Human Intestinal Epithelial Cells Research Articles

VP1 of human and murine noroviruses recognizes glycolipid sulfatide via the P domain.

Noroviruses are a prevalent cause of human viral gastroenteritis, yet the precise mechanisms underlying their infection cycle, particularly their interactions with and entry into cells, remain poorly understood. Human norovirus (HuNoV) primarily targets human small intestinal epithelial cells, within which 3-O sulfogalactosylceramide (sulfatide) ranks among the most abundant glycosphingolipids (GSLs). While sulfatide involvement in the binding and infection mechanism of several viruses has been documented, its interaction with noroviruses remains underexplored. This study investigated whether noroviruses interact with sulfatide. We found that the recombinant viral capsid protein VP1 of HuNoV (genogroups I and II) and murine norovirus (genogroup V) exhibited robust binding to sulfatide compared with other tested GSLs using ELISA, TLC binding assay, and qRT-PCR binding assay. Notably, we found that sulfatide is a novel binding target for norovirus particles. Overall, our findings reveal a previously unknown norovirus-sulfatide interaction, proposing sulfatide as a potential candidate for norovirus infection receptors.

Modulation of miR-155-5p signalling via 5-ASA for the prevention of high microsatellite instability: an in vitro study using human epithelial cell lines.

5-aminosalicylic acid (5-ASA) is a first-line treatment for maintaining colitis remission. It is a highly effective, safe, and well-tolerated drug with anti-inflammatory and chemo-preventive properties. While patients with primary sclerosing cholangitis (PSC) with concomitant ulcerative colitis are treated with 5-ASA, the molecular mechanisms underlying the drug's chemo-preventive effects are not entirely understood. We previously reported that bile acids and lipopolysaccharide-induced miR-155 expression was associated with downregulating mismatch repair (MMR) proteins in CACO-2 cell lines. Therefore, in this investigation, a set of in vitro functional studies was performed to show the possible mechanisms behind the epigenetic relationship between miR-155 and 5-ASA's prevention of high microsatellite instability (MSI-H). In transient transfection with miR-155Mimic, which behaves like endogenous miRNA, we confirmed the relationships between miR-155 and its target MMR in three human intestinal epithelial cell lines: CACO-2, NCM460D and HT-29. We have shown, for the first time, that 5-ASA modulates MLH1, MSH2, MSH6 in miR-155 transfected cells. These findings underline that chemoprotective 5-ASA therapy can effectively attenuate the expression of miR-155 and potentially prevent a development of MSI-H in a subset of colorectal cancers associated with PSC.

Modulation of miR-155-5p signalling via 5-ASA for the prevention of high microsatellite instability: an in vitro study using human epithelial cell lines.

5-aminosalicylic acid (5-ASA) is a first-line treatment for maintaining colitis remission. It is a highly effective, safe, and well-tolerated drug with anti-inflammatory and chemo-preventive properties. While patients with primary sclerosing cholangitis (PSC) with concomitant ulcerative colitis are treated with 5-ASA, the molecular mechanisms underlying the drug's chemo-preventive effects are not entirely understood. We previously reported that bile acids and lipopolysaccharide-induced miR-155 expression was associated with downregulating mismatch repair (MMR) proteins in CACO-2 cell lines. Therefore, in this investigation, a set of in vitro functional studies was performed to show the possible mechanisms behind the epigenetic relationship between miR-155 and 5-ASA's prevention of high microsatellite instability (MSI-H). In transient transfection with miR-155Mimic, which behaves like endogenous miRNA, we confirmed the relationships between miR-155 and its target MMR in three human intestinal epithelial cell lines: CACO-2, NCM460D and HT-29. We have shown, for the first time, that 5-ASA modulates MLH1, MSH2, MSH6 in miR-155 transfected cells. These findings underline that chemoprotective 5-ASA therapy can effectively attenuate the expression of miR-155 and potentially prevent a development of MSI-H in a subset of colorectal cancers associated with PSC.

BTB and CNC homology 1 deficiency disrupts intestinal IgA secretion through regulation of polymeric immunoglobulin receptor expression.

Immunoglobulin A (IgA)-mediated mucosal immunity is important for the host because it contributes to reducing infection risk and to establishing host-microbe symbiosis. BTB and CNC homology 1 (Bach1) is a transcriptional repressor with physiological and pathophysiological functions that are of particular interest for their relation to gastrointestinal diseases. However, Bach1 effects on IgA-mediated mucosal immunity remain unknown. For this study using Bach1-deficient (Bach1-/-) mice, we investigated the function of Bach1 in IgA-mediated mucosal immunity. Intestinal mucosa, feces, and plasma IgA were examined using immunosorbent assay. After cell suspensions were prepared from Peyer's patches and colonic lamina propria, they were examined using flow cytometry. The expression level of polymeric immunoglobulin receptor (pIgR), which plays an important role in the transepithelial transport of IgA, was evaluated using Western blotting, quantitative real-time PCR, and immunohistochemistry. Although no changes in the proportions of IgA-producing cells were observed, the amounts of IgA in the intestinal mucosa were increased in Bach1-/- mice. Furthermore, plasma IgA was increased in Bach1-/- mice, but fecal IgA was decreased, indicating that Bach1-/- mice have abnormal secretion of IgA into the intestinal lumen. In fact, Bach1 deficiency reduced pIgR expression in colonic mucosa at both the protein and mRNA levels. In the human intestinal epithelial cell line LS174T, suppression of Bach1 reduced pIgR mRNA stability. In contrast, overexpression of Bach1 increased pIgR mRNA stability. These results demonstrate that Bach1 deficiency causes abnormal secretion of IgA into the intestinal lumen via suppression of pIgR expression.

BTB and CNC homology 1 deficiency disrupts intestinal IgA secretion through regulation of polymeric immunoglobulin receptor expression.

Immunoglobulin A (IgA)-mediated mucosal immunity is important for the host because it contributes to reducing infection risk and to establishing host-microbe symbiosis. BTB and CNC homology 1 (Bach1) is a transcriptional repressor with physiological and pathophysiological functions that are of particular interest for their relation to gastrointestinal diseases. However, Bach1 effects on IgA-mediated mucosal immunity remain unknown. For this study using Bach1-deficient (Bach1-/-) mice, we investigated the function of Bach1 in IgA-mediated mucosal immunity. Intestinal mucosa, feces, and plasma IgA were examined using immunosorbent assay. After cell suspensions were prepared from Peyer's patches and colonic lamina propria, they were examined using flow cytometry. The expression level of polymeric immunoglobulin receptor (pIgR), which plays an important role in the transepithelial transport of IgA, was evaluated using Western blotting, quantitative real-time PCR, and immunohistochemistry. Although no changes in the proportions of IgA-producing cells were observed, the amounts of IgA in the intestinal mucosa were increased in Bach1-/- mice. Furthermore, plasma IgA was increased in Bach1-/- mice, but fecal IgA was decreased, indicating that Bach1-/- mice have abnormal secretion of IgA into the intestinal lumen. In fact, Bach1 deficiency reduced pIgR expression in colonic mucosa at both the protein and mRNA levels. In the human intestinal epithelial cell line LS174T, suppression of Bach1 reduced pIgR mRNA stability. In contrast, overexpression of Bach1 increased pIgR mRNA stability. These results demonstrate that Bach1 deficiency causes abnormal secretion of IgA into the intestinal lumen via suppression of pIgR expression.

Coactosin-like protein 1 regulates integrity and repair of model intestinal epithelial barriers via actin binding dependent and independent mechanisms

The actin cytoskeleton regulates the integrity and repair of epithelial barriers by mediating the assembly of tight junctions (TJs), and adherens junctions (AJs), and driving epithelial wound healing. Actin filaments undergo a constant turnover guided by numerous actin-binding proteins, however, the roles of actin filament dynamics in regulating intestinal epithelial barrier integrity and repair remain poorly understood. Coactosin-like protein 1 (COTL1) is a member of the ADF/cofilin homology domain protein superfamily that binds and stabilizes actin filaments. COTL1 is essential for neuronal and cancer cell migration, however, its functions in epithelia remain unknown. The goal of this study is to investigate the roles of COTL1 in regulating the structure, permeability, and repair of the epithelial barrier in human intestinal epithelial cells (IEC). COTL1 was found to be enriched at apical junctions in polarized IEC monolayers in vitro. The knockdown of COTL1 in IEC significantly increased paracellular permeability, impaired the steady state TJ and AJ integrity, and attenuated junctional reassembly in a calcium-switch model. Consistently, downregulation of COTL1 expression in Drosophila melanogaster increased gut permeability. Loss of COTL1 attenuated collective IEC migration and decreased cell-matrix attachment. The observed junctional abnormalities in COTL1-depleted IEC were accompanied by the impaired assembly of the cortical actomyosin cytoskeleton. Overexpression of either wild-type COTL1 or its actin-binding deficient mutant tightened the paracellular barrier and activated junction-associated myosin II. Furthermore, the actin-uncoupled COTL1 mutant inhibited epithelial migration and matrix attachment. These findings highlight COTL1 as a novel regulator of the intestinal epithelial barrier integrity and repair.

Identification of human host factors required for beta-defensin-2 expression in intestinal epithelial cells upon a bacterial challenge

The human intestinal tract is colonized with microorganisms, which present a diverse array of immunological challenges. A number of antimicrobial mechanisms have evolved to cope with these challenges. A key defense mechanism is the expression of inducible antimicrobial peptides (AMPs), such as beta-defensins, which rapidly inactivate microorganisms. We currently have a limited knowledge of mechanisms regulating the inducible expression of AMP genes, especially factors from the host required in these regulatory mechanisms. To identify the host factors required for expression of the beta-defensin-2 gene (HBD2) in intestinal epithelial cells upon a bacterial challenge, we performed a RNAi screen using a siRNA library spanning the whole human genome. The screening was performed in duplicate to select the strongest 79 and 110 hit genes whose silencing promoted or inhibited HBD2 expression, respectively. A set of 57 hits selected among the two groups of genes was subjected to a counter-screening and a subset was subsequently validated for its impact onto HBD2 expression. Among the 57 confirmed hits, we brought out the TLR5-MYD88 signaling pathway, but above all new signaling proteins, epigenetic regulators and transcription factors so far unrevealed in the HBD2 regulatory circuits, like the GATA6 transcription factor involved in inflammatory bowel diseases. This study represents a significant step toward unveiling the key molecular requirements to promote AMP expression in human intestinal epithelial cells, and revealing new potential targets for the development of an innovative therapeutic strategy aiming at stimulating the host AMP expression, at the era of antimicrobial resistance.

An in vitro platform for quantifying cell cycle phase lengths in primary human intestinal epithelial cells.

The intestinal epithelium dynamically controls cell cycle, yet no experimental platform exists for directly analyzing cell cycle phases in non-immortalized human intestinal epithelial cells (IECs). Here, we present two reporters and a complete platform for analyzing cell cycle phases in live primary human IECs. We interrogate the transcriptional identity of IECs grown on soft collagen, develop two fluorescent cell cycle reporter IEC lines, design and 3D print a collagen press to make chamber slides for optimal imaging while supporting primary human IEC growth, live image cell cycle dynamics, then assemble a computational pipeline building upon free-to-use programs for semi-automated analysis of cell cycle phases. The PIP-FUCCI construct allows for assigning cell cycle phase from a single image of living cells, and our PIP-H2A construct allows for semi-automated direct quantification of cell cycle phase lengths using our publicly available computational pipeline. Treating PIP-FUCCI IECs with oligomycin demonstrates that inhibiting mitochondrial respiration lengthens G1 phase, and PIP-H2A cells allow us to measure that oligomycin differentially lengthens S and G2/M phases across heterogeneous IECs. These platforms provide opportunities for future studies on pharmaceutical effects on the intestinal epithelium, cell cycle regulation, and more.

Enteroaggregative Escherichia coli: Frequent, yet underdiagnosed pathotype among E. coli O111 strains isolated from children with gastrointestinal disorders in the Czech Republic

Enteroaggregative Escherichia coli (EAEC) strains including those of serogroup O111 are important causes of diarrhea in children. In the Czech Republic, no information is available on the etiological role of EAEC in pediatric diarrhea due to the lack of their targeted surveillance. To fill this gap, we determined the proportion of EAEC among E. coli O111 isolates from children with gastrointestinal disorders ≤ 2 years of age submitted to the National Reference Laboratory for E. coli and Shigella during 2013–2022. EAEC accounted for 177 of 384 (46.1 %) E. coli O111 isolates, being the second most frequent E. coli O111 pathotype. Most of them (75.7 %) were typical EAEC that carried aggR, usually with aaiC and aatA marker genes; the remaining 24.3 % were atypical EAEC that lacked aggR but carried aaiC and/or aatA. Whole genome sequencing of 11 typical and two atypical EAEC O111 strains demonstrated differences in serotypes, sequence types (ST), virulence gene profiles, and the core genomes between these two groups. Typical EAEC O111:H21/ST40 strains resembled by their virulence profiles including the presence of the aggregative adherence fimbriae V (AAF/V)-encoding cluster to such strains from other countries and clustered with them in the core genome multilocus sequence typing (cgMLST). Atypical EAEC O111:H12/ST10 strains lacked virulence genes of typical EAEC and differed from them in cgMLST. All tested EAEC O111 strains displayed stacked-brick aggregative adherence to human intestinal epithelial cells. The AAF/V-encoding cluster was located on a plasmid of 95,749 bp or 93,286 bp (pAAO111) which also carried aggR, aap, aar, sepA, and aat cluster. EAEC O111 strains were resistant to antibiotics, in particular to aminopenicillins and cephalosporins; 88.3 % produced AmpC β-lactamase, and 4.1 % extended spectrum β-lactamase. We conclude that EAEC are frequent among E. coli O111 strains isolated from children with gastrointestinal disorders in the Czech Republic. To reliably assess the etiological role of EAEC in pediatric diarrhea, a serotype-independent, PCR-based pathotype surveillance system needs to be implemented in the future.

Structural characterisation and anti-colon cancer activity of an arabinogalactan RSA-1 from Raphani semen

RSA-1 is a polysaccharide obtained from Raphani semen with a relatively clear structure and anti-colon cancer activity. In this study, high-performance liquid chromatography (HPLC), gas chromatography–mass spectrometry (GC–MS), and nuclear magnetic resonance (NMR) spectroscopy were applied to characterise the complex chain structure of RSA-1. Subsequently, the inhibitory effect on colon cancer growth through apoptosis induction in colon cancer cells was explored. The findings indicate that the main chain of RSA-1 consists of →3)-β-D-Galp-(1 → and 3,6)-β-D-Galp-(1 → substituted at C-6 with branched α-L-Araf side chains. RSA-1 disrupts the Bax/Bcl-2 ratio and thus inhibits the viability of colon cancer cells in vitro. Furthermore, it inhibits colon cancer migration by attenuating epithelial-mesenchymal transition. Notably, RSA-1 exhibited negligible impact on the growth of human intestinal epithelial cells within a relevant concentration range. This study establishes a theoretical foundation and provides technical support for the prospective development and application of RSA-1 as a dual-purpose anti-colon cancer drug and functional food.

Intestinal lysozyme engagement of Salmonella Typhimurium stimulates the release of barrier-impairing InvE and Lpp1

Lysozyme is a β-1,4-glycosidase that hydrolyzes the polysaccharide backbone of bacterial cell walls. With an additional bactericidal function mediated by a separate protein domain, lysozyme is considered as a uniquely important antimicrobial molecule contributing to the host's innate immune response to infection. Elevated lysozyme production is found in various inflammatory conditions while patients with genetic risks for inflammatory bowel diseases demonstrate abnormal lysozyme expression, granule packaging, and secretion in Paneth cells. However, it remains unclear how a gain- or loss-of-function in host lysozyme may impact the host inflammatory responses to pathogenic infection. We challenged Lyz1-/- and ectopic Lyz1-expressing (Villin-Lyz1TG) mice with S. Typhimurium then comprehensively assessed the inflammatory disease progression. We conducted proteomics analysis to identify molecules derived from human lysozyme-mediated processing of live Salmonella. We examined the barrier-impairing effects of these identified molecules in human intestinal epithelial cell monolayer and enteroids. Lyz1-/- mice are protected from infection in terms of morbidity, mortality, and barrier integrity, whereas Villin-Lyz1TG mice demonstrate exacerbated infection and inflammation. The growth and invasion of Salmonella in vitro are not affected by human or chicken lysozyme, whereas lysozyme encountering of live Salmonella stimulates the release of barrier-disrupting factors, InvE-sipC and Lpp1, which directly or indirectly impair the tight junctions. The direct engagement of host intestinal lysozyme with an enteric pathogen such as Salmonella promotes the release of virulence factors that are barrier-impairing and pro-inflammatory. Controlling lysozyme function may help alleviate the inflammatory progression.

Collagen hydrogel protects intestinal epithelial cells from indomethacin-induced damage: results of an in vitro experiment

INTRODUCTION. Indomethacin is a derivative of indoleacetic acid and has anti-inflammatory, analgesic and antipyretic effects. However, the results of numerous studies show that indomethacin, like many other nonsteroidal anti-inflammatory drugs (NSAIDs), have an inhibitory effect on the viability and functional activity of enterocytes. In this regard, the search for new ways to reduce the severity of side effects from the use of NSAIDs remains relevant. One of these approaches may be to enrich patients’ diets with non-drug biologically active compounds, including proteins. However, the effect of dietary proteins and biologically active peptides on NSAID-induced damage to the wall of the small intestine and stomach has not been sufficiently studied. AIM. To evaluate the ability of a collagen-containing dietary supplement to protect human duodenal epithelial cells (HuTu-80 line) from indomethacin-induced damage. MATERIALS AND METHODS. The composite collagen-containing hydrogel was provided by «FIRST ALIVE COLLAGEN» LLC (Russia) and is a registered dietary supplement. The work used a commercial culture of human skin fibroblast cells and human duodenal epithelial cells (line HuTu-80). The viability of intestinal cells and fibroblasts was assessed using light and fluorescence microscopy and flow cytometry methods. RESULTS AND DISCUSSION. It has been established that indomethacin inhibits cell growth, causes apoptosis and death of enterocytes, and also leads to the accumulation of cells in the S-phase, which indicates a disruption in the regulation of the cell cycle. It was revealed that collagen hydrogel prevents cell death caused by indomethacin and reduces the number of apoptotic cells in the population. The protective effect of collagen hydrogel is characterized by normalization of the cell cycle of enterocytes and restoration of their growth and proliferative activity. CONCLUSION. Thus, collagen hydrogel, in vitro, is able to reduce the pathogenic effect of indomethacin on human intestinal epithelial cells. The protective effect of collagen hydrogel is characterized by maintaining viability, inhibiting apoptotic processes, and maintaining cell cycle stability. The results obtained indicate the prospects of using a dietary supplement based on a composite collagen hydrogel as a prophylactic agent to reduce the risk of NSAID-associated gastrointestinal diseases. However, to confirm the therapeutic effectiveness of the dietary supplement, further research is necessary, both using experimental animal modeling of NSAID-associated diseases of the human gastrointestinal tract, and clinical studies.

Outer Membrane Vesicles Derived from Adherent-Invasive Escherichia coli Induce Inflammatory Response and Alter the Gene Expression of Junction-Associated Proteins in Human Intestinal Epithelial Cells.

Adherent-invasive Escherichia coli (AIEC) pathobionts, which are characterized by their ability to adhere to and invade intestinal epithelial cells, are associated with the etiopathogenesis of inflammatory bowel diseases (IBDs). Outer membrane vesicles (OMVs) released by AIEC strains can facilitate the interaction of these bacteria with host cells through delivering bacterial effectors. The aim of this study was to determine the ability of OMVs derived from AIEC strain LF82 to induce the host immune response, leading to production of proinflammatory cytokines and also altering the gene expression of junction-associated proteins in the human epithelial colorectal adenocarcinoma Caco-2 cell line. OMVs were extracted from AIEC strain LF82, and the cell viability of Caco-2 cells treated with these vesicles was assessed by MTT assay. The morphology and size distribution of vesicles were analyzed using transmission electron microscopy and dynamic light scattering, respectively. Gene expression of occludin, ZO-1, claudin-2, E-cadherin, TLR-2, and TLR-4 in response to OMVs was assessed in Caco-2 cells by RT-qPCR. Moreover, the secretion of IL-8 and TNF-α into the supernatant of Caco-2 cells upon treatment with OMVs was measured using ELISA. Our results demonstrated that OMVs upregulated the gene expression level of TLRs and also altered the gene expression level of junction-associated proteins. OMVs derived from AIEC may play a major role in the promotion of intestinal inflammation and epithelial barrier dysfunction. However, further investigations are needed to elucidate the putative role of OMVs in the pathogenesis of AIEC and IBD.

Engineered urolithin A-laden functional polymer-lipid hybrid nanoparticles prevent cisplatin-induced proximal tubular injury in vitro

Functional polymer-lipid hybrid nanoparticles (H-NPs) are a promising class of nanocarriers that combine the benefits of polymer and lipid nanoparticles, offering biocompatibility, structural stability, high loading capacity, and, most importantly, superior surface functionalization. Here, we report the synthesis and design of highly functional H-NPs with specificity toward the transferrin receptor (TfR), using a small molecule ligand, gambogic acid (GA). A fluorescence study revealed the molecular orientation of H-NPs, where the lipid-dense core is surrounded by a polymer exterior, functionalized with GA. Urolithin A, an immunomodulator and anti-inflammatory agent, served as a model drug-like compound to prepare H-NPs via traditional emulsion-based techniques, where H-NPs led to smaller particles (132 nm) and superior entrapment efficiencies (70 % at 10 % drug loading) compared to GA-conjugated polymeric nanoparticles (P-NPs) (157 nm and 52 % entrapment efficiency) and solid lipid nanoparticles (L-NPs) (186 nm and 29 % entrapment efficiency). H-NPs showed superior intracellular accumulation compared to individual NPs using human small intestinal epithelial (FHs 74) cells. The in vitro efficacy was demonstrated by flow cytometry analysis, in which UA-laden H-NPs showed excellent anti-inflammatory properties in cisplatin-induced injury in healthy human proximal tubular cell (HK2) model by decreasing the TLR4, NF-κβ, and IL-β expression. This preliminary work highlights the potential of H-NPs as a novel functional polymer-lipid drug delivery system, establishing the foundation for future research on its therapeutic potential in addressing chemotherapy-induced acute kidney injury in cancer patients.

Common food additive carrageenan inhibits proglucagon expression and GLP-1 secretion by human enteroendocrine L-cells

Proglucagon mRNA expression and GLP-1 secretion by cultured human L-cells (NCI-H716) were inhibited following exposure to λ-carrageenan, a commonly used additive in processed foods. Carrageenan is composed of sulfated or unsulfated galactose residues linked in alternating alpha-1,3 and beta-1,4 bonds and resembles the endogenous sulfated glycosaminoglycans. However, carrageenan has unusual alpha-1,3-galactosidic bonds, which are not innate to human cells and are implicated in immune responses. Exposure to carrageenan predictably causes inflammation, and carrageenan impairs glucose tolerance and contributes to insulin resistance. When cultured human L-cells were deprived overnight of glucose and serum and then exposed to high glucose, 10% FBS, and λ-carrageenan (1 µg/ml) for 10 minutes, 1 h, and 24 h, mRNA expression of proglucagon and secretion of GLP-1 were significantly reduced, compared to control cells not exposed to carrageenan. mRNA expression of proglucagon by mouse L-cells (STC-1) was also significantly reduced and supports the findings in the human cells. Exposure of co-cultured human intestinal epithelial cells (LS174T) to the spent media of the carrageenan-treated L-cells led to a decline in mRNA expression of GLUT-2 at 24 h. These findings suggest that ingestion of carrageenan-containing processed foods may impair the production of GLP-1, counteract the effect of GLP-1 receptor agonists and induce secondary effects on intestinal epithelial cells.

Cholesterol uptake in the intestine is regulated by the LASP1-AKT-NPC1L1 signaling pathway.

Cholesterol is essential for the stability and architecture of the plasma membrane and a precursor of bile acids and steroid hormones in mammals. Excess dietary cholesterol uptake leads to hypercholesterolemia and atherosclerosis and plays a role in cancer development. The role of actin-binding scaffolding protein LIM and SH3 protein 1 (LASP1) in cholesterol trafficking has not been investigated previously. Cholesterol levels, its uptake, and excretion were studied in mice deficient for low-density lipoprotein receptor and Lasp1 (Ldlr-/-Lasp1-/- mice) upon feeding a high-fat diet, and in LASP1-knockdown, differentiated human intestinal epithelial CaCo-2 cells. When compared with diet-fed Ldlr-/- control mice, Ldlr-/-Lasp1-/- mice displayed a reduction in serum cholesterol levels. Mechanistically, we identified a new role of LASP1 in controlling the translocation of the intestinal cholesterol transporter Niemann-Pick C1-like 1 (NPC1L1) to the apical cell surface, which was limited in LASP1-knockdown human CaCo-2 enterocytes and in the intestine of Ldlr-/- Lasp1-/- compared with Ldlr-/- mice, linked to LASP1-pAKT signaling but not CDC42 activation. In line, a reduction in cholesterol reabsorption was noted in LASP1-knockdown CaCo-2 cells in vitro, and an enhanced cholesterol excretion via the feces was observed in Ldlr-/- Lasp1-/- mice. These data uncover a novel function of Lasp1 in cholesterol trafficking, promoting cholesterol reabsorption in the intestine. Targeting LASP1 locally could thus represent a novel targeting strategy to ameliorate hypercholesterolemia and associated diseases.NEW & NOTEWORTHY We here uncovered LASP1 as a novel regulator of the shuttling of the sterol transporter NPC1L1 to the cell surface in enterocytes to control cholesterol absorption. Accordingly, LASP1-deficient mice displayed lowered serum cholesterol levels under dietary cholesterol supplementation.

Air-liquid intestinal cell culture allows in situ rheological characterization of intestinal mucus.

Intestinal health heavily depends on establishing a mucus layer within the gut with physical properties that strike a balance between being sufficiently elastic to keep out harmful pathogens yet viscous enough to flow and turnover the contents being digested. Studies investigating dysfunction of the mucus layer in the intestines are largely confined to animal models, which require invasive procedures to collect the mucus fluid. In this work, we develop a nondestructive method to study intestinal mucus. We use an air-liquid interface culture of primary human intestinal epithelial cells that exposes their apical surface to allow in situ analysis of the mucus layer. Mucus collection is not only invasive but also disrupts the mucus microstructure, which plays a crucial role in the interaction between mucus and the gut microbiome. Therefore, we leverage a noninvasive rheology technique that probes the mechanical properties of the mucus without removal from the culture. Finally, to demonstrate biomedical uses for this cell culture system, we characterize the biochemical and biophysical properties of intestinal mucus due to addition of the cytokine IL-13 to recapitulate the gut environment of Nippostrongylus brasiliensis infection.

Differential Effect of JAK1/2 and TYK2 Inhibitors on Type III Interferon Induced Antiviral Defence in Human Intestinal Epithelial Cells

Plain Language SummaryCytokines are small molecules that play a crucial role in causing inflammatory diseases. Janus kinase (JAK) and tyrosine kinase (TYK) are proteins that help these messengers communicate with cells. JAK inhibitors (JAKis) are drugs that block these proteins and are becoming more and more commonly used to treat inflammatory diseases. A common side effect of JAKis is an increased risk for viral infections. In this study, it is shown that a TYK2 inhibitor drug may not weaken our defences against viruses in our intestines as much as a JAK1/2 inhibitor. As this inhibitor blocks the disease causing cytokines, this inhibitor drug may be better than the other and be associated with lower risk of infections with viruses that enter the body through the gut.

Cdc42 Rho GTPase Activity Sustains Epithelial Barrier Function in the Cystic Fibrosis Intestine

Increased paracellular permeability is a common manifestation of cystic fibrosis (CF) intestinal disease in people with CF and in CF mouse models. Increased paracellular permeability has been found in CF airway epithelium cultured in the absence of inflammatory factors, suggesting an inherent defect in CF epithelial barrier function (Weiser et al., Cell Physiol Biochem. 2011;28(2):289-96). CF transmembrane conductance regulator knockout ( Cftr KO) mouse intestinal crypts demonstrate an increase in epithelial cell proliferation, which correlates with an alkaline intracellular pH (pHi), a pHi-dependent increase in lateral cell membrane localization of the Wnt transducer Disheveled (Dvl), and increased Wnt/β-catenin signaling (Strubberg et al., Cell Mol Gastroenterol Hepatol. 2017 Dec 7;5(3):253-271). We hypothesized that the alkaline pHi of Cftr KO crypts also facilitates Dvl-dependent noncanonical Wnt signaling to increase Rho GTPase activity and thereby tight junction remodeling during increased proliferation. In Dvl2-eGFP rescue mice, Dvl2-eGFP intensity near the apical membrane of Cftr KO enteroid crypts was greater versus wild-type (WT) at both the crypt base and the transit-amplifying zone. Immunofluorescence studies of both freshly isolated crypts and early passage enteroids also found increased accumulation of the Rho GTPase Cdc42 at the tight junctions of Cftr KO versus WT crypts. Live imaging revealed a small increase in leak pathway permeability (3kD dextran) in Cftr KO as compared to WT murine enteroids. Increased leak permeability and Cdc42 tight junction localization were normalized in Cftr KO enteroids by suppressing proliferation through inhibition of Wnt/β-catenin signaling. There was no difference in Cdc42 expression in Cftr KO fresh crypts or enteroids, but initial studies show increased Cdc42 GTP-loaded activity in Cftr KO enteroids relative to WT. Similarly, CFTR KO human intestinal epithelial Caco-2 cells, which also show an alkaline pHi, increased Wnt/β-catenin signaling and a hyperproliferative phenotype, demonstrated a significant increase in GTP-loaded Cdc42 activity compared to WT cells. Inhibition of Cdc42 activity with ML141 (R&D Systems) resulted in a significant disruption of the Cftr KO epithelial barrier, whereas WT enteroid permeability was not affected by Cdc42 inhibition, Wnt inhibition or Wnt3a-induced proliferation. We conclude that increased Cdc42 Rho GTPase activity is cell-autonomous and is necessary for maintaining barrier integrity of the Cftr KO crypt epithelium. This work was supported by the Cystic Fibrosis Foundation grant CLARKE20G0 and the Crohn’s and Colitis Foundation grant #693938. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

TLR7-dependent eosinophil degranulation links psoriatic skin inflammation to small intestinal inflammatory changes in mice

Recent evidence of gut microbiota dysbiosis in the context of psoriasis and the increased cooccurrence of inflammatory bowel disease and psoriasis suggest a close relationship between skin and gut immune responses. Using a mouse model of psoriasis induced by the Toll-like receptor (TLR) 7 ligand imiquimod, we found that psoriatic dermatitis was accompanied by inflammatory changes in the small intestine associated with eosinophil degranulation, which impaired intestinal barrier integrity. Inflammatory responses in the skin and small intestine were increased in mice prone to eosinophil degranulation. Caco-2 human intestinal epithelial cells were treated with media containing eosinophil granule proteins and exhibited signs of inflammation and damage. Imiquimod-induced skin and intestinal changes were attenuated in eosinophil-deficient mice, and this attenuation was counteracted by the transfer of eosinophils. Imiquimod levels and the distribution of eosinophils were positively correlated in the intestine. TLR7-deficient mice did not exhibit intestinal eosinophil degranulation but did exhibit attenuated inflammation in the skin and small intestine following imiquimod administration. These results suggest that TLR7-dependent bidirectional skin-to-gut communication occurs in psoriatic inflammation and that inflammatory changes in the intestine can accelerate psoriasis.