Fluorescence Microscopy Journal Club

STAT6 induces expression of Gas6 in macrophages to clear apoptotic neutrophils and resolve inflammation

by Saroj Nepal, Chinnaswamy Tiruppathi, Yoshikazu Tsukasaki, Joseph Farahany, Manish Mittal, Jalees Rehman, Darwin J. Prockop and Asrar B. Malik

PNAS, 2019, 116, pp. 16513-16518

When the lung is injured and inflamed, alveolar macrophages clear apoptotic neutrophils, tissue debris, and pathogenic bacteria from the alveolar space in the lung through a process known as “efferocytosis.” The role of macrophages in efferocytosis is critical for the resolution of lung injury since inflammation will continue and injury will worsen if apoptotic neutrophils, tissue debris, and bacteria accumulate in the alveolar space. 

Imaging efferocytosis in living mice was made possible using a Bruker Ultima resonant-scanning two-photon microscope with an Olympus PlanFl 20× (NA 1.00) lens to collect dual-color images (emission filter: 460/50 nm for Brilliant Violet 421, 525/50 nm for Alexa 488, and 595/50 for Cell Tracker Red CMTPX) with 820 nm excitation. The surgical methods for gaining access to living lungs for two-photon imaging were based on a method devised by Looney et al. (Nature Methods 2011: 8, 91–96. doi:10.1038/nmeth.1543). 

Living macrophages were isolated from bone marrow, stained with Cell Tracker Red, and transferred into experimental mice. Before surgery to access the lungs of the living mice using the Looney method, fluorescently labeled antibodies were injected to label apoptotic neutrophils (Brilliant Violet 421-labeled LY6G antibody) and lung vessels (Alexa 488-labeled CD31 antibody). Multi-label images of living lung tissue, including alveolar macrophages, apoptotic neutrophils, and lung vessels, were collected using two-photon microscopy. Images of the lung tissue from control and experimental mice were subsequently analyzed using ImageJ and LabVIEW. 

The faster resonant scanning and the ability to image deep into the living lung tissue afforded by the Bruker Ultima two-photon microscope were essential to the positive outcome of this work. This study demonstrated that induction of alveolar macrophages activates the transcription factor STAT6, which promotes resolution of lung injury by the increased expression of Gas6, the ligand for efferocytosis. This has opened up opportunities for developing drugs for treating inflammatory lung injury by targeting the STAT6 activation pathway in alveolar macrophages by increasing Gas6 expression.