Plasmolipin regulates basolateral-to-apical transcytosis of ICAM-1 and leukocyte adhesion in polarized hepatic epithelial cells

Abstract

Apical localization of Intercellular Adhesion Receptor (ICAM)-1 regulates the adhesion and guidance of leukocytes across polarized epithelial barriers. Here, we investigate the molecular mechanisms that determine ICAM-1 localization into apical membrane domains of polarized hepatic epithelial cells, and their effect on lymphocyte-hepatic epithelial cell interaction. We had previously shown that segregation of ICAM-1 into apical membrane domains, which form bile canaliculi and bile ducts in hepatic epithelial cells, requires basolateral-to-apical transcytosis. Searching for protein machinery potentially involved in ICAM-1 polarization we found that the SNARE-associated protein plasmolipin (PLLP) is expressed in the subapical compartment of hepatic epithelial cells in vitro and in vivo. BioID analysis of ICAM-1 revealed proximal interaction between this adhesion receptor and PLLP. ICAM-1 colocalized and interacted with PLLP during the transcytosis of the receptor. PLLP gene editing and silencing increased the basolateral localization and reduced the apical confinement of ICAM-1 without affecting apicobasal polarity of hepatic epithelial cells, indicating that ICAM-1 transcytosis is specifically impaired in the absence of PLLP. Importantly, PLLP depletion was sufficient to increase T-cell adhesion to hepatic epithelial cells. Such an increase depended on the epithelial cell polarity and ICAM-1 expression, showing that the epithelial transcytotic machinery regulates the adhesion of lymphocytes to polarized epithelial cells. Our findings strongly suggest that the polarized intracellular transport of adhesion receptors constitutes a new regulatory layer of the epithelial inflammatory response.

Molecular Insights of Cholestasis in MDR2 Knockout Murine Liver Organoids

“…Differentiated organoids formed spheroids (Figure A,B). In addition to secreting albumin and processing CFDA (not shown), , electron microscopy analyses confirmed that organoid cells formed lateral cavities between them, which have been shown to functionally resemble bile canaliculi upon differentiation …”

“…(B) Differentiated organoids from WT and MDR2KO mice were fixed and processed for transmission electron microscopy. Cells are organized in sheets facing big central lumens (left and central images) and form smaller cavities between their lateral membranes containing cell–cell junctions and microvilli (right images), which have been shown to have functional features resembling bile canaliculi and contain canalicular markers …”

“…Liver organoids from WT and MDR2KO (MDR2–/−) mice (FVB.129P2-Abcb4tm1Bor/J) were generated and cultured as described. , Briefly, livers were isolated from 3 month-old WT and MDR2KO mice and digested using a mix of collagenase and Dispase II. Isolated hepatic bipotent adult stem cells were embedded in Matrigel (BD Bioscience) and cultured in expansion medium containing a cocktail of liver-specific growth factors such as EGF, FGF10, Rspo1, HGF, and nicotinamide.…”

ICAM-1 nanoclusters regulate hepatic epithelial cell polarity by leukocyte adhesion-independent control of apical actomyosin

“…We had previously shown that ICAM-1 is expressed in sinusoids but also concentrates in the bile canaliculi and bile ducts of polarized hepatic epithelial cells of the human liver parenchyma (Figure 1A) (Reglero-Real et al , 2014). HepG2 cells are spontaneously polarized human hepatic epithelial cells that form actin-rich, bile canalicular-like structures (BCs) and are thus a prototypical in vitro model for studying hepatic apicobasal polarity (Reglero-Real et al , 2014; Lazaro-Dieguez & Musch, 2017; Madrid et al , 2010; van Ijzendoorn et al , 1997; Cacho-Navas et al , 2022). To investigate the role of ICAM-1 on hepatic epithelial cell polarity, we first generated HepG2 cells in which the ICAM-1 gene was edited using CRISPR/CAS9 (ICAM-1_KO cells) (Figure 1B and C).…”

“…The apical membrane domains of hepatocytes and cholangiocytes form bile canaliculi and bile ducts, respectively, which drain bile acids and other hepatic molecules into the gastrointestinal tract. The composition of the apical plasma membrane domains in these small channels differs from that of basolateral membranes and from plasma membranes in non-polarized cells, and is determined by an actin cytoskeletal network, which concentrates in these apical domains, and by mechanisms of polarized intracellular sorting of proteins and lipids towards these membrane regions (Meyer et al , 2020; Tsukada et al , 1995; Fu et al , 2010; Cacho-Navas et al , 2022; Musch, 2014). The filamentous actin cytoskeleton in the bile canaliculus forms small membrane protrusions, called microvilli, which contain actin but not myosin.…”