Dysfunction of epithelial permeability barrier induced by HMGB1 in 2.5D cultures of human epithelial cells

ABSTRACT

Airway and intestinal epithelial permeability barriers are crucial in epithelial homeostasis. High mobility group box 1 (HMGB1), increased by various stimuli, is involved in the induction of airway inflammation, as well as the pathogenesis of inflammatory bowel disease. HMGB1 enhances epithelial hyperpermeability. Two-and-a-half dimensional (2.5D) culture assays are experimentally convenient and induce cells to form a more physiological tissue architecture than 2D culture assays for molecular transfer mechanism analysis. In 2.5D culture, treatment with HMGB1 induced permeability of FITC-dextran into the lumen formed by human lung, nasal and intestinal epithelial cells. The tricellular tight junction molecule angulin-1/LSR is responsible for the epithelial permeability barrier at tricellular contacts and contributes to various human airway and intestinal inflammatory diseases. In this review, we indicate the mechanisms including angulin-1/LSR and multiple signaling in dysfunction of the epithelial permeability barrier induced by HMGB1 in 2.5D culture of human airway and intestinal epithelial cells.

KEYWORDS:

• 2.5d matrigel cultures
• epithelial permeability barriers
• hmgb1
• proinflammatory cytokines
• tight junctions
• claudins
• angulin-1/LSR
• tricellulin
• TGF-β
• EW-7197
• PYK2
• PF431396
• tnfα-antibody
• p63
• normal human pulmonary epithelial cells
• normal human nasal epithelial cells
• human intestinal epithelial cell line CACO-2
• cell metabolism

Introduction

The airway and gut epithelia interface with the environment, where they act as a physiological barrier critical for protecting the body from environmental allergens, pathogenic microbes and viruses, toxins, volatile organic compounds and other pollutants.^1–3 The epithelial permeability barriers are crucial in epithelial homeostasis. High mobility group box 1 (HMGB1), increased by various stimuli, is involved in the induction of airway inflammation and inflammatory bowel disease. Two-and-a-half dimensional (2.5D) culture assays are convenient and induce cells to form a more physiological tissue architecture than 2D culture assays for molecular transfer mechanism analysis (Figure 1a). In 2.5D culture, treatment with HMGB1 induced permeability of FITC-dextran into the lumen formed by human lung, nasal and intestinal epithelial cells (Figure 2a, Figure 2d). The tricellular tight junction molecule angulin-1/LSR is responsible for the epithelial permeability barrier at tricellular contacts and contributes to various human airway and intestinal inflammatory diseases as well as other tight junction molecules (Figure 1b, Figure 1c). In this review, we demonstrated the mechanisms including angulin-1/LSR, multiple signaling and cellular metabolism in dysfunction of the epithelial permeability barrier induced by HMGB1 in 2.5D culture of human airway and intestinal epithelial cells (primary cultures of human lung epithelial (HLE) cells and human nasal epithelial (HNE) cells and human colorectal adenocarcinoma cell line Caco-2).

Figure 1. Schematic representation of (a) methods and (b) localization of tight junction proteins in 2D and 2.5D cultures. (c) Schematic representation and images in localization of OCLN and angulin-1/LSR in 2.5D culture of Caco-2.

Figure 2. (a) Schematic representation of FD4 in 2.5D culture of HLE cells and HNE cells pretreated with EW-7197 (EW) before treatment with HMGB1. (b) Schematic representation of OCLN and LSR in 2.5D culture of HLE cells and HNE cells pretreated with EW before treatment with HMGB1. (c) Schematic representation of OCLN and angulin-1/LSR in 2.5D culture of Caco-2 cells pretreated with EW before treatment with HMGB1. (d) Schematic representation of FD4 in 2.5D culture of Caco-2 cells pretreated with PF431396 (PF43) before treatment with HMGB1. (e) Schematic representation of phosphorylated and non-phosphorylated angulin-1/LSR and Ac-tubulin in 2.5D culture of Caco-2 cells pretreated with PF43 before treatment with HMGB1.

Tight junctions

The tight junction (TJ) is an epithelial cell–cell junction that regulates the flow of solutes through paracellular pathways and maintains cell polarity.⁴ TJs are involved in various signal transduction pathways that regulate epithelial cell proliferation, gene expression, differentiation and morphogenesis.^5,6 TJ proteins are closely involved in cancer, innate immunity and infectious diseases.⁷ They are composed of claudins (CLDNs), occludins (OCLN), JAMs and scaffold proteins such as ZO.^8,9 CLDNs are major components of TJs in epithelial and endothelial cells.^10,11 CLDN-2 is expressed in the tight junctions of leaky epithelia, where it forms cation-selective and water permeable paracellular channels.¹² Its expression is modulated by a variety of conditions.¹³

Angulin-1/lipolysis-stimulated lipoprotein receptor (LSR) and tricellulin (TRIC) seal the extracellular spaces where three epithelial cells come into contact and have various functions.¹⁴ The family of angulins consists of angulin-1/LSR, angulin-2, (immunoglobulin-like domain-containing receptor 1 [ILDR1]) and angulin-3 (ILDR2).¹⁵ LSR recruits TRIC to tTJs, and both proteins are required for the full barrier function of epithelial cellular sheets.¹⁵ Downregulation of LSR influences not only the barrier function but also various kinds of signaling.^16–19 The c-Jun N-terminal kinase (JNK) pathway is involved in the regulation of tricellular tight junctions, including TRIC expression and the barrier function.^16,20 Protein tyrosine kinase 2β (Pyk2) phosphorylates angulin-1/LSR and enhances the localization of angulin-1/LSR and TRIC at tTJs.^21,22 PF431396 (PF43), which is an inhibitor of Pyk2, suppresses the recruitment of LSR and TRIC from tTJs to bTJs and cytoplasmic regions.²¹

Tight junctions in lung

The airways are broadly classified into conducting and respiratory regions. The conducting airways consist of the trachea, bronchi, and bronchioles, while respiratory regions consist of respiratory bronchioles and alveoli. The conducting airways are lined by a continuous layer of bronchiolar epithelial cells, which act as a first barrier for inhaled materials and play an important role in protection from external pathogens such as bacteria, viruses, chemical substances and allergic components.^23,24 On the other hand, alveolar epithelial cells, which are vital for gas exchanges, form a barrier for an air-liquid interface.²⁵ To maintain the air-liquid interface and control gas exchanges, alveolar epithelial cells provide a proper permeability barrier.²⁶ The main characteristic of epithelial tissues is that epithelial cells are arranged in monolayers or stratified layers and tightly adhere to each other. This sheet-like structure provides a barrier function that separates the apical and basolateral compartments of various tissues, and maintains homeostasis.

In the normal lung, there are various reports about expression of CLDNs in bronchiolar epithelial cells and alveolar epithelial cells. In human bronchiolar epithelium, expression of CLDN-1, −2, −3, −4, −5, −7 and −8 is reported,^27–31 whereas 14 CLDNs are expressed in alveolar epithelium and CLDN-3, −4 and −18 are the major components of type II pneumocytes.^30,32,33 Angulin-1/LSR, expressed at tTJ of most epithelial tissues,^18,19,34 is closely associated with lung epithelial permeability barriers.^35,36

Asthma

Asthma is characterized by chronic airway inflammation induced by allergens, chemical mediators and other external pathogens, and the airway epithelial barrier plays an important role in protection against these factors.^24,37 This airway epithelial barrier is attenuated by changing expression of CLDNs in asthma. Some reports have discussed the relationship of the lung-specific TJ protein CLDN-18.1 and airway epithelial barrier dysfunction in asthma.^37–39 Interleukin-13 (IL-13), which is a cytokine secreted by T helper type 2 (Th2) cells, is highly expressed in Th2 cells in asthma, and decreases trans-epithelial electronical resistance (TEER) and increases the permeability of FITC-conjugated dextran by decreasing expression of CLDN-18.1 in 16HBE cells.^38,39 Another report showed that overexpression of orosomucoid-like protein isoform 3 (ORMDL3), which is a gene closely associated with childhood onset asthma, decreased expression of CLDN-18.1 in human bronchial epithelial 16HBE cells.⁴⁰ As for the other CLDNs, high-mobility group box 1 protein (HMGB1), a non-histone chromatin-associated protein involved in pathological process in asthma, decreases TEER and increases permeability to 4 kDa FITC-dextran by decreasing expression of CLDN-1⁴¹. Transforming growth factor-β (TGF-β), which contributes to epithelial permeability,^41,42 is a major mediator of airway tissue remodeling during atopic asthma, and it increases epithelial permeability via redistribution of CLDN-3 from TJ into cell nuclei.²⁵ Furthermore, house dust mite allergen Der p1, an important factor in asthma, increases expression of CLDN-4 and decreases TEER in normal human bronchial epithelial cells.⁴³ In addition, exposure to diesel exhaust particles, a major component of airborne particulate matter (PM), decreases expression of the tTJ protein tricellulin, decreases TEER and increases permeability to 4 kDa FITC-dextran in 16HBE cells.⁴⁴

Chronic obstructive pulmonary disease (COPD)

Chronic obstructive pulmonary disease (COPD), which is one of the most common chronic respiratory diseases, causes slow progressive and irreversible alveolar emphysema and airway inflammation. The main risk factor for COPD is cigarette smoking, including indirect and passive exposures, and some cases are caused by inhalation of toxic gases and particulates due to air pollution and occupational diseases. The epithelial barrier disruption induced by cigarette smoke is associated with not only the pathogenesis of COPD, but also viral and bacterial infections in COPD.⁴⁵ In COPD, some reports have revealed the relationship between cigarette smoke exposure and disruption of the epithelial barrier inducing by changing expressions of TJ proteins. The cigarette smoke exposure induces expression of CLDN-3 as an early inflammatory reaction.⁴⁶

Idiopathic pulmonary fibrosis (IPF)

Idiopathic pulmonary fibrosis (IPF) is one of the idiopathic interstitial pneumonias, a pulmonary disease in which severe fibrosis of the lungs is the main pathology, resulting in restrictive ventilatory impairment.⁴³ The mechanisms leading to IPF are unclear, but one of the causes of pathogenesis in IPF is considered to be epithelial injury, and disruption of epithelial barrier is important in the pathogenesis of IPF. Recent reports have focused on TGF-β and HMGB1 as factors in the pathogenesis of IPF. TGF-β is highly expressed in fibrotic lungs,⁴⁷ and HMGB1 levels in sputum and serum are higher in patients with asthma, COPD and IPF than in the healthy lung.⁴⁸ As we mentioned in the previous section, both TGF-β and HMGB1 contribute to epithelial permeability.^41,42,49 In addition, TGF-β induces differentiation of alveolar epithelial cells into fibroblasts via epithelial-to-mesenchymal transition (EMT)⁵⁰ and this process also plays a crucial role in the progression of IPF.⁵¹ HMGB1 impairs airway epithelial barrier function through the activation of the RAGE/ERK pathway.⁴⁹ Furthermore, expression of HMGB1 is increased by TGF-β and knockdown of HMGB1 reverses EMT induced by TGF-β in human alveolar epithelial cell line A549 and normal human bronchial epithelial cell line BEAS-2B.⁵² Importantly, these two factors may affect the epithelial barrier via TJ proteins in IPF.

Acute lung injury (ALI)/acute respiratory distress syndrome (ARDS)

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are acute and severe respiratory failures secondary to various diseases. Injury of the alveolar epithelium in ALI/ARDS increases epithelial permeability and leads to pulmonary edema.^53,54 Therefore, the disruption of the TJ complex in alveolar epithelial cells can be one of the major causes. Most of the investigations about the relationship between changing TJ molecules and epithelial hyperpermeability in ALI/ARDS have focused on the expression of CLDN-4 and CLDN-18.1, which are highly expressed in alveolar epithelial cells. CLDN-4 is a sealing type of CLDN that plays an important role in the alveolar epithelial barrier.^32,55 Studies have revealed that an increase of CLDN-4 expression decreases paracellular permeability,²⁷ whereas knockout of CLDN-4 increases solute permeability of the alveolar epithelium and, as a result, increases susceptibility to lung injury.⁵⁶ In an experiment using a ventilator-induced lung injury model in mice, expression of CLDN-4 was increased after 3 h of moderate or high tidal ventilation, indicating that expression of CLDN-4 is increased in the early stage of ALI.⁵⁷ The increase of CLDN-4 expression in ARDS may be a compensatory response of the alveolar epithelium and alteration of CLDN-4 expression can be a potential target for therapy. On the other hand, CLDN-18.1 is a lung-specific TJ molecule that is highly expressed in normal alveolar epithelium and the downregulation of its expression impairs epithelial barrier function.⁵⁸ Some studies have indicated that expression of CLDN-18.1 is decreased in ALI/ARDS.^42,59,60 Knockout of CLDN-18.1 expression increases solute permeability and alveolar fluid clearance, and furthermore, expression of CLDN-3 and CLDN-4 is remarkably increased.⁶¹ CLDN-18.1 may be associated with permeability and alveolar fluid homeostasis, either directly or by altering expression of other CLDNs.

Tight junctions in nasal epithelium

In the human nasal epithelium, OCLN, JAM-A, ZO-1, ZO-2, CLDN-1, −4, −7, −8, −12, −13, −14 at bicellular TJs (bTj) and TRIC and angulin-1/LSR at tricellular TJs (tTJ) are detected on the surfaces of cells.^12,6 Nasal epithelial barrier dysfunction contributes to various nasal diseases, including allergies.^62–66

Tight junctions in intestine

TJs play a major role in maintaining the integrity and impermeability of the intestinal barrier, which is required for the maintenance of mucosal homeostasis. In the human small intestine, CLDN-1, −2, −3, −4, −5, −7, −8, −12 and −15 are expressed.⁶⁷ In addition, CLDN-2 is expressed along the crypt-villus axis, whereas in the fetal colon the expression is limited to the crypt base and in the adult colon tissue it is even absent.⁶⁸ Tricellular junctions regulate intestinal stem cell behavior to maintain homeostasis.⁶⁹

Inflammatory bowel disease (IBD)

TJ molecules play important roles in the intestinal epithelial barrier of inflammatory bowel disease (IBD).⁷⁰ In addition, an abundance of CLDN-2 has been widely observed in ulcerative colitis (UC) and Crohn’s disease (CD).^71,72 TNFα and IL-13 have been linked to the observed CLDN-2 increase.^71,72 TNFα also causes dislocalization of CLDN-5 and CLDN-8 from the TJ to sub-TJ membrane components and into endosomes.⁷² CLDN-4 and −7 are downregulated in UC.⁷ In CD, CLDN-5 and −8 are downregulated.⁷¹ In IBD, recent studies have indicated that zinc is important for the maintenance of the mucosal barrier.⁷³ In UC, downregulation of TRIC and CLDN-4, and upregulation of CLDN-2 are observed.^74,75 The downregulation of TRIC in active UC is recovered in remission from UC, while angulin-1/LSR expression is in remission UC at the same level as in the control.⁷⁶ In CD, TRIC expression level is unchanged, but its localization shifts from crypts to surface epithelium.⁷⁶

HMGB1

High mobility group box 1 (HMGB1), a chromatin-associated protein, is one of the damage-associated molecular patterns (DAMPs) and is also a proinflammatory mediator that belongs to the alarmin family.⁷⁷ HMGB1 is abundantly and widely expressed in a variety of cell nuclei and plays a role in gene transcription in various human diseases, including autoimmune diseases, inflammatory diseases and cancer.⁸ It promotes the induction of inflammatory cytokines in the pathogenesis of various inflammatory diseases.⁷⁸

HMGB1 is involved in the induction of airway inflammation and injury in patients with allergy, asthma, chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF) and respiratory virus infections.^79–81 The HMGB1 levels in sputum and serum are higher in patients with asthma, COPD, and IPF than in healthy subjects.^48,79 It impairs airway epithelial barrier function through activation of the receptor for advanced glycation end-products (RAGE)/ERK pathway.⁴⁹

HMGB1 is also involved with allergic rhinitis (AR), chronic rhinosinusitis (CRS) and eosinophilic chronic rhinosinusitis (ECRS).^82–84 It induces hyperpermeability of human nasal epithelial cells by downregulation of ZO-1, OCLN and CLDN-1 via hypoxia.⁸⁵

HMGB1 protein is involved in the development of IBD.^86,87 In a UC mouse model prepared with dextran sulfate sodium (DSS), colonic expression of HMGB1 and its receptor, RAGE, was significantly higher than in control mice.⁸⁸ Coincubation of the cytokines TNFα, IFNγ and IL-1β with human colon cancer Caco-2 cells and rat intestinal epithelial cells (IECs) revealed that supernatant concentrations of HMGB1 in both cell cultures were elevated.⁸⁹ Incubation of Caco-2 enterocytic monolayer cells with HMGB1 or B-box increases NO expression and hyperpermeability of Caco-2 cells.⁹⁰

TGF-β and EW-7197

TGF-β1 induces epithelial permeability in respiratory epithelial cells.⁴¹ HMGB1 expression is increased by TGF-β1 and knockdown of HMGB1 reverses TGF-β1-induced epithelial–mesenchymal transition (EMT) in human alveolar epithelial cell line A549 and normal human bronchial epithelial cell line BEAS-2B.⁹¹ The disruption of the epithelial barrier induced by HMGB1 and inflammatory cytokines contributes to TGF-β/EGF signaling in Caco-2 cells.⁹² EW-7197 is a TGF-β type I receptor kinase inhibitor with potential anti-inflammatory and antifibrotic properties.⁹³ EW-7197 prevents ulcerative colitis-associated fibrosis and inflammation⁹⁴ and prevents changes in the distribution of angulin-1/LSR and the epithelial barrier function by TGF-β in a pancreatic cancer cell line.¹⁹

p63

Transcription factor p63, which is a member of the p53 family, plays a crucial role in the proliferation and differentiation of various epithelial basal cells.⁹⁵ It regulates the epithelial barrier via various signaling pathways in the upper airway epithelium.^96,97 p63 is upregulated in the epithelium of CRS and nasal polyps and contributes to the formation and maintenance of differentiated pseudostratified bronchial epithelium and epithelial remodeling.⁹⁸ In bronchioles of IPF lungs, the numbers of p63-positive cells are increased.⁹⁹ Some p63-positive basal cells undergo EMT, and knockdown of p63 prevents the phenotypic switch in bronchial epithelial cells.¹⁰⁰

Analysis of epithelial permeability barriers in 2.5 D culture of human airway and intestinal epithelial cells

The addition of basement membrane proteins such as Matrigel to the medium in 2D cultures is sufficient to induce tissue-specific differentiation of diverse epithelial cells, whereas epithelial tissues often lose their differentiated state and migrate individually when cultured in a stromal matrix such as collagen I (Figure 1a). Two-and-a-half dimensional (2.5D) culture in which the cells are plated with additional 10% Matrigel in the medium on top of 100% Matrigel, induces cells to form a more physiological tissue architecture than 2D assays and the cells remain accessible for molecular analysis (Figure 1a, Figure 1c). In our system, 35-mm culture glass-coated dishes were coated with 100% Matrigel at 4 C° and incubated at 37 C° for 30 min. The cells (5 × 10⁴) were plated in the medium with 10% Matrigel and cultured for 4 days. The epithelial permeability barrier and the factors controlling it can be assessed in vitro by measurement of transepithelial electrical resistance (TEER) and fluorescein isothiocyanate (FITC) permeability assay. The permeability of fluorescein isothiocyanate (FITC)-dextran (FD4, MW 4.0 kDa) from the outside into the spheroid lumen was examined by using 2.5D Matrigel culture of the cells on 35-mm glass-coated dishes (Figure 1a, Figure 1d). In our culture system, the cells were incubated in the medium with 1% FD4 at 37 C° for 2 h. Ten spheroids of all experiments were photographed and measured using a confocal laser scanning microscope with imaging software.

Dysfunction of epithelial permeability barriers induced by HMGB1 in 2.5D culture of human lung epithelial (HLE) cells

In 2.5D culture of HLE cells, multi-row columnar epithelial cells were observed to have bronchial-like morphology. In 2.5D culture of HLE cells, treatment with HMGB1 induced permeability of FITC-dextran (FD4) into the lumen, whereas pretreatment with EW-7197 prevented this hyperpermeability (Figure 2a). In treatment with HMGB1, LSR was decreased at the membranes, while OCLN was detected at the luminal surface (Figure 2b). Treatment with HMGB1 decreased expression of angulin-1/LSR, TRIC and CLDN-1, −4, −7 and increased that of CLDN-2 (Figure 2c). Pretreatment with EW-7197 prevented the changes of all tight junction molecules induced by HMGB1 (Figure 2c). In 2.5D culture, treatment with TGF-β1 induced permeability of FD-4 and HDAC inhibitors TSA and Quisinostat prevented the hyperpermeability induced by TGF-β1³⁶.

In 2.5D culture, knockdown of transcription factor p63 prevents the hyperpermeability induced by HMGB1 as well as pretreatment with EW-7197³⁵. In 2D culture of HLE cells with HMGB1, knockdown of p63 increases the levels of angulin-1/LSR and CLDN-4, while pretreatment with EW-7197 enhances the increase of CLDN-4 induced by knockdown of p63³⁵. Immunohistochemical analysis of IPF, CLDN-2, HMGB1 and p63 has revealed that their levels are higher in regenerative epithelium of the terminal bronchial region than in normal epithelium.³⁵ HMGB1 induces epithelial permeability of HLE cells via tight junctions and p63/TGF-β and HDAC signaling. Thus, EW-7197 and HDAC inhibitors may have potential for use in therapy for IPF.

Dysfunction of epithelial permeability barriers induced by HMGB1 in 2.5D culture of human nasal epithelial (HNE) cells

2.5D culture of HNE cells was formed by simple columnar epithelial cells. In 2.5D culture, treatment with HMGB1 induced the permeability of FD4 into the lumen and pretreatment with EW-7197 prevented the hyperpermeability of FD4 into the lumen induced by HMGB1 (Figure 2a). ¹⁰¹ Treatment with EW-7197 prevented the downregulation of angulin-1/LSR, TRIC, CLDN-4 and p63 induced by treatment with HMGB1¹⁰⁴. HMGB1 induces epithelial permeability of HNE cells via tight junctions and p63/TGF-β signaling.¹⁰¹ EW-7197 may have potential for use in therapy for AR, CRS and ECRS, to which HMGB1 is closely related.

Dysfunction of epithelial permeability barriers induced by HMGB1 in 2.5D culture of human intestinal epithelial cells Caco-2

2.5D culture of Caco-2 cells was formed by simple columnar epithelial cells. In the spheroid cells, OCLN and LSR were strongly expressed at the luminal surface and LSR was also expressed throughout the basolateral membranes (Figure 1b). In 2.5D culture, EW-7197 prevented hyperpermeability of FD4 induced by HMGB1 (Figure 1a). In treatment with HMGB1, the cells expressed OCLN and LSR throughout the basolateral membranes from the luminal surfaces (Figure 1c). Pretreatment with EW-7197 prevented the changes in expression of TJs caused by HMGB1⁹⁵. Transmission electron microscopic (TEM) analysis revealed that “kissing points”, indistinguishable distances between two adjacent cell membranes, were loosened by treatment with HMGB1 in spheroid cells, and this change was prevented by treatment with EW-7197⁹⁵. Treatment with HMGB1 decreased the expression of TRIC and CLDN-1, and pretreatment with EW-7197 prevented the change in expression induced by HMGB1⁹⁵.

HMGB1 affected cilia formation in 2.5D culture. Treatment with HMGB1 decreased Ac-tubulin-positive cilia at the luminal surfaces (Figure 2e). TEM analysis showed that the number of cilia was decreased by treatment with HMGB1⁹⁵. Treatment with EW-7197 prevented these changes caused by HMGB1⁹⁵. The disruption of the epithelial barrier including cilia formation induced by HMGB1 contributed to TGF-β signaling in Caco-2 cells.

To investigate the roles of Pyk2 phosphorylated angulin-1/LSR and TRIC in the intestinal epithelial barrier, 2D and 2.5D cultures of Caco-2 cells were treated with the Pyk2 inhibitor PF43 with or without HMGB1. Treatment with PF43 increased expression of angulin-1/LSR, phosphorylated AMPK and phosphorylated MAPK and decreased that of phosphorylated JNK, with upregulation of the epithelial barrier.²² Treatment with PF43 prevented the downregulation of the epithelial barrier by HMGB in 2D culture.²² Treatment with PF43 prevented the epithelial hyperpermeability and the decrease of cilia induced by HMGB in 2.5D culture (Figure 2e). Treatment with HMGB1 inhibited phosphorylation of the serine of angulin-1/LSR (Figure 2e). Treatment with PF-43 induced phosphorylation of the serine of angulin-1/LSR (Figure 2e).

HMGB1 induces epithelial permeability of Caco-2 cells via tight junctions and TGF-β and Pyk2 signaling. The immunohistochemical results for IBD showed that in the ductal structural area of regenerative colonic epithelium, the expression of HMGB1 was higher than that of the normal region and angulin-1/LSR was not detected in regenerative colonic epithelium.⁹² EW-7197 and PF43 may have potential for use in therapy for IBD.

Effects of HMGB1 on epithelial permeability barriers via cellular metabolism

In bronchial epithelial Calu-3 cells, knockdown of angulin-1/LSR upregulates the expression of the tight junction molecule claudin-2, AMPK activity, and the cellular metabolism indicated as mitochondrial respiration, and downregulates the epithelial barrier.¹⁰² To examine how HMGB1 contributes to the cellular metabolism indicated as mitochondrial respiration, bronchial epithelial Calu-3 cells and intestinal epithelial Caco-2 cells were pretreated with EW-7197 before treatment with HMGB1.^95,102 In Calu-3 cells, treatment with HMGB1 increased baseline OCR, maximal OCR, proton leak and ATP production, and decreased spare respiratory capacity with downregulation of the epithelial barrier (Supplemental Figure 1). EW7197 prevented the changes induced by HMGB1 (Supplemental Figure 1). In Caco-2 cells, treatment with HMGB1 increases baseline OCR, proton leaks and ATP production with downregulation of the epithelial barrier.⁹² EW-7197 prevents the changes caused by treatment with HMGB1⁹⁵. In Caco-2 cells, PF-43 increased baseline OCR and ATP production with upregulation of the epithelial barrier.⁹² HMGB1 in part induces the dysfunction of the epithelial permeability barriers of airway and intestinal epithelial cells via cellular metabolism.

Conclusions

In conclusion, our findings indicate dysfunction of the epithelial permeability barriers induced by HMGB1 via expression of phosphorylated angulin-1/LSR, TRIC and claudins dependent on various signaling pathways (MAPK, AMPK, JNK, Pyk2, HDAC, p63/TGF-β) in 2.5D culture of human airway and intestinal epithelial cells (Figure 3). Furthermore, cell metabolism induced by HMGB1 may also contribute to dysfunction of the epithelial permeability barriers (Figure 3). TGF-β type I receptor kinase inhibitor EW7197 and Pyk2 inhibitor PF431396 have potential for use in therapy for inflammatory diseases such as IPF, nasal allergies and IBD via not only epithelial barrier function but also cellular metabolism.^103–105 The 2.5D culture is similar to human organoids in vivo and the changes induced by various stimuli are sensitive compared to those of 2D culture. Thus, 2.5D culture may be a useful in vitro model for studying the mechanisms of human diseases.

Figure 3. Schematic representation illustrating the possible mechanisms of action of HMGB1 and proinflammatory cytokines.

Future considerations

In patients with severe case of COVID-19, serum HMGB is elevated and exogenous HMGB1 induces the expression of SARS-CoV-2 entry receptor ACE2 in alveolar epithelial cells in an AGER-dependent manner.¹⁰⁶ An increased concentration of HMGB1 in the serum may contribute to several infectious and inflammatory diseases of the airway and intestine.¹⁰⁷ In HNECs treated with HMGB1, upregulation of the COVID-19-related genes cathepsin L (CTSL) and furin is observed¹⁰⁴. Cysteine protease CTSL expression is upregulated during chronic inflammation and is involved in processing the COVID-19 spike protein.¹⁰⁸¹⁰⁹ The protease furin is also involved in mediating SARS-CoV-2 entry.¹⁰⁹ It is possible that HMGB1 induced by COVID-19 infection may increase in HNECs and disrupt the epithelial permeability barriers. Thus, HMGB1 in the airway and intestine may be a therapeutic target in the severe inflammation of COVID-19.

Ethics statement

The protocol for human study was reviewed and approved by the ethics committee of the Sapporo Medical University School of Medicine. Written informed consent was obtained from each patient who participated in the investigation. All experiments were carried out in accordance with the approved guidelines and the Declaration of Helsinki.

Disclosure Statement

The authors declare no competing financial interests.

Supplementary material

Supplemental data for this article can be accessed on the publisher’s website

Additional information

Funding

This work was supported by JSPS KAKENHI [Grant Numbers 19K07464 and 20K17186] and GSK Japan Research 2019 (Konno T).