Respiratory Enteroviruses, Inflammasome Activation and Innate Immune Cells

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Project Summary Respiratory enteroviruses, including rhinoviruses (RVs) and enterovirus D68 (EV-D68), cause acute respiratory tract infections and asthma exacerbations. We previously identified the role of exudative macrophages in the development of airways inflammation and hyperresponsiveness (AHR) following RV-A1B infection. However, mechanisms by which macrophages contribute to airway responses have not been fully explored. We recently found that RV-A1B triggers Nod-like receptor protein 3 (NLRP3) inflammasome activation and IL-1β production in naïve and allergen-sensitized and challenged mice. Macrophages were required and sufficient for inflammasome activation, and NLRP3 null mice showed reduced airway inflammation and AHR, suggesting that macrophage NLRP3 inflammasome activation is required for RV-A1B-induced responses. EV-D68 caused an outbreak of severe respiratory illness in 2014 and is responsible for 14% of acute respiratory illnesses in children biannually. At the same time, RV-C was linked to severe respiratory infections and asthma exacerbations. Our pilot data show that, compared to RV-A1B, inflammasome activation and type 3 innate lymphoid cells (ILC3s) are increased in the lungs of EV-D68-infected mice. In contrast, inflammasome activation is decreased, and type 2 innate lymphoid cells (ILC2s) increased, in RV-C15-infected mice. We propose that the level of macrophage NLRP3 inflammasome activation and IL-1β produced by different respiratory enteroviruses determines their divergent innate immune cell responses and airway outcomes. EV-D68 triggers intense inflammasome activation and IL-1β-driven ILC3s, leading to IL-17-dependent neutrophilic inflammation. RV-C15 infection elicits minimal inflammasome activation, thus the predominant response is ILC2 expansion and IL-13-dependent eosinophilic inflammation. RV-A1B triggers an intermediate response. In Specific Aim 1, we will determine the mechanisms by which respiratory enteroviruses EV-D68, RV-A1B and RV-C15 differentially activate the macrophage NLRP3 inflammasome. We hypothesize that: 1) NLRP3 is required for EV-D68-induced inflammasome activation; 2) inflammasome priming is dependent on TLR2 and viral protein 4 (VP4); 3) RV-C inflammasome priming requires clathrin-mediated endocytosis; 4) EV-D68 and RV-A1B inflammasome activation is triggered by viral RNA; 5) RV-C15 viral genome is insufficient for inflammasome activation; and 6) EV-D68 viral protein 2B is sufficient for inflammasome activation. In Specific Aim 2, we will determine the role of the NLRP3 inflammasome in the activation of lung innate immune cells. We hypothesize that: 1) NLRP3 inflammasome activation is required for EV-D68-induced expansion of lung ILC3s, neutrophilic inflammation and AHR; 2) RV-C15-induced eosinophilic inflammation requires ILC2s; 3) in the context of allergic airways disease, IL-1β, in combination with IL-25 and IL-33, promotes enterovirus-induced ILC2 activation and eosinophilic inflammation; and 4) human infection with respiratory enteroviruses induces virus-specific inflammasome activation and expansion of nasal ILCs.