here, we outline the development of a co-culture model of in vitro differentiated 'gut-like' DCs with small intestinal organoids (enteroids). Sequential exposure of murine bone marrow progenitors to Flt3L, granulocyte macrophage colony-stimulating factor (GM-CSF) and all-trans-retinoic acid (RA) resulted in the generation of a distinct population of conventional DCs expressing CD11b+SIRPα+CD103+/- (cDC2) exhibiting retinaldehyde dehydrogenase (RALDH) activity. These 'gut-like' DCs extended transepithelial dendrites across the intact epithelium of enteroids. 'Gut-like' DC in co-culture with enteroids can be utilized to define how epithelial cells and cDCs communicate in the intestine under a variety of different physiological conditions, including exposure to different nutrients, natural products, components of the microbiota, or pathogens. Surprisingly, we found that co-culture with enteroids resulted in a loss of RALDH activity in 'gut-like' DCs. Continued provision of GM-CSF and RA during co-culture was required to oppose putative negative signals from the enteroid epithelium. Our data contribute to a growing understanding of how intestinal cDCs assess environmental conditions to ensure appropriate activation of the immune response.

Corrigendum: Unraveling the interplay between norovirus infection, gut microbiota, and novel antiviral approaches: a comprehensive review.

[This corrects the article DOI: 10.3389/fmicb.2023.1212582.].