Background The long-term effects of SARS-CoV-2 infection on pulmonary structure and function remain incompletely characterized. Purpose To test whether SARS-CoV-2 infection leads to small airways ...disease in patients with persistent symptoms. Materials and Methods In this single center study at a university teaching hospital, adults with confirmed COVID-19 who remained symptomatic >30 days following diagnosis were prospectively enrolled between June and December 2020 and compared to healthy participants (controls) prospectively enrolled between March and August 2018. Participants with post-acute sequelae of COVID-19 (PASC) were classified as ambulatory, hospitalized, or requiring the intensive care unit (ICU) based on the highest level of care received during acute infection. Symptoms, pulmonary function tests, and chest CT images were collected, and quantitative CT analysis was performed using supervised machine-learning to measure regional ground glass opacities (GGO) and inspiratory and expiratory image-matching to measure regional air trapping. Univariable analyses and multivariable linear regression were used to compare groups. Results 100 participants with PASC (median age, 48 years; 66 women) were evaluated and compared with 106 matched healthy controls. Sixty-seven percent (67/100) of the participants with PASC were classified as ambulatory, 17% (17/100) wer e hospitalized and 16% (16/100) required care in the ICU. Among the hospitalized and ICU groups, the mean percent of total lung classified as GGO was 13.2% and 28.7%, respectively, and was higher than in the ambulatory group (3.7%, p<.001 for both comparisons). The mean percentage of total lung affected by air trapping was 25.4%, 34.6%, and 27.3% in the ambulatory, hospitalized, and ICU groups and 7.2% in healthy controls (p<.001). Air trapping correlated with the residual volume to total lung capacity rati o (RV/TLC; r=0.6, p<.001). Conclusions In survivors of COVID-19, small airways disease occurred independently of initial infection severity. The long-term consequences are unknown. See also the editorial by Elicker.
Vitamin D supplementation has been suggested to enhance immunity during respiratory infection season. We tested the effect of active vitamin D (calcitriol) supplementation on key airway innate immune ...mechanisms in vitro.
Primary human airway epithelial cells (hAECs) grown at the air liquid interface were supplemented with 10-7 M calcitriol for 24 hours (or a time course) and their antimicrobial airway surface liquid (ASL) was tested for pH, viscoscity, and antibacterial and antiviral properties. We also tested hAEC ciliary beat frequency (CBF). Next, we assessed alterations to hAEC gene expression using RNA sequencing, and based on results, we measured neutrophil migration across hAECs.
Calcitriol supplementation enhanced ASL bacterial killing of Staphylococcus aureus (p = 0.02) but did not enhance its antiviral activity against 229E-CoV. It had no effect on ASL pH or viscosity at three timepoints. Lastly, it did not affect hAEC CBF or neutrophil migration, although there was a trend of enhanced migration in the presence of a neutrophil chemokine (p = 0.09). Supplementation significantly altered hAEC gene expression, primarily of AMP-related genes including CAMP and TREM1.
While vitamin D supplementation did not have effects on many airway innate immune mechanisms, it may provide a useful tool to resolve respiratory bacterial infections.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Cystic fibrosis (CF) is a life-shortening disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Although bacterial lung infection and the resulting ...inflammation cause most of the morbidity and mortality, how the loss of CFTR function first disrupts airway host defence has remained uncertain. To investigate the abnormalities that impair elimination when a bacterium lands on the pristine surface of a newborn CF airway, we interrogated the viability of individual bacteria immobilized on solid grids and placed onto the airway surface. As a model, we studied CF pigs, which spontaneously develop hallmark features of CF lung disease. At birth, their lungs lack infection and inflammation, but have a reduced ability to eradicate bacteria. Here we show that in newborn wild-type pigs, the thin layer of airway surface liquid (ASL) rapidly kills bacteria in vivo, when removed from the lung and in primary epithelial cultures. Lack of CFTR reduces bacterial killing. We found that the ASL pH was more acidic in CF pigs, and reducing pH inhibited the antimicrobial activity of ASL. Reducing ASL pH diminished bacterial killing in wild-type pigs, and, conversely, increasing ASL pH rescued killing in CF pigs. These results directly link the initial host defence defect to the loss of CFTR, an anion channel that facilitates HCO(3)(-) transport. Without CFTR, airway epithelial HCO(3)(-) secretion is defective, the ASL pH falls and inhibits antimicrobial function, and thereby impairs the killing of bacteria that enter the newborn lung. These findings suggest that increasing ASL pH might prevent the initial infection in patients with CF, and that assaying bacterial killing could report on the benefit of therapeutic interventions.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, KISLJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Measles virus (MeV) is the most contagious human virus. Unlike most respiratory viruses, MeV does not directly infect epithelial cells upon entry in a new host. MeV traverses the epithelium within ...immune cells that carry it to lymphatic organs where amplification occurs. Infected immune cells then synchronously deliver large amounts of virus to the airways. However, our understanding of MeV replication in airway epithelia is limited. To model it, we use well-differentiated primary cultures of human airway epithelial cells (HAE) from lung donors. In HAE, MeV spreads directly cell-to-cell forming infectious centers that grow for ~3-5 days, are stable for a few days, and then disappear. Transepithelial electrical resistance remains intact during the entire course of HAE infection, thus we hypothesized that MeV infectious centers may dislodge while epithelial function is preserved. After documenting by confocal microscopy that infectious centers progressively detach from HAE, we recovered apical washes and separated cell-associated from cell-free virus by centrifugation. Virus titers were about 10 times higher in the cell-associated fraction than in the supernatant. In dislodged infectious centers, ciliary beating persisted, and apoptotic markers were not readily detected, suggesting that they retain functional metabolism. Cell-associated MeV infected primary human monocyte-derived macrophages, which models the first stage of infection in a new host. Single-cell RNA sequencing identified wound healing, cell growth, and cell differentiation as biological processes relevant for infectious center dislodging. 5-ethynyl-2'-deoxyuridine (EdU) staining located proliferating cells underneath infectious centers. Thus, cells located below infectious centers divide and differentiate to repair the dislodged infected epithelial patch. As an extension of these studies, we postulate that expulsion of infectious centers through coughing and sneezing could contribute to MeV's strikingly high reproductive number by allowing the virus to survive longer in the environment and by delivering a high infectious dose to the next host.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Zoonotically transmitted coronaviruses are responsible for three disease outbreaks since 2002, including the current COVID-19 pandemic, caused by SARS-CoV-2. Its efficient transmission and range of ...disease severity raise questions regarding the contributions of virus-receptor interactions. ACE2 is a host ectopeptidase and the receptor for SARS-CoV-2. Numerous reports describe ACE2 mRNA abundance and tissue distribution; however, mRNA abundance is not always representative of protein levels. Currently, there is limited data evaluating ACE2 protein and its correlation with other SARS-CoV-2 susceptibility factors.
We systematically examined the human upper and lower respiratory tract using single-cell RNA sequencing and immunohistochemistry to determine receptor expression and evaluated its association with risk factors for severe COVID-19.
Our results reveal that ACE2 protein is highest within regions of the sinonasal cavity and pulmonary alveoli, sites of presumptive viral transmission and severe disease development, respectively. In the lung parenchyma, ACE2 protein was found on the apical surface of a small subset of alveolar type II cells and colocalized with TMPRSS2, a cofactor for SARS-CoV2 entry. ACE2 protein was not increased by pulmonary risk factors for severe COVID-19. Additionally, ACE2 protein was not reduced in children, a demographic with a lower incidence of severe COVID-19.
These results offer new insights into ACE2 protein localization in the human respiratory tract and its relationship with susceptibility factors to COVID-19.
Diabetes mellitus predisposes the host to bacterial infections. Moreover, hyperglycemia has been shown to be an independent risk factor for respiratory infections. The luminal surface of airway ...epithelia is covered by a thin layer of airway surface liquid (ASL) and is normally sterile despite constant exposure to bacteria. The balance between bacterial growth and killing in the airway determines the outcome of exposure to inhaled or aspirated bacteria: infection or sterility. We hypothesized that restriction of carbon sources--including glucose--in the ASL is required for sterility of the lungs. We found that airway epithelia deplete glucose from the ASL via a novel mechanism involving polarized expression of GLUT-1 and GLUT-10, intracellular glucose phosphorylation, and low relative paracellular glucose permeability in well-differentiated cultures of human airway epithelia and in segments of airway epithelia excised from human tracheas. Moreover, we found that increased glucose concentration in the ASL augments growth of P. aeruginosa in vitro and in the lungs of hyperglycemic ob/ob and db/db mice in vivo. In contrast, hyperglycemia had no effect on intrapulmonary bacterial growth of a P. aeruginosa mutant that is unable to utilize glucose as a carbon source. Our data suggest that depletion of glucose in the airway epithelial surface is a novel mechanism for innate immunity. This mechanism is important for sterility of the airways and has implications in hyperglycemia and conditions that result in disruption of the epithelial barrier in the lung.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Organotypic cultures of primary human airway epithelial cells have been used to investigate the morphology, ion and fluid transport, innate immunity, transcytosis, infection, inflammation, signaling, ...cilia, and repair functions of this complex tissue. However, we do not know how closely these cultures resemble the airway surface epithelium in vivo. In this study, we examined the genome-wide expression profile of tracheal and bronchial human airway epithelia in vivo and compared it with the expression profile of primary cultures of human airway epithelia grown at the air-liquid interface. For comparison, we also investigated the expression profile of Calu-3 cells grown at the air-liquid interface and primary cultures of human airway epithelia submerged in nutrient media. We found that the transcriptional profile of differentiated primary cultures grown at the air-liquid interface most closely resembles that of in vivo airway epithelia, suggesting that the use of primary cultures and the presence of an air-liquid interface are important to recapitulate airway epithelia biology. We describe a high level of similarity between cells of tracheal and bronchial origin within and between different human donors, which suggests a very robust expression profile that is specific to airway cells.
Coronavirus disease 2019 (COVID-19) is especially severe in aged populations
. Vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are highly effective, but vaccine efficacy ...is partly compromised by the emergence of SARS-CoV-2 variants with enhanced transmissibility
. The emergence of these variants emphasizes the need for further development of anti-SARS-CoV-2 therapies, especially for aged populations. Here we describe the isolation of highly virulent mouse-adapted viruses and use them to test a new therapeutic drug in infected aged animals. Many of the alterations observed in SARS-CoV-2 during mouse adaptation (positions 417, 484, 493, 498 and 501 of the spike protein) also arise in humans in variants of concern
. Their appearance during mouse adaptation indicates that immune pressure is not required for selection. For murine SARS, for which severity is also age dependent, elevated levels of an eicosanoid (prostaglandin D
(PGD
)) and a phospholipase (phospholipase A2 group 2D (PLA
G2D)) contributed to poor outcomes in aged mice
. mRNA expression of PLA
G2D and prostaglandin D
receptor (PTGDR), and production of PGD
also increase with ageing and after SARS-CoV-2 infection in dendritic cells derived from human peripheral blood mononuclear cells. Using our mouse-adapted SARS-CoV-2, we show that middle-aged mice lacking expression of PTGDR or PLA
G2D are protected from severe disease. Furthermore, treatment with a PTGDR antagonist, asapiprant, protected aged mice from lethal infection. PTGDR antagonism is one of the first interventions in SARS-CoV-2-infected animals that specifically protects aged animals, suggesting that the PLA
G2D-PGD
/PTGDR pathway is a useful target for therapeutic interventions.
Key points
Cl− and HCO3− had similar paracellular permeabilities in human airway epithelia.
PCl/PNa of airway epithelia was unaltered by pH 7.4 vs. pH 6.0 solutions.
Under basal conditions, ...calculated paracellular HCO3− flux was secretory.
Cytokines that increased airway surface liquid pH decreased or reversed paracellular HCO3− flux.
HCO3− flux through the paracellular pathway may counterbalance effects of cellular H+ and HCO3− secretion.
Airway epithelia control the pH of airway surface liquid (ASL), thereby optimizing respiratory defences. Active H+ and HCO3− secretion by airway epithelial cells produce an ASL that is acidic compared with the interstitial space. The paracellular pathway could provide a route for passive HCO3− flux that also modifies ASL pH. However, there is limited information about paracellular HCO3− flux, and it remains uncertain whether an acidic pH produced by loss of cystic fibrosis transmembrane conductance regulator anion channels or proinflammatory cytokines might alter the paracellular pathway function. To investigate paracellular HCO3− transport, we studied differentiated primary cultures of human cystic fibrosis (CF) and non‐CF airway epithelia. The paracellular pathway was pH‐insensitive at pH 6.0 vs. pH 7.4 and was equally permeable to Cl− and HCO3−. Under basal conditions at pH ∼6.6, calculated paracellular HCO3− flux was weakly secretory. Treating epithelia with IL‐17 plus TNFα alkalinized ASL pH to ∼7.0, increased paracellular HCO3− permeability, and paracellular HCO3− flux was negligible. Applying IL‐13 increased ASL pH to ∼7.4 without altering paracellular HCO3− permeability, and calculated paracellular HCO3− flux was absorptive. These results suggest that HCO3− flux through the paracellular pathway counterbalances, in part, changes in the ASL pH produced via cellular mechanisms. As the pH of ASL increases towards that of basolateral liquid, paracellular HCO3− flux becomes absorptive, tempering the alkaline pH generated by transcellular HCO3− secretion.
Key points
Cl− and HCO3− had similar paracellular permeabilities in human airway epithelia.
PCl/PNa of airway epithelia was unaltered by pH 7.4 vs. pH 6.0 solutions.
Under basal conditions, calculated paracellular HCO3− flux was secretory.
Cytokines that increased airway surface liquid pH decreased or reversed paracellular HCO3− flux.
HCO3− flux through the paracellular pathway may counterbalance effects of cellular H+ and HCO3− secretion.
Vitamin D deficiency is a risk factor for exacerbation of obstructive airway disease, a hallmark of which is mucus dehydration and plugging. Calcitriol (the active form of vitamin D) deficiency in ...cultured human airway epithelia resulted in increased
and
mRNAs encoding subunits of ENaC and the Na-K pump compared with supplemented epithelia. These drive the absorption of airway surface liquid. Consistently, calcitriol-deficient epithelia absorbed liquid faster than supplemented epithelia. Calcitriol deficiency also increased amiloride-sensitive
and
without altering Na-K pump activity, indicating the changes in amiloride-sensitivity arose from ENaC. ENaC activity can be regulated by trafficking, proteases, and channel abundance. We found the effect was likely not induced by changes to endocytosis of ENaC given that calcitriol did not affect the half-lives of amiloride-sensitive
and
. Furthermore, trypsin nominally increased
produced by epithelia ± calcitriol, suggesting calcitriol did not affect proteolytic activation of ENaC. Consistent with mRNA and functional data, calcitriol deficiency resulted in increased γENaC protein. These data indicate that the vitamin D receptor response controls ENaC function and subsequent liquid absorption, providing insight into the relationship between vitamin D deficiency and respiratory disease.
It is unknown why calcitriol (active vitamin D) deficiency worsens pulmonary disease outcomes. Results from mRNA, immunoblot, Ussing chamber, and absorption experiments indicate that calcitriol deficiency increases ENaC activity in human airway epithelia, decreasing apical hydration. Given that epithelial hydration is required for mucociliary transport and airway innate immune function, the increased ENaC activity observed in calcitriol-deficient epithelia may contribute to respiratory pathology observed in vitamin D deficiency.
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