Innate generation of thrombin and intracellular oxidants in airway epithelium by allergen Der p 1
Authors: Jihui Zhang, Jie Chen, Kimberley Allen-Philbey, Chathuri Perera Baruhupolage, Theresa Tachie-Menson, Shannon C. Mangat, David R. Garrod, Clive Robinson
Journal: Journal of Allergy and Clinical Immunology (2016)

Group 1 cysteine protease allergens from house dust mites (HDMs) are targets of a new class of drugs known as allergen delivery inhibitors (ADIs), which are entering development for asthma therapy.1Newton G.K. Perrior T.R. Jenkins K. Major M.R. Key R.E. Stewart M.R. et al.The discovery of potent, selective, and reversible inhibitors of the house dust mite peptidase allergen Der p 1: an innovative approach to the treatment of allergic asthma.J Med Chem. 2014; 57: 9447-9462Crossref PubMed Scopus (21) Google Scholar In studying pro-inflammatory signaling by protease allergens, attention has focused on their direct cleavage of protease-activated receptors (PARs).2Asokananthan N. Graham P.T. Stewart D.J. Bakker A.J. Eidne K.A. Thompson P.J. et al.House dust mite allergens induce proinflammatory cytokines from respiratory epithelial cells: the cysteine protease allergen, Der p 1, activates protease-activated receptor (PAR)-2 and inactivates PAR-1.J Immunol. 2002; 169: 4572-4578Crossref PubMed Scopus (284) Google Scholar We have explored an alternative view, namely, that a key HDM allergen triggers the activation of thrombin and stimulates the production of intracellular reactive oxygen species (ROS) and the extracellular release of ATP. ROS have significance because they orchestrate an allergic polarization of immune responses, and both heightened ROS production and a broad deficit in antioxidant defenses are characteristics of asthma.3Comhair S.A.A. Erzurum S.C. Redox control of asthma: molecular mechanisms and therapeutic opportunities.Antioxid Redox Signal. 2010; 12: 93-124Crossref PubMed Scopus (179) Google Scholar To investigate the production of intracellular ROS we loaded human airway epithelial cells (primary cultures and established lines) with dihydrorhodamine 123 and exposed them to a natural mixture of Dermatophagoides pteronyssinus allergens. This resulted in a sustained generation of ROS (Fig 1, A and B) associated with mitochondria and nuclei (Fig 1, C and D; see the Methods section and Fig E1, A-D, in this article's Online Repository at www.jacionline.org). ADZ 51,457 and ADZ 51,529, which are reversible ADIs targeting group 1 HDM protease allergens,1Newton G.K. Perrior T.R. Jenkins K. Major M.R. Key R.E. Stewart M.R. et al.The discovery of potent, selective, and reversible inhibitors of the house dust mite peptidase allergen Der p 1: an innovative approach to the treatment of allergic asthma.J Med Chem. 2014; 57: 9447-9462Crossref PubMed Scopus (21) Google Scholar substantially reduced ROS generation (Fig 1, E). Purified natural Der p 1 replicated ROS production and was fully inhibited by ADZ 51,457 (Fig 1, F). In contrast, an irreversible inhibitor of serine proteases had no effect on ROS production and purified Der p 2 conspicuously failed to elicit ROS generation (see Fig E2, A and B, in this article's Online Repository at www.jacionline.org). Thus, among natural HDM allergens, the initiators of intracellular ROS generation are the group 1 cysteine proteases. Surprisingly, ROS production by HDM allergens was transduced through PAR1 and PAR4, with only a small contribution from PAR2 (Fig 1, G and H; see Fig E3, A-E, in this article's Online Repository at www.jacionline.org). These responses required the opening of pannexons, which are inter alia conduits for ATP release (Fig 1, I). Interestingly, the viral RNA surrogate polyinosinic:polycytidylic acid (poly i:c) also caused pannexon-dependent ROS production (Fig 1, I). Although HDM allergens and poly i:c initiated ROS production differently (see Fig E4, A and B, in this article's Online Repository at www.jacionline.org), their signaling converges at pannexons (Fig 1, I), with the extracellular release of ATP and activation of mechanisms sensitive to the allosteric P2X7 receptor modulator, AZ 10606120 (Fig 2, A and B). Stimulation of PAR1 and PAR4 has not previously been associated with Der p 1,2Asokananthan N. Graham P.T. Stewart D.J. Bakker A.J. Eidne K.A. Thompson P.J. et al.House dust mite allergens induce proinflammatory cytokines from respiratory epithelial cells: the cysteine protease allergen, Der p 1, activates protease-activated receptor (PAR)-2 and inactivates PAR-1.J Immunol. 2002; 169: 4572-4578Crossref PubMed Scopus (284) Google Scholar so we were interested in determining whether this involved the generation of thrombin, their canonical activator. The thrombin inhibitor argatroban inhibited ROS generation by HDM allergens, whereas the Factor Xa inhibitor apixaban was without effect (Fig 2, C and D; see Fig E5, A-C, in this article's Online Repository at www.jacionline.org), thus excluding thrombin formation by the full coagulation cascade. Interestingly, both argatroban and a PAR1 antagonist were effective inhibitors of poly i:c (Fig 2, E and F). Incubation of prothrombin with mixed HDM allergens caused the appearance of prethrombin-1, the zymogen form of meizothrombin desF1, and the B chain of thrombin as major products. This process was inhibited by ADZ 50,000, an irreversible active site titrant analogue of ADZ 51,457 and ADZ 51,5291Newton G.K. Perrior T.R. Jenkins K. Major M.R. Key R.E. Stewart M.R. et al.The discovery of potent, selective, and reversible inhibitors of the house dust mite peptidase allergen Der p 1: an innovative approach to the treatment of allergic asthma.J Med Chem. 2014; 57: 9447-9462Crossref PubMed Scopus (21) Google Scholar (Fig 2, G). Formation of thrombin by Der p 1 provides further insight into the PAR siRNA data (Fig 1, G and H) and a possible explanation of the extensive antagonism of ROS formation by PAR1 antagonists (SCH 79797, FR 171113) and the PAR4 antagonist, tcY-NH2 (see Fig E3, A-C). Heterodimerization of PAR1 and PAR4 is precedented, providing a mechanism for thrombin bound to PAR1 through exosite 1 to cleave PAR4 (which cannot bind) more efficiently.4Arachiche A. Mumaw M.M. de la Fuente M. Nieman M.T. Protease-activated receptor 1 (PAR1) and PAR4 heterodimers are required for PAR1-enhanced cleavage of PAR4 by alpha-thrombin.J Biol Chem. 2013; 288: 32553-32562Crossref PubMed Scopus (62) Google Scholar The formation of a ternary complex would thus render ROS generation sensitive to antagonism of both receptors and imply that the main effector of Der p 1–stimulated ROS production might be PAR4, which is notably associated with epithelial-mesenchymal transition in airway cells. Hitherto, PAR1 and PAR4 have not been considered activatable by group 1 HDM allergens,2Asokananthan N. Graham P.T. Stewart D.J. Bakker A.J. Eidne K.A. Thompson P.J. et al.House dust mite allergens induce proinflammatory cytokines from respiratory epithelial cells: the cysteine protease allergen, Der p 1, activates protease-activated receptor (PAR)-2 and inactivates PAR-1.J Immunol. 2002; 169: 4572-4578Crossref PubMed Scopus (284) Google Scholar but in revealing the Der p 1–dependent cleavage of prothrombin we have identified their canonical activation with subsequent intracellular ROS formation via ATP release. Extracellular ATP is elevated in asthma, which is noteworthy because it stimulates dendritic cells and triggers the release of IL-33, which is genetically linked to asthma susceptibility and a key activator of cytokine production by iH2 nuocytes.5Muller T. Vieira R.P. Grimm M. Durk T. Cicko S. Zeiser R. et al.A potential role for P2X7R in allergic airway inflammation in mice and humans.Am J Respir Cell Mol Biol. 2011; 44: 456-464Crossref PubMed Scopus (116) Google Scholar Thrombin is present in airway surface liquid in asthma at levels sufficiently elevated to drive cell proliferation and is also increased following respiratory virus infection.6Terada M. Kelly E.A. Jarjour N.N. Increased thrombin activity after allergen challenge: a potential link to airway remodeling?.Am J Respir Crit Care Med. 2004; 169: 373-377Crossref PubMed Google Scholar Although it is generally assumed that these changes are associated with tissue repair following inflammation, our data implicate thrombin-mediated signaling as both an innate strategic initiator and an effector-perpetuator of allergic sensitization through its direct generation by inhaled Der p 1. That the Toll-like receptor 3 ligand poly i:c operates ROS generation through a mechanism that converges with Der p 1 signaling at pannexons is interesting because interactions between allergens and respiratory viruses precipitate exacerbations of asthma and allergy-polarizing transcription factors are redox sensitive. PAR1 contributes to the pathogenicity of influenza A,7Antoniak S. Owens III, A.P. Baunacke M. Williams J.C. Lee R.D. Weithauser A. et al.PAR-1 contributes to the innate immune response during viral infection.J Clin Invest. 2013; 123: 1310-1322Crossref PubMed Scopus (100) Google Scholar PAR1 and Toll-like receptor 3 are both upregulated by respiratory virus infections,8Groskreutz D.J. Monick M.M. Powers L.S. Yarovinsky T.O. Look D.C. Hunninghake G.W. Respiratory syncytial virus induces TLR3 protein and protein kinase R, leading to increased double-stranded RNA responsiveness in airway epithelial cells.J Immunol. 2006; 176: 1733-1740Crossref PubMed Scopus (212) Google Scholar ATP promotes TH2 immunity, and P2X7 expression is upregulated in asthma.5Muller T. Vieira R.P. Grimm M. Durk T. Cicko S. Zeiser R. et al.A potential role for P2X7R in allergic airway inflammation in mice and humans.Am J Respir Cell Mol Biol. 2011; 44: 456-464Crossref PubMed Scopus (116) Google Scholar It will therefore be of interest to investigate the operational role of pannexons as a signaling nexus in allergic sensitization and the triggering of disease exacerbations. The sensitivity of Toll-like receptor 3–mediated activation to argatroban or PAR1 antagonists (see Fig E2, E-F) suggests that events downstream of pannexon opening involve the endogenous activation of thrombin, creating a cyclical process. These findings reveal a surprising primary trigger for thrombin production that further emphasize its contribution to inflammatory lung responses. Although an oral thrombin inhibitor, albeit with bioavailability and protein binding which may preclude significant airway access from the systemic circulation, has only moderate improving effect on HDM-induced pathology in a murine model,9de Boer J.D. Berkhout L.C. de Stoppelaar S.F. Yang J. Ottenhoff R. Meijers J.C.M. et al.Effect of the oral thrombin inhibitor dabigatran on allergic lung inflammation induced by repeated house dust mite administration in mice.Am J Physiol Lung Cell Mol Physiol. 2015; 309: L768-L775PubMed Google Scholar our data suggest that it would be of interest to explore similar effects of ADIs, especially as these molecules have been optimized with the pharmaceutical credentials for inhaled delivery. 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