621.8 - Influenza A Virus Causes Shedding of the Alveolar Epithelial Glycocalyx through Activation of Sheddases
Sunday, April 3, 2022
9:30 AM – 9:45 AM
Room: 203 B - Pennsylvania Convention Center
Introduction: Mortality in critically ill patients has decreased due to improvements in hemodynamic support, mechanical ventilation strategies and bundled care. However, patients surviving to discharge have staggering rates of mortality, ~50%, during the next year of life. Furthermore, survivors have significant morbidity due to end-organ dysfunction. An NHBLI report suggests two erroneous paradigms that have limited our understanding of the role of pneumonia on health: that pneumonia is a 1) lt;emgt;localizedlt;/emgt; and 2) lt;emgt;acutelt;/emgt; disease. In fact, pneumonia causes end-organ injury by poorly understood mechanisms and the clinical manifestations persist long after resolution of the primary infection. Biological changes occurring during pneumonia that induce long-term morbidity are poorly understood. In summary, we propose to discuss exciting new mechanisms and hypotheses elucidating mechanisms of long-term morbidity and end-organ dysfunction in survivors of pneumonia that suffer from chronic critical illness.
Kaitlyn Schaaf (Vanderbilt University), Charity Buggs (Vanderbilt University Medical Center), Nathan Putz (Vanderbilt University Medical Center), Christophe Langouet-Astrie (University of Colorado School of Medicine), Christopher Jetter (Vanderbilt University Medical Center), Nicolas Nigretti (Vanderbilt University Medical Center), Jennifer Sucre (Vanderbilt University Medical Center), Eric Schmidt (University of Colorado School of Medicine), Julie Bastarache (Vanderbilt University Medical Center), Ciara Shaver (Vanderbilt University Medical Center)
Objective: The healthy alveolar epithelium is protected by a heparan sulfate rich, glycosaminoglycan layer called the epithelial glycocalyx. Our group found that the epithelial glycocalyx is shed in patients with acute respiratory distress syndrome (ARDS). In murine models of LPS- or bleomycin-induced acute lung injury, sheddases (membrane-bound enzymes that cleave extracellular potions of transmembrane proteins) are upregulated and associated with glycocalyx shedding and increased lung permeability. ARDS is commonly caused by viral infections including influenza A (IAV). In murine models, IAV causes massive and persistent glycocalyx shedding into the airspace but the mechanisms by which this occurs are unknown. The objective of this work is to determine the molecular processes underlying IAV-induced shedding of the glycocalyx.
Hypothesis: We hypothesize that IAV causes glycocalyx shedding through induction of host sheddases.
Methods: We examined the literature and curated a list of sheddases associated with IAV with potential to cleave the glycocalyx (MMP-7, -2, -9 and their inhibitors TIMP-1 and -2). C57BL/6 mice were infected intranasally with A/PR/8/34 (H1N1) at 30,000 PFU/mouse and bronchoalveolar lavage and lung tissue were collected at day 1, 3, and 7 post infection. Sheddase expression was assessed by RT-qPCR and RNAscope was used to localize lung sheddase expression in infected and uninfected lungs. MLE-12 mouse lung epithelial cells were infected with viable or heat-inactivated (56C for 30 min) A/PR/8/34 (H1N1) at a MOI of 1 and sheddase expression measured by RT-qPCR.
Results: Mice infected with IAV develop significant lung inflammation (increased BAL inflammatory cells), lung permeability (increased BAL protein), and increased glycocalyx shedding. MMP-7 is upregulated in infected vs. uninfected lungs at day 1 and 3 post infection, then returns to baseline levels by day 7. MMP-7 is only expressed in cells that are directly infected by IAV. Expression of the MMP-7 inhibitor TIMP-1 is similar to uninfected lungs on day 1, but increases 50-fold on day 3. In contrast, MMP-2 and MMP-9, as well as their inhibitor TIMP-2 are not upregulated in the first 7 days after IAV infection. Preliminary studies in lung epithelial cells suggest that heat-inactivated IAV fails to upregulate MMP-7.
Conclusions: Together, these data suggest that localized IAV infection increases MMP-7 in a murine model of IAV infection, but has no effect on several other sheddases. This suggests that MMP-7 may modulate IAV-induced glycocalyx shedding. Future studies will explore the mechanisms of IAV induced glycocalyx shedding which could provide molecular targets for clinical intervention in IAV-ARDS pathogenesis.
