C96-07 Neutrophil-Epithelial Interactions in Live Alveoli of Influenza-Infected Lungs

Abstract Rationale In people with influenza infection, secondary lung infection by S. aureus (SA) causes alveolar barrier dysfunction, leading to pulmonary edema formation. Neutrophils are essential to antibacterial lung defense but may cause barrier dysfunction. How neutrophil-alveolar interactions contribute to barrier loss mechanisms is not clear. We addressed this issue using live lung imaging. Methods We intranasally-instilled mice with PBS or influenza (A/PR/8/1934). After 3 days, we excised, inflated, perfused, and viewed the live lungs by confocal microscopy. We identified neutrophils by adding fluorophore-tagged anti-Ly6G Ab to the lung perfusate solution or microinstilling alveolar airspaces with anti-CD11b Ab. All Ab instillations were followed by buffer washout. Results Confocal images show Ly6G+ neutrophils in alveolar microvessels of PBS- and influenza-instilled lungs. Neutrophil numbers were greater in influenza-infected lungs (50±6 vs 21±4 per field of 60 alveoli, p 0.05), indicating influenza induced microvascular neutrophil recruitment. By contrast, Ly6G+ neutrophils were nearly absent from alveolar airspaces, indicating influenza induced little airspace neutrophil recruitment in the first 3 days of infection. To define whether SA coinfection induces neutrophil transmigration from microvessels into airspaces, we carried out a multistep experiment in which we added anti-Ly6G Ab to the perfusate solution, microinstilled airspaces with GFP-labeled SA, waited 2h, and microinstilled airspaces with anti-CD11b Ab. Addition of fluorophore-tagged dextran (20 kD) to the perfusate showed alveolar barrier function was intact, ruling out the possibility that anti-Ly6G Ab in the perfusate leaked into airspaces. Our findings show SA rapidly formed microaggregates in alveoli. In airspaces of some SA-containing alveoli, dual-labelled Ly6G+CD11b+ cells marked neutrophils that transmigrated in response to SA (11±3 cells per field). No neutrophils transmigrated in response to airspace microinstillation of mutant SA that lacked alpha-hemolysin toxin (p 0.05), indicating transmigration resulted from toxin-mediated, SA-epithelial interactions. Repeat addition of dextran to the perfusate at 3h after SA microinstillation showed dextran fluorescence in airspaces, indicating SA caused alveolar barrier dysfunction. Barrier dysfunction occurred only in imaging fields of alveoli that contained transmigrated neutrophils, while airspaces of neighboring, SA-containing alveoli that lacked transmigrated neutrophils remained dextran-free (p 0.05). Conclusions Our findings show SA microinstillation in alveoli caused alveolar barrier dysfunction in influenza-infected lungs. Surprisingly, barrier loss was restricted to alveoli in which SA-epithelial interactions induced neutrophil transmigration from microvessels into airspaces. We interpret that alveolar barrier dysfunction in lungs with influenza-SA coinfection is spatially heterogenous and associates with neutrophil transmigration. Better understanding of transmigrating neutrophil populations may reveal new therapeutic targets. This abstract is funded by: Stony Wold-Herbert Fund Fellowship Award (JZ), R01HL164821 (JH) and American Lung Association COVID-19 and Emerging Respiratory Viruses Research Award 1031520 (JH)