Abstract
BackgroundMammalian small intestinal tight junctions (TJ) link epithelial cells to one another and function as a permselective barrier, strictly modulating the passage of ions and macromolecules through the pore and leak pathways, respectively, thereby preventing the absorption of harmful compounds and microbes while allowing regulated transport of nutrients and electrolytes. Small intestinal epithelial permeability is ascribed primarily to the properties of TJs between adjoining enterocytes (ENTs), because there is almost no information on TJ composition and the paracellular permeability of nonenterocyte cell types that constitute a small but significant fraction of the intestinal epithelia.ResultsHere we directed murine intestinal crypts to form specialized organoids highly enriched in intestinal stem cells (ISCs), absorptive ENTs, secretory goblet cells, or Paneth cells. The morphological and morphometric characteristics of these cells in organoids were similar to those in vivo. The expression of certain TJ proteins varied with cell type: occludin and tricellulin levels were high in both ISCs and Paneth cells, while claudin-1, -2, and -7 expression was greatest in Paneth cells, ISCs, and ENTs, respectively. In contrast, the distribution of claudin-15, zonula occludens 1 (ZO-1), and E-cadherin was relatively homogeneous. E-cadherin and claudin-7 marked mainly the basolateral membrane, while claudin-2, ZO-1, and occludin resided in the apical membrane. Remarkably, organoids enriched in ENTs or goblet cells were over threefold more permeable to 4 and 10 kDa dextran compared to those containing stem and Paneth cells. The TJ-regulator larazotide prevented the approximately tenfold increases in dextran flux induced by the TJ-disrupter AT1002 into organoids of different cell types, indicating that this ZO toxin nonselectively increases permeability. Forced dedifferentiation of mature ENTs results in the reacquisition of ISC-like characteristics in TJ composition and dextran permeability, suggesting that the post-differentiation properties of TJs are not hardwired.ConclusionsDifferentiation of adult intestinal stem cells into mature secretory and absorptive cell types causes marked, but potentially reversible, changes in TJ composition, resulting in enhanced macromolecular permeability of the TJ leak pathway between ENTs and between goblet cells. This work advances our understanding of how cell differentiation affects the paracellular pathway of epithelia.
Highlights
Mammalian small intestinal tight junctions (TJ) link epithelial cells to one another and function as a permselective barrier, strictly modulating the passage of ions and macromolecules through the pore and leak pathways, respectively, thereby preventing the absorption of harmful compounds and microbes while allowing regulated transport of nutrients and electrolytes
Tight junction proteins and the leak pathway mRNA expression Since there are numerous TJ proteins, we focused on ones that have been shown to be expressed significantly in the small intestine, to vary in expression along the crypt–villus axis, or potentially to regulate changes in paracellular permeability [1, 6]. mRNA levels of all organoid claudins were within an order of magnitude of those in isolated crypts or mucosal homogenates, suggesting that expression was similar to that in vivo
Since shaped and sized ENT and Paneth cells (PANs) organoids accumulated dextrans at vastly different rates, while differently shaped and sized PAN and intestinal stem cells (ISCs) organoids both accumulated dextrans at limited rates, these findings suggest that the shapes of organoids likely do not affect the accumulation of dextran, and by extension, of our estimates of permeability of the leak pathway
Summary
Mammalian small intestinal tight junctions (TJ) link epithelial cells to one another and function as a permselective barrier, strictly modulating the passage of ions and macromolecules through the pore and leak pathways, respectively, thereby preventing the absorption of harmful compounds and microbes while allowing regulated transport of nutrients and electrolytes. The small intestinal cells contain digestive enzymes and transporters that process nutrients and electrolytes, but the barrier between cells is essential because the lumen contains large amounts of bacteria, viruses, food allergens, toxins, and excreted metabolic byproducts, as well as varying concentrations of ions and solutes that can be markedly different from those of internal fluids This barrier, which is permselectively sealed by tight junctions (TJs), has at least two types of pathways: the high-capacity pore pathway regulating mainly the transport of ions and small molecules, and the low-capacity leak pathway modulating the flux of large uncharged macromolecules [1, 2]. Adherens junctions and desmosomes are found between cells but these exhibit mainly structural functions
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