Marked differences in tight junction composition and macromolecular permeability among different intestinal cell types

Abstract

Mammalian small intestinal (SI) tight junctions (TJ) link epithelial cells to one another and function as aperm selective barrier, strictly modulating the passage of ions and macromolecules through the “pore” and “leak” pathways, respectively, thereby preventing absorption of harmful compounds and microbes while allowing regulated transport of nutrients and electrolytes. SI epithelial permeability is ascribed primarily to properties of TJ between adjoiningenterocytes (ENT), because there is almost no information on TJ composition and paracellular permeability of non‐enterocyte cell types that constitute a small but significant fraction of the intestinal epithelia. In the current study we directed murine intestinal crypts to form specialized organoids highly enrichedin stem cells (ISC), absorptive ENTs, secretory goblet (GOB) or Paneth cells(PAN). Morphological and morphometriccharacteristics of these cells in organoids were similar to those in vivo. Expression of certain TJ genes varied with cell type. Occludin and tricellulin levels were high in both ISC and PAN organoids, while claudin‐1, junctional adhesion molecule‐1, and cingulin were greatest mainly in PAN. Claudin‐2 was greatest in ISC and claudin‐7 expression was greatest in ENT organoids. In contrast, claudin‐15, zonula occludens 1 (ZO‐1) and e‐cadherin distribution were relatively homogeneous among cell types. Immunofluorescent staining showed that e‐cadherin and claudin‐7 marked mainly the basolateral membrane, while claudin‐2, ZO‐1 and occludin resided apically. Remarkably, organoids enrichedin ENTs and GOBs, respectively were 3.7‐fold and 3.4‐fold more permeable to 4 kda dextran thanthose enriched in ISCs (P<0.05). Similarly, ENT and GOB were 2.9 and2.6‐fold more permeable to 10 kda dextran compared to ISC organoids. The trend was similar (>2‐fold)for ENT and GOB vs. PAN organoids for both 4 and 10 kda dextran flux (P<0.05). As was expected, for all cell types 10 kda dextran flux was significantly lower than 4 kda (P < 0.05). A time course of dextran flux in ENT and PAN organoids revealed that 4 kda dextran entered the lumen of ENT by 10 min and highest intensity was observed by 30 min (P<0.05). In contrast, dextran flux was nearly absent in PAN organoids, regardless of time point. Additionally, the TJ regulator larazotide prevented the ~10‐fold increases in dextran flux induced by the TJ disrupter AT1002 in both ISC and ENT organoids (P < 0.05), indicating that AT1002 non‐selectively increases permeability. Lastly, dedifferentiation of mature ENTs results inreacquisition of ISC‐like characteristics in TJ composition and dextranpermeability, suggesting that post‐differentiation properties of TJs are not hard wired. In conclusion, differentiation 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 paracellularpathway of epithelia.Support or Funding InformationSupported by NSF Grants No. IOS‐1121049 (RPF) and 1456673 (RPF) and by NIH Grant R01‐DK‐102934 (NG).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.