Increase In Transepithelial Electrical Resistance Research Articles

Evaluation of Kefir Grain Microbiota, Grain Viability, and Kefir Bioactivity from Fermenting Dairy Processing By-Products

Four dairy foods processing by-products (acid whey permeate (AWP), buttermilk (BM), sweet whey permeate (SWP), and sweet whey permeate with added milk fat globule ingredient (SWP+MFGM)) were fermented for 4 weeks and compared with traditional kefir milks for production of novel kefir-like dairy products. AU: Sweet whey permeates and SWP supplemented with 1.5% milk fat globule membrane (MFGM) showed to be the most viable by-products for kefir grain fermentation, exhibiting diverse abundance of traditional kefir microorganisms and positive indicators of bioactive properties. Grain viability was assessed with shotgun metagenomics, texture profile analysis, live cell counts, and scanning electron microscopy. Assessed bioactivities of the kefir-like products included antibacterial, antioxidant, potential anticancerogenic properties, and membrane barrier effect on human colorectal adenocarcinoma Caco-2 cells. All kefir grains were most abundant in Lactobacillus kefiranofaciens when analyzed with shotgun metagenomics. When analyzed with live cell counts on selective media, AWP kefir-like product had no countable Lactococcus spp. indicating suboptimal conditions for kefir grain microbiota survival and application for fermented dairy starter culture bacterium. Live cell counts were affirmed with kefir grain surface scanning electron microscopy images. SWP had the most adhesive kefir grain surface while SWP+MFGM had the largest exopolysaccharide (EPS) yield from grain extraction. All kefir and kefir-like products were able to achieve a 6-log reduction against Listeria innocua and Escherichia coli. Traditional milk kefirs had the highest antioxidant capacity for 2,2-diphenyl-1-picrylhydrazyl (DPPH) and the 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay. AWP had a significantly higher DPPH antioxidant activity and SWP had the lowest Trolox equivalence concentration in the ABTS assay. Sweet whey and supplemented milk fat sweet whey had upregulation of Cldn-1 and Ocln-1 gene expression, which correspond with a significant increase in transepithelial electrical resistance.

Modelling bronchial epithelial-fibroblast cross-talk in idiopathic pulmonary fibrosis (IPF) using a human-derived in vitro air liquid interface (ALI) culture

Idiopathic Pulmonary Fibrosis (IPF) is a devastating form of respiratory disease with a life expectancy of 3–4 years. Inflammation, epithelial injury and myofibroblast proliferation have been implicated in disease initiation and, recently, epithelial-fibroblastic crosstalk has been identified as a central driver. However, the ability to interrogate this crosstalk is limited due to the absence of in vitro models that mimic physiological conditions. To investigate IPF dysregulated cross-talk, primary normal human bronchial epithelial (NHBE) cells and primary normal human lung fibroblasts (NHLF) or diseased human lung fibroblasts (DHLF) from IPF patients, were co-cultured in direct contact at the air–liquid interface (ALI). Intercellular crosstalk was assessed by comparing cellular phenotypes of co-cultures to respective monocultures, through optical, biomolecular and electrical methods. A co-culture-dependent decrease in epithelium thickness, basal cell mRNA (P63, KRT5) and an increase in transepithelial electrical resistance (TEER) was observed. This effect was significantly enhanced in DHLF co-cultures and lead to the induction of epithelial to mesenchymal transition (EMT) and increased mRNA expression of TGFβ-2, ZO-1 and DN12. When stimulated with exogenous TGFβ, NHBE and NHLF monocultures showed a significant upregulation of EMT (COL1A1, FN1, VIM, ASMA) and senescence (P21) markers, respectively. In contrast, direct NHLF/NHBE co-culture indicated a protective role of epithelial-fibroblastic cross-talk against TGFβ-induced EMT, fibroblast-to-myofibroblast transition (FMT) and inflammatory cytokine release (IL-6, IL-8, IL-13, IL-1β, TNF-α). DHLF co-cultures showed no significant phenotypic transition upon stimulation, likely due to the constitutively high expression of TGFβ isoforms prior to any exogenous stimulation. The model developed provides an alternative method to generate IPF-related bronchial epithelial phenotypes in vitro, through the direct co-culture of human lung fibroblasts with NHBEs. These findings highlight the importance of fibroblast TGFβ signaling in EMT but that monocultures give rise to differential responses compared to co-cultures, when exposed to this pro-inflammatory stimulus. This holds implications for any translation conclusions drawn from monoculture studies and is an important step in development of more biomimetic models of IPF. In summary, we believe this in vitro system to study fibroblast-epithelial crosstalk, within the context of IPF, provides a platform which will aid in the identification and validation of novel targets.

Anti-Salmonella Defence and Intestinal Homeostatic Maintenance In Vitro of a Consortium Containing Limosilactobacillus fermentum 3872 and Ligilactobacillus salivarius 7247 Strains in Human, Porcine, and Chicken Enterocytes.

Limosilactobacillus fermentum strain 3872 (LF3872) was originally isolated from the breast milk of a healthy woman during lactation and the breastfeeding of a child. Ligilactobacillus salivarius strain 7247 (LS7247) was isolated at the same time from the intestines and reproductive system of a healthy woman. The genomes of these strains contain genes responsible for the production of peptidoglycan-degrading enzymes and factors that increase the permeability of the outer membrane of Gram-negative pathogens. In this work, the anti-Salmonella and intestinal homeostatic features of the LF3872 and LS7247 consortium were studied. A multi-drug resistant (MDR) strain of Salmonella enteritidis (SE) was used in the experiments. The consortium effectively inhibited the adhesion of SE to intact and activated human, porcine, and chicken enterocytes and reduced invasion. The consortium had a bactericidal effect on SE in 6 h of co-culturing. A gene expression analysis of SE showed that the cell-free supernatant (CFS) of the consortium inhibited the expression of virulence genes critical for the colonization of human and animal enterocytes. The CFS stimulated the production of an intestinal homeostatic factor-intestinal alkaline phosphatase (IAP)-in Caco-2 and HT-29 enterocytes. The consortium decreased the production of pro-inflammatory cytokines IL-8, TNF-α, and IL-1β, and TLR4 mRNA expression in human and animal enterocytes. It stimulated the expression of TLR9 in human and porcine enterocytes and stimulated the expression of TLR21 in chicken enterocytes. The consortium also protected the intestinal barrier functions through the increase of transepithelial electrical resistance (TEER) and the inhibition of paracellular permeability in the monolayers of human and animal enterocytes. The results obtained suggest that a LF3872 and LS7247 consortium can be used as an innovative feed additive to reduce the spread of MDR SE among the population and farm animals.

Toxicological assessment of E-cigarette flavored E-liquids aerosols using Calu-3 cells: A 3D lung model approach

Scientific progress and ethical considerations are increasingly shifting the toxicological focus from in vivo animal models to in vitro studies utilizing physiologically relevant cell cultures. Consequently, we evaluated and validated a three-dimensional (3D) model of the human lung using Calu-3 cells cultured at an air-liquid interface (ALI) for 28 days. Assessment of seven essential genes of differentiation and transepithelial electrical resistance (TEER) measurements, in conjunction with mucin (MUC5AC) staining, validated the model. We observed a time-dependent increase in TEER, genetic markers of mucus-producing cells (muc5ac, muc5b), basal cells (trp63), ciliated cells (foxj1), and tight junctions (tjp1). A decrease in basal cell marker krt5 levels was observed. Subsequently, we utilized this validated ALI-cultured Calu-3 model to investigate the adversity of the aerosols generated from three flavored electronic cigarette (EC) e-liquids: cinnamon, vanilla tobacco, and hazelnut. These aerosols were compared against traditional cigarette smoke (3R4F) to assess their relative toxicity. The aerosols generated from PG/VG vehicle control, hazelnut and cinnamon e-liquids, but not vanilla tobacco, significantly decreased TEER and increased lactate dehydrogenase (LDH) release compared to the incubator and air-only controls. Compared to 3R4F, there were no significant differences in TEER or LDH with the tested flavored EC aerosols other than vanilla tobacco. This starkly contrasted our expectations, given the common perception of e-liquids as a safer alternative to cigarettes. Our study suggests that these results depend on flavor type. Therefore, we strongly advocate for further research, increased user awareness regarding flavors in ECs, and rigorous regulatory scrutiny to protect public health.

Cannabidiol attenuates inflammatory impairment of intestinal cells expanding biomaterial-based therapeutic approaches

Cannabis-based biomaterials have the potential to deliver anti-inflammatory therapeutics specifically to desired cells, tissues, and organs, enhancing drug delivery and the effectiveness of anti-inflammatory treatment while minimizing toxicity. As a major component of Cannabis, Cannabidiol (CBD) has gained major attention in recent years because of its potential therapeutic properties, e.g., for restoring a disturbed barrier resulting from inflammatory conditions. The aim of this study was to test the hypothesis that CBD has beneficial effects under normal and inflammatory conditions in the established non-transformed intestinal epithelial cell model IPEC-J2. CBD induced a significant increase in transepithelial electrical resistance (TER) values and a decrease in the paracellular permeability of [³H]-D-Mannitol, indicating a strengthening effect on the barrier. Under inflammatory conditions induced by tumor necrosis factor alpha (TNFα), CBD stabilized the TER and mitigated the increase in paracellular permeability. Additionally, CBD prevented the barrier-disrupting effects of TNFα on the distribution and localization of sealing TJ proteins. CBD also affected the expression of TNF receptors. These findings demonstrate the potential of CBD as a component of Cannabis-based biomaterials used in the development of novel therapeutic approaches against inflammatory pathogenesis.

Distinct Organotypic Platforms Modulate Rainbow Trout (Oncorhynchus mykiss) Intestinal Cell Differentiation In Vitro.

In vitro organotypic cell-based intestinal platforms, able to faithfully recapitulate the complex functions of the organ in vivo, would be a great support to search for more sustainable feed ingredients in aquaculture. We previously demonstrated that proliferation or differentiation of rainbow trout intestinal cell lines is dictated by the culture environment. The aim of the present work was to develop a culture platform that can efficiently promote cell differentiation into mature enterocytes. We compared four options, seeding the RTpiMI cell line derived from the proximal intestine on (1) polyethylene terephthalate (PET) culture inserts ThinCert™ (TC), (2) TC coated with the solubilized basement membrane matrix Matrigel® (MM), (3) TC with the rainbow trout fibroblast cell line RTskin01 embedded within the Matrigel® matrix (MMfb), or (4) the highly porous polystyrene scaffold Alvetex® populated with the abovementioned fibroblast cell line (AV). We evaluated the presence of columnar cells with a clear polarization of brush border enzymes, the formation of an efficient barrier with a significant increase in transepithelial electrical resistance (TEER), and its ability to prevent the paracellular flux of large molecules but allow the transit of small compounds (proline and glucose) from the apical to the basolateral compartment. All parameters improved moving from the simplest (TC) through the more complex platforms. The presence of fibroblasts was particularly effective in enhancing epithelial cell differentiation within the AV platform recreating more closely the complexity of the intestinal mucosa, including the presence of extracellular vesicles between fibroblasts and epithelial cells.

Cis-Nerolidol Inhibits MAP Kinase and NF-κB Signaling Pathways and Prevents Epithelial Tight Junction Dysfunction in Colon Inflammation: In Vivo and In Vitro Studies

Inflammation of the GI tract leads to compromised epithelial barrier integrity, which increases intestine permeability. A compromised intestinal barrier is a critical event that leads to microbe entry and promotes inflammatory responses. Inflammatory bowel diseases that comprise Crohn's disease (CD) and ulcerative colitis (UC) show an increase in intestinal permeability. Nerolidol (NED), a naturally occurring sesquiterpene alcohol, has potent anti-inflammatory properties in preclinical models of colon inflammation. In this study, we investigated the effect of NED on MAPKs, NF-κB signaling pathways, and intestine epithelial tight junction physiology using in vivo and in vitro models. The effect of NED on proinflammatory cytokine release and MAPK and NF-κB signaling pathways were evaluated using lipopolysaccharides (LPS)-stimulated RAW 264.7 macrophages. Subsequently, the role of NED on MAPKs, NF-κB signaling, and the intestine tight junction integrity were assessed using DSS-induced colitis and LPS-stimulated Caco-2 cell culture models. Our result indicates that NED pre-treatment significantly inhibited proinflammatory cytokine release, expression of proteins involved in MAP kinase, and NF-κB signaling pathways in LPS-stimulated RAW macrophages and DSS-induced colitis. Furthermore, NED treatment significantly decreased FITC-dextran permeability in DSS-induced colitis. NED treatment enhanced tight junction protein expression (claudin-1, 3, 7, and occludin). Time-dependent increases in transepithelial electrical resistance (TEER) measurements reflect the formation of healthy tight junctions in the Caco-2 monolayer. LPS-stimulated Caco-2 showed a significant decrease in TEER. However, NED pre-treatment significantly prevented the fall in TEER measurements, indicating its protective role. In conclusion, NED significantly decreased MAPK and NF-κB signaling pathways and decreased tight junction permeability by enhancing epithelial tight junction protein expression.

Human Colonoid–Myofibroblast Coculture for Study of Apical Na+/H+ Exchangers of the Lower Cryptal Neck Region

Cation and anion transport in the colonocyte apical membrane is highly spatially organized along the cryptal axis. Because of lack of experimental accessibility, information about the functionality of ion transporters in the colonocyte apical membrane in the lower part of the crypt is scarce. The aim of this study was to establish an in vitro model of the colonic lower crypt compartment, which expresses the transit amplifying/progenitor (TA/PE) cells, with accessibility of the apical membrane for functional study of lower crypt-expressed Na+/H+ exchangers (NHEs). Colonic crypts and myofibroblasts were isolated from human transverse colonic biopsies, expanded as three-dimensional (3D) colonoids and myofibroblast monolayers, and characterized. Filter-grown colonic myofibroblast-colonic epithelial cell (CM-CE) cocultures (myofibroblasts on the bottom of the transwell and colonocytes on the filter) were established. The expression pattern for ion transport/junctional/stem cell markers of the CM-CE monolayers was compared with that of nondifferentiated (EM) and differentiated (DM) colonoid monolayers. Fluorometric pHi measurements were performed to characterize apical NHEs. CM-CE cocultures displayed a rapid increase in transepithelial electrical resistance (TEER), paralleled by downregulation of claudin-2. They maintained proliferative activity and an expression pattern resembling TA/PE cells. The CM-CE monolayers displayed high apical Na+/H+ exchange activity, mediated to >80% by NHE2. Human colonoid-myofibroblast cocultures allow the study of ion transporters that are expressed in the apical membrane of the nondifferentiated colonocytes of the cryptal neck region. The NHE2 isoform is the predominant apical Na+/H+ exchanger in this epithelial compartment.

Hesperidin enhances intestinal barrier function in Caco-2 cell monolayers via AMPK-mediated tight junction-related proteins.

The intestinal epithelium is a single-cell layer on the mucosal surface that absorbs food-derived nutrients and functions as a barrier that protects mucosal integrity. Hesperidin (hesperetin-7-rhamnoglucoside) is a flavanone glycoside composed of the flavanone hesperetin and the disaccharide rutinose, which has various physiological benefits, including antioxidative, anti-inflammatory, and antiallergic effects. Here, we used human intestinal Caco-2 cell monolayers to examine the effect of hesperidin on intestinal barrier function. Hesperidin-treated Caco-2 cell monolayers displayed enhanced intestinal barrier integrity, as indicated by an increase in transepithelial electrical resistance (TEER) and a decreased apparent permeability (Papp ) for fluorescein. Hesperidin elevated the mRNA and protein levels of occludin, MarvelD3, JAM-1, claudin-1, and claudin-4, which are encoded by tight junction (TJ)-related genes. Moreover, hesperidin significantly increased the phosphorylation of AMP-activated protein kinase (AMPK), indicating improved intestinal barrier function. Thus, our results suggest that hesperidin enhances intestinal barrier function by increasing the expression of TJ-related occludin, MarvelD3, JAM-1, and claudin-1 via AMPK activation in human intestinal Caco-2 cells.

Wild blueberry (V. angustifolium) improves TNFα-induced cell barrier permeability through claudin-1 and oxidative stress modulation in Caco-2 cells.

Increasing evidence links the impairment of intestinal permeability (IP), a feature of the intestinal barrier, to numerous dysmetabolic and dysfunctional conditions. Several host and environmental factors, including dietary factors, can negatively and/or positively affect IP. In this regard, polyphenol-rich foods including berries have been proposed as potential IP modulators. However, the exact mechanisms involved are not yet fully elucidated. The aim of the present study was to evaluate the effect of a wild blueberry (WB; V. angustifolium) powder, naturally rich in polyphenols, to affect Caco-2 cell monolayer permeability and to identify the potential mechanisms in modulating the IP process. Caco-2 cells were incubated with TNF-α (10 ng mL-1), as a pro-inflammatory stimulus, and supplemented for 24 hours with different concentrations (1 and 5 mg mL-1) of WB powder. The integrity of the intestinal cell monolayer was evaluated by measuring the transepithelial electrical resistance (TEER) and the paracellular transport of FITC-dextran. In addition, the production of the tight junction proteins, such as claudin-1 and occludin, as well as protein carbonyl and 8-hydroxy 2 deoxyguanosine, as oxidative stress markers, were quantified in the supernatant by ELISA kits. Overall, the treatment with WB powder (5 mg mL-1) mitigated the loss of Caco-2 cell barrier integrity, as documented by an increase in TEER and a reduction in FITC values. This modulation was accompanied by an upregulation of claudin-1 and a reduction of 8-OHdG. Conversely, no effect was documented for the lower concentration (1 mg mL-1) and the other IP markers, as well as oxidative stress markers analysed. In conclusion, our findings suggest a potential role of WB in the modulation of cell barrier integrity. This modulation process could be attributed to an increase in claudin-1 expression and a reduction in 8-OHdG. Further studies should be performed to corroborate the results obtained. In addition, since the effects were observed at doses of WB achievable with the diet, these findings should be substantiated also through in vivo approaches.

Potentially probiotic Limosilactobacillus reuteri from human milk strengthens the gut barrier in T84 cells and a murine enteroid model.

At conception, the infant gut barrier is immature, gradually developing with regular intake of maternal milk. This study addressed whether the barrier-strengthening effect of breast feeding might be attributable, at least in part, to autochthonous beneficial human milk bacteria. Twelve bacterial strains from the breast milk of Pakistani mothers who underwent cesarean delivery (NPL-88, NPL-157, NPL-179, NPL-181, NPL-388 (Limosilactobacillus reuteri), NPL-76, NPL-495, NPL-504 (Limosilactobacillus fermentum), NPL-415 (Lactobacillus pentosus), NPL-412, NPL-416 (Lactiplantibacilllus plantarum) and NPL-374 (Bifidobacterium longum) were shortlisted based on their tolerance to acidic pH (2.8-4.2) and bile (0.1-0.3%). The effect of these bacteria on gut barrier function in the presence and absence of pathogens was assessed as changes in transepithelial electrical resistance (TEER) in the human T84 colonic epithelial cell line and in murine enteroid-derived monolayers (EDMs). The TEER of T84 cells monolayers rose in the presence of most of the human milk strains, being most pronounced in case of L. reuteri NPL-88 (34% within five h), exceeding the effect of the well-known probiotic L. acidophilus (20%). qRT-PCR, western blot and immunofluorescent staining associated the increase in TEER with enhanced expression of tight junction proteins. Pretreatment of murine EDMs with NPL-88 also largely prevented the ability of the pathogen, Salmonella, to decrease TEER (87±1.50%; P<0.0001, n=4). Human milk lactic acid bacteria are potential probiotics that can strengthen gut barrier function and protect breastfed neonates against enteric infections.

Investigation into the anti-inflammatory mechanism of coffee leaf extract in LPS-induced Caco-2/U937 co-culture model through cytokines and NMR-based untargeted metabolomics analyses

In this study, lipopolysaccharide (LPS)-induced Caco-2/U937 co-culture was used to investigate the anti-inflammatory mechanism of coffee leaf extract (CLE). HPLC analysis found that 5-caffeoylquinic acid (5-CQA) and epicatechin were degraded by 96.66 % and 85.35 %, respectively after 24 h incubation, while rutin and trigonelline remained unchanged. The absorption efficiency of 4,5-dicaffeoylquinic acid (4,5-diCQA) and mangiferin were higher than the other phytochemicals, reaching 46.90 % and 37.65 %, respectively. CLE significantly inhibited TNF-α, IL-1β, and IL-8 produced by LPS-induced U937 cells in the basolateral side as well as IL-8 produced by apical Caco-2 cells, thereby inhibiting the intestinal monolayer leakage evidenced by the increase of transepithelial electrical resistance (TEER) values. CLE ameliorated some of the LPS-induced impaired cellular immunometabolism, including amino acid and energy metabolisms. Our study indicated that CLE inhibited the pro-inflammatory cytokines and regulated the metabolites in the co-culture system, thus recovering the disrupted intestinal monolayer caused by inflammation.

A bioactive bovine whey protein extract improves intestinal barrier function in vitro

The human intestine plays an important role as a barrier against the ingress of pathogens and other harmful antigens. Accordingly, proper regulation of the intestinal barrier is essential for optimal health. Intestinal barrier function is regulated in part by the interactions between dietary compounds and the intestinal immune system. Bioactive whey proteins from bovine milk (such as lactoferrin, lactoperoxidase, and immunoglobulins) are known to exert a range of physiological functions, including modulation of the immune system, and thus have the potential to regulate intestinal barrier function. While the effects of individual whey proteins on intestinal barrier function have been studied to some extent, less is known about the potentially synergistic properties of whey protein mixtures. Here we investigated the effects of a bioactive bovine whey protein (BWP) extract containing all whey proteins with an isoelectric point >6.8 on intestinal barrier function in vitro. Intestinal epithelial cell (Caco-2) monolayers were treated with BWP before measuring the barrier integrity over 48 h by means of trans-epithelial electrical resistance (TEER). Treatment of epithelial monolayers with 1 mg/mL BWP resulted in an increase in TEER compared with untreated epithelial monolayers. To determine whether BWP could mitigate immune-mediated intestinal barrier dysfunction, we challenged differentiated Caco-2 cell monolayers with tumor necrosis factor α (TNFα) to obtain an in vitro model of a “leaky” intestinal epithelium. The TNFα challenge led to a decrease in TEER over time across untreated control monolayers, indicating a loss of barrier function. This loss of barrier function was mitigated in monolayers treated with 1 mg/mL BWP, but not monolayers treated with the equivalent amount of lactoferrin present in 1 mg/mL BWP. These data suggest that naturally co-occurring bioactive proteins together may enhance intestinal barrier integrity and protect against inflammation-induced barrier dysfunction to a greater extent than lactoferrin alone. Further work is required to determine the key proteins and protein combinations within BWP, and the mechanisms through which BWP modulates intestinal barrier function.

426 Effect of L-4-thiazolylalanine on skin barrier strength

Skin barrier properties are critical for maintaining epidermal water content, protecting from environmental factors, and providing first line of defense against pathogens. In this study, the effect of a non-proteinogenic amino acid, L-4-Thiazolylalanine (L4), on this important aspect of skin health was evaluated by in vitro and in vivo biomarkers. 3D human skin models were treated with 0.3% L4. After 48h, RNA samples were collected and gene expression analyzed. Significant increase in the expression of ALOXE3 (protein eLOX-3) vs. vehicle (VEH) (+27%) was measured. eLOX-3 is a member of the lipoxygenase family required in the epidermis for the covalent linkage of ceramides to proteins of the cornified envelope, an important step in establishing skin barrier function. To further evaluate skin barrier integrity, trans-epithelial electrical resistance (TEER) was measured in the 3D tissues at basal level as well as upon barrier disruption. TEER is a quantitative method that allows barrier strength evaluation. TEER values are highly related to the tight junction barrier function and decrease upon disruption induced by calcium depletion. L4 treatment induced a significant increase in TEER vs. vehicle and protected from low calcium-induced barrier decrease. Clinically, skin treated with L4 for 4 weeks had better barrier integrity vs. vehicle control. This was shown via instrumentation and evaluation of biomarkers in skin samples collected via tape stripping. A fluorescence-based assay was used to quantify the enzymatic activity of 12R-LOX, another lipoxygenase critical for maturation of skin barrier ceramides. Significant increase in 12R-LOX activity was measured after L4 treatment vs. vehicle (+31%) with a stronger effect in subjects 40 years-old and older (+45%). In conclusion, L4 led to modulation of in vitro and in vivo biomarkers critical for skin barrier strength, making it desirable ingredient for topical treatments.

Evaluation of Caco-2 and human intestinal epithelial cells as in vitro models of colonic and small intestinal integrity

Although the colonic cell line Caco-2 is widely used as a model of the small intestinal barrier function, it has limitations such as overestimated transepithelial electrical resistance (TEER) compared to in vivo conditions. Therefore, we investigated Human Intestinal Epithelial Cells (HIECs) as an alternative in vitro model.We explored whether cell seeding number of HIEC-6, and the number of incubation days for HIEC and Caco-2 cells had an impact on TEER, and tight junction expression was examined for both cell lines via immunofluorescence in the presence and absence of probiotic bacteria.We observed no significant difference in TEER readings for either cell lines when cultured for different days. Further, the HIEC TEER readings did not change with increased seeding number and were not significantly different from a control with no cells. HIECs expressed Claudin-1 and Zonula Occludens-1 but not Occludin. Caco-2 co-culture with probiotic bacteria demonstrated a significant increase in TEER, particularly for the lactobacillus strains, whereas HIEC TEER did not respond to bacterial co-incubation.Our study shows that although HIECs express certain TJ proteins, a significant TEER was not observed, likely due to the embryonic origin of the cells, which limits the application of this cell line as a suitable model for small intestinal barrier function.

Human iPSC colon organoid function is improved by exposure to fecal fermentates.

The host–microbe interaction is critical for intestinal homeostasis. By‐products from microbial metabolism of unabsorbed dietary components have been studied increasingly as potential contributors to health and disease. In vitro fermentation systems provide a way to simulate microbial activity and by‐product production of the colon using human fecal samples. Objectives of the study were to determine how clarified supernatants from two different fermentation conditions affect markers of cell proliferation, differentiation, barrier function, and immune function in a human‐induced pluripotent (iPSC) colon organoid model. SCFA and BCFA's of the supernatants were analyzed and were similar to known in vivo concentrations. Molecular results showed 25% of the clarified supernatant from batch fermentation led to a more physiological intestinal phenotype including increased markers of differentiation, including alkaline phosphatase, chromogranin A, SCFA transport monocarboxylate transporter‐1, (6.2‐fold, 2.1‐fold, and 1.8‐fold, respectively; p < 0.05). Mucin production (mucin‐2, mucin‐4) was increased in cells treated with 25% supernatant, as observed by confocal microscopy. In addition, increased tight junction expression (claudin‐3) was noted by immunofluorescence in 25% supernatant‐ treated cells. A dose–response increase in barrier function was observed over the 72‐h time course, with a twofold increase in transepithelial electrical resistance (TER) in the 25% group compared to the control group (p < 0.05). To further investigate host effects, clarified supernatants from a continuous multistage fermentation representing the ascending (AC), transverse (TC), and descending (DC) colonic domains were utilized and some regional differences were observed including increased markers of inflammation (IL‐1β, 6.15 pg/ml; IL‐6, 27.58 pg/ml; TNFα, 4.49 pg/ml; p < 0.05) in DC‐treated samples only. Overall, clarified supernatants represent a valuable model to examine effects of microbial by‐products on host intestinal development and function and future efforts will be designed to further understand microbial communities and metabolites, along with additional host response measures.

Human iPSC Colonoid Function is Improved by Exposure to Fecal Fermentates

The host-microbe interaction is critical for intestinal homeostasis. By-products from microbial metabolism of unabsorbed dietary components have been studied increasingly as potential contributors to health and disease. In vitro fermentation systems provide a way to simulate microbial activity and by-product production of the colon using human fecal samples. Objectives of the study were to determine how clarified supernatants from two different fermentation conditions affect markers of cell proliferation, differentiation, barrier function, and immune function in a human induced pluripotent (iPSC) colon organoid model. Short-chain and branch-chain fatty acid concentrations of the supernatants were analyzed and were similar to known in vivo concentrations. Molecular results showed 25% of the clarified supernatant from batch fermentation led to a more physiological intestinal phenotype including increased markers of differentiation, including alkaline phosphatase, chromogranin-A, SCFA transport monocarboxylate transporter-1, (6.2-fold, 2.1-fold, 1.8-fold, respectively; P<0.05). Mucin production (mucin-2, mucin-4) was increased in cells treated with 25% supernatant, as observed by confocal microscopy. In addition, increased tight junction expression (claudin-3) was noted by immunofluorescence in 25% supernatant treated cells. A dose-response increase in barrier function was observed over the 72-hour time course, with a 2-fold increase in transepithelial electrical resistance (TER) in the 25% group compared to the control group (P<0.05). To further investigate host effects, clarified supernatants from a continuous multi-stage fermentation representing the ascending (AC), transverse (TC), and descending (DC) colonic domains were utilized and some regional differences were observed including increased markers of inflammation (IL-1β, 6.15pg/mL; IL-6, 27.58pg/mL; TNFα, 4.49pg/mL; P < 0.05) in DC treated samples only. Overall, clarified supernatants represent a valuable model to examine effects of microbial by-products on host intestinal development and function and future efforts will be designed to further understand microbial communities and metabolites, along with additional host response measures.

In-vitro study of Limosilactobacillus fermentum PCC adhesion to and integrity of the Caco-2 cell monolayers as affected by pectins

• Adhesion of L. fermentum PCC to Caco-2 cells was effected by pectins. • L. fermentum PCC and in combination with pectins increase TEER across the monolayer. • Increases in TEER are related to the differential expression of the TJ genes. This study investigated the effect of four citrus pectins on adhesion of the probiotic Limosilactobacillus fermentum PCC and integrity of the intestinal epithelial monolayers using the Caco-2 cell model. Adhesion of L. fermentum PCC was enhanced (from 35% to 54–73%) in the presence of pectins with high and moderate degree of esterification (DE), whereas it was reduced (to 2%) by a pectin with low DE. Transepithelial electrical resistance (TEER) was significantly increased upon exposure to L. fermentum PCC alone (by 33%) and, to a greater extent, by combinations with pectins (by 44 – 48%), suggesting synergistic action of bacteria and pectins. Increases in TEER were related to transcriptional responses of the tight junction genes, encoding claudin-4 and claudin-2 proteins. The overall results indicated that pectins, when applied in synbiotic combination with L. fermentum PCC, have potential to improve bacterial adhesion and the intestinal epithelial barrier.

Retinoic acid improves baseline barrier function and attenuates TNF-α-induced barrier leak in human bronchial epithelial cell culture model, 16HBE 14o.

Retinoic acid (RA) has been shown to improve epithelial and endothelial barrier function and development and even suppress damage inflicted by inflammation on these barriers through regulating immune cell activity. This paper thus sought to determine whether RA could improve baseline barrier function and attenuate TNF-α-induced barrier leak in the human bronchial epithelial cell culture model, 16HBE14o- (16HBE). We show for the first time that RA increases baseline barrier function of these cell layers indicated by an 89% increase in transepithelial electrical resistance (TER) and 22% decrease in 14C-mannitol flux. A simultaneous, RA-induced 70% increase in claudin-4 attests to RA affecting the tight junctional (TJ) complex itself. RA was also effective in alleviating TNF-α-induced 16HBE barrier leak, attenuating 60% of the TNF-α-induced leak to 14C-mannitol and 80% of the leak to 14C-inulin. Interleukin-6-induced barrier leak was also reduced by RA. Treatment of 16HBE cell layers with TNF-α resulted in dramatic decrease in immunostaining for occludin and claudin-4, as well as a downward “band-shift” in occludin Western immunoblots. The presence of RA partially reversed TNF-α’s effects on these select TJ proteins. Lastly, RA completely abrogated the TNF-α-induced increase in ERK-1,2 phosphorylation without significantly decreasing the TNF-driven increase in total ERK-1,2. This study suggests RA could be effective as a prophylactic agent in minimizing airway barrier leak and as a therapeutic in preventing leak triggered by inflammatory cascades. Given the growing literature suggesting a “cytokine storm” may be related to COVID-19 morbidity, RA may be a useful adjuvant for use with anti-viral therapies.

Epithelial barrier function properties of the 16HBE14o- human bronchial epithelial cell culture model.

The human bronchial epithelial cell line, 16HBE14o- (16HBE), is widely used as a model for respiratory epithelial diseases and barrier function. During differentiation, transepithelial electrical resistance (TER) increased to approximately 800 Ohms × cm2, while 14C-d-mannitol flux rates (Jm) simultaneously decreased. Tight junctions (TJs) were shown by diffusion potential studies to be anion-selective with PC1/PNa = 1.9. Transepithelial leakiness could be induced by the phorbol ester, protein kinase C (PKC) activator, 12-O-tetradecanoylphorbol-13-acetate (TPA), and the proinflammatory cytokine, tumor necrosis factor-α (TNF-α). Basal barrier function could not be improved by the micronutrients, zinc, or quercetin. Of methodological significance, TER was observed to be more variable and to spontaneously, significantly decrease after initial barrier formation, whereas Jm did not significantly fluctuate or increase. Unlike the strong inverse relationship between TER and Jm during differentiation, differentiated cell layers manifested no relationship between TER and Jm. There was also much greater variability for TER values compared with Jm. Investigating the dependence of 16HBE TER on transcellular ion conductance, inhibition of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) chloride channel with GlyH-101 produced a large decrease in short-circuit current (Isc) and a slight increase in TER, but no significant change in Jm. A strong temperature dependence was observed not only for Isc, but also for TER. In summary, research utilizing 16HBE as a model in airway barrier function studies needs to be aware of the complexity of TER as a parameter of barrier function given the influence of CFTR-dependent transcellular conductance on TER.