The Study Of High-mobility Group Box-1Protein (HMGB1) Induced Barrier Dysfunction In Airway Epithelial Cells

Asthma is a chronic inflammatory airway disorder involved multiple cells (eosnophils, neutrophils, T lymphocytes and airway epithelium) and cellular element, characterized by airway hyper-responsiveness, airflow obstruction and airway remodeling. There has been a sharp increase in the global prevalence, morbidity, mortality, and economic burden associated with asthma over recent years.The bronchial epithelium is central to the pathogenesis of asthma and plays a important role in immune regulation during initiation of allergic responses. The epithelial barrier function is dependent on cellular integrity, as well as coordinate expression and interaction of proteins in cellular junctional complexes, including apical tight junctions and adherens junctions. Tight juctional proteins localize apically and are considered to be the main regulators of paracellular permeability and movement of ions and solutes between epithelial cells. Adherent junction localizes below tight junction and is composed of a calcium-dependent E-cadherin and various kinds of catenin proteins. E-cadherin is thought to provide the architecture that is required to form other junctional complexes, including tight junctions.It is now clear that the pro-inflammatory cytokine release drive airway pathology in asthma. A number of observations suggest that airway epithelial barrier function is compromised in asthma. These observations have revealed structural abnormalities in apical junctional complexes of asthmatics, which comprise the interacting proteins occludin and claudins, and adherens junction proteins E-cadherin and β-catenin, compared with that in healthy subjects. The junction proteins are supposed to be gatekeeper in charge of different aspects of epithelial barrier function. These structural and functional abnormalities of junction proteins might lead to enhanced signaling between the epithelium and underlying immune and structural cells.High-mobility group box-1protein (HMGB1) has been known to be a nuclear DNA-binding protein, participating in many pathological processes. HMGB1is not only actively secreted by innate immune cells such as macrophages and monocytes, but also passively released by injured and necrotic cells. It is secreted into the extracellular milieu and acts as a pro-inflammatory cytokine. By binding to cytokines such as IL-1β, IFN-γ and TNF-α, HMGB1protein can enhance pro-inflammatory and immune response. There are accumulating evidences that HMGB1is involved in pathological processes including inflammatory disorders of the respiratory tract. When airway epithelial cells were attacked by allergen and microorganism, it can release endogenous dangerous signaling molecule, named damage associated pattern molecules (DAMPs). HMGB1, a typical molecule of DAMPs, can be released into the extracellular milieu by the damaged epithelial cells and binds pattern-recognition receptors (PRRs) such as RAGE, TLR2and TLR4. They can function in an autocrine manner (secreted and acting on airway epithelial cell).In a previous study, we investigated the expression of HMGB1in plasma and induced sputum of patients with asthma and explored the relationship with lung function. We demonstrate that a potential role of HMGB1in the development of asthma. However, it has remained undefined how HMGB1participates in the pathogenesis of asthma. Recent studies further suggest that both HMGB1and RAGE contributes to pathogenesis of asthma. Blocking HMGB1activity decreased magnitude of the allergic response in either HDM or OVA induced mouse asthma model. RAGE knockout mice exhibit attenuated airway hypersensitivity, eosinophilic inflammation, and airway remodeling. To date, there appears to have no studies about the role of HMGB1/RAGE axle on epithelial barrier function.Therefore, the aims of our study were to determine the effects of HMGB1on epithelial barrier structure and function and to characterize possible mechanisms involved in these modulatory properties. Our findings indicate that HMGB1not only increases barrier permeability, but also selectively disrupts expression of occludin and claudin-2, dislocates E-cadherin and P-catenin via RAGE/ERK1/2signal pathways. In addition, the presence of IL-1β may facilitate HMGB1induced epithelial barrier dysfunction.Objectives:1. To investigate the effect of HMGB1induced barrier function defect in airway cells;2. To investigate the possible signaling pathway involved in HMGB1mediacted epithelial barrier dysfunction.3. To study the effect of HMGB1in synergy with IL-1β on epithelial barrier property.Methods:1. Epithelial Cell lines (16HBE and A549) culture.2. Methyltetrazolium (MTT) assay was used to assess the16HBE and A549 cell viabilities under different concentration (100,200and400ng/ml) of HMGB1stimulation.3. To study the dosage effect of HMGB1on airway epithelial barrier function impairment.16HBE or A549cell monolayer was treated with100,200and400ng/ml HMGB1for24h, respectively. And then the indicators as follows were measured:a) Transepithelial electrical resistance (TER) was measured in real-time using MILLICELL-ERS Voltohmmeter. Fluorescein isothiocyanate-dextran flux (FITC) across monolayers of cultured epithelial cells was evaluated using a fluorescent plate reader.b) Western bolt was used to analyze protein expression of junction proteins.c) Immunofluorescence microscopy was measured to evaluate the delocalization of junction proteins (E-cadhrein, β-catenin occludin and claudin-2).4. To study time effct of HMGB1on airway epithelial barrier function impairment.16HBE or A549cell monolayer was treated with400ng/ml HMGB1for0,1,3,6,12,24,48h, respectively. And then the indicators the same as method3were measured.5. To study the effect of HMGB1in synergy with IL-1βon barrier properties in16HBE cells. Groups as follows:the normal control group,100ng/ml HMGB1stimulation group,2.5ng/ml IL-1βstimulation group and HMGB1in synergy with IL-1βstimulation group. After stimulated for24h, ion and macromolecular barrier permeability were measured and Western blot and immunohistochemistry were used to detect the expression and distribution of junctional proteins.6. To investigate the crucial role of tight junction protein occludin in the HMGB1induced barrier function defect in16HBE cells. Plasmid containing wild type of full length occludin gene were constructed, amplified, extracted and identified. Plasmid was extracted by alkaline lysis method.After the agar gel electrophoresis and DNA sequencing were used to identify the target gene. Titer of occludin gene was measured. Wild type of full length occludin gene was transfected to16HBE cells by Lipofectin Reagent. QPCR and western bolt were used to analyze transfection efficiency.a) To investigate the effect of over-expressing occludin protein on barrier function and other junction protein expression (E-cadhrein, β-catenin and claudin-2) in the16HBE cells. Groups as follows:the normal vector group and over-expressing occludin group. Ion and macromolecular barrier permeability were measured and Western blot were used to detect the expression of other junctional proteins (E-cadhrein, β-catenin and claudin-2).b) To investigate the crucial role of tight junction protein occludin in the HMGB1induced barrier function defect in16HBE cells. Over-expressing and normal-expressing occludin protein in16HBE cells were stimulated with400ng/ml HMGB1and then the change of epithelial barrier properties were measured.7. To explore the possible signaling pathway involved in HMGB1induced epithelial barrier function defect. Western bolt was used to detect the expression of main membrane receptors (TLR2, TLR2and RAGE) and activation of down-stream MAPK signaling. Based on the above experiments, anti-membrane receptors antibody or inhibitor of MAPK signaling was used to detect the effect on epithelial barrier dysfunction and junction proteins disruption.Results:1. HMGB1induced an increase in ion and macromolecular barrier permeabilityIon Barrier PermeabilityThe ion barrier permeability effect of HMGB1was manifested in a concentration-and time-dependent manner. The transmembrane resistance (TER) levels started to decline at6h, with a significant effect after stimulation with HMGB1for24h(A549cells:F=82.815,P=0.001;16HBE cells:F=57.887,P=0.002). Additions of400ng/ml HMGB1induced a more significant decline in TER than the other two low concentrations HMGB1treated groups and control group (all P value＜0.001, n=3). This indicates high concentration of HMGB1displays potent ability to changes in ion barrier permeability. In addition, A549cells were more susceptible to the stimulation of HMGB1, compared with16HBE cells (maximal mean+SEM decrease,59+7% and42+2%, respectively; n=3).Macromolecular Barrier PermeabilityIn addition to its effects on ion barrier permeability, HMGB1also increased macromolecular transmission. We found that addition of HMGB1induced an increase in macromolecular permeability to FITC-labeled4-kDa dextran. A549and16HBE cells monolayer treated with400ng/ml HMGB1for48h showed an over50%increase in macromolecular permeability (all P value<0.001, n=3),compared with controls. Concentration-and time-dependent changes in FITC dextran permeability were also observed.2. HMGB1mediated disruption in the expression and localization of junction proteins.To investigate the mechanism of HMGB1induced epithelial barrier dysfunction, we studied whether the changes in barrier function were paralleled by changes in intercellular junction proteins. First we treated16HBE cells with100,200, and400ng/ml HMGB1respectively in culture medium for24h. Low expression of tight junction proteins occludin and claudin-2was observed with incubation of400ng/ml HMGB1, other than100and200ng/ml HMGB1(all P value<0.05). Then both two epithelial cell line monolayers were treated with400ng/ml HMGB1for1,6, 12,24and48h, respectively. Western blot analyses showed variable changes in tight junction proteins and adhesion junction proteins in response to HMGB1. HMGB1caused a time dependent down-regulation of occludin and claudin-2, which was already detectable at12h in A549cells and24h in16HBE cells. An even stronger time-dependent effect on tight junction proteins down-regulation was observed when cell monolayers were treated with HMGB1for48h. These modifications were in accordance with changes in ion and macromolecular barrier permeability.Surprisingly, no similar changes in the expression of E-cadherin and β-catenin were seen between the two cell lines. Down-regulated expression of E-cadherin and P-catenin was observed at48h in A549cells, while HMGB1had no effect on the expression of the two adhesion junction proteins in16HBE cells. However, Immunofluorescent staining showed delocalization of E-cadherin from cell membrane to cell plasma and disruption of β-catenin in HMGB1treated16HBE, suggesting the function of E-cadherin and β-catenin may also be affected by HMGB1and the redistribution of these proteins was the main effect3. Tight junction protein occludin play a crucial role in HMGB1induced epithelial barrier function defect.Compared with normal16HBE cells, the over-expression occludin16HBE cells showed an increase in transmembrane resistance (TER) levels and an decrease in macromolecular permeability to FITC-labeled4-kDa dextran (all P values<0.001, n=6).Howerver, over-expression of occludin had no effect on the expression of E-cadherin,p-catenine and claudin-2. Overexpression of occludin protein can partially inhibited HMGB1induced the increase in transmembrane resistance (TER) levels and the increase in macromolecular permeability to FITC-labeled4-kDa dextran in16HBE, the differences of transmembrane resistance (TER) levels and macromolecular permeability between two group had statistical significance(all P values<0.001, n=5).4. Involvement of RAGE/ERK cascade in HMGB1induced barrier dysfunction and junction proteins modification.To unravel the mechanisms underlying HMGB1induced epithelial barrier dysfunction, we considered that Pattern Recognition Receptors (PRRs) as well as downstream MAPK signalling may contribute to this type of epithelia barrier function defect. Western blot analysis was performed to evaluate the activation of PRRs [Tolls like receptor (TLR)2, TLR4and RAGE], downstream Mitogen-activated protein kinase (MAPK) signaling [p38MAPK (p38), c-Jun N-terminal Kinase (JNK) and Extracellular Signal-Regulated Kinase (ERK)] and PI3kinase/Akt signalling in16HBE cells treated with400ng/ml HMGB1for different time periods. Present study showed HMGB1increased the protein expression of RAGE at1h and reached a maximum level at12h but decreased at24h, no changes in the protein levels of TLR2and TLR4were observed. In line with this, HMGB1increased threonine/tyrosine phosphorylation of ERK, JNK and Akt, which were noticeable at6h,5h and7h, respectively.Previous studies reported HMGB1and RAGE contributes to immune and inflammatory and responses and we supposed RAGE signaling pathways may involve in HMGB1induced defect in epithelial barrier function. As expected, compared with HMGB1stimulaiton group, pretreatment with RAGE antibody (5ug/ml) significantly attenuated permeability-increasing effect (TER:P=0.001; FITC:P=0.002, n=3). We next investigated whether MAPK and PI3kinase/Akt signalling inhibition could improve HMGB1induced epithelial barrier dysfunction. Treatment of16HBE cells with the ERK1/2tyrosine kinase inhibitor U0126significantly rescued HMGB1induced changes in TER and FITC dextran permeability, while there were no such effects with treatment of the PI3K tyrosine kinase inhibitor LY294002and JNK kinase inhibitor SP600125. We also found that ERK1/2downstream signalling was dependent on RAGE, as HMGB1induced phosphorylated-ERKl/2response was suppressed by anti-RAGE antibody. In line with the involvement of tyrosine phosphorylation of ERK1/2in HMGB1induced barrier permeability dysfunction; we found that U0126inhibited HMGB1mediated down-regulation of occludin and claudin-2. As prior experiment showed, redistribution of proteins was the main effect of HMGB1induced disruption of E-cadherin and P-catenin. We assessed adhesion junction proteins distribution in response to HMGB1with U0126. In control cells, immunostaining for E-cadherin and β-catenin showed a similar plasma membrane distribution. Although HMGB1treatment promoted loss of immunostaining for E-cadherin, this was prevented by treatment with U0126. These suggested that RAGE/ERK1/2signalling contributed to HMGB1induced epithelial barrier dysfunction.5. HMGB1in synergy with IL-1β had more significant effect on barrier dysfunction than HMGB1aloneSince previous studies reported HMGB1forming specific complexes with Damage associated molecular pattern (PAMPs) and cytokines has greater pro-inflammatory activity than HMGB1alone, we next investigated whether there was a similar effect on barrier function. Compared with control group and HMB1stimulation group, treatment with of HMGB1(100ng/ml) in synergy with IL-1β (2.5ng/ml) induced a lower TER and higher FITC dextran permeability changes (all P values<0.05). Consistent with this effect, Western blot analyses showed lower expression of occludin, claudin-2and E-cadherin (all P values<0.01). Immunofluorescent staining of16HBE for E-cadherin showed a redistribution of staining from membrane to cytoplasm. HMGB1and in synergy with IL-1β caused loss and interruption in β-catenin staining at plasma membrane. This indicates HMGB1treatment induced epithelial barrier dysfunction was enhanced by IL-1β.Conclusions:1. HMGB1induce barrier function defect in a time and dosage response manner in human bronchial epithelial16HBE and A549cells,largely due to disruption of cell-cell contacts.2. HMGB1induced epithelial barrier function impairment were paralleled by disruption of tight junction proteins occludin and claudin-2.Occludin protein played a crucial role in HMGB1induced epithelial barrier dysfunction. In a addition, HMGB1can cause delocalization and disruption of adhesion junction protein E-cadherin and β-catenin.3. Application of the specific ERK inhibitor U0125and anti-RAGE antibody showed that RAGE/ERK activation contributes to the HMGB1-induced defects in16HBE cells.RAGE/ERK signaling pathway was involved in HMGB1induced barrier function defect and abnormal expression and distribution of junctional proteins.