SARS-CoV-2 replication in airway epithelia requires motile cilia and microvillar reprogramming.

How SARS-CoV-2 penetrates the airway barrier of mucus and periciliary mucins to infect nasal epithelium remains unclear. Using primary nasal epithelial organoid cultures, we found that the virus attaches to motile cilia via the ACE2 receptor. SARS-CoV-2 traverses the mucus layer, using motile cilia as tracks to access the cell body. Depleting cilia blocks infection for SARS-CoV-2 and other respiratory viruses. SARS-CoV-2 progeny attach to airway microvilli 24 h post-infection and trigger formation of apically extended and highly branched microvilli that organize viral egress from the microvilli back into the mucus layer, supporting a model of virus dispersion throughout airway tissue via mucociliary transport. Phosphoproteomics and kinase inhibition reveal that microvillar remodeling is regulated by p21-activated kinases (PAK). Importantly, Omicron variants bind with higher affinity to motile cilia and show accelerated viral entry. Our work suggests that motile cilia, microvilli, and mucociliary-dependent mucus flow are critical for efficient virus replication in nasal epithelia.

Human Anti-fungal Th17 Immunity and Pathology Rely on Cross-Reactivity against Candida albicans.

Th17 cells provide protection at barrier tissues but may also contribute to immune pathology. The relevance and induction mechanisms of pathologic Th17 responses in humans are poorly understood. Here, we identify the mucocutaneous pathobiont Candida albicans as the major direct inducer of human anti-fungal Th17 cells. Th17 cells directed against other fungi are induced by cross-reactivity to C. albicans. Intestinal inflammation expands total C. albicans and cross-reactive Th17 cells. Strikingly, Th17 cells cross-reactive to the airborne fungus Aspergillus fumigatus are selectively activated and expanded in patients with airway inflammation, especially during acute allergic bronchopulmonary aspergillosis. This indicates a direct link between protective intestinal Th17 responses against C. albicans and lung inflammation caused by airborne fungi. We identify heterologous immunity to a single, ubiquitous member of the microbiota as a central mechanism for systemic induction of human anti-fungal Th17 responses and as a potential risk factor for pulmonary inflammatory diseases.

Primary nasal influenza infection rewires tissue-scale memory response dynamics.

The nasal mucosa is often the initial site of respiratory viral infection, replication, and transmission. Understanding how infection shapes tissue-scale primary and memory responses is critical for designing mucosal therapeutics and vaccines. We generated a single-cell RNA-sequencing atlas of the murine nasal mucosa, sampling three regions during primary influenza infection and rechallenge. Compositional analysis revealed restricted infection to the respiratory mucosa with stepwise changes in immune and epithelial cell subsets and states. We identified and characterized a rare subset of Krt13+ nasal immune-interacting floor epithelial (KNIIFE) cells, which concurrently increased with tissue-resident memory T (TRM)-like cells. Proportionality analysis, cell-cell communication inference, and microscopy underscored the CXCL16-CXCR6 axis between KNIIFE and TRM cells. Secondary influenza challenge induced accelerated and coordinated myeloid and lymphoid responses without epithelial proliferation. Together, this atlas serves as a reference for viral infection in the upper respiratory tract and highlights the efficacy of local coordinated memory responses.

Antibiotic-driven dysbiosis in early life disrupts indole-3-propionic acid production and exacerbates allergic airway inflammation in adulthood.

Antibiotic use in early life disrupts microbial colonization and increases the risk of developing allergies and asthma. We report that mice given antibiotics in early life (EL-Abx), but not in adulthood, were more susceptible to house dust mite (HDM)-induced allergic airway inflammation. This susceptibility was maintained even after normalization of the gut microbiome. EL-Abx decreased systemic levels of indole-3-propionic acid (IPA), which induced long-term changes to cellular stress, metabolism, and mitochondrial respiration in the lung epithelium. IPA reduced mitochondrial respiration and superoxide production and altered chemokine and cytokine production. Consequently, early-life IPA supplementation protected EL-Abx mice against exacerbated HDM-induced allergic airway inflammation in adulthood. These results reveal a mechanism through which EL-Abx can predispose the lung to allergic airway inflammation and highlight a possible preventative approach to mitigate the detrimental consequences of EL-Abx.

Dopamine inhibits group 2 innate lymphoid cell-driven allergic lung inflammation by dampening mitochondrial activity.

Neuronal signals have emerged as pivotal regulators of group 2 innate lymphoid cells (ILC2s) that regulate tissue homeostasis and allergic inflammation. The molecular pathways underlying the neuronal regulation of ILC2 responses in lungs remain to be fully elucidated. Here, we found that the abundance of neurotransmitter dopamine was negatively correlated with circulating ILC2 numbers and positively associated with pulmonary function in humans. Dopamine potently suppressed lung ILC2 responses in a DRD1-receptor-dependent manner. Genetic deletion of Drd1 or local ablation of dopaminergic neurons augmented ILC2 responses and allergic lung inflammation. Transcriptome and metabolic analyses revealed that dopamine impaired the mitochondrial oxidative phosphorylation (OXPHOS) pathway in ILC2s. Augmentation of OXPHOS activity with oltipraz antagonized the inhibitory effect of dopamine. Local administration of dopamine alleviated allergen-induced ILC2 responses and airway inflammation. These findings demonstrate that dopamine represents an inhibitory regulator of ILC2 responses in allergic airway inflammation.

The epigenetic state of IL-4-polarized macrophages enables inflammatory cistromic expansion and extended synergistic response to TLR ligands.

Prior exposure to microenvironmental signals could fundamentally change the response of macrophages to subsequent stimuli. It is believed that T helper-2 (Th2)-cell-type cytokine interleukin-4 (IL-4) and Toll-like receptor (TLR) ligand-activated transcriptional programs mutually antagonize each other, and no remarkable convergence has been identified between them. In contrast, here, we show that IL-4-polarized macrophages established a hyperinflammatory gene expression program upon lipopolysaccharide (LPS) exposure. This phenomenon, which we termed extended synergy, was supported by IL-4-directed epigenomic remodeling, LPS-activated NF-κB-p65 cistrome expansion, and increased enhancer activity. The EGR2 transcription factor contributed to the extended synergy in a macrophage-subtype-specific manner. Consequently, the previously alternatively polarized macrophages produced increased amounts of immune-modulatory factors both in vitro and in vivo in a murine Th2 cell-type airway inflammation model upon LPS exposure. Our findings establish that IL-4-induced epigenetic reprogramming is responsible for the development of inflammatory hyperresponsiveness to TLR activation and contributes to lung pathologies.

Overlapping and distinct features of viral and allergen immunity in the human lung.

Immunity in the human respiratory tract is provided by a diverse range of tissue-resident cells, including specialized epithelial and macrophage populations and a network of innate and innate-like lymphocytes, such as natural killer cells, innate lymphoid cells, and invariant T cells. Lung-resident memory T and B cells contribute to this network following initial exposure to antigenic stimuli. This review explores how advances in the study of human immunology have shaped our understanding of this resident immune network and its response to two of the most commonly encountered inflammatory stimuli in the airways: viruses and allergens. It discusses the many ways in which pathogenic infection and allergic inflammation mirror each other, highlighting the key checkpoints at which they diverge and how this can result in a lifetime of allergic exacerbation versus protective anti-viral immunity.

Pannexin 1 channels facilitate communication between T cells to restrict the severity of airway inflammation.

Allergic airway inflammation is driven by type-2 CD4+ T cell inflammatory responses. We uncover an immunoregulatory role for the nucleotide release channel, Panx1, in T cell crosstalk during airway disease. Inverse correlations between Panx1 and asthmatics and our mouse models revealed the necessity, specificity, and sufficiency of Panx1 in T cells to restrict inflammation. Global Panx1-/- mice experienced exacerbated airway inflammation, and T-cell-specific deletion phenocopied Panx1-/- mice. A transgenic designed to re-express Panx1 in T cells reversed disease severity in global Panx1-/- mice. Panx1 activation occurred in pro-inflammatory T effector (Teff) and inhibitory T regulatory (Treg) cells and mediated the extracellular-nucleotide-based Treg-Teff crosstalk required for suppression of Teff cell proliferation. Mechanistic studies identified a Salt-inducible kinase-dependent phosphorylation of Panx1 serine 205 important for channel activation. A genetically targeted mouse expressing non-phosphorylatable Panx1S205A phenocopied the exacerbated inflammation in Panx1-/- mice. These data identify Panx1-dependent Treg:Teff cell communication in restricting airway disease.

S-nitrosylation is required for ß2AR desensitization and experimental asthma.

The ß2-adrenergic receptor (ß2AR), a prototypic G-protein-coupled receptor (GPCR), is a powerful driver of bronchorelaxation, but the effectiveness of ß-agonist drugs in asthma is limited by desensitization and tachyphylaxis. We find that during activation, the ß2AR is modified by S-nitrosylation, which is essential for both classic desensitization by PKA as well as desensitization of NO-based signaling that mediates bronchorelaxation. Strikingly, S-nitrosylation alone can drive ß2AR internalization in the absence of traditional agonist. Mutant ß2AR refractory to S-nitrosylation (Cys265Ser) exhibits reduced desensitization and internalization, thereby amplifying NO-based signaling, and mice with Cys265Ser mutation are resistant to bronchoconstriction, inflammation, and the development of asthma. S-nitrosylation is thus a central mechanism in ß2AR signaling that may be operative widely among GPCRs and targeted for therapeutic gain.

Physiology and pathophysiology of human airway mucus.

The mucus clearance system is the dominant mechanical host defense system of the human lung. Mucus is cleared from the lung by cilia and airflow, including both two-phase gas-liquid pumping and cough-dependent mechanisms, and mucus transport rates are heavily dependent on mucus concentration. Importantly, mucus transport rates are accurately predicted by the gel-on-brush model of the mucociliary apparatus from the relative osmotic moduli of the mucus and periciliary-glycocalyceal (PCL-G) layers. The fluid available to hydrate mucus is generated by transepithelial fluid transport. Feedback interactions between mucus concentrations and cilia beating, via purinergic signaling, coordinate Na+ absorptive vs Cl- secretory rates to maintain mucus hydration in health. In disease, mucus becomes hyperconcentrated (dehydrated). Multiple mechanisms derange the ion transport pathways that normally hydrate mucus in muco-obstructive lung diseases, e.g., cystic fibrosis (CF), chronic obstructive pulmonary disease (COPD), non-CF bronchiectasis (NCFB), and primary ciliary dyskinesia (PCD). A key step in muco-obstructive disease pathogenesis is the osmotic compression of the mucus layer onto the airway surface with the formation of adherent mucus plaques and plugs, particularly in distal airways. Mucus plaques create locally hypoxic conditions and produce airflow obstruction, inflammation, infection, and, ultimately, airway wall damage. Therapies to clear adherent mucus with hydrating and mucolytic agents are rational, and strategies to develop these agents are reviewed.

Lipid-Droplet Formation Drives Pathogenic Group 2 Innate Lymphoid Cells in Airway Inflammation.

Innate lymphoid cells (ILCs) play an important role in the control and maintenance of barrier immunity. However, chronic activation of ILCs results in immune-mediated pathology. Here, we show that tissue-resident type 2 ILCs (ILC2s) display a distinct metabolic signature upon chronic activation. In the context of allergen-driven airway inflammation, ILC2s increase their uptake of both external lipids and glucose. Externally acquired fatty acids are transiently stored in lipid droplets and converted into phospholipids to promote the proliferation of ILC2s. This metabolic program is imprinted by interleukin-33 (IL-33) and regulated by the genes Pparg and Dgat1, which are both controlled by glucose availability and mTOR signaling. Restricting dietary glucose by feeding mice a ketogenic diet largely ablated ILC2-mediated airway inflammation by impairing fatty acid metabolism and the formation of lipid droplets. Together, these results reveal that pathogenic ILC2 responses require lipid metabolism and identify ketogenic diet as a potent intervention strategy to treat airway inflammation.

Calcitonin Gene-Related Peptide Negatively Regulates Alarmin-Driven Type 2 Innate Lymphoid Cell Responses.

Neuroimmune interactions have emerged as critical modulators of allergic inflammation, and type 2 innate lymphoid cells (ILC2s) are an important cell type for mediating these interactions. Here, we show that ILC2s expressed both the neuropeptide calcitonin gene-related peptide (CGRP) and its receptor. CGRP potently inhibited alarmin-driven type 2 cytokine production and proliferation by lung ILC2s both in vitro and in vivo. CGRP induced marked changes in ILC2 expression programs in vivo and in vitro, attenuating alarmin-driven proliferative and effector responses. A distinct subset of ILCs scored highly for a CGRP-specific gene signature after in vivo alarmin stimulation, suggesting CGRP regulated this response. Finally, we observed increased ILC2 proliferation and type 2 cytokine production as well as exaggerated responses to alarmins in mice lacking the CGRP receptor. Together, these data indicate that endogenous CGRP is a critical negative regulator of ILC2 responses in vivo.

Impact of Respiratory Syncytial Virus Infection on Host Functions: Implications for Antiviral Strategies.

Respiratory syncytial virus (RSV) is one of the leading causes of viral respiratory tract infection in infants, the elderly, and the immunocompromised worldwide, causing more deaths each year than influenza. Years of research into RSV since its discovery over 60 yr ago have elucidated detailed mechanisms of the host-pathogen interface. RSV infection elicits widespread transcriptomic and proteomic changes, which both mediate the host innate and adaptive immune responses to infection, and reflect RSV's ability to circumvent the host stress responses, including stress granule formation, endoplasmic reticulum stress, oxidative stress, and programmed cell death. The combination of these events can severely impact on human lungs, resulting in airway remodeling and pathophysiology. The RSV membrane envelope glycoproteins (fusion F and attachment G), matrix (M) and nonstructural (NS) 1 and 2 proteins play key roles in modulating host cell functions to promote the infectious cycle. This review presents a comprehensive overview of how RSV impacts the host response to infection and how detailed knowledge of the mechanisms thereof can inform the development of new approaches to develop RSV vaccines and therapeutics.

Amphiregulin-Producing Pathogenic Memory T Helper 2 Cells Instruct Eosinophils to Secrete Osteopontin and Facilitate Airway Fibrosis.

Memory T cells provide long-lasting protective immunity, and distinct subpopulations of memory T cells drive chronic inflammatory diseases such as asthma. Asthma is a chronic allergic inflammatory disease with airway remodeling including fibrotic changes. The immunological mechanisms that induce airway fibrotic changes remain unknown. We found that interleukin-33 (IL-33) enhanced amphiregulin production by the IL-33 receptor, ST2hi memory T helper 2 (Th2) cells. Amphiregulin-epidermal growth factor receptor (EGFR)-mediated signaling directly reprogramed eosinophils to an inflammatory state with enhanced production of osteopontin, a key profibrotic immunomodulatory protein. IL-5-producing memory Th2 cells and amphiregulin-producing memory Th2 cells appeared to cooperate to establish lung fibrosis. The analysis of polyps from patients with eosinophilic chronic rhinosinusitis revealed fibrosis with accumulation of amphiregulin-producing CRTH2hiCD161hiCD45RO+CD4+ Th2 cells and osteopontin-producing eosinophils. Thus, the IL-33-amphiregulin-osteopontin axis directs fibrotic responses in eosinophilic airway inflammation and is a potential target for the treatment of fibrosis induced by chronic allergic disorders.

Airway Epithelial Cell-Derived Colony Stimulating Factor-1 Promotes Allergen Sensitization.

Airway epithelial cells (AECs) secrete innate immune cytokines that regulate adaptive immune effector cells. In allergen-sensitized humans and mice, the airway and alveolar microenvironment is enriched with colony stimulating factor-1 (CSF1) in response to allergen exposure. In this study we found that AEC-derived CSF1 had a critical role in the production of allergen reactive-IgE production. Furthermore, spatiotemporally secreted CSF1 regulated the recruitment of alveolar dendritic cells (DCs) and enhanced the migration of conventional DC2s (cDC2s) to the draining lymph node in an interferon regulatory factor 4 (IRF4)-dependent manner. CSF1 selectively upregulated the expression of the chemokine receptor CCR7 on the CSF1R+ cDC2, but not the cDC1, population in response to allergen stimuli. Our data describe the functional specification of CSF1-dependent DC subsets that link the innate and adaptive immune responses in T helper 2 (Th2) cell-mediated allergic lung inflammation.

Chitinases and chitinase-like proteins in asthma.

Despite the lack of endogenous chitin synthesis, mammalian genomes encode two enzymatically active true chitinases (chitotriosidase and acidic mammalian chitinase) and a variable number of chitinase-like proteins (CLPs) that have no enzyme activity but bind chitin. Chitinases and CLPs are prominent components of type-2 immune response-mediated respiratory diseases. However, despite extensive research into their role in allergic airway disease, there is still no agreement on whether they are mere biomarkers of disease or actual disease drivers. Functions ascribed to chitinases and CLPs include, but are not limited to host defense against chitin-containing pathogens, directly promoting inflammation, and modulating tissue remodeling and fibrosis. Here, we discuss in detail the chitin-dependent and -independent roles of chitinases and CLPs in the context of allergic airway disease, and recent advances and emerging concepts in the field that might identify opportunities for new therapies.

Tropism for ciliated cells is the dominant driver of influenza viral burst size in the human airway.

Influenza viruses pose a significant burden on global human health. Influenza has a broad cellular tropism in the airway, but how infection of different epithelial cell types impacts replication kinetics and burden in the airways is not fully understood. Using primary human airway cultures, which recapitulate the diverse epithelial cell landscape of the human airways, we investigated the impact of cell type composition on virus tropism and replication kinetics. Cultures were highly diverse across multiple donors and 30 independent differentiation conditions and supported a range of influenza replication. Although many cell types were susceptible to influenza, ciliated and secretory cells were predominantly infected. Despite the strong tropism preference for secretory and ciliated cells, which consistently make up 75% or more of infected cells, only ciliated cells were associated with increased virus production. Surprisingly, infected secretory cells were associated with overall reduced virus output. The disparate response and contribution to influenza virus production could be due to different pro- and antiviral interferon-stimulated gene signatures between ciliated and secretory populations, which were interrogated with single-cell RNA sequencing. These data highlight the heterogeneous outcomes of influenza virus infections in the complex cellular environment of the human airway and the disparate impacts of infected cell identity on multiround burst size, even among preferentially infected cell types.

Pharmacological expansion of type 2 alveolar epithelial cells promotes regenerative lower airway repair.

Type 2 alveolar epithelial cells (AEC2s) are stem cells in the adult lung that contribute to lower airway repair. Agents that promote the selective expansion of these cells might stimulate regeneration of the compromised alveolar epithelium, an etiology-defining event in several pulmonary diseases. From a high-content imaging screen of the drug repurposing library ReFRAME, we identified that dipeptidyl peptidase 4 (DPP4) inhibitors, widely used type 2 diabetes medications, selectively expand AEC2s and are broadly efficacious in several mouse models of lung damage. Mechanism of action studies revealed that the protease DPP4, in addition to processing incretin hormones, degrades IGF-1 and IL-6, essential regulators of AEC2 expansion whose levels are increased in the luminal compartment of the lung in response to drug treatment. To selectively target DPP4 in the lung with sufficient drug exposure, we developed NZ-97, a locally delivered, lung persistent DPP4 inhibitor that broadly promotes efficacy in mouse lung damage models with minimal peripheral exposure and good tolerability. This work reveals DPP4 as a central regulator of AEC2 expansion and affords a promising therapeutic approach to broadly stimulate regenerative repair in pulmonary disease.

Interventional Pulmonology: Extending the Breadth of Thoracic Care.

Interventional pulmonary medicine has developed as a subspecialty focused on the management of patients with complex thoracic disease. Leveraging minimally invasive techniques, interventional pulmonologists diagnose and treat pathologies that previously required more invasive options such as surgery. By mitigating procedural risk, interventional pulmonologists have extended the reach of care to a wider pool of vulnerable patients who require therapy. Endoscopic innovations, including endobronchial ultrasound and robotic and electromagnetic bronchoscopy, have enhanced the ability to perform diagnostic procedures on an ambulatory basis. Therapeutic procedures for patients with symptomatic airway disease, pleural disease, and severe emphysema have provided the ability to palliate symptoms. The combination of medical and procedural expertise has made interventional pulmonologists an integral part of comprehensive care teams for patients with oncologic, airway, and pleural needs. This review surveys key areas in which interventional pulmonologists have impacted the care of thoracic disease through bronchoscopic intervention.

CD45 alleviates airway inflammation and lung fibrosis by limiting expansion and activation of ILC2s.

Group 2 innate lymphoid cells (ILC2s) are critical for the immune response against parasite infection and tissue homeostasis and involved in the pathogenesis of allergy and inflammatory diseases. Although multiple molecules positively regulating ILC2 development and activation have been extensively investigated, the factors limiting their population size and response remain poorly studied. Here, we found that CD45, a membrane-bound tyrosine phosphatase essential for T cell development, negatively regulated ILC2s in a cell-intrinsic manner. ILC2s in CD45-deficient mice exhibited enhanced proliferation and maturation in the bone marrow and hyperactivated phenotypes in the lung with high glycolytic capacity. Furthermore, CD45 signaling suppressed the type 2 inflammatory response by lung ILC2s and alleviated airway inflammation and pulmonary fibrosis. Finally, the interaction with galectin-9 influenced CD45 signaling in ILC2s. These results demonstrate that CD45 is a cell-intrinsic negative regulator of ILC2s and prevents lung inflammation and fibrosis via ILC2s.