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Airway hyperresponsiveness (AHR) is a critical feature of wheezing and asthma in children, but the initiating immune mechanisms remain unconfirmed. We demonstrate that both recombinant interleukin-33 (rIL-33) and allergen [house dust mite (HDM) or Alternaria alternata] exposure from day 3 of life resulted in significantly increased pulmonary IL-13+CD4+ T cells, which were indispensable for the development of AHR. In contrast, adult mice had a predominance of pulmonary LinnegCD45+CD90+IL-13+ type 2 innate lymphoid cells (ILC2s) after administration of rIL-33. HDM exposure of neonatal IL-33 knockout (KO) mice still resulted in AHR. However, neonatal CD4creIL-13 KO mice (lacking IL-13+CD4+ T cells) exposed to allergen from day 3 of life were protected from AHR despite persistent pulmonary eosinophilia, elevated IL-33 levels, and IL-13+ ILCs. Moreover, neonatal mice were protected from AHR when inhaled Acinetobacter lwoffii (an environmental bacterial isolate found in cattle farms, which is known to protect from childhood asthma) was administered concurrent with HDM. A. lwoffii blocked the expansion of pulmonary IL-13+CD4+ T cells, whereas IL-13+ ILCs and IL-33 remained elevated. Administration of A. lwoffii mirrored the findings from the CD4creIL-13 KO mice, providing a translational approach for disease protection in early life. These data demonstrate that IL-13+CD4+ T cells, rather than IL-13+ ILCs or IL-33, are critical for inception of allergic AHR in early life.

Background Alveolar macrophages are sentinels of the airways that must exhibit immune restraint to innocuous antigens but elicit a robust inflammatory response to pathogenic threats. How distinction between these dichotomous functions is controlled is poorly defined. Neutrophils are the first responders to infection, and we hypothesised that they may free alveolar macrophages from their hyporesponsive state, promoting their activation. Activation of the inflammasome and interleukin (IL)-1β release is a key early inflammatory event that must be tightly regulated. Thus, the role of neutrophils in defining inflammasome activation in the alveolar macrophage was assessed. Methods Mice were infected with the X31 strain of influenza virus and the role of neutrophils in alveolar macrophage activation established through administration of a neutrophil-depleting (1A8) antibody. Results Influenza elicited a robust IL-1β release that correlated (r=0.6849; p

Mucosal surfaces are under constant bombardment from potentially antigenic particles and so must maintain a balance between homeostasis and inappropriate immune activation and consequent pathology. Epithelial cells have a vital role orchestrating pulmonary homeostasis and defense against pathogens. TGF-β regulates an array of immune responses—both inflammatory and regulatory—however, its function is highly location- and context-dependent. We demonstrate that epithelial-derived TGF-β acts as a pro-viral factor suppressing early immune responses during influenza A infection. Mice specifically lacking bronchial epithelial TGF-β1 (epTGFβKO) displayed marked protection from influenza-induced weight loss, airway inflammation, and pathology. However, protection from influenza-induced pathology was not associated with a heightened lymphocytic immune response. In contrast, the kinetics of interferon beta (IFNβ) release into the airways was significantly enhanced in epTGFβKO mice compared with control mice, with elevated IFNβ on day 1 in epTGFβKO compared with control mice. This induced a heighted antiviral state resulting in impaired viral replication in epTGFβKO mice. Thus, epithelial-derived TGF-β acts to suppress early IFNβ responses leading to increased viral burden and pathology. This study demonstrates the importance of the local epithelial microenvironmental niche in shaping initial immune responses to viral infection and controlling host disease.

Background Genome-wide association studies have identified the ORM (yeast)-like protein isoform 3 (ORMDL3) gene locus on human chromosome 17q to be a highly significant risk factor for childhood-onset asthma. Objective We sought to investigate in vivo the functional role of ORMDL3 in disease inception. Methods An Ormdl3-deficient mouse was generated and the role of ORMDL3 in the generation of allergic airways disease to the fungal aeroallergen Alternaria alternata was determined. An adeno-associated viral vector was also used to reconstitute ORMDL3 expression in airway epithelial cells of Ormdl3 knockout mice. Results Ormdl3 knockout mice were found to be protected from developing allergic airways disease and showed a marked decrease in pathophysiology, including lung function and airway eosinophilia induced by Alternaria. Alternaria is a potent inducer of cellular stress and the unfolded protein response, and ORMDL3 was found to play a critical role in driving the activating transcription factor 6–mediated arm of this response through Xbp1 and downstream activation of the endoplasmic reticulum–associated degradation pathway. In addition, ORMDL3 mediated uric acid release, another marker of cellular stress. In the knockout mice, reconstitution of Ormdl3 transcript levels specifically in the bronchial epithelium resulted in reinstatement of susceptibility to fungal allergen–induced allergic airways disease. Conclusions This study demonstrates that ORMDL3, an asthma susceptibility gene identified by genome-wide association studies, contributes to key pathways that promote changes in airway physiology during allergic immune responses., Graphical abstract

BackgroundThe fungal allergen Alternaria alternata is implicated in severe asthma and rapid onset life-threatening exacerbations of disease. However, the mechanisms that underlie this severe pathogenicity remain unclear.ObjectiveWe sought to investigate the mechanism whereby Alternaria was capable of initiating severe, rapid onset allergic inflammation.MethodsIL-33 levels were quantified in wild-type and ST2−/− mice that lacked the IL-33 receptor given inhaled house dust mite, cat dander, or Alternaria, and the effect of inhibiting allergen-specific protease activities on IL-33 levels was assessed. An exacerbation model of allergic airway disease was established whereby mice were sensitized with house dust mite before subsequently being challenged with Alternaria (with or without serine protease activity), and inflammation, remodeling, and lung function assessed 24 hours later.ResultsAlternaria, but not other common aeroallergens, possessed intrinsic serine protease activity that elicited the rapid release of IL-33 into the airways of mice through a mechanism that was dependent upon the activation of protease activated receptor-2 and adenosine triphosphate signaling. The unique capacity of Alternaria to drive this early IL-33 release resulted in a greater pulmonary inflammation by 24 hours after challenge relative to the common aeroallergen house dust mite. Furthermore, this Alternaria serine protease–IL-33 axis triggered a rapid, augmented inflammation, mucus release, and loss of lung function in our exacerbation model.ConclusionAlternaria-specific serine protease activity causes rapid IL-33 release, which underlies the development of a robust TH2 inflammation and exacerbation of allergic airway disease.