Differentiated transplant derived airway epithelial cell cytokine secretion is not regulated by cyclosporine

Background

While lung transplantation is an increasingly utilized therapy for advanced lung diseases, chronic rejection in the form of Bronchiolitis Obliterans Syndrome (BOS) continues to result in significant allograft dysfunction and patient mortality. Despite correlation of clinical events with eventual development of BOS, the causative pathophysiology remains unknown. Airway epithelial cells within the region of inflammation and fibrosis associated with BOS may have a participatory role.

Methods

Transplant derived airway epithelial cells differentiated in air liquid interface culture were treated with IL-1β and/or cyclosporine, after which secretion of cytokines and growth factor and gene expression for markers of epithelial to mesenchymal transition were analyzed.

Results

Secretion of IL-6, IL-8, and TNF-α, but not TGF-β1, was increased by IL-1β stimulation. In contrast to previous studies using epithelial cells grown in submersion culture, treatment of differentiated cells in ALI culture with cyclosporine did not elicit cytokine or growth factor secretion, and did not alter IL-6, IL-8, or TNF-α production in response to IL-1β treatment. Neither IL-1β nor cyclosporine elicited expression of markers of the epithelial to mesenchymal transition E-cadherin, EDN-fibronectin, and α-smooth muscle actin.

Conclusion

Transplant derived differentiated airway epithelial cell IL-6, IL-8, and TNF-α secretion is not regulated by cyclosporine in vitro; these cells thus may participate in local inflammatory responses in the setting of immunosuppression. Further, treatment with IL-1β did not elicit gene expression of markers of epithelial to mesenchymal transition. These data present a model of differentiated airway epithelial cells that may be useful in understanding epithelial participation in airway inflammation and allograft rejection in lung transplantation.     

Differential effects of cytokines and corticosteroids on Toll-like receptor 2 expression and activity in human airway epithelia

Background

The recognition of microbial molecular patterns via Toll-like receptors (TLRs) is critical for mucosal defenses.

Methods

Using well-differentiated primary cultures of human airway epithelia, we investigated the effects of exposure of the cells to cytokines (TNF-α and IFN-γ) and dexamethasone (dex) on responsiveness to the TLR2/TLR1 ligand Pam3CSK4. Production of IL-8, CCL20, and airway surface liquid antimicrobial activity were used as endpoints.

Results

Microarray expression profiling in human airway epithelia revealed that first response cytokines markedly induced TLR2 expression. Real-time PCR confirmed that cytokines (TNF-α and IFN-γ), dexamethasone (dex), or cytokines + dex increased TLR2 mRNA abundance. A synergistic increase was seen with cytokines + dex. To assess TLR2 function, epithelia pre-treated with cytokines ± dex were exposed to the TLR2/TLR1 ligand Pam3CSK4 for 24 hours. While cells pre-treated with cytokines alone exhibited significantly enhanced IL-8 and CCL20 secretion following Pam3CSK4, mean IL-8 and CCL20 release decreased in Pam3CSK4 stimulated cells following cytokines + dex pre-treatment. This marked increase in inflammatory gene expression seen after treatment with cytokines followed by the TLR2 ligand did not correlate well with NF-κB, Stat1, or p38 MAP kinase pathway activation. Cytokines also enhanced TLR2 agonist-induced beta-defensin 2 mRNA expression and increased the antimicrobial activity of airway surface liquid. Dex blocked these effects.

Conclusion

While dex treatment enhanced TLR2 expression, co-administration of dex with cytokines inhibited airway epithelial cell responsiveness to TLR2/TLR1 ligand over cytokines alone. Enhanced functional TLR2 expression following exposure to TNF-α and IFN-γ may serve as a dynamic means to amplify epithelial innate immune responses during infectious or inflammatory pulmonary diseases.     

Culture of Airway Epithelial Cells from Neonates Sampled within 48-Hours of Birth

Introduction

Little is known about how neonatal airway epithelial cell phenotype impacts on respiratory disease in later life. This study aimed to establish a methodology to culture and characterise neonatal nasal epithelial cells sampled from healthy, non-sedated infants within 48 hours of delivery.

Methods

Nasal epithelial cells were sampled by brushing both nostrils with an interdental brush, grown to confluence and sub-cultured. Cultured cells were characterised morphologically by light and electron microscopy and by immunocytochemistry. As an exemplar pro-inflammatory chemokine, IL-8 concentrations were measured in supernatants from unstimulated monolayers and after exposure to IL-1β/TNF-α or house dust mite extract.

Results

Primary cultures were successfully established in 135 (91%) of 149 neonatal samples seeded, with 79% (n  =  117) successfully cultured to passage 3. The epithelial lineage of the cells was confirmed by morphological analysis and immunostaining. Constitutive IL-8 secretion was observed and was upregulated by IL-1β/TNF-α or house dust mite extract in a dose dependent manner.

Conclusion

We describe a safe, minimally invasive method of culturing nasal epithelial cells from neonates suitable for functional cell analysis offering an opportunity to study “naïve” cells that may prove useful in elucidating the role of the epithelium in the early origins of asthma and/or allergic rhinitis.     

Extracellular Glutathione Decreases the Ability of Burkholderia cenocepacia to Penetrate into Epithelial Cells and to Induce an Inflammatory Response

Background

The airway surface liquid (ASL) of Cystic Fibrosis (CF) patients contains a lower concentration of reduced glutathione (GSH) with respect to healthy people. It is not known whether this defect may favor lung colonization by opportunistic pathogens.

Principal Findings

We have analyzed the effects of extracellular GSH on the ability of Burkholderia cenocepacia to penetrate and multiply in epithelial respiratory cells. Extracellular GSH proved to be able to drastically reduce the pathogen ability to adhere and invade airway epithelial cells. This effect is correlated to a GSH-dependent increase in the number of free thiols on the surface of epithelial cells, suggestive of a change in the oxidoreductive status of membrane proteins involved in B. cenocepacia recognition. Moreover, treatments with GSH led to a consistent reduction of the expression of IL-8, TNF-α and IL-1β in response to B. cenocepacia infection.

Conclusions and Significance

Extracellular GSH modulates the interaction between B. cenocepacia and epithelial respiratory cells and inhibits the bacterial invasion into these cells. This suggests that therapies aimed at restoring normal levels of GSH in the ASL might be beneficial to control CF lung infections.     

Epidermal growth factor receptor reactivation induced by E-prostanoid-3 receptor- and tumor necrosis factor-alpha-converting enzyme-dependent feedback exaggerates interleukin-8 production in airway cancer (NCI-H292) cells

Airway epithelial cancer cells produce increased amounts of the chemokine interleukin-8 (IL-8), inducing pro-tumor responses. Multiple stimuli induce airway epithelial IL-8 production epidermal growth factor receptor (EGFR) dependently, but the mechanisms that exaggerate IL-8 production in airway cancers remain unknown. Here we show that direct activation of EGFR (EGFR-P) by its ligand transforming growth factor (TGF)-alpha induces a second EGFR-P in human airway (NCI-H292) cancer cells but not in normal human bronchial epithelial (NHBE) cells, exaggerating IL-8 production in these cancer cells. The second EGFR-P in NCI-H292 cells was caused by metalloprotease TNF-alpha-converting enzyme (TACE)-dependent cleavage of EGFR pro-ligands and was responsible for most of the total IL-8 induced by TGF-alpha. In NCI-H292 cells, TGF-alpha induced cyclooxygenase (COX)-2-dependent prostaglandin (PG)E2 production and release. PGE2 increased the second EGFR-P and IL-8 production via binding to its Gi-protein-coupled E-prostanoid (EP)3 receptor. In NHBE cells, TGF-alpha-induced EGFR-P did not lead to PGE2 production or to a second EGFR-P, and less IL-8 was produced. Thus, we conclude that a positive feedback pathway involving COX-2/PGE2/EP3 receptor-dependent EGFR reactivation exaggerates IL-8 production in NCI-H292 cancer cells but not in NHBE (normal) cells.     

6-Mercaptopurine reduces cytokine and Muc5ac expression involving inhibition of NFκB activation in airway epithelial cells

Background

Mucus hypersecretion and excessive cytokine synthesis is associated with many of the pathologic features of chronic airway diseases such as asthma. 6-Mercaptopurine (6-MP) is an immunosuppressive drug that is widely used in several inflammatory disorders. Although 6-MP has been used to treat asthma, its function and mechanism of action in airway epithelial cells is unknown.

Methods

Confluent NCI-H292 and MLE-12 epithelial cells were pretreated with 6-MP followed by stimulation with TNFα or PMA. mRNA levels of cytokines and mucins were measured by RT-PCR. Western blot analysis was performed to assess the phosphorylation of IκBα and luciferase assays were performed using an NFκB reporter plasmid to determine NFκB activity. Periodic Acid Schiff staining was used to assess the production of mucus.

Results

6-MP displayed no effect on cell viability up to a concentration of 15 μM. RT-PCR analysis showed that 6-MP significantly reduces TNFα- and PMA-induced expression of several proinflammatory cytokines in NCI-H292 and MLE-12 cells. Consistent with this, we demonstrated that 6-MP strongly inhibits TNFα-induced phosphorylation of IκBα and thus attenuates NFκB luciferase reporter activity. In addition, 6-MP decreases Rac1 activity in MLE-12 cells. 6-MP down-regulates gene expression of the mucin Muc5ac, but not Muc2, through inhibition of activation of the NFκB pathway. Furthermore, PMA- and TNFα-induced mucus production, as visualized by Periodic Acid Schiff (PAS) staining, is decreased by 6-MP.

Conclusions

Our data demonstrate that 6-MP inhibits Muc5ac gene expression and mucus production in airway epithelial cells through inhibition of the NFκB pathway, and 6-MP may represent a novel therapeutic target for mucus hypersecretion in airway diseases.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-015-0236-0) contains supplementary material, which is available to authorized users.     

IL-13 induces a bronchial epithelial phenotype that is profibrotic

Background

Inflammatory cytokines (e.g. IL-13) and mechanical perturbations (e.g. scrape injury) to the epithelium release profibrotic factors such as TGF-β₂, which may, in turn, stimulate subepithelial fibrosis in asthma. We hypothesized that prolonged IL-13 exposure creates a plastic epithelial phenotype that is profibrotic through continuous secretion of soluble mediators at levels that stimulate subepithelial fibrosis.

Methods

Normal human bronchial epithelial cells (NHBE) were treated with IL-13 (0, 0.1, 1, or 10 ng/ml) for 14 days (day 7 to day 21 following seeding) at an air-liquid interface during differentiation, and then withdrawn for 1 or 7 days. Pre-treated and untreated NHBE were co-cultured for 3 days with normal human lung fibroblasts (NHLF) embedded in rat-tail collagen gels during days 22–25 or days 28–31.

Results

IL-13 induced increasing levels of MUC5AC protein, and TGF-β₂, while decreasing β-Tubulin IV at day 22 and 28 in the NHBE. TGF-β₂, soluble collagen in the media, salt soluble collagen in the matrix, and second harmonic generation (SHG) signal from fibrillar collagen in the matrix were elevated in the IL-13 pre-treated NHBE co-cultures at day 25, but not at day 31. A TGF-β₂ neutralizing antibody reversed the increase in collagen content and SHG signal.

Conclusion

Prolonged IL-13 exposure followed by withdrawal creates an epithelial phenotype, which continuously secretes TGF-β₂ at levels that increase collagen secretion and alters the bulk optical properties of an underlying fibroblast-embedded collagen matrix. Extended withdrawal of IL-13 from the epithelium followed by co-culture does not stimulate fibrosis, indicating plasticity of the cultured airway epithelium and an ability to return to a baseline. Hence, IL-13 may contribute to subepithelial fibrosis in asthma by stimulating biologically significant TGF-β₂ secretion from the airway epithelium.     

Cigarette smoke induces IL-8, but inhibits eotaxin and RANTES release from airway smooth muscle

Background

Cigarette smoke is the leading risk factor for the development of chronic obstructive pulmonary disease (COPD) an inflammatory condition characterised by neutrophilic inflammation and release of proinflammatory mediators such as interleukin-8 (IL-8). Human airway smooth muscle cells (HASMC) are a source of proinflammatory cytokines and chemokines. We investigated whether cigarette smoke could directly induce the release of chemokines from HASMC.

Methods

HASMC in primary culture were exposed to cigarette smoke extract (CSE) with or without TNFα. Chemokines were measured by enzyme-linked immunosorbent assay (ELISA) and gene expression by real time polymerase chain reaction (PCR). Data were analysed using one-way analysis of variance (ANOVA) followed by Bonferroni''s t test

Results

CSE (5, 10 and 15%) induced IL-8 release and expression without effect on eotaxin or RANTES release. At 20%, there was less IL-8 release. TNFα enhanced CSE-induced IL-8 release and expression. However, CSE (5–30%) inhibited TNFα-induced eotaxin and RANTES production. The effects of CSE on IL-8 release were inhibited by glutathione (GSH) and associated with the induction of the oxidant sensing protein, heme oxygenase-1.

Conclusion

Cigarette smoke may directly cause the release of IL-8 from HASMC, an effect enhanced by TNF-α which is overexpressed in COPD. Inhibition of eotaxin and RANTES by cigarette smoke is consistent with the predominant neutrophilic but not eosinophilic inflammation found in COPD.     

25-hydroxycholesterol enhances cytokine release and toll-like receptor 3 response in airway epithelial cells

Background

25-hydroxycholesterol (25-HC) is one of the oxysterols, which are oxidized derivatives of cholesterol, and has been reported to be involved in the pathogenesis of atherosclerosis and Alzheimer’s disease. In lung, the possible involvement of 25-HC in airway diseases has been revealed. In the present study, we examined whether 25-HC affects the release of cytokines and also modulates the responses of toll-like receptor 3 (TLR3) in airway epithelial cells.

Methods

The effect of 25-HC on the release of cytokines from primary human bronchial epithelial cells after stimulation with or without polyinosine-polycytidylic acid [poly(I:C)], a ligand for TLR3, and the signal transduction were examined.

Results

25-HC significantly potentiated the release of interleukin-8 (IL-8) and IL-6 from the cells. This effect was more potent compared with that of other oxysterols, 22-HC and 27-HC. GW3965 and TO901317, synthetic agonists of liver X receptors that are receptors for oxysterols, did not augment the IL-8 release. 25-HC enhanced the nuclear factor-kappa B (NF-κB) DNA binding activity and translocation of phosphorylated c-Jun into the nucleus. The release of IL-8 was inhibited by the NF-κB inhibitor, caffeic acid phenethyl ester (CAPE), an inhibitor of nuclear factor kappa-B alpha (IκBα) inhibitor, BAY 11–7085, and an inhibitor of nuclear factor kappa-B kinase-2 (IKK-2) inhibitor, SC-514, but not by a c-Jun N-terminal kinase (JNK) inhibitory peptide, L-JNKi1. 25-HC significantly potentiated IL-8 release in poly(I:C)-treated cells and the augmentation was inhibited by CAPE, BAY 11–7085, and SC-514. Furthermore, 25-HC potentiated the translocation of interferon regulatory factor 3 into the nucleus and the release of interferon-beta (IFN-β) in poly(I:C)-treated cells.

Conclusions

These data demonstrated that 25-HC augments the release of IL-8 and IL-6 via NF-κB signalling pathway and enhances the release of IL-8 and IFN-β after stimulation of TLR3 in airway epithelial cells. 25-HC may be involved in the neutrophilic airway inflammation through the stimulant effect of IL-8 and IL-6 release and also potentiate the TLR3-mediated innate immunity in airway diseases.     

Macrophage activation state determines the response to rhinovirus infection in a mouse model of allergic asthma

Background

The mechanisms by which viruses cause asthma exacerbations are not precisely known. Previously, we showed that, in ovalbumin (OVA)-sensitized and -challenged mice with allergic airway inflammation, rhinovirus (RV) infection increases type 2 cytokine production from alternatively-activated (M2) airway macrophages, enhancing eosinophilic inflammation and airways hyperresponsiveness. In this paper, we tested the hypothesis that IL-4 signaling determines the state of macrophage activation and pattern of RV-induced exacerbation in mice with allergic airways disease.

Methods

Eight week-old wild type or IL-4 receptor knockout (IL-4R KO) mice were sensitized and challenged with OVA and inoculated with RV1B or sham HeLa cell lysate.

Results

In contrast to OVA-treated wild-type mice with both neutrophilic and eosinophilic airway inflammation, OVA-treated IL-4R KO mice showed increased neutrophilic inflammation with few eosinophils in the airways. Like wild-type mice, IL-4R KO mice showed OVA-induced airway hyperreactivity which was further exacerbated by RV. There was a shift in lung cytokines from a type 2-predominant response to a type 1 response, including production of IL-12p40 and TNF-α. IL-17A was also increased. RV infection of OVA-treated IL-4R KO mice further increased neutrophilic inflammation. Bronchoalveolar macrophages showed an M1 polarization pattern and ex vivo RV infection increased macrophage production of TNF-α, IFN-γ and IL-12p40. Finally, lung cells from OVA-treated IL-4R KO mice showed reduced CD206+ CD301+ M2 macrophages, decreased IL-13 and increased TNF-α and IL-17A production by F4/80+, CD11b+ macrophages.

Conclusions

OVA-treated IL-4R KO mice show neutrophilic airway inflammation constituting a model of allergic, type 1 cytokine-driven neutrophilic asthma. In the absence of IL-4/IL-13 signaling, RV infection of OVA-treated mice increased type 1 cytokine and IL-17A production from conventionally-activated macrophages, augmenting neutrophilic rather than eosinophilic inflammation. In mice with allergic airways inflammation, IL-4R signaling determines macrophage activation state and the response to subsequent RV infection.     

Nitric oxide gas phase release in human small airway epithelial cells

Background

Asthma is a chronic airway inflammatory disease characterized by an imbalance in both Th1 and Th2 cytokines. Exhaled nitric oxide (NO) is elevated in asthma, and is a potentially useful non-invasive marker of airway inflammation. However, the origin and underlying mechanisms of intersubject variability of exhaled NO are not yet fully understood. We have previously described NO gas phase release from normal human bronchial epithelial cells (NHBEs, tracheal origin). However, smaller airways are the major site of morbidity in asthma. We hypothesized that IL-13 or cytomix (IL-1β, TNF-α, and IFN-γ) stimulation of differentiated small airway epithelial cells (SAECs, generation 10–12) and A549 cells (model cell line of alveolar type II cells) in culture would enhance NO gas phase release.

Methods

Confluent monolayers of SAECs and A549 cells were cultured in Transwell plates and SAECs were allowed to differentiate into ciliated and mucus producing cells at an air-liquid interface. The cells were then stimulated with IL-13 (10 ng/mL) or cytomix (10 ng/mL for each cytokine). Gas phase NO release in the headspace air over the cells was measured for 48 hours using a chemiluminescence analyzer.

Results

In contrast to our previous result in NHBE, baseline NO release from SAECs and A549 is negligible. However, NO release is significantly increased by cytomix (0.51 ± 0.18 and 0.29 ± 0.20 pl^.s^-1.cm^-2, respectively) reaching a peak at approximately 10 hours. iNOS protein expression increases in a consistent pattern both temporally and in magnitude. In contrast, IL-13 only modestly increases NO release in SAECs reaching a peak (0.06 ± 0.03 pl^.s^-1.cm^-2) more slowly (30 to 48 hours), and does not alter NO release in A549 cells.

Conclusion

We conclude that the airway epithelium is a probable source of NO in the exhaled breath, and intersubject variability may be due, in part, to variability in the type (Th1 vs Th2) and location (large vs small airway) of inflammation.     

WW Domain Containing E3 Ubiquitin Protein Ligase 1 (WWP1) Negatively Regulates TLR4-Mediated TNF-α and IL-6 Production by Proteasomal Degradation of TNF Receptor Associated Factor 6 (TRAF6)

Background

Toll-like receptors (TLRs) play a pivotal role in the defense against invading pathogens by detecting pathogen-associated molecular patterns (PAMPs). TLR4 recognizes lipopolysaccharides (LPS) in the cell walls of Gram-negative bacteria, resulting in the induction and secretion of proinflammatory cytokines such as TNF-α and IL-6. The WW domain containing E3 ubiquitin protein ligase 1 (WWP1) regulates a variety of cellular biological processes. Here, we investigated whether WWP1 acts as an E3 ubiquitin ligase in TLR-mediated inflammation.

Methodology/Results

Knocking down WWP1 enhanced the TNF-α and IL-6 production induced by LPS, and over-expression of WWP1 inhibited the TNF-α and IL-6 production induced by LPS, but not by TNF-α. WWP1 also inhibited the IκB-α, NF-κB, and MAPK activation stimulated by LPS. Additionally, WWP1 could degrade TRAF6, but not IRAK1, in the proteasome pathway, and knocking down WWP1 reduced the LPS-induced K48-linked, but not K63-linked, polyubiquitination of endogenous TRAF6.

Conclusions/Significance

We identified WWP1 as an important negative regulator of TLR4-mediated TNF-α and IL-6 production. We also showed that WWP1 functions as an E3 ligase when cells are stimulated with LPS by binding to TRAF6 and promoting K48-linked polyubiquitination. This results in the proteasomal degradation of TRAF6.     

Expression of myoferlin in human airway epithelium and its role in cell adhesion and zonula occludens-1 expression

Background

Normal airway epithelial barrier function is maintained by cell-cell contacts which require the translocation of adhesion proteins at the cell surface, through membrane vesicle trafficking and fusion events. Myoferlin and dysferlin, members of the multiple-C2-domain Ferlin superfamily, have been implicated in membrane fusion processes through the induction of membrane curvature. The objectives of this study were to examine the expression of dysferlin and myoferlin within the human airway and determine the roles of these proteins in airway epithelial homeostasis.

Methods

The expression of dysferlin and myoferlin were evaluated in normal human airway sections by immunohistochemistry, and primary human airway epithelial cells and fibroblasts by immuno blot. Localization of dysferlin and myoferlin in epithelial cells were determined using confocal microscopy. Functional outcomes analyzed included cell adhesion, protein expression, and cell detachment following dysferlin and myoferlin siRNA knock-down, using the human bronchial epithelial cell line, 16HBE.

Results

Primary human airway epithelial cells express both dysferlin and myoferlin whereas fibroblasts isolated from bronchi and the parenchyma only express myoferlin. Expression of dysferlin and myoferlin was further localized within the Golgi, cell cytoplasm and plasma membrane of 16HBE cells using confocal micrscopy. Treatment of 16HBE cells with myoferlin siRNA, but not dysferlin siRNA, resulted in a rounded cell morphology and loss of cell adhesion. This cell shedding following myoferlin knockdown was associated with decreased expression of tight junction molecule, zonula occludens-1 (ZO-1) and increased number of cells positive for apoptotic markers Annexin V and propidium iodide. Cell shedding was not associated with release of the innate inflammatory cytokines IL-6 and IL-8.

Conclusions/Significance

This study demonstrates the heterogeneous expression of myoferlin within epithelial cells and fibroblasts of the respiratory airway. The effect of myoferlin on the expression of ZO-1 in airway epithelial cells indicates its role in membrane fusion events that regulate cell detachment and apoptosis within the airway epithelium.     

Increased Baseline Proinflammatory Cytokine Production in Chronic Hepatitis C Patients with Rapid Virological Response to Peginterferon Plus Ribavirin

Background

Chronic hepatitis C (CHC) patients achieving rapid virological response (RVR) on PEG-IFN/ribavirin (P/R) therapy have high chance of sustained virological response (SVR). To analyze host immunological factors associated with RVR, viral kinetics, phenotype distribution and Th1/Th2 cytokine production by peripheral blood mononuclear cells (PBMC) were studied prior to and during P/R therapy.

Methods

TNF-α, IFN-γ, IL-2, IL-6, IL-4 and IL-10 production by PBMC were measured after Toll-like receptor 4 (TLR-4) or phorbol myristate acetate/Ionomycin stimulation in 20 healthy controls and in 50 CHC patients before receiving and during P/R therapy. RVR was achieved by 14, complete early virological response (cEVR) by 19 patients and 17 patients were null-responders (NR).

Results

Patients with RVR showed an increased baseline TNF-α and IL-6 production by TLR-4 activated monocytes and increased IFN-γ, decreased IL-4 and IL-10 production by lymphocytes compared to non-RVR patients. SVR was also associated with increased baseline TNF-α production and decreased IL-10 levels compared to patients who did not achieve SVR. Baseline IL-2 production was higher in cEVR compared to NR patients. Antiviral treatment increased TNF-α, IL-6 production by monocytes and IFN-γ secretion by lymphocytes and decreased IL-4 and IL-10 production by lymphocytes in cEVR compared to NR patients.

Conclusion

RVR was associated with increased baseline proinflammatory cytokine production by TLR-4 stimulated monocytes and by activated lymphocytes. In null-responders and in patients who did not achieve SVR both TLR-4 sensing function and proinflammatory cytokine production were impaired, suggesting that modulation of TLR activity and controlled induction of inflammatory cytokine production may provide further therapeutic strategy for CHC patients non-responding to P/R treatment.     

Polarized Secretion of Interleukin (IL)-6 and IL-8 by Human Airway Epithelia 16HBE14o- Cells in Response to Cationic Polypeptide Challenge

Background

The airway epithelium participates in asthmatic inflammation in many ways. Target cells of the epithelium can respond to a variety of inflammatory mediators and cytokines. Damage to the surface epithelium occurs following the secretion of eosinophil-derived, highly toxic cationic proteins. Moreover, the surface epithelium itself is responsible for the synthesis and release of cytokines that cause the selective recruitment, retention, and accumulation of various inflammatory cells. To mimic the damage seen during asthmatic inflammation, the bronchial epithelium can be challenged with highly charged cationic polypeptides such as poly-l-arginine.

Methodology/Principal Findings

In this study, human bronchial epithelial cells, 16HBE14o- cells, were “chemically injured” by exposing them to poly-l-arginine as a surrogate of the eosinophil cationic protein. Cytokine antibody array data showed that seven inflammatory mediators were elevated out of the 40 tested, including marked elevation in interleukin (IL)-6 and IL-8 secretion. IL-6 and IL-8 mRNA expression levels were elevated as measured with real-time PCR. Cell culture supernatants from apical and basolateral compartments were collected, and the IL-6 and IL-8 production was quantified with ELISA. IL-6 and IL-8 secretion by 16HBE14o- epithelia into the apical compartment was significantly higher than that from the basolateral compartment. Using specific inhibitors, the production of IL-6 and IL-8 was found to be dependent on p38 MAPK, ERK1/2 MAPK, and NF-κB pathways.

Conclusions/Significance

The results clearly demonstrate that damage to the bronchial epithelia by poly-l-arginine stimulates polarized IL-6 and IL-8 secretion. This apically directed secretion of cytokines may play an important role in orchestrating epithelial cell responses to inflammation.     

The Hemorrhagic Coli Pilus (HCP) of Escherichia coli O157:H7 Is an Inducer of Proinflammatory Cytokine Secretion in Intestinal Epithelial Cells

Background

Enterohemorrhagic Escherichia coli (EHEC) O157:H7, the causative agent of hemorrhagic colitis and the hemolytic uremic syndrome (HUS), produces long bundles of type IV pili (TFP) called hemorrhagic coli pili (HCP). HCP are capable of mediating several phenomena associated with pathogenicity: i) adherence to human and bovine epithelial cells; ii) invasion of epithelial cells; iii) hemagglutination of rabbit erythrocytes; iv) biofilm formation; v) twitching motility; and vi) specific binding to laminin and fibronectin. HCP are composed of a 19 kDa pilin subunit (HcpA) encoded by the hcpA chromosomal gene (called prepilin peptidase-dependent gene [ppdD] in E. coli K-12).

Methodology/Principal Findings

In this study we investigated the potential role of HCP of E. coli O157:H7 strain EDL933 in activating the release of pro- and anti-inflammatory cytokines from a variety of host epithelial cells. We found that purified HCP and a recombinant HcpA protein induced significant release of IL-8 and TNF-α, from cultured polarized intestinal cells (T84 and HT-29 cells) and non-intestinal HeLa cells. Levels of proinflammatory IL-8 and TNF-α, but not IL-2, IL6, or IL-10 cytokines, were increased in the presence of HCP and recombinant HcpA after 6 h of incubation with ≥50 ng/ml of protein, suggesting that stimulation of IL-8 and TNF-α are dose and time-dependent. In addition, we also demonstrated that flagella are potent inducers of cytokine production. Furthermore, MAPK activation kinetics studies showed that EHEC induces p38 phosphorylation under HCP-producing conditions, and ERK1/2 and JNK activation was detectable after 3 h of EHEC infection. HT-29 cells were stimulated with epidermal growth factor stimulation of HT-29 cells for 30 min leading to activation of three MAPKs.

Conclusions/Significance

The HcpA pilin monomer of the HCP produced by EHEC O157:H7 is a potent inducer of IL-8 and TNF-α release, an event which could play a significant role in the pathogenesis of hemorrhagic colitis caused by this pathogen.