Host and environmental factors shape upper airway microbiota and respiratory health across the human lifespan.

Our understanding of the normal variation in the upper respiratory tract (URT) microbiota across the human lifespan and how these relate to host, environment, and health is limited. We studied the microbiota of 3,104 saliva (<10 year-olds)/oropharynx (≥10 year-olds) and 2,485 nasopharynx samples of 3,160 Dutch individuals 0-87 years of age, participating in a cross-sectional population-wide study (PIENTER-3) using 16S-rRNA sequencing. The microbiota composition was strongly related to age, especially in the nasopharynx, with maturation occurring throughout childhood and adolescence. Clear niche- and age-specific associations were found between the microbiota composition and host/environmental factors and health outcomes. Among others, social interaction, sex, and season were associated with the nasopharyngeal microbial community. By contrast, the oral microbiota was more related to antibiotics, tobacco, and alcohol use. We present an atlas of the URT microbiota across the lifespan in association with environment and health, establishing a baseline for future research.

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.

African lungfish genome sheds light on the vertebrate water-to-land transition.

Lungfishes are the closest extant relatives of tetrapods and preserve ancestral traits linked with the water-to-land transition. However, their huge genome sizes have hindered understanding of this key transition in evolution. Here, we report a 40-Gb chromosome-level assembly of the African lungfish (Protopterus annectens) genome, which is the largest genome assembly ever reported and has a contig and chromosome N50 of 1.60 Mb and 2.81 Gb, respectively. The large size of the lungfish genome is due mainly to retrotransposons. Genes with ultra-long length show similar expression levels to other genes, indicating that lungfishes have evolved high transcription efficacy to keep gene expression balanced. Together with transcriptome and experimental data, we identified potential genes and regulatory elements related to such terrestrial adaptation traits as pulmonary surfactant, anxiolytic ability, pentadactyl limbs, and pharyngeal remodeling. Our results provide insights and key resources for understanding the evolutionary pathway leading from fishes to humans.

The interferon landscape along the respiratory tract impacts the severity of COVID-19.

Severe coronavirus disease 2019 (COVID-19) is characterized by overproduction of immune mediators, but the role of interferons (IFNs) of the type I (IFN-I) or type III (IFN-III) families remains debated. We scrutinized the production of IFNs along the respiratory tract of COVID-19 patients and found that high levels of IFN-III, and to a lesser extent IFN-I, characterize the upper airways of patients with high viral burden but reduced disease risk or severity. Production of specific IFN-III, but not IFN-I, members denotes patients with a mild pathology and efficiently drives the transcription of genes that protect against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In contrast, compared to subjects with other infectious or noninfectious lung pathologies, IFNs are overrepresented in the lower airways of patients with severe COVID-19 that exhibit gene pathways associated with increased apoptosis and decreased proliferation. Our data demonstrate a dynamic production of IFNs in SARS-CoV-2-infected patients and show IFNs play opposing roles at distinct anatomical sites.

Visualizing in deceased COVID-19 patients how SARS-CoV-2 attacks the respiratory and olfactory mucosae but spares the olfactory bulb.

Anosmia, the loss of smell, is a common and often the sole symptom of COVID-19. The onset of the sequence of pathobiological events leading to olfactory dysfunction remains obscure. Here, we have developed a postmortem bedside surgical procedure to harvest endoscopically samples of respiratory and olfactory mucosae and whole olfactory bulbs. Our cohort of 85 cases included COVID-19 patients who died a few days after infection with SARS-CoV-2, enabling us to catch the virus while it was still replicating. We found that sustentacular cells are the major target cell type in the olfactory mucosa. We failed to find evidence for infection of olfactory sensory neurons, and the parenchyma of the olfactory bulb is spared as well. Thus, SARS-CoV-2 does not appear to be a neurotropic virus. We postulate that transient insufficient support from sustentacular cells triggers transient olfactory dysfunction in COVID-19. Olfactory sensory neurons would become affected without getting infected.

SARS-CoV-2 Reverse Genetics Reveals a Variable Infection Gradient in the Respiratory Tract.

The mode of acquisition and causes for the variable clinical spectrum of coronavirus disease 2019 (COVID-19) remain unknown. We utilized a reverse genetics system to generate a GFP reporter virus to explore severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pathogenesis and a luciferase reporter virus to demonstrate sera collected from SARS and COVID-19 patients exhibited limited cross-CoV neutralization. High-sensitivity RNA in situ mapping revealed the highest angiotensin-converting enzyme 2 (ACE2) expression in the nose with decreasing expression throughout the lower respiratory tract, paralleled by a striking gradient of SARS-CoV-2 infection in proximal (high) versus distal (low) pulmonary epithelial cultures. COVID-19 autopsied lung studies identified focal disease and, congruent with culture data, SARS-CoV-2-infected ciliated and type 2 pneumocyte cells in airway and alveolar regions, respectively. These findings highlight the nasal susceptibility to SARS-CoV-2 with likely subsequent aspiration-mediated virus seeding to the lung in SARS-CoV-2 pathogenesis. These reagents provide a foundation for investigations into virus-host interactions in protective immunity, host susceptibility, and virus pathogenesis.

Tracking Changes in SARS-CoV-2 Spike: Evidence that D614G Increases Infectivity of the COVID-19 Virus.

A SARS-CoV-2 variant carrying the Spike protein amino acid change D614G has become the most prevalent form in the global pandemic. Dynamic tracking of variant frequencies revealed a recurrent pattern of G614 increase at multiple geographic levels: national, regional, and municipal. The shift occurred even in local epidemics where the original D614 form was well established prior to introduction of the G614 variant. The consistency of this pattern was highly statistically significant, suggesting that the G614 variant may have a fitness advantage. We found that the G614 variant grows to a higher titer as pseudotyped virions. In infected individuals, G614 is associated with lower RT-PCR cycle thresholds, suggestive of higher upper respiratory tract viral loads, but not with increased disease severity. These findings illuminate changes important for a mechanistic understanding of the virus and support continuing surveillance of Spike mutations to aid with development of immunological interventions.

Airway-resident T cells from unexposed individuals cross-recognize SARS-CoV-2.

T cells can contribute to clearance of respiratory viruses that cause acute-resolving infections such as SARS-CoV-2, helping to provide long-lived protection against disease. Recent studies have suggested an additional role for T cells in resisting overt infection: pre-existing cross-reactive responses were preferentially enriched in healthcare workers who had abortive infections1, and in household contacts protected from infection2. We hypothesize that such early viral control would require pre-existing cross-reactive memory T cells already resident at the site of infection; such airway-resident responses have been shown to be critical for mediating protection after intranasal vaccination in a murine model of SARS-CoV3. Bronchoalveolar lavage samples from the lower respiratory tract of healthy donors obtained before the COVID-19 pandemic revealed airway-resident, SARS-CoV-2-cross-reactive T cells, which correlated with the strength of human seasonal coronavirus immunity. We therefore demonstrate the potential to harness functional airway-resident SARS-CoV-2-reactive T cells in next-generation mucosal vaccines.

The Stromal Intervention: Regulation of Immunity and Inflammation at the Epithelial-Mesenchymal Barrier.

The immune system safeguards organ integrity by employing a balancing act of inflammatory and immunosuppressive mechanisms designed to neutralize foreign invaders and resolve injury. Maintaining or restoring a state of immune homeostasis is particularly challenging at barrier sites where constant exposure to immunogenic environmental agents may induce destructive inflammation. Recent studies underscore the role of epithelial and mesenchymal barrier cells in regulating immune cell function and local homeostatic and inflammatory responses. Here, we highlight immunoregulatory circuits engaging epithelial and mesenchymal cells in the intestine, airways, and skin and discuss how immune communications with hematopoietic cells and the microbiota orchestrate local immune homeostasis and inflammation.

Bias Factor and Therapeutic Window Correlate to Predict Safer Opioid Analgesics.

Biased agonism has been proposed as a means to separate desirable and adverse drug responses downstream of G protein-coupled receptor (GPCR) targets. Herein, we describe structural features of a series of mu-opioid-receptor (MOR)-selective agonists that preferentially activate receptors to couple to G proteins or to recruit ßarrestin proteins. By comparing relative bias for MOR-mediated signaling in each pathway, we demonstrate a strong correlation between the respiratory suppression/antinociception therapeutic window in a series of compounds spanning a wide range of signaling bias. We find that ßarrestin-biased compounds, such as fentanyl, are more likely to induce respiratory suppression at weak analgesic doses, while G protein signaling bias broadens the therapeutic window, allowing for antinociception in the absence of respiratory suppression.

Upper airway and brain protection by plasma cells: A local affair.

Protecting the upper airways and brain from viral invasion through the olfactory mucosa is critical. Wellford et al. describe a barrier that restricts the passage of circulating antibodies and prevents them from reaching the olfactory mucosa. Instead, plasma cells are recruited into this site and prevent viral infection of the airways and the brain through local antibody production.

Immuno-proteomic profiling reveals aberrant immune cell regulation in the airways of individuals with ongoing post-COVID-19 respiratory disease.

Some patients hospitalized with acute COVID-19 suffer respiratory symptoms that persist for many months. We delineated the immune-proteomic landscape in the airways and peripheral blood of healthy controls and post-COVID-19 patients 3 to 6 months after hospital discharge. Post-COVID-19 patients showed abnormal airway (but not plasma) proteomes, with an elevated concentration of proteins associated with apoptosis, tissue repair, and epithelial injury versus healthy individuals. Increased numbers of cytotoxic lymphocytes were observed in individuals with greater airway dysfunction, while increased B cell numbers and altered monocyte subsets were associated with more widespread lung abnormalities. A one-year follow-up of some post-COVID-19 patients indicated that these abnormalities resolved over time. In summary, COVID-19 causes a prolonged change to the airway immune landscape in those with persistent lung disease, with evidence of cell death and tissue repair linked to the ongoing activation of cytotoxic T cells.

Cellular crosstalk in the development and regeneration of the respiratory system.

The respiratory system, including the peripheral lungs, large airways and trachea, is one of the most recently evolved adaptations to terrestrial life. To support the exchange of respiratory gases, the respiratory system is interconnected with the cardiovascular system, and this interconnective nature requires a complex interplay between a myriad of cell types. Until recently, this complexity has hampered our understanding of how the respiratory system develops and responds to postnatal injury to maintain homeostasis. The advent of new single-cell sequencing technologies, developments in cellular and tissue imaging and advances in cell lineage tracing have begun to fill this gap. The view that emerges from these studies is that cellular and functional heterogeneity of the respiratory system is even greater than expected and also highly adaptive. In this Review, we explore the cellular crosstalk that coordinates the development and regeneration of the respiratory system. We discuss both the classic cell and developmental biology studies and recent single-cell analysis to provide an integrated understanding of the cellular niches that control how the respiratory system develops, interacts with the external environment and responds to injury.

Smooth-talking T cells leave asthma breathless.

The plasma membrane channel PANX1 mediates release of bio-active adenine nucleotides; however, its function in immune cells is unknown. In this issue of Immunity, Medina et al. show that PANX1 mediates adenosine-dependent communication between regulatory and effector CD4+ T cells during allergic airway inflammation.

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.

A serpin shapes the extracellular environment to prevent influenza A virus maturation.

Interferon-stimulated genes (ISGs) act in concert to provide a tight barrier against viruses. Recent studies have shed light on the contribution of individual ISG effectors to the antiviral state, but most have examined those acting on early, intracellular stages of the viral life cycle. Here, we applied an image-based screen to identify ISGs inhibiting late stages of influenza A virus (IAV) infection. We unraveled a directly antiviral function for the gene SERPINE1, encoding plasminogen activator inhibitor 1 (PAI-1). By targeting extracellular airway proteases, PAI-1 inhibits IAV glycoprotein cleavage, thereby reducing infectivity of progeny viruses. This was biologically relevant for IAV restriction in vivo. Further, partial PAI-1 deficiency, attributable to a polymorphism in human SERPINE1, conferred increased susceptibility to IAV in vitro. Together, our findings reveal that manipulating the extracellular environment to inhibit the last step in a virus life cycle is an important mechanism of the antiviral response.

Prevention of respiratory virus transmission by resident memory CD8+ T cells.

An ideal vaccine both attenuates virus growth and disease in infected individuals and reduces the spread of infections in the population, thereby generating herd immunity. Although this strategy has proved successful by generating humoral immunity to measles, yellow fever and polio, many respiratory viruses evolve to evade pre-existing antibodies1. One approach for improving the breadth of antiviral immunity against escape variants is through the generation of memory T cells in the respiratory tract, which are positioned to respond rapidly to respiratory virus infections2-6. However, it is unknown whether memory T cells alone can effectively surveil the respiratory tract to the extent that they eliminate or greatly reduce viral transmission following exposure of an individual to infection. Here we use a mouse model of natural parainfluenza virus transmission to quantify the extent to which memory CD8+ T cells resident in the respiratory tract can provide herd immunity by reducing both the susceptibility of acquiring infection and the extent of transmission, even in the absence of virus-specific antibodies. We demonstrate that protection by resident memory CD8+ T cells requires the antiviral cytokine interferon-γ (IFNγ) and leads to altered transcriptional programming of epithelial cells within the respiratory tract. These results suggest that tissue-resident CD8+ T cells in the respiratory tract can have important roles in protecting the host against viral disease and limiting viral spread throughout the population.

The respiratory system influences flight mechanics in soaring birds.

The subpectoral diverticulum (SPD) is an extension of the respiratory system in birds that is located between the primary muscles responsible for flapping the wing1,2. Here we survey the pulmonary apparatus in 68 avian species, and show that the SPD was present in virtually all of the soaring taxa investigated but absent in non-soarers. We find that this structure evolved independently with soaring flight at least seven times, which indicates that the diverticulum might have a functional and adaptive relationship with this flight style. Using the soaring hawks Buteo jamaicensis and Buteo swainsoni as models, we show that the SPD is not integral for ventilation, that an inflated SPD can increase the moment arm of cranial parts of the pectoralis, and that pectoralis muscle fascicles are significantly shorter in soaring hawks than in non-soaring birds. This coupling of an SPD-mediated increase in pectoralis leverage with force-specialized muscle architecture produces a pneumatic system that is adapted for the isometric contractile conditions expected in soaring flight. The discovery of a mechanical role for the respiratory system in avian locomotion underscores the functional complexity and heterogeneity of this organ system, and suggests that pulmonary diverticula are likely to have other undiscovered secondary functions. These data provide a mechanistic explanation for the repeated appearance of the SPD in soaring lineages and show that the respiratory system can be co-opted to provide biomechanical solutions to the challenges of flight and thereby influence the evolution of avian volancy.

The Role of Lung and Gut Microbiota in the Pathology of Asthma.

Asthma is a common chronic respiratory disease affecting more than 300 million people worldwide. Clinical features of asthma and its immunological and molecular etiology vary significantly among patients. An understanding of the complexities of asthma has evolved to the point where precision medicine approaches, including microbiome analysis, are being increasingly recognized as an important part of disease management. Lung and gut microbiota play several important roles in the development, regulation, and maintenance of healthy immune responses. Dysbiosis and subsequent dysregulation of microbiota-related immunological processes affect the onset of the disease, its clinical characteristics, and responses to treatment. Bacteria and viruses are the most extensively studied microorganisms relating to asthma pathogenesis, but other microbes, including fungi and even archaea, can potently influence airway inflammation. This review focuses on recently discovered connections between lung and gut microbiota, including bacteria, fungi, viruses, and archaea, and their influence on asthma.