Burn Pit Smoke Condensate-Mediated Toxicity in Human Airway Epithelial Cells.

Burn pits are a method of open-air waste management that was common during military operations in Iraq, Afghanistan, and other regions in Southwest Asia. Veterans returning from deployment have reported respiratory symptoms, potentially from exposure to burn pit smoke, yet comprehensive assessment of such exposure on pulmonary health is lacking. We have previously shown that exposure to condensates from burn pit smoke emissions causes inflammation and cytotoxicity in mice. In this study, we explored the effects of burn pit smoke condensates on human airway epithelial cells (HAECs) to understand their impact on cellular targets in the human lung. HAECs were cultured at the air-liquid interface (ALI) and exposed to burn pit waste smoke condensates (plywood, cardboard, plastic, mixed, and mixed with diesel) generated under smoldering and flaming conditions. Cytotoxicity was evaluated by measuring transepithelial electrical resistance (TEER) and lactate dehydrogenase (LDH) release; toxicity scores (TSs) were quantified for each exposure. Pro-inflammatory cytokine release and modulation of gene expression were examined for cardboard and plastic condensate exposures. Burn pit smoke condensates generated under flaming conditions affected cell viability, with flaming mixed waste and plywood exhibiting the highest toxicity scores. Cardboard and plastic smoke condensates modulated cytokine secretion, with GM-CSF and IL-1ß altered in more than one exposure group. Gene expression of detoxifying enzymes (ALDH1A3, ALDH3A1, CYP1A1, CYP1B1, NQO1, etc.), mucins (MUC5AC, MUC5B), and cytokines was affected by several smoke condensates. Particularly, expression of IL6 was elevated following exposure to all burn pit smoke condensates, and polycyclic aromatic hydrocarbon acenaphthene was positively associated with the IL-6 level in the basolateral media of HAECs. These observations demonstrate that exposure to smoke condensates of materials present in burn pits adversely affects HAECs and that aberrant cytokine secretion and altered gene expression profiles following burn pit material smoke exposure could contribute to the development of airway disease.

SARS-CoV-2 variant of concern fitness and adaptation in primary human airway epithelia.

The severe acute respiratory syndrome coronavirus 2 pandemic is characterized by the emergence of novel variants of concern (VOCs) that replace ancestral strains. Here, we dissect the complex selective pressures by evaluating variant fitness and adaptation in human respiratory tissues. We evaluate viral properties and host responses to reconstruct forces behind D614G through Omicron (BA.1) emergence. We observe differential replication in airway epithelia, differences in cellular tropism, and virus-induced cytotoxicity. D614G accumulates the most mutations after infection, supporting zoonosis and adaptation to the human airway. We perform head-to-head competitions and observe the highest fitness for Gamma and Delta. Under these conditions, RNA recombination favors variants encoding the B.1.617.1 lineage 3' end. Based on viral growth kinetics, Alpha, Gamma, and Delta exhibit increased fitness compared to D614G. In contrast, the global success of Omicron likely derives from increased transmission and antigenic variation. Our data provide molecular evidence to support epidemiological observations of VOC emergence.

Prolonged airway explant culture enables study of health, disease, and viral pathogenesis.

In vitro models play a major role in studying airway physiology and disease. However, the native lung's complex tissue architecture and non-epithelial cell lineages are not preserved in these models. Ex vivo tissue models could overcome in vitro limitations, but methods for long-term maintenance of ex vivo tissue has not been established. We describe methods to culture human large airway explants, small airway explants, and precision-cut lung slices for at least 14 days. Human airway explants recapitulate genotype-specific electrophysiology, characteristic epithelial, endothelial, stromal and immune cell populations, and model viral infection after 14 days in culture. These methods also maintain mouse, rabbit, and pig tracheal explants. Notably, intact airway tissue can be cryopreserved, thawed, and used to generate explants with recovery of function 14 days post-thaw. These studies highlight the broad applications of airway tissue explants and their use as translational intermediates between in vitro and in vivo studies.

Host range, transmissibility and antigenicity of a pangolin coronavirus.

The pathogenic and cross-species transmission potential of SARS-CoV-2-related coronaviruses (CoVs) remain poorly characterized. Here we recovered a wild-type pangolin (Pg) CoV GD strain including derivatives encoding reporter genes using reverse genetics. In primary human cells, PgCoV replicated efficiently but with reduced fitness and showed less efficient transmission via airborne route compared with SARS-CoV-2 in hamsters. PgCoV was potently inhibited by US Food and Drug Administration approved drugs, and neutralized by COVID-19 patient sera and SARS-CoV-2 therapeutic antibodies in vitro. A pan-Sarbecovirus antibody and SARS-CoV-2 S2P recombinant protein vaccine protected BALB/c mice from PgCoV infection. In K18-hACE2 mice, PgCoV infection caused severe clinical disease, but mice were protected by a SARS-CoV-2 human antibody. Efficient PgCoV replication in primary human cells and hACE2 mice, coupled with a capacity for airborne spread, highlights an emergence potential. However, low competitive fitness, pre-immune humans and the benefit of COVID-19 countermeasures should impede its ability to spread globally in human populations.

Small-molecule eRF3a degraders rescue CFTR nonsense mutations by promoting premature termination codon readthrough.

The vast majority of people with cystic fibrosis (CF) are now eligible for CF transmembrane regulator (CFTR) modulator therapy. The remaining individuals with CF harbor premature termination codons (PTCs) or rare CFTR variants with limited treatment options. Although the clinical modulator response can be reliably predicted using primary airway epithelial cells, primary cells carrying rare CFTR variants are scarce. To overcome this obstacle, cell lines can be created by overexpression of mouse Bmi-1 and human TERT (hTERT). Using this approach, we developed 2 non-CF and 6 CF airway epithelial cell lines, 3 of which were homozygous for the W1282X PTC variant. The Bmi-1/hTERT cell lines recapitulated primary cell morphology and ion transport function. The 2 F508del-CFTR cell lines responded robustly to CFTR modulators, which was mirrored in the parent primary cells and in the cell donors' clinical response. Cereblon E3 ligase modulators targeting eukaryotic release factor 3a (eRF3a) rescued W1282X-CFTR function to approximately 20% of WT levels and, when paired with G418, rescued G542X-CFTR function to approximately 50% of WT levels. Intriguingly, eRF3a degraders also diminished epithelial sodium channel (ENaC) function. These studies demonstrate that Bmi-1/hTERT cell lines faithfully mirrored primary cell responses to CFTR modulators and illustrate a therapeutic approach to rescue CFTR nonsense mutations.