Effects of Oxidative Stress on Airway Epithelium Permeability in Asthma and Potential Implications for Patients with Comorbid Obesity

Figures & data

Figure 1 Disruption of the balance in ROS + RNS to antioxidants leads to oxidative stress. Under conditions of oxidative stress, oxygen (O₂) is reduced to superoxide (·O₂⁻), hydrogen peroxide (H₂O₂), or hydroxyl radical (·OH). Hypochlorite (OCl⁻)is formed through the reaction of H₂O₂ with chloride ions (Cl⁻). Nitric oxide (NO) is rapidly oxidized to form nitrite (NO₂⁻) and in the presence of oxyhemoglobin, nitrate (NO₃⁻). Increased production of ROS and RNS causes cell damage through lipid peroxidation, DNA damage, and oxidation of proteins and enzymes. Created with BioRender.com.

Abbreviations: ROS, reactive oxygen species; RNS, reactive nitrogen species; O₂, oxygen; ·O₂⁻, superoxide ion; H₂O₂, hydrogen peroxide; OCl⁻, hypochlorite; ·OH, hydroxyl radical; NO, nitric oxide; NO₃⁻, nitrate; NO₂⁻, nitrite; DNA, deoxyribonucleic acid.

Figure 2 Oxidative stress in patients with asthma and comorbid obesity contributes to persistent immune cell recruitment and inflammation, leading to airway epithelium permeability. Left: The airway epithelium of healthy patients is comprised of several different cell types, including club cells, basal cells, ionocytes, ciliated epithelial cells, goblet cells, tuft cells, and neuroendocrine cells, which are attached to the basement membrane. The pseudostratified epithelium provides a sealed barrier to prevent environmental particles from infiltrating the lung tissue. The submucosal area lies between the basement membrane and blood vessels. Immune cells, such as macrophages, lymphocytes, eosinophils, and basophils are located within both the submucosal area and blood vessels. Right: In the presence of allergenic, viral, or air pollution particles that exacerbate asthma, immune cells are recruited from the blood stream into the submucosal area. These activated immune cells produce ROS in response to these provocative agents. Recurrent exacerbations and oxidative stress cause more inflammation and damage to the epithelial cells, resulting in epithelium permeability. In patients with obesity and asthma, inflamed adipose tissue serves as an additional source of oxidative stress, which may accelerate epithelial damage and barrier permeability. Created with BioRender.com.

Abbreviation: ROS, Reactive oxygen species.

Figure 3 Comparison of postulated apical junctional protein organization in patients with asthma and comorbid obesity versus healthy patients. Airway epithelial cells are kept in close contact with adjacent epithelial cells through an organized arrangement of tight junction (ZO-1, claudin, occludin) and adherens junction proteins. People with asthma and comorbid obesity may have more severe asthma exacerbations caused by allergens, viral and bacterial pathogens, or pollutants, contributing to elevated oxidative stress. This may disrupt the organization expression levels of junction proteins, resulting in airway epithelium permeability. Created with Biorender.com.

Abbreviations: ZO-1, Zona occludens 1; JAM, Junctional adhesion molecule.

Table 1 Allergens, ROS, RNS, and Cytokines Affecting the Airway Epithelium in Human Cells

Authors	Type	Treatment	TEER	Permeability	Junction Protein Expression
Sweerus et al, 2017^Citation115	Bronchial brushing epithelial cells from patients with and without asthma	None	ND	ND	Decreased claudin-18 mRNA
	Claudin-18 KO 16HBEC	None	ND	Increased to 0.5 kDa pyranine	Decreased
	Primary human airway epithelial cells	IL-13	Decrease	ND	Decreased claudin-18 mRNA and protein; No change in claudin-1, −4, −7 mRNA
Hardyman et al, 2013^Citation82	Human bronchial brushing epithelial cells	TNF-α	Decrease	4kDa FITC-dextran permeability increase	Occludin, claudin-3, −4, −8 staining decrease
Xiao et al, 2011^Citation44	Human bronchial biopsies from patients with and without asthma	None	Decrease on day 21 of differentiation of ALI cultures	Increase permeability to 4kDa and 20kDa FITC-dextran	Decreased ZO-1 protein expression
Tan et al, 2019^Citation9	HBEC	IL-1β	Decrease	FITC-dextran increase
	16HBEC	TNF-α	Decrease	Mannitol increase	Decreased claudin-4, occludin
Callaghan et al, 2020^Citation89	16HBEC	TNF-α + Retinoic Acid	Increase	Decrease (compared to TNF-α treated cells)	Preserved expression of claudin-4, occludin
	HBECs of patients with and without asthma	NO2	Decrease	Increase in ^14C-BSA	ND
Bayram et al, 2002^Citation50	HBECs of patients with and without asthma	Ozone	Decrease	Increase in ^14C-BSA	ND
	16HBE14o-	IL-4	Decrease	3kDa dextran increase	ND
Saatian et al, 2013^Citation116	16HBE14o-	IL-13	Decrease	3kDa dextran increase	ND
Sajjan et al, 2008^Citation67	Primary human tracheal epithelial cells	RV16, RV39, or RV1B	Decrease	ND	Loss of ZO-1 around cell periphery
	16HBEC14o-	RV39 or RV1B	Decrease	Increase to inulin	Loss of ZO-1 around cell periphery
	Calu-3	RB39	Decrease	ND	Loss of ZO-1
Singh et al, 2007^Citation68	Primary HBECs	RSV	Decrease		Reduced actin polymerization
Heijink et al, 2010^Citation117	16HBEC	HDM	Decrease	ND	Disrupted ZO-1 and E-cadherin arrangement around airways
Trautmann et al, 2005^Citation118	Bronchial biopsies of asthma and healthy patients	None	ND	ND	Staining for E-cadherin decreased
Petecchia et al, 2012^Citation119	Calu-3	TNF-α, IL-4, or INFγ	Decrease in TEER measured using paracellular conductance (1/TEER)	ND	Decrease in ZO-1 and occludin expression
Post et al, 2012^Citation120	16HBE14o-	HDM extracts with high protease activity (Citeq, ALK) or high chitinase activity (Greer)	Acute exposure to Greer extract decreases TEER within 20 minutes; 24-hour exposure to Citeq decreases TEER	ND	HDM disrupts E-cadherin, ZO-1, occludin localization
Wan et al, 1999^Citation121	16HBE14o-, MDCK cells	Der p 1	ND	Increase in mannitol permeability and Der p 1	ZO-1 disruption, decrease in occludin
Kim et al, 2018^Citation57	NHBE cells	2 ppm ozone or filtered air	Decrease in TEER after 3-hour ozone exposure		Increase in claudin-3, decrease in claudin-4

Abbreviations: ALI, Air-liquid interface; BSA, bovine serum albumin; Calu-3, non-small-cell lung cancer epithelial cell line; FITC, fluorescein isothiocyanate; HBEC, human bronchial epithelial cells; HDM, house dust mite allergen; IL-1β, interleukin-1 beta; IL-4, interleukin-4; IL-13, interleukin-13; IFNγ, interferon gamma; KO, knockout; MDCK, Madin-Darby canine kidney; ND, Not determined; NHBE, Normal human bronchial epithelial; NO₂, nitrite; ppm, parts per million; RSV, respiratory syncytial virus; RV, rhinovirus; TEER, transepithelial electrical resistance.

Box 1 Future Areas of Study for Clinical and Basic Science Research on Asthma and Obesity

Questions for Future Research
Which oxidative stress-provoked inflammatory cytokines are significantly involved in epithelial barrier dysfunction? How does the interaction of obesity and asthma pathobiology alter airway epithelial barrier and tight junction protein expression and activity? What are the effects of dietary fat or fiber on the airway epithelium? Are patients with metabolic syndrome more susceptible than metabolically healthy individuals to airway epithelium permeability?
How does excess circulating leptin affect airway epithelial barrier integrity in the presence of allergic and non-allergic triggers of asthma? How does the excess leptin or other adipokines affect expression of occludins, claudins, E-cadherins, and other cell adhesion molecules present between airway epithelial cells? Can antioxidants, such as adiponectin, improve epithelium permeability by reducing oxidative stress?
Following an ozone exposure (either acute or chronic), is airway epithelium permeability higher among patients who have asthma and obesity, compared to healthy patients or those with asthma alone? Does the cellular composition of the airway epithelium differ between patients with asthma versus patients with asthma and obesity?

Notes: Many questions remain to be investigated and answered, especially in regard to understanding the different factors that may be contributing to treatment resistance in patient populations with asthma and obesity. More explorations of how the airway epithelial cells and epithelium are damaged, how tight junction complexes are affected, and how oxidative stress damages the airways are needed.

Table 2 Allergens and ROS Affecting the Airway Epithelium in Mouse Models

Authors	Type	Treatment	TEER	Permeability	Junction Protein Expression
Sweerus et al, 2017^Citation115	Claudin-18 KO mice	None	ND	Tracheal permeability increased to 0.5 kDa pyranine	ND
Tan et al, 2019^Citation9	Balb/c mice	HDM	ND	ND	Decreased claudin-18, ZO-1 expression
Kim et al, 2018^Citation57	Balb/c mice	0.1, 1, and 2 ppm ozone or filtered air	ND	ND	Increased claudin-4 and claudin-3, decreased claudin-14
Michaudel et al, 2017^Citation58	ST2-deficient C57BL/6J mice	1 ppm ozone exposure for 1 hour	ND	Increase in Evans Blue leakage into BAL fluid	Decreased ZO-1, claudin-4, and E-cadherin expression

Abbreviations: HDM, house dust mite allergen; kDa, kilodaltons; KO, knockout; ND, not determined; OVA, ovalbumin; ppm, parts per million; SCID, severe combined immunodeficiency mutation; ST2, IL-33 receptor; ZO, zona occludens.

Sweerus K, Lachowicz-Scroggins M, Gordon E, et al. Claudin-18 deficiency is associated with airway epithelial barrier dysfunction and asthma. J Allergy Clin Immunol. 2017;139(1):72–81e1. doi:10.1016/j.jaci.2016.02.035

 PubMed Web of Science ®Google Scholar

Hardyman MA, Wilkinson E, Martin E, et al. TNF-alpha-mediated bronchial barrier disruption and regulation by src-family kinase activation. J Allergy Clin Immunol. 2013;132(3):665–675 e8. doi:10.1016/j.jaci.2013.03.005

 PubMed Web of Science ®Google Scholar

Xiao C, Puddicombe SM, Field S, et al. Defective epithelial barrier function in asthma. J Allergy Clin Immunol. 2011;128(3):549–612. doi:10.1016/j.jaci.2011.05.038

 PubMed Web of Science ®Google Scholar

Tan HT, Hagner S, Ruchti F, et al. Tight junction, mucin, and inflammasome-related molecules are differentially expressed in eosinophilic, mixed, and neutrophilic experimental asthma in mice. Allergy. 2019;74(2):294–307. doi:10.1111/all.13619

 PubMed Web of Science ®Google Scholar

Callaghan PJ, Rybakovsky E, Ferrick B, Thomas S, Mullin JM. Retinoic acid improves baseline barrier function and attenuates TNF-alpha-induced barrier leak in human bronchial epithelial cell culture model, 16HBE 14o. PLoS One. 2020;15(12):e0242536. doi:10.1371/journal.pone.0242536

 PubMed Web of Science ®Google Scholar

Bayram H, Rusznak C, Khair OA, Sapsford RJ, Abdelaziz MM. Effect of ozone and nitrogen dioxide on the permeability of bronchial epithelial cell cultures of non-asthmatic and asthmatic subjects. Clin Exp Allergy. 2002;32(9):1285–1292. doi:10.1046/j.1365-2745.2002.01435.x

 PubMed Web of Science ®Google Scholar

Saatian B, Rezaee F, Desando S, et al. Interleukin-4 and interleukin-13 cause barrier dysfunction in human airway epithelial cells. Tissue Barriers. 2013;1(2):e24333. doi:10.4161/tisb.24333

 PubMedGoogle Scholar

Sajjan U, Wang Q, Zhao Y, Gruenert DC, Hershenson MB. Rhinovirus disrupts the barrier function of polarized airway epithelial cells. Am J Respir Crit Care Med. 2008;178(12):1271–1281. doi:10.1164/rccm.200801-136OC

 PubMed Web of Science ®Google Scholar

Singh D, McCann KL, Imani F. MAPK and heat shock protein 27 activation are associated with respiratory syncytial virus induction of human bronchial epithelial monolayer disruption. Am J Physiol Lung Cell Mol Physiol. 2007;293(2):L436–L445. doi:10.1152/ajplung.00097.2007

 PubMed Web of Science ®Google Scholar

Heijink IH, van Oosterhout A, Kapus A. Epidermal growth factor receptor signalling contributes to house dust mite-induced epithelial barrier dysfunction. Eur Respir J. 2010;36(5):1016–1026. doi:10.1183/09031936.00125809

 PubMed Web of Science ®Google Scholar

Trautmann A, Kruger K, Akdis M, et al. Apoptosis and loss of adhesion of bronchial epithelial cells in asthma. Int Arch Allergy Immunol. 2005;138(2):142–150. doi:10.1159/000088436

 PubMed Web of Science ®Google Scholar

Petecchia L, Sabatini F, Usai C, Caci E, Varesio L, Rossi GA. Cytokines induce tight junction disassembly in airway cells via an EGFR-dependent MAPK/ERK1/2-pathway. Lab Invest. 2012;92(8):1140–1148. doi:10.1038/labinvest.2012.67

 PubMed Web of Science ®Google Scholar

Post S, Nawijn MC, Hackett TL, et al. The composition of house dust mite is critical for mucosal barrier dysfunction and allergic sensitisation. Thorax. 2012;67(6):488–495. doi:10.1136/thoraxjnl-2011-200606

 PubMed Web of Science ®Google Scholar

Wan H, Winton HL, Soeller C, et al. Der p 1 facilitates transepithelial allergen delivery by disruption of tight junctions. J Clin Invest. 1999;104(1):123–133. doi:10.1172/JCI5844

 PubMed Web of Science ®Google Scholar

Kim BG, Lee PH, Lee SH, Park CS, Jang AS. Impact of ozone on claudins and tight junctions in the lungs. Environ Toxicol. 2018;33(7):798–806. doi:10.1002/tox.22566

 PubMed Web of Science ®Google Scholar

Michaudel C, Mackowiak C, Maillet I, et al. Ozone exposure induces respiratory barrier biphasic injury and inflammation controlled by IL-33. J Allergy Clin Immunol. 2018;142(3):942–958. doi:10.1016/j.jaci.2017.11.044

 PubMed Web of Science ®Google Scholar