Pathways of airway oxidant formation by house dust mite allergens and viral RNA converge through myosin motors, pannexons and Toll‐like receptor 4

Abstract

IntroductionIntracellular reactive oxidant species (ROS) are generated in human airway epithelial cells by the prothrombinase action of Group 1 house dust mite (HDM) allergens and by ligation of viral RNA sensor Toll‐like receptors (TLRs). We explored signaling convergence between HDM allergens and TLRs in ROS generation because epithelial cells form the primary barrier against inhaled substances and dictate host responses to allergens and viruses.MethodsROS formation by Calu‐3 human airway cells was studied by measuring dihydrorhodamine 123 oxidation after activation by polyinosinic:polycytidylic acid (to activate TLR3), CL097 (to activate TLR7), a natural mixture of HDM allergens, or BzATP.ResultsTLR4 activation was identified as an indispensable response element for all stimuli, operating downstream from myosin motor activation, pannexon gating for ATP release and the endogenous activation of prothrombin. Exogenous prothrombin activation by HDM allergens was prevented by SGUL 1733, a novel inhibitor of the proteolytic activity of Group 1 HDM allergens, which thus prevented TLR4 from being activated at source.ConclusionsOur data identify for the first time that endogenously‐generated prothrombin and TLR4 form a shared effector mechanism essential to intracellular ROS generation activated by a group 1 HDM allergen (itself a prothrombinase) or by ligation of viral RNA‐sensing TLRs. These stimuli operate a confluent signaling pathway in which myosin motors, gating of pannexons, and ADAM 10 lead to prothrombin‐dependent activation of TLR4 with a recycling activation of pannexons.

Allergen Delivery Inhibitors: Characterisation of Potent and Selective Inhibitors of Der p 1 and Their Attenuation of Airway Responses to House Dust Mite Allergens

“…In severe asthma, exhaled breath condensate measurements suggest that pH may be reduced [ 31 ], but not below levels likely to materially impair enzymatic activity of Der p 1 based on assessment of its pH optimum. In fact, any such acidification would be expected to augment the protease-dependent effects of Der p 1 mediated through the generation of reactive oxygen and nitrogen species [ 2 , 32 ].…”

Allergen Delivery Inhibitors: A Rationale for Targeting Sentinel Innate Immune Signaling of Group 1 House Dust Mite Allergens through Structure-Based Protease Inhibitor Design

“…HDM 0.1, 1 refer to a natural mixture of allergens containing 0.1 or 1 µ g ml −1 , respectively. Further methodological details concerning the studies in (C–I) are available ( Zhang et al, 2016 , 2018 ), and these form the basis of previously unpublished or recomposited data shown here. RFU, relative fluorescence units.…”

“…Unexpectedly, one of the proteolytic features of Der p 1 is its ability to behave as a prothrombinase, forming thrombin from prothrombin independently from classic coagulation pathways ( Zhang et al, 2016 ) ( Table 2 ). In airway epithelial cells, this effect of Der p 1 is augmented by the subsequent activation of an endogenous prothrombinase, which plays a major role in controlling the generation of reactive signaling molecules ( Zhang et al, 2018 ). These recent discoveries cast an exciting new perspective on how group 1 HDM allergens upregulate cytokine expression through the operation of a signaling cycle which interlinks components having a pleiotropic role in allergy specifically, or innate immunity generally ( Fig.…”

“…At the time of writing, the exact relationships between many key events in this cycle remain to be defined, but what is known suggests that group 1 HDM allergens are, by means of their proteolytic activity, the ignition keys of fundamental innate responses. Consequently, novel molecular entities which inhibit the proteolytic activity of group 1 HDM allergens affect a broad spectrum of events relevant to allergy initiation and maintenance ( Zhang et al, 2018 ).…”

“…Although the underlying reasons for this dichotomy are not established, it may reflect poor accessibility of apically generated ligands to TLR4 in a polarized epithelium. The figure was revised and updated with added mechanistic details from the scheme originally published by Zhang et al (2018) . (B) Gene silencing production of the α -chain of fibrinogen in calu-3 airway epithelial cells inhibits ROS generation activated by ligation of P 2 X 7 (2′(3′)-O-(4-benzoylbenzoyl)adenosine 5′-triphosphate, BzATP) or P 2 Y 2 (uridine-5′-tetraphosphate δ-phenyl ester tetrasodium salt, MRS2768) receptors.…”