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To protect against the constant threat of inhaled pathogens, the lung is equipped with cellular defenders. In coordination with resident and recruited immune cells, this defence is initiated by the airway and alveolar epithelium following their infection with respiratory viruses. Further support for viral clearance and infection resolution is provided by adjacent endothelial and stromal cells. However, even with these defence mechanisms, respiratory viral infections are a significant global health concern, causing substantial morbidity, socioeconomic losses, and mortality, underlining the need to develop effective vaccines and antiviral medications. In turn, the identification of new treatment options for respiratory infections is critically dependent on the availability of tractable in vitro experimental models that faithfully recapitulate key aspects of lung physiology. For such models to be informative, it is important these models incorporate human-derived, physiologically relevant versions of all cell types that normally form part of the lungs anti-viral response. This review proposes a guideline using human induced pluripotent stem cells (iPSCs) to create all the disease-relevant cell types. iPSCs can be differentiated into lung epithelium, innate immune cells, endothelial cells, and fibroblasts at a large scale, recapitulating in vivo functions and providing genetic tractability. We advocate for building comprehensive iPSC-derived in vitro models of both proximal and distal lung regions to better understand and model respiratory infections, including interactions with chronic lung diseases. [ABSTRACT FROM AUTHOR]

There is an urgent need to understand how SARS-CoV-2 infects the airway epithelium and in a subset of individuals leads to severe illness or death. Induced pluripotent stem cells (iPSCs) provide a near limitless supply of human cells that can be differentiated into cell types of interest, including airway epithelium, for disease modeling. We present a human iPSC-derived airway epithelial platform, composed of the major airway epithelial cell types, that is permissive to SARS-CoV-2 infection. Subsets of iPSC-airway cells express the SARS-CoV-2 entry factors angiotensin-converting enzyme 2 (ACE2), and transmembrane protease serine 2 (TMPRSS2). Multiciliated cells are the primary initial target of SARS-CoV-2 infection. On infection with SARS-CoV-2, iPSC-airway cells generate robust interferon and inflammatory responses, and treatment with remdesivir or camostat mesylate causes a decrease in viral propagation and entry, respectively. In conclusion, iPSC-derived airway cells provide a physiologically relevant in vitro model system to interrogate the pathogenesis of, and develop treatment strategies for, COVID-19 pneumonia. [ABSTRACT FROM AUTHOR]

Rationale: The alarmins IL-33 and HMGB1 (high mobility group box 1) contribute to type 2 inflammation and asthma pathogenesis. Objectives: To determine whether P2Y13-R (P2Y13 receptor), a purinergic GPCR (G protein-coupled receptor) and risk allele for asthma, regulates the release of IL-33 and HMGB1. Methods: Bronchial biopsy specimens were obtained from healthy subjects and subjects with asthma. Primary human airway epithelial cells (AECs), primary mouse AECs, or C57Bl/6 mice were inoculated with various aeroallergens or respiratory viruses, and the nuclear-to-cytoplasmic translocation and release of alarmins was measured by using immunohistochemistry and an ELISA. The role of P2Y13-R in AEC function and in the onset, progression, and exacerbation of experimental asthma was assessed by using pharmacological antagonists and mice with P2Y13-R gene deletion. Measurements and Main Results: Aeroallergen exposure induced the extracellular release of ADP and ATP, nucleotides that activate P2Y13-R. ATP, ADP, and aeroallergen (house dust mite, cockroach, or Alternaria antigen) or virus exposure induced the nuclear-to-cytoplasmic translocation and subsequent release of IL-33 and HMGB1, and this response was ablated by genetic deletion or pharmacological antagonism of P2Y13. In mice, prophylactic or therapeutic P2Y13-R blockade attenuated asthma onset and, critically, ablated the severity of a rhinovirus-associated exacerbation in a high-fidelity experimental model of chronic asthma. Moreover, P2Y13-R antagonism derepressed antiviral immunity, increasing IFN-λ production and decreasing viral copies in the lung. Conclusions: We identify P2Y13-R as a novel gatekeeper of the nuclear alarmins IL-33 and HMGB1 and demonstrate that the targeting of this GPCR via genetic deletion or treatment with a small-molecule antagonist protects against the onset and exacerbations of experimental asthma. [ABSTRACT FROM AUTHOR]

Chronic obstructive pulmonary disease (COPD) is a leading cause of death worldwide, however our understanding of cell specific mechanisms underlying COPD pathobiology remains incomplete. Here, we analyze single-cell RNA sequencing profiles of explanted lung tissue from subjects with advanced COPD or control lungs, and we validate findings using single-cell RNA sequencing of lungs from mice exposed to 10 months of cigarette smoke, RNA sequencing of isolated human alveolar epithelial cells, functional in vitro models, and in situ hybridization and immunostaining of human lung tissue samples. We identify a subpopulation of alveolar epithelial type II cells with transcriptional evidence for aberrant cellular metabolism and reduced cellular stress tolerance in COPD. Using transcriptomic network analyses, we predict capillary endothelial cells are inflamed in COPD, particularly through increased CXCL-motif chemokine signaling. Finally, we detect a high-metallothionein expressing macrophage subpopulation enriched in advanced COPD. Collectively, these findings highlight cell-specific mechanisms involved in the pathobiology of advanced COPD. Chronic obstructive pulmonary disease is a leading cause of death worldwide, while our understanding of cell-specific mechanisms underlying its pathobiology remains incomplete. Here the authors perform scRNA-seq of human lung tissue to identify transcriptional changes in alveolar niche cells associated with the disease. [ABSTRACT FROM AUTHOR]

Type-2 immunity elicits tissue repair and homeostasis, however dysregulated type-2 responses cause aberrant tissue remodelling, as observed in asthma. Severe respiratory viral infections in infancy predispose to later asthma, however, the processes that mediate tissue damage-induced type-2 inflammation and the origins of airway remodelling remain ill-defined. Here, using a preclinical mouse model of viral bronchiolitis, we find that increased epithelial and mesenchymal high-mobility group box 1 (HMGB1) expression is associated with increased numbers of IL-13-producing type-2 innate lymphoid cell (ILC2s) and the expansion of the airway smooth muscle (ASM) layer. Anti-HMGB1 ablated lung ILC2 numbers and ASM growth in vivo, and inhibited ILC2-mediated ASM cell proliferation in a co-culture model. Furthermore, we identified that HMGB1/RAGE (receptor for advanced glycation endproducts) signalling mediates an ILC2-intrinsic IL-13 auto-amplification loop. In summary, therapeutic targeting of the HMGB1/RAGE signalling axis may act as a novel asthma preventative by dampening ILC2-mediated type-2 inflammation and associated ASM remodelling. Author summary: Asthma can start at any time in life, although most often begins in early childhood. Wheezy viral bronchiolitis is a major independent risk factor for subsequent asthma. However, key knowledge gaps exist in relation to the sequelae of severe viral bronchiolitis and the pathogenic processes that promote type-2 inflammation and airway wall remodelling, cardinal features of asthma. Our study addresses this gap by identifying high-mobility group box 1 as a pathogenic cytokine that contributes to group 2 innate lymphoid cell-induced airway smooth muscle growth. [ABSTRACT FROM AUTHOR]

Rationale: Respiratory syncytial virus (RSV) bronchiolitis causes significant infant mortality. Bronchiolitis is characterized by airway epithelial cell (AEC) death; however, the mode of death remains unknown.Objectives: To determine whether necroptosis contributes to RSV bronchiolitis pathogenesis via HMGB1 (high mobility group box 1) release.Methods: Nasopharyngeal samples were collected from children presenting to the hospital with acute respiratory infection. Primary human AECs and neonatal mice were inoculated with RSV and murine Pneumovirus, respectively. Necroptosis was determined via viability assays and immunohistochemistry for RIPK1 (receptor-interacting protein kinase-1), MLKL (mixed lineage kinase domain-like pseudokinase) protein, and caspase-3. Necroptosis was blocked using pharmacological inhibitors and RIPK1 kinase-dead knockin mice.Measurements and Main Results: HMGB1 levels were elevated in nasopharyngeal samples of children with acute RSV infection. RSV-induced epithelial cell death was associated with increased phosphorylated RIPK1 and phosphorylated MLKL but not active caspase-3 expression. Inhibition of RIPK1 or MLKL attenuated RSV-induced HMGB1 translocation and release, and lowered viral load. MLKL inhibition increased active caspase-3 expression in a caspase-8/9-dependent manner. In susceptible mice, Pneumovirus infection upregulated RIPK1 and MLKL expression in the airway epithelium at 8 to 10 days after infection, coinciding with AEC sloughing, HMGB1 release, and neutrophilic inflammation. Genetic or pharmacological inhibition of RIPK1 or MLKL attenuated these pathologies, lowered viral load, and prevented type 2 inflammation and airway remodeling. Necroptosis inhibition in early life ameliorated asthma progression induced by viral or allergen challenge in later life.Conclusions: Pneumovirus infection induces AEC necroptosis. Inhibition of necroptosis may be a viable strategy to limit the severity of viral bronchiolitis and break its nexus with asthma. [ABSTRACT FROM AUTHOR]

RSV-induced prostaglandin D2 release contributes to disease severity by impairing IFN-λ production. RSV gives innate immunity the runaround: Asthma can be exacerbated by pathogens such as respiratory syncytial virus (RSV); prostaglandin D2 (PGD2) is also important in asthma and is being investigated as a therapeutic target. Werder et al. used multiple models to examine how RSV infection may perturb immune responses and influence asthma pathogenesis. Samples from infants with bronchiolitis or primary pediatric epithelial cells infected with RSV had elevated PGD2. Modulating PGD2 signaling in a mouse model of severe bronchiolitis improved antiviral immunity and dampened asthmatic symptoms later in life. This protection was not due to preventing type 2 immunity but instead a restoration of IFN-λ production. Their findings shed light on this host-pathogen interaction and suggest new therapeutic avenues. Prostaglandin D2 (PGD2) signals through PGD2 receptor 2 (DP2, also known as CRTH2) on type 2 effector cells to promote asthma pathogenesis; however, little is known about its role during respiratory syncytial virus (RSV) bronchiolitis, a major risk factor for asthma development. We show that RSV infection up-regulated hematopoietic prostaglandin D synthase expression and increased PGD2 release by cultured human primary airway epithelial cells (AECs). Moreover, PGD2 production was elevated in nasopharyngeal samples from young infants hospitalized with RSV bronchiolitis compared to healthy controls. In a neonatal mouse model of severe viral bronchiolitis, DP2 antagonism decreased viral load, immunopathology, and morbidity and ablated the predisposition for subsequent asthma onset in later life. This protective response was abolished upon dual DP1/DP2 antagonism and replicated with a specific DP1 agonist. Rather than mediating an effect via type 2 inflammation, the beneficial effects of DP2 blockade or DP1 agonism were associated with increased interferon-λ (IFN-λ) [interleukin-28A/B (IL-28A/B)] expression and were lost upon IL-28A neutralization. In RSV-infected AEC cultures, DP1 activation up-regulated IFN-λ production, which, in turn, increased IFN-stimulated gene expression, accelerating viral clearance. Our findings suggest that DP2 antagonists or DP1 agonists may be useful antivirals for the treatment of viral bronchiolitis and possibly as primary preventatives for asthma. [ABSTRACT FROM AUTHOR]

Severe viral lower respiratory infections are a major cause of infant morbidity. In developing countries, respiratory syncytial virus (RSV)-bronchiolitis induces significant mortality, whereas in developed nations the disease represents a major risk factor for subsequent asthma. Susceptibility to severe RSV-bronchiolitis is governed by gene-environmental interactions that affect the host response to RSV infection. Emerging evidence suggests that the excessive inflammatory response and ensuing immunopathology, typically as a consequence of insufficient immunoregulation, leads to long-term changes in immune cells and structural cells that render the host susceptible to subsequent environmental incursions. Thus, the initial host response to RSV may represent a tipping point in the balance between long-term respiratory health or chronic disease (e.g., asthma). The composition and diversity of the microbiota, which in humans stabilizes in the first year of life, critically affects the development and function of the immune system. Hence, perturbations to the maternal and/or infant microbiota are likely to have a profound impact on the host response to RSV and susceptibility to childhood asthma. Here, we review recent insights describing the effects of the microbiota on immune system homeostasis and respiratory disease and discuss the environmental factors that promote microbial dysbiosis in infancy. Ultimately, this knowledge will be harnessed for the prevention and treatment of severe viral bronchiolitis as a strategy to prevent the onset and development of asthma. [ABSTRACT FROM AUTHOR]

Individuals homozygous for the "Z" mutation in alpha-1 antitrypsin deficiency are known to be at increased risk for liver disease. It has also become clear that some degree of risk is similarly conferred by the heterozygous state. A lack of model systems that recapitulate heterozygosity in human hepatocytes has limited the ability to study the impact of a single Z alpha-1 antitrypsin (ZAAT) allele on hepatocyte biology. Here, we describe the derivation of syngeneic induced pluripotent stem cells (iPSCs) engineered to determine the effects of ZAAT heterozygosity in iPSC-hepatocytes (iHeps). We find that heterozygous MZ iHeps exhibit an intermediate disease phenotype and share with ZZ iHeps alterations in AAT protein processing and downstream perturbations including altered endoplasmic reticulum (ER) and mitochondrial morphology, reduced mitochondrial respiration, and branch-specific activation of the unfolded protein response in cell subpopulations. Our model of MZ heterozygosity thus provides evidence that a single Z allele is sufficient to disrupt hepatocyte homeostatic function. [Display omitted] • Syngeneic iPSCs effectively model SERPINA1 heterozygosity • Heterozygous MZ iHeps exhibit an intermediate phenotype versus MM and ZZ controls • MZ and ZZ iHeps have impaired metabolic and mitochondrial function • MZ and ZZ iHeps are transcriptionally heterogenous with UPR branch-specific activation Kaserman et al. show that syngeneic iPSCs from individuals with alpha-1 antitrypsin deficiency (AATD) model MZ heterozygosity. They find that MZ iPSC-derived hepatocytes (iHeps) exhibit an intermediate phenotype but share with ZZ iHeps alterations in AATD processing, metabolic and mitochondrial dysregulation, and cellular heterogeneity with UPR branch-specific activation. [ABSTRACT FROM AUTHOR]