Basolateral Secretion Research Articles

Eicosapentaenoic acid inhibits intestinal β-carotene absorption by downregulation of lipid transporter expression via PPAR-α dependent mechanism

The involvement of lipid transporters, the scavenger receptor class B, type I (SR-BI) and Niemann–Pick type C1 Like 1 protein (NPC1L1) in carotenoid absorption is demonstrated in intestinal cells and animal models. Dietary ω-3 fatty acids are known to possess antilipidemic properties, which could be mediated by activation of PPAR family transcription factors. The present study was conducted to determine the effect of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), on intestinal β-carotene absorption. β-carotene uptake in Caco-2/TC7 cells was inhibited by EPA (p < 0.01) and PPARα agonist (P < 0.01), but not by DHA, PPARγ or PPARδ agonists. Despite unaltered β-carotene uptake, both DHA and PPARδ agonists inhibited the NPC1L1 expression. Further, EPA also induced the expression of carnitine palmitoyl transferase 1A (CPT1A) expression, a PPARα target gene. Interestingly, EPA induced inhibition of β-carotene uptake and SR B1 expression were abrogated by specific PPARα antagonist, but not by PPARδ antagonist. EPA and PPARα agonist also inhibited the basolateral secretion of β-carotene from Caco-2 cells grown on permeable supports. These results suggest that EPA inhibits intestinal β-carotene absorption by down regulation of SR B1 expression via PPARα dependent mechanism and provide an evidence for dietary modulation of intestinal β-carotene absorption.

Human Adult Retinal Pigment Epithelial Stem Cell–Derived RPE Monolayers Exhibit Key Physiological Characteristics of Native Tissue

We tested what native features have been preserved with a new culture protocol for adult human RPE. We cultured RPE from adult human eyes. Standard protocols for immunohistochemistry, electron microscopy, electrophysiology, fluid transport, and ELISA were used. Confluent monolayers of adult human RPE cultures exhibit characteristics of native RPE. Immunohistochemistry demonstrated polarized expression of RPE markers. Electron microscopy illustrated characteristics of native RPE. The mean transepithelial potential (TEP) was 1.19 ± 0.24 mV (mean ± SEM, n = 31), apical positive, and the mean transepithelial resistance (RT) was 178.7 ± 9.9 Ω·cm2 (mean ± SEM, n = 31). Application of 100 μM adenosine triphosphate (ATP) apically increased net fluid absorption (Jv) by 6.11 ± 0.53 μL·cm2·h-1 (mean ± SEM, n = 6) and TEP by 0.33 ± 0.048 mV (mean ± SEM, n = 25). Gene expression of cultured RPE was comparable to native adult RPE (n = 5); however, native RPE RNA was harvested between 24 and 40 hours after death and, therefore, may not accurately reflect healthy native RPE. Vascular endothelial growth factor secreted preferentially basally 2582 ± 146 pg/mL/d, compared to an apical secretion of 1548 ± 162 pg/mL/d (n = 14, P < 0.01), while PEDF preferentially secreted apically 1487 ± 280 ng/mL/d compared to a basolateral secretion of 864 ± 132 ng/mL/d (n = 14, P < 0.01). The new culture model preserves native RPE morphology, electrophysiology, and gene and protein expression patterns, and may be a useful model to study RPE physiology, disease, and transplantation.

Secretion of angiopoietin-like 4 protein from intestinal cells

Background: Angiopoietin-like 4 (ANGPTL4) has been suggested to play a role in lipid metabolism as a regulatory protein of lipoprotein lipase activity. Intestinal secretion of ANGPTL4, which is regulated by fatty acids, may inhibit the activity of circulating lipoprotein lipase; but, recent studies suggest that it could also inhibit pancreatic lipase in the gut and thereby reduce intestinal uptake of lipids. Secretion of the ANGPTL4 protein to either the lumen or tissue/blood side of the intestinal epithelial layer would indicate possible modes of action.Methods: Caco-2 cells were grown on permeable membranes and cultured for 21 days to spontaneously differentiate into an intact monolayer of intestinal cells, mimicking the epithelial cell layer lining the intestinal wall. Cells were treated with 9 mM butyrate and the time dependent gene expression and protein secretion to the apical and basolateral side was analysed over a time-course of 24 hours. Possible feedback from ANGPTL4 protein was investigated by adding 0.25 ng/ml recombinant ANGPTL4 protein to culture media.Results: Butyrate-induced ANGPTL4 gene expression increased in Caco-2 cells after 2 hours, reaching a plateau of approximately 6 fold after 6-24 hours, while the ANGPTL4 protein secretion to both the apical and basolateral sides was increased 18-24 hours after stimulation. A negative feedback on apical and basolateral secretion was observed in the presence of recombinant ANGPTL4 on the apical and basolateral sides, respectively.Conclusion: The present study indicates that, upon exposure to butyrate, the monolayer of epithelial cells secretes the ANGPTL4 protein to both the tissue/blood (basolateral) side and the luminal (apical) side of the monolayer which, in an in vivo situation, may be interpreted as potential inhibition of both the circulating and pancreatic lipase.Keywords: Angiopoietin-like 4, Caco-2, Permeable membrane, Protein, Secretion, Intestine, Negative feedback

Anti-inflammatory activity of the basolateral fraction of Caco-2 cells exposed to a rosemary supercritical extract

The anti-inflammatory activity of the basolateral fraction of Caco-2 cells exposed to a rosemary supercritical extract was examined. Uptake of rosemary extract fractions was tested on Caco-2 cell monolayers (2–12 h incubation times) and the quantification of carnosic acid and carnosol was performed by UPLC-MS/MS. Human macrophages were treated with the basolateral fractions and secretion of TNF-α, IL-1β, IL-6 and IL-10 was measured by ELISA. The fractions obtained after 8 and 12 h in absorption experiments caused a significant reduction in excretion of the pro-inflammatory cytokines. This reduction in cytokine secretion levels corresponded to the amounts of carnosic acid and carnosol in the basolateral fractions. Thus, the basolateral fraction of a rosemary supercritical extract showed an important anti-inflammatory activity, providing the basis for increasing the use of supercritical rosemary extracts for the prevention of inflammatory diseases.

Basolateral secretion of Wnt5a in polarized epithelial cells is required for apical lumen formation.

Wnt5a regulates planar cell polarity in epithelial cells, but it remains to be determined whether Wnt5a and its receptors are sorted apically or basolaterally, and how Wnt5a signaling is involved in apical and basolateral polarization. We found that Wnt5a was secreted basolaterally in polarized kidney epithelial cells. The basolateral secretion of Wnt5a required Wntless (Wls), clathrin and adaptor protein 1 (AP-1). Wnt5a receptors were also localized to the basolateral membranes, but their sorting did not require Wls. Wnt5a-induced signaling was stimulated more efficiently at the basolateral side than the apical side of epithelial cells. Knockdown of Wnt5a delayed apical lumen formation of the epithelial cyst, and these phenotypes were rescued by wild-type Wnt5a, but not by a Wnt5a mutant that is secreted apically. Although apoptosis was not required for apical lumen formation in a wild-type cyst, apoptosis was necessary for eliminating luminal cells in a Wnt5a-depleted cyst. These results suggest that Wnt5a and its receptors are sorted to their correct destination by different mechanisms and that the basolateral secretion of Wnt5a is necessary for apical lumen formation in the epithelial cyst.

Age-Related Changes of Cystatin C Expression and Polarized Secretion by Retinal Pigment Epithelium: Potential Age-Related Macular Degeneration Links

Cystatin C, a potent cysteine proteinase inhibitor, is abundantly secreted by the RPE and may contribute to regulating protein turnover in the Bruch's membrane (BrM). A cystatin C variant associated with increased risk of developing AMD and Alzheimer's disease (AD) presents reduced secretion levels from RPE. The purpose of this study was to analyze the effects of age and the accumulation of advanced glycation end-products (AGEs) on the expression and secretion of cystatin C by the RPE. Confluent monolayers of human fetal RPE (hfRPE) cells were cultured using an in vitro model mimicking extracellular AGE accumulation. Cystatin C expression, secretion, and its polarity were analyzed following culture on AGE-containing BrM mimics (AGEd versus non-AGEd). Monolayer barrier properties were assessed by transepithelial resistance measurements. The relative level of cystatin C protein expression in human RPE in situ was assessed immunohistochemically in relation to age. Advanced glycation end product-exposed RPE monolayers presented significantly decreased cystatin C expression and secretion. Basolateral secretion was fully established by week 8 in non-AGEd conditions. In AGEd cultures, polarity of secretion was impaired despite maintenance of physiological barrier properties of the monolayer. In the macula region of RPE/choroid segments from human eyes, the level of cystatin C protein was reduced with increasing donor age. Exposure to AGEs reduces expression of cystatin C and affects its normal secretion in cultured RPE. Age-related changes of cystatin C in the RPE from the posterior pole may compromise its extracellular functions, potentially contributing to AMD pathogenesis.

Unsaturated fatty acids promote bioaccessibility and basolateral secretion of carotenoids and α-tocopherol by Caco-2 cells

Bioavailability of carotenoids and tocopherols from foods is determined by the efficiency of transfer from food/meal to mixed micelles during digestion, incorporation into chylomicrons for trans-epithelial transport to lymphatic/blood system, and distribution to target tissues. Fats and oils are important factors for facilitating the absorption of lipophilic compounds. However, dietary fats and oils are composed of various types of saturated and unsaturated fatty acids which may differentially impact the bioavailability of carotenoids and tocopherols from foods. We have investigated the effects of several common commercial lipids on bioavailability using an in vitro digestion model and Caco-2 human intestinal cells. Meals consisted of mixed salad vegetables containing a single test lipid. Micellarization and cellular uptake of β-carotene (βC) and lycopene (LYC) during small intestinal digestion was increased by lipids rich in unsaturated fatty acids: soybean oil > olive > canola > butter. In contrast, type of lipid minimally affected the bioaccessibility of lutein (LUT) and zeaxanthin (ZEA). To examine the influence of type of dietary triglyceride on uptake and basolateral secretion of carotenoids, Caco-2 cells grown on Transwell membranes were incubated with micellar mixtures of fatty acids (1.0 mM) mimicking the types and ratio of saturated to unsaturated (mono- + poly-unsaturated) fatty acids (FA) present in butter (70 : 30), olive oil (7 : 93) and soybean oil (11 : 89). Cells were exposed to micelles containing βC, LUT, α-tocopherol (α-TC) and a mixture of test fatty acids. Uptake and basolateral secretion of βC, LUT and α-TC were greater in cells pre-treated with mixtures enriched in unsaturated compared to saturated FA and these effects were mediated by increased assembly and secretion of chylomicrons. These results suggest that dietary fats/oils rich in unsaturated fatty acids promote carotenoid and α-TC bioavailability by enhancing their micellarization during digestion and intestinal transport.

Vitamin E Secretion by Caco-2 Monolayers to APOA1, but Not to HDL, Is Vitamer Selective1,2

The aim of this study was to characterize the pathways of basolateral secretion of common dietary tocopherols from polarized Caco-2 monolayers, a model of intestinal absorption. Given differences in structure and physical properties, we hypothesized that secretion may differ between different forms of vitamin E, thus potentially contribute to the selectivity seen in vivo. Monolayers were incubated apically and simultaneously with 10 μmol/L α-, γ-, and δ-tocopherol (1:1:1) in lipid micelles. Treatment with the microsomal triglyceride transfer protein inhibitor BMS201038 revealed that the triglyceride-rich particle secretory pathway (apolipoprotein B–dependent pathway) accounted for ∼80% of total tocopherol secretion, without selectivity among the three forms of vitamin E. Apolipoprotein B–independent secretion of tocopherols (and cholesterol) was greatly enhanced by the liver X receptor agonist T0901317. T0901317 induced ATP-binding cassette transporter A1 (ABCA1) protein expression and basolateral secretion of tocopherols to apolipoprotein A1. ABCA1-dependent secretion demonstrated vitamer selectivity such that efficiency of secretion of α- and γ-tocopherols exceeded that of δ-tocopherol. Basal addition of HDL stimulated vitamin E secretion but without selectivity among the three forms, whereas LDL had no effect. Basal addition of scavenger receptor class B member I (SR-BI) blocking antibody, which inhibits the interaction between SR-BI and HDL, increased basal accumulation of all tocopherols, demonstrating a role for SR-BI in cellular re-uptake of secreted vitamin E. These findings demonstrated that vitamin E and cholesterol utilize common pathways of secretion and that secretion via the ABCA1 pathway favors certain forms of vitamin E.

Response of Differentiated Human Airway Epithelia to Alcohol Exposure and Klebsiella Pneumoniae Challenge.

Alcohol abuse has been associated with increased susceptibility to pulmonary infection. It is not fully defined how alcohol contributes to the host defense compromise. Here primary human airway epithelial cells were cultured at an air-liquid interface to form a differentiated and polarized epithelium. This unique culture model allowed us to closely mimic lung infection in the context of alcohol abuse by basolateral alcohol exposure and apical live bacterial challenge. Application of clinically relevant concentrations of alcohol for 24 hours did not significantly alter epithelial integrity or barrier function. When apically challenged with viable Klebsiella pneumoniae, the cultured epithelia had an enhanced tightness which was unaffected by alcohol. Further, alcohol enhanced apical bacterial growth, but not bacterial binding to the cells. The cultured epithelium in the absence of any treatment or stimulation had a base-level IL-6 and IL-8 secretion. Apical bacterial challenge significantly elevated the basolateral secretion of inflammatory cytokines including IL-2, IL-4, IL-6, IL-8, IFN-γ, GM-CSF, and TNF-α. However, alcohol suppressed the observed cytokine burst in response to infection. Addition of adenosine receptor agonists negated the suppression of IL-6 and TNF-α. Thus, acute alcohol alters the epithelial cytokine response to infection, which can be partially mitigated by adenosine receptor agonists.

Apicobasal secretion of Wnt11 and Wnt3a in polarized epithelial cells is regulated by distinct mechanisms

Wnts are glycan- and lipid-modified morphogens that are important for cellular responses, but how Wnts are secreted in polarized epithelial cells remains unclear. Although Wntless (Wls) has been shown to interact with Wnts and support their secretion, the role of Wls in the sorting of Wnts to the final destination in polarized epithelial cells have not been clarified. Glycosylation was shown to be important for the sorting of some transmembrane and secreted proteins, but glycan profiles and their roles in the polarized secretion of Wnts has not yet been demonstrated. Here we show the apical and basolateral secretion of Wnts is regulated by different mechanisms. Wnt11 and Wnt3a were secreted apically and basolaterally, respectively, in polarized epithelial cells. Wls was localized to the basolateral membrane. Mass-spectrometric analyses revealed that Wnt11 is modified with complex/hybrid(Asn40)-, high-mannose(Asn90)- and high-mannose/hybrid(Asn300)-type glycans and that Wnt3a is modified with two high-mannose-type glycans (Asn87 and Asn298). Glycosylation processing at Asn40 and galectin-3 were required for the apical secretion of Wnt11, whereas clathrin and adaptor protein-1 were required for the basolateral secretion of Wnt3a. By the fusion of the Asn40 glycosylation site of Wnt11, Wnt3a was secreted apically. The recycling of Wls by AP-2 was necessary for the basolateral secretion of Wnt3a but not for the apical secretion of Wnt11. These results suggest that Wls has different roles in the polarized secretion of Wnt11 and Wnt3a and that glycosylation processing of Wnts decides their secretory routes.

Intracellular Na+, K+ and Cl− activities in Acheta domesticus Malpighian tubules and the response to a diuretic kinin neuropeptide

The mechanism of primary urine production and the activity of a diuretic kinin, Achdo-KII, were investigated in malpighian tubules of Acheta domesticus by measuring intracellular Na(+), K(+) and Cl(-) activities, basolateral membrane voltage (V(b)), fluid secretion and transepithelial ion transport. Calculated electrochemical gradients for K(+) and Cl(-) across the basolateral membrane show they are actively transported into principal cells, and basolateral Ba(2+)-sensitive K(+) channels do not contribute to net transepithelial K(+) transport and fluid secretion. A basolateral Cl(-) conductance was revealed after the blockade of K(+) channels with Ba(2+), and a current carried by the passive outward movement of Cl(-) accounts for the hyperpolarization of V(b) in response to Ba(2+). Ion uptake via Na(+)/K(+)/2Cl(-) cotransport, driven by the inwardly directed Na(+) electrochemical gradient, is thermodynamically feasible, and is consistent with the actions of bumetanide, which reduces fluid secretion and both Na(+) and K(+) transport. The Na(+) gradient is maintained by its extrusion across the apical membrane and by a basolateral ouabain-sensitive Na(+)/K(+)-ATPase. Achdo-KII has no significant effect on the intracellular ion activities or V(b). Electrochemical gradients across the apical membrane were estimated from previously published values for the levels of Na(+), K(+) and Cl(-) in the secreted fluid. The electrochemical gradient for Cl(-) favours passive movement into the lumen, but falls towards zero after stimulation by Achdo-KII. This coincides with a twofold increase in Cl(-) transport, which is attributed to the opening of an apical Cl(-) conductance, which depolarises the apical membrane voltage.

Phytosterols Reduce Cholesterol Absorption by Inhibition of 27-Hydroxycholesterol Generation, Liver X Receptor α Activation, and Expression of the Basolateral Sterol Exporter ATP-Binding Cassette A1 in Caco-2 Enterocytes1–3

Phytosterol-enriched foods are increasingly marketed to lower cholesterol levels and atherosclerosis in the general population. Phytosterols reduce cholesterol absorption, but the molecular mechanism is controversial. We therefore investigated the phytosterol effects on cholesterol metabolism in human enterocyte, hepatocyte, and macrophage models relevant for sterol absorption, reverse transport, and excretion. Isomolar sitosterol (50 μmol/L) was less effectively taken up by enterocytes than cholesterol but suppressed apical cholesterol uptake by 50% (P < 0.01) and basolateral secretion by two-thirds (P < 0.01) whether added in micelles or ethanol or complexed to cyclodextrin. In contrast, enterocytes handled nanomolar 3H-sitosterol similarly to cholesterol. Enterocytes selectively oxidized all sterols to 27-hydroxy- and 27-carboxy-sterols. Conversion rates were much lower for sitosterol (0.05 ± 0.02 nmol/mg protein) and campesterol (0.48 ± 0.10) compared with cholesterol (3.73 ± 0.60) (P < 0.001). 27-Hydroxycholesterol (27OH-C) activated liver-X-receptor alpha (LXRα) (P < 0.01) and stimulated ATP-binding cassette transporter (ABC) A1 expression (P < 0.001) and basolateral systemic cholesterol secretion from enterocytes (P < 0.05). In co-incubations, phytosterols inhibited 27OH-C generation by sterol 27-hydroxylase (P < 0.001) and reduced LXRα-mediated ABCA1 expression (P < 0.01) and basolateral systemic cholesterol secretion. In contrast, ABCG8 transcription and apical sterol resecretion was unchanged by LXRα activation in human enterocytes. Exogenous LXRα agonists reverted sterol selectivity and phytosterol cholesterol interaction. Due to constitutive apical expression of ABCG5/G8 and LXRα-enhanced basolateral expression of ABCA1 in enterocytes, interference of phytosterols with the generation of the dominating LXRα-agonist 27OH-C blocks the self-priming component of cholesterol absorption. This local LXRα antagonism of dietary phytosterols contributes to sterol selectivity and reduces fractional cholesterol absorption and preloading of nascent HDL with dietary cholesterol.

Increase in Campylobacter jejuni invasion of intestinal epithelial cells under low-oxygen coculture conditions that reflect the in vivo environment.

Campylobacter jejuni infection often results in bloody, inflammatory diarrhea, indicating bacterial disruption and invasion of the intestinal epithelium. While C. jejuni infection can be reproduced in vitro using intestinal epithelial cell (IEC) lines, low numbers of bacteria invading IECs do not reflect these clinical symptoms. Performing in vitro assays under atmospheric oxygen conditions neither is optimal for microaerophilic C. jejuni nor reflects the low-oxygen environment of the intestinal lumen. A vertical diffusion chamber (VDC) model system creates microaerobic conditions at the apical surface and aerobic conditions at the basolateral surface of cultured IECs, producing an in vitro system that closely mimics in vivo conditions in the human intestine. Ninefold increases in interacting and 80-fold increases in intracellular C. jejuni 11168H wild-type strain bacteria were observed after 24-h coculture with Caco-2 IECs in VDCs under microaerobic conditions at the apical surface, compared to results under aerobic conditions. Increased bacterial interaction was matched by an enhanced and directional host innate immune response, particularly an increased basolateral secretion of the proinflammatory chemokine interleukin-8 (IL-8). Analysis of the invasive ability of a nonmotile C. jejuni 11168H rpoN mutant in the VDC model system indicates that motility is an important factor in the early stages of bacterial invasion. The first report of the use of a VDC model system for studying the interactions of an invasive bacterial pathogen with IECs demonstrates the importance of performing such experiments under conditions that represent the in vivo situation and will allow novel insights into C. jejuni pathogenic mechanisms.

Luminal-Applied Flagellin Is Internalized by Polarized Intestinal Epithelial Cells and Elicits Immune Responses via the TLR5 Dependent Mechanism

Bacteria release flagellin that elicits innate responses via Toll-like receptor 5 (TLR5). Here, we investigated the fate of apically administrated full length flagellin from virulent and avirulent bacteria, along with truncated recombinant flagellin proteins in intestinal epithelial cells and cellular responses. Flagellin was internalized by intestinal epithelial cell (IEC) monolayers of IEC-18. Additionally, apically applied flagellin was internalized by polarized human Caco-2BBe and T-84 cells in a TLR5 dependent mechanism. More, flagellin exposure did not affect the integrity of intestinal monolayers. With immunofluorescent staining, internalized flagellin was detected in both early endosomes as well as lysosomes. We found that apical exposure of polarized Caco-2BBe and T-84 to flagellin from purified Salmonella, Escherichia coli O83:H1 (isolate from Crohn’s lesion) or avirulent E. coli K12 induced comparable levels of basolateral IL-8 secretion. A recombinant protein representing the conserved amino (N) and carboxyl (C) domains (D) of the flagellin protein (ND1/2ECHCD2/1) induced IL-8 secretion from IEC similar to levels elicited by full-length flagellins. However, a recombinant flagellin protein containing only the D3 hypervariable region elicited no IL-8 secretion in both cell lines compared to un-stimulated controls. Silencing or blocking TLR5 in Caco-2BBe cells resulted in a lack of flagellin internalization and decreased IL-8 secretion. Furthermore, apical exposure to flagellin stimulated transepithelial migration of neutrophils and dendritic cells. The novel findings in this study show that luminal-applied flagellin is internalized by normal IEC via TLR5 and co-localizes to endosomal and lysosomal compartments where it is likely degraded as flagellin was not detected on the basolateral side of IEC cultures.

Essential roles of snap-29 in C. elegans

SNARE domain proteins are key molecules mediating intracellular fusion events. SNAP25 family proteins are unique target-SNAREs possessing two SNARE domains. Here we report the genetic, molecular, and cell biological characterization of C. elegans SNAP-29. We found that snap-29 is an essential gene required throughout the life-cycle. Depletion of snap-29 by RNAi in adults results in sterility associated with endomitotic oocytes and pre-meiotic maturation of the oocytes. Many of the embryos that are produced are multinucleated, indicating a defect in embryonic cytokinesis. A profound defect in secretion by oocytes and early embryos in animals lacking SNAP-29 appears to be the underlying defect connecting these phenotypes. Further analysis revealed defects in basolateral and apical secretion by intestinal epithelial cells in animals lacking SNAP-29, indicating a broad requirement for this protein in the secretory pathway. A SNAP-29-GFP fusion protein was enriched on recycling endosomes, and loss of SNAP-29 disrupted recycling endosome morphology. Taken together these results suggest a requirement for SNAP-29 in the fusion of post-Golgi vesicles with the recycling endosome for cargo to reach the cell surface.

IL-17A and TNF-α Exert Synergistic Effects on Expression of CXCL5 by Alveolar Type II Cells In Vivo and In Vitro

CXCL5, a member of the CXC family of chemokines, contributes to neutrophil recruitment during lung inflammation, but its regulation is poorly understood. Because the T cell-derived cytokine IL-17A enhances host defense by triggering production of chemokines, particularly in combination with TNF-α, we hypothesized that IL-17A would enhance TNF-α-induced expression of CXCL5. Intratracheal coadministration of IL-17A and TNF-α in mice induced production of CXCL1, CXCL2, and CXCL5, which was associated with increased neutrophil influx in the lung at 8 and 24 h. The synergistic effects of TNF-α and IL17A were greatly attenuated in Cxcl5(-/-) mice at 24 h, but not 8 h, after exposure, a time when CXCL5 expression was at its peak in wild-type mice. Bone marrow chimeras produced using Cxcl5(-/-) donors and recipients demonstrated that lung-resident cells were the source of CXCL5. Using differentiated alveolar epithelial type II (ATII) cells derived from human fetal lung, we found that IL-17A enhanced TNF-α-induced CXCL5 transcription and stabilized TNF-α-induced CXCL5 transcripts. Whereas expression of CXCL5 required activation of NF-κB, IL-17A did not increase TNF-α-induced NF-κB activation. Apical costimulation of IL-17A and TNF-α provoked apical secretion of CXCL5 by human ATII cells in a transwell system, whereas basolateral costimulation led to both apical and basolateral secretion of CXCL5. The observation that human ATII cells secrete CXCL5 in a polarized fashion may represent a mechanism to recruit neutrophils in host defense in a fashion that discriminates the site of initial injury.

Cytopathogenesis of Sendai Virus in Well-Differentiated Primary Pediatric Bronchial Epithelial Cells

Sendai virus (SeV) is a murine respiratory virus of considerable interest as a gene therapy or vaccine vector, as it is considered nonpathogenic in humans. However, little is known about its interaction with the human respiratory tract. To address this, we developed a model of respiratory virus infection based on well-differentiated primary pediatric bronchial epithelial cells (WD-PBECs). These physiologically authentic cultures are comprised of polarized pseudostratified multilayered epithelium containing ciliated, goblet, and basal cells and intact tight junctions. To facilitate our studies, we rescued a replication-competent recombinant SeV expressing enhanced green fluorescent protein (rSeV/eGFP). rSeV/eGFP infected WD-PBECs efficiently and progressively and was restricted to ciliated and nonciliated cells, not goblet cells, on the apical surface. Considerable cytopathology was evident in the rSeV/eGFP-infected cultures postinfection. This manifested itself by ciliostasis, cell sloughing, apoptosis, and extensive degeneration of WD-PBEC cultures. Syncytia were also evident, along with significant basolateral secretion of proinflammatory chemokines, including IP-10, RANTES, tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), interleukin 6 (IL-6), and IL-8. Such deleterious responses are difficult to reconcile with a lack of pathogenesis in humans and suggest that caution may be required in exploiting replication-competent SeV as a vaccine vector. Alternatively, such robust responses might constitute appropriate normal host responses to viral infection and be a prerequisite for the induction of efficient immune responses.

Respiratory Syncytial Virus F and G Proteins Induce Interleukin 1α, CC, and CXC Chemokine Responses by Normal Human Bronchoepithelial Cells

Human respiratory syncytial virus (RSV) is a ubiquitous respiratory virus that causes serious lower respiratory tract disease in infants and young children worldwide. Studies have shown that RSV infection modulates chemokine expression patterns, suggesting that particular cytokine expression profiles may be indicators of disease severity. In this study, we show that RSV F or G protein treatment of fully differentiated primary normal human bronchial epithelial cells induces apical and basolateral secretion of interleukin 8 (IL-8), interferon-inducible protein 10 (IP-10), monocyte chemotactic protein 1 (MCP-1), and RANTES (regulated on activation, normal T cell expressed and secreted). Purified RSV G (attachment) protein was shown to stimulate the secretion of interleukin 1alpha and RANTES, whereas purified F (fusion) protein elicited the production of IL-8, IP-10, and RANTES. Studies of ultraviolet-inactivated RSV showed that treatment of normal human bronchial epithelial cells induces apical IL-8, IP-10, and MCP-1 secretion independent of infection, suggesting that RSV proteins alone modify the chemokine response pattern, which may affect the early immune response before infection.

Dietary fats with increased ratio of unsaturated to saturated fatty acids enhance absorption of carotenoid and vitamin E by increasing both efficiency of micellarization and lipoprotein secretion.

Although it is well established that dietary fat promotes the bioavailability of carotenoids and other dietary lipophiles, insights regarding the effects of different lipid sources on processes associated with absorption of these plant compounds remain unclear. We previously reported that triacylglycerides (TG) with higher ratios of unsaturated to saturated fatty acids (FA) increased the efficiency of micellarization of carotenes. Here we examined the influence of FA composition of TG on uptake of β‐carotene (BC), lutein (Lut) and αTC and their basolateral secretion in chylomicrons by Caco‐2 cells. Differentiated Caco‐2 cells were pretreated 4h/d for 4d by apical exposure of cultures to mixtures of saturated, mono‐unsaturated and poly‐unsaturated FA similar to those present in butter (76:24:0) and olive(12:74:14), canola(9:56:35) and soybean oil (16:24:60). On the following day, micelles containing βC, Lut and αTC were included along with the FA mixtures in apical medium. Uptake of test compounds was unaffected by micelle FA composition, whereas micelles rich in unsaturated FA strongly promoted basolateral secretion of BC, Lut and αTC. The number and size of chylomicrons (total TG and TG to apoB48/100 ratio) also were significantly increased by chronic exposure to micelles with FA composition mimicking vegetable oils. Therefore, it appears that ingestion of TG enriched in with unsaturated FA enhances bioavailability of carotenoids and αTC by increasing micellarization during digestion and transepithelial transport by absorptive epithelial cells. Supported by USDA NRI Grant # 2007‐02313 and OARDC.

Inhibitory effects of Lactobacillus casei subsp. rhamnosus on Salmonella lipopolysaccharide-induced inflammation and epithelial barrier dysfunction in a co-culture model using Caco-2/peripheral blood mononuclear cells

In this study, we investigated the anti-inflammatory and reinforcing barrier effects of Lactobacillus casei subsp. rhamnosus (Lcr35) on Caco-2 intestinal epithelial cells already exposed to Salmonella LPS. Using the Transwell co-culture model, Salmonella LPS was apically added to polarized Caco-2 cells co-cultured with peripheral blood mononuclear cells (PBMCs) in the basolateral compartment. LPS-stimulated Caco-2 cells were incubated with Lcr35 for 1, 6, 24 or 48 h. Apical inoculation of Lcr35 after 48 h significantly inhibited the basolateral secretion of interleukin-8 (IL-8) in the Caco-2/PBMC co-culture. The PCR analysis showed that Lcr35 significantly downregulated mRNA expression of monocyte chemoattractant protein 1 (MCP-1) (P<0.05) and had a trend of decreasing mRNA expression of IL-8 (P=0.05), but did not alter mRNA expression of transforming growth factor-beta1 in LPS-stimulated Caco-2 cells at 48 h after addition of Lcr35. Compared to non-LPS-pretreated controls, transepithelial electrical resistance (TEER) of the polarized Caco-2 cell monolayers pretreated with LPS for 48 h was decreased by 9.9 % (P<0.05). Additionally, compared to those cells only treated with LPS, apical co-incubation with Lcr35 showed biphasic TEER levels increased by 12.1 % (P<0.001), 5.7 % (P<0.05) and 86.8 % (P<0.001) in the Caco-2 cell monolayers compared to those without Lcr35 treatment after 1, 6 and 48 h, respectively. In conclusion, Lcr35 can exert anti-inflammatory effects and ameliorate barrier dysfunction in the Salmonella LPS-pretreated inflamed intestinal epithelium in vitro.