Introduction/Aim:
Methicillin-resistant Staphylococcus aureus (MRSA) is a feature of both hospital- and community-acquired pneumonia, but translatable models to investigate early pathogenesis and potential treatments are limited. Precision-cut lung slices (PCLS), containing all resident structural and immune cells of the lung, may provide a novel approach for dynamic time-course studies of MRSA infection with higher throughput than in vivo mouse models.
Methods:
To establish infection, PCLS from agarose-inflated lungs of male C57BL/6 mice (8 -10 weeks old) were incubated for 2 h with MRSA (dsRED-expressing, 105 colony-forming units (CFU)) and transferred to fresh media. Up to 48 h post-infection (hpi), MRSA were visualised by fluorescent microscopy or quantitated as CFU in intact or homogenised PCLS. MRSA association with alveolar macrophages (AMs) and dendritic cells (DCs) were detected by flow cytometry. Media was assayed for lactate dehydrogenase activity (viability measure) or released cytokines by BioLegend LEGENDplex™ (inflammation).
Results:
Viability was similar in uninfected and infected PCLS. Diffuse MRSA colonies were evident across all structures (airways, arteries, parenchyma) in PCLS, increasing CFU >100,000 fold by 48 hpi (n=6, P<0.001). The increased proportion of CD45+ immune cells associated with MRSA over time (24hpi 9±2%; 48hpi 74±10%, n=4, P<0.05) was evident in AMs and DCs but not non-phagocytic B cells. The inflammatory cytokines TNF-α, GM-CSF and IL-6 were increased in MRSA-infected PCLS within 24 hpi (n=6, P<0.05).
Conclusion:
Mouse PCLS provide an ex vivo model of MRSA infection involving intact resident immune cells and inflammation. Our findings support the future application of human PCLS for the assessment of drugs to prevent infection or limit the replication of resistant bacterial strains.