Abstract Rationale Pneumonia is a severe respiratory illness, with immune-experience (immunity from prior infections) as a key variable affecting prognosis. How pneumonia progresses in real-time and how systemic (blood-borne) vs. pulmonary (lung-resident) factors impact experience remain poorly understood. Here, we develop paracorporeal cross-circulation with crystal ribcage to 1) continuously probe real-time pneumonia progression at high spatiotemporal resolution for 24-hours and 2) dissect the influence of pulmonary vs. systemic immunity in pneumonia. Methods 24-hour immunogenic murine paracorporeal cross-circulation was established between awake “donor” mouse and S. pneumoniae infected ex-vivo “recipient” lung. Recipients were housed inside a transparent Crystal Ribcage, enabling real-time, continuous imaging of fluorescent immune events across 24-hours infection. Pulmonary vs. systemic immunity against pneumonia was dissected by mismatching experience vs. naive backgrounds between donors vs. recipients, with intact vs. depleted lung-resident CD4+ T-cells, and analyzed via immunohistochemistry and immunofluorescence. Single immune-naïve donor was linked to double recipients mismatched of experience vs. naïve condition to investigate effect of immune-experience on alveolar macrophage dynamic response. Results We established the first 24-hour immunogenic paracorporeal cross-circulation between donor and recipient systems enabling continuous long-term imaging of disease progression and biological mismatch between systemic vs. pulmonary factors (Fig.A). Whole-blood cross-circulation preserves alveolar macrophage motility compared to RPMI, allowing us to study their differential motility in infected, uninfected, and liquid-filled lungs across 24h (B,C). We show neutrophils are recruited inside alveoli and swarm in clusters to combat infection (Fig.D-G). By mismatching experience vs. naïve immunity between donor vs. recipients, we mechanistically reveal how experienced pulmonary factors alone generate a significant immune response against pneumonia (Fig.H), with resident CD4+ T-cells central to this trained immunity (Fig.I), in contrast to systemic factors. Thereafter, cross-circulating single naïve donor into double recipients with mismatched pulmonary immunity (Fig.J), we show that trained lung-resident alveolar macrophages exhibit amplified motility during infection, vs. naive (Fig.K-M). Motility cluster (MC) analysis revealed 9 MCs, with trained alveolar macrophages exhibiting more MC8, dominated by greater speed and angle covariance, compared to naïve (Fig.N-P). Conclusion Using paracorporeal crystal ribcage, we reveal how macrophage and neutrophil immunity evolves in early pneumonia infection. We discover early immune defense is orchestrated by pulmonary, not systemic, factors, mediated by lung-resident memory CD4+ T-cells. We reveal trained alveolar macrophages are more motile than naïve by coupling double recipients. Together, our paracorporeal crystal ribcage allows long-term cellular-resolution and mechanistic mismatch studies of the lung investigating immune-experience, and in future, aging. This abstract is funded by: DP2HL168562, Beckman Young Investigator Award, NSF CAREER Award.
Ling et al. (Fri,) studied this question.