Background: The fungal cell-wall constituent 1,3-beta-D-glucan (BDG) is a pathogen-associated molecular pattern that can stimulate innate immunity. We hypothesized that BDG from colonizing fungi in critically-ill patients may translocate into the systemic circulation and be associated with host inflammation and outcomes. Methods:We enrolled 453 mechanically-ventilated patients with acute respiratory failure (ARF) without invasive fungal infection and measured BDG, innate immunity and epithelial permeability biomarkers in serially collected plasma samples.Results: Compared to healthy controls, ARF patients had significantly higher BDG levels (median [interquartile-range] 26 [15-49]pg/ml, p<0.001), whereas ARF patients with high BDG levels (≥40pg/ml, 31%) had higher odds for assignment to the prognostically adverse hyperinflammatory subphenotype (odds ratio [confidence interval] 2.88 [1.83-4.54], p<0.001). Baseline BDG levels were predictive of fewer ventilator-free days and worse 30-day survival (adjusted p<0.05). Integrative analyses of fungal colonization and epithelial barrier disruption suggested that BDG may translocate from either the lung or gut compartment. We validated the associations between plasma BDG and host inflammatory responses in 97 hospitalized patients with COVID-19.Conclusions: BDG measurements offered prognostic information in critically-ill patients without fungal infections. Further research in the mechanisms of translocation and innate immunity recognition and stimulation may offer new therapeutic opportunities in critical illness.
Aquaporin 5 (AQP5) is expressed in several cell types in the lung and regulates water transport, which contributes to barrier function during injury and the composition of glandular secretions. Reduced AQP5 expression is associated with barrier dysfunction during acute lung injury, and strategies to enhance its expression are associated with favorable phenotypes. Thus, pharmacologically enhancing AQP5 expression could be beneficial. Here, we optimized a high-throughput assay designed to detect AQP5 abundance using a cell line stably expressing bioluminescent-tagged AQP5. We then screened a library of 1153 compounds composed of FDA-approved drugs for their effects on AQP5 abundance. We show compounds Niclosamide, Panobinostat, and Candesartan Celexitil increased AQP5 abundance, and show that Niclosamide has favorable cellular toxicity profiles. We determine that AQP5 levels are regulated in part by ubiquitination and proteasomal degradation in lung epithelial cells, and mechanistically Niclosamide increases AQP5 levels by reducing AQP5 ubiquitination and proteasomal degradation. Functionally, Niclosamide stabilized AQP5 levels in response to hypotonic stress, a stimulus known to reduce AQP5 levels. In complementary assays, Niclosamide increased endogenous AQP5 in both A549 cells and in primary, polarized human bronchial epithelial cells compared to control-treated cells. Further, we measured rapid cell volume changes in A549 cells in response to osmotic stress, an effect controlled by aquaporin channels. Niclosamide-treated A549 cell volume changes occurred more rapidly compared to control-treated cells, suggesting that increased Niclosamide-mediated increases in AQP5 expression affects functional water transport. Taken together, we describe a strategy to identify repurposed compounds for their effect on AQP5 protein abundance. We validated the effects of Niclosamide on endogenous AQP5 levels and in regulating cell-volume changes in response to tonicity changes. Our findings highlight a unique approach to screen for drug effects on protein abundance, and our workflow can be applied broadly to study compound effects on protein abundance in lung epithelial cells.
Accumulation of excessive extracellular matrix (ECM) components from lung fibroblasts is a feature of Systemic Sclerosis-associated Interstitial Lung Disease (SSc-ILD), and there is increasing evidence that innate immune signaling pathways contribute to these processes. Toll-Like Receptors (TLRs) are innate immune sensors activated by danger signals derived from pathogens or host molecular patterns. Several Damage-Associated Molecular Pattern (DAMP) molecules are elevated in SSc-ILD plasma, including ligands that activate TLR9, an innate immune sensor recently implicated in driving profibrotic responses in fibroblasts. Fibronectin and the isoform Fibronectin-Extra Domain A (FN-EDA) is prominent in pathologic extracellular matrix accumulation, but mechanisms promoting FN-EDA accumulation are only partially understood. Here, we show that TLR9 activation increases FN-EDA accumulation in MRC5 and SSc-ILD fibroblasts, but that this effect is independent of changes in FN-EDA gene transcription. Rather, we describe a novel mechanism where TLR9 activation inhibits FN-EDA turnover via reduced FN-EDA ubiquitination. TLR9 ligand ODN2006 reduces ubiquitinated FN-EDA destined for lysosomal degradation, an effect abrogated with TLR9 knockdown or inhibition. Taken together, these results provide rationale for 1) disrupting the TLR9 signaling axis or FN-EDA degradation pathways to reduce FN-EDA accumulation in SSc-ILD fibroblasts. More broadly, enhancing intracellular degradation of ECM components through TLR9 inhibition or enhanced ECM turnover could be a novel strategy to attenuate pathogenic ECM accumulation in SSc-ILD.
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