Dissecting the role of systemic vs pulmonary immunity against pneumonia via extracorporeal cross-circulation coupled with Crystal Ribcage

Introduction: Pneumonia is a serious result of respiratory infection and a leading global burden of disease, even prior to COVID. The course of respiratory infection depends more on variations among hosts than among microbes, with immune experience (i.e., history of exposure to prior infection) being a key variable that affects both systemic (blood-borne) and pulmonary (local resident) defenses against infection. However, discriminating the effects of experience on discrete immune components (e.g., systemic vs. pulmonary) has remained a long-standing challenge. To dissect the contribution of systemic vs pulmonary immune defenses, and how immune experience differentially affects systemic vs. pulmonary immunity, key unmet needs include (i) lack of model systems to probe immune response at the cellular resolution and in real-time, and (ii) lack of model systems to mechanistically dissect the role of systemic (from the circulation) vs. pulmonary (from the resident tissue) immunity. To address these challenges, we developed a novel extracorporeal cross-circulation system coupled with the Crystal Ribcage (Nature Methods 2023) to 1) longitudinally probe pneumonia progression in real-time at single-cell resolution, and importantly 2) demonstrate that lung-resident immunity is key to the profound effects of prior microbial experience on the defense against respiratory infection.

Materials and

Methods: Long-term extracorporeal cross-circulation of whole-blood from an awake “donor” mouse into an ex vivo “recipient” lung housed in the Crystal Ribcage was established as follows. Donor system arteriovenous vascular access was established between the carotid artery and external jugular vein, and then connected to the pulmonary circulation of the recipient system. Extracorporeal circuit components were optimized for long-term cross-circulation (up to 24h or longer). To overcome high blood pressures caused by extracorporeal tubing, a major challenge in whole-blood murine cross-circulation, a proportional-integral-derivative controller coupled with in-line peristaltic pumps were developed to automatically adjust the perfusion pressures within the ex vivo lung to physiologically-relevant pressures. Cross-circulation of whole blood between the systems was maintained under normothermia. Immune-naïve or immune-experience experimental arms were generated as previously described in wildtype C57BL/6 mice (Mucusal Immunology 2020). Briefly, mice were infected with self-limiting left-lobar pneumococcal pneumonia (S. pneumoniae serotype Sp19), allowed to fully recover, and confirmed for immunity against future infections of heterologous serotypes (e.g. Sp3). In a pair-wise fashion, lungs with immune-naïve vs. -experience statuses were inoculated left-lobar with Sp3 pneumococci, and immediately harvested as the recipient system under the Crystal Ribcage, subsequently cross-circulated with whole-blood from immune-naive mTmG-MRP8 donor mice for 8 hours. Recipient lungs were subject to protective ventilation regimen and imaged with ultra-fast confocal microscopy (up to 2000 fps). At experiment termination, recipient systems were analyzed by immunohistochemistry imaging for inflammation such as neutrophil recruitment. Statistical tests for paired comparisons were conducted with paired t-test, otherwise two-sample t-test for unpaired samples.

Results, Conclusions, and Discussions: For the first time, we report the robust cross-circulation of fresh whole blood from an awake “donor” mouse to preserve the status of Sp3-infected “recipient” lungs ex vivo longitudinally to study pneumonia pathogenesis in real-time at high spatiotemporal resolution. Importantly, by mismatching immune-naïve donors with recipients from immune-naïve vs. -experienced backgrounds, our model can dissect the contributions of systemic (from donor) vs resident pulmonary (from recipient) factors towards immune-experience against pneumonia (Fig.1A). We first show differential neutrophil recruitment from donor mice into recipient lungs infected with Sp3 pneumonia versus those without (healthy) across 8h of cross-circulation (Fig.1B), demonstrating donor biology is sensitive and responsive to pathological perturbation of recipient biology. To first investigate immune-experience conferred by tissue-resident pulmonary factors, recipient lungs of immune-naïve vs. -experienced statuses were cross-circulated with immune-naïve donors. Post-experimental immunohistochemistry (IHC) against neutrophil elastase in recipient lungs revealed significantly increased donor neutrophil recruitment into recipients from experienced backgrounds, compared to naïve backgrounds (Fig.1C). To complement these findings in real-time, we imaged the dynamics of donor MRP8+ neutrophils cross-circulating into the Sp3-infected recipient, whose resident neutrophils exhibit no intrinsic fluorescence. We discovered that recipient systems of experienced backgrounds differentially elicit accelerated neutrophil recruitment by 4hr of cross-circulation and onwards, compared to naïve backgrounds. Interestingly, we observed increased neutrophilia in the uninfected recipient right lungs of experience arms than naïve, suggesting that resident experience influences baseline lung inflammation independently of local bacterial presence in lobar pneumonia. Comparing between timepoints, while both experimental arms showed significant neutrophil infiltration between hours 0-4, only the experienced arm shows sustained neutrophil recruitment between hours 4-8 (Fig.1D).

For the first time ever, we use our transformative technology to isolate the components of immune-experience from the tissue-resident pulmonary population and to demonstrate their central role in hastening the immune response against pneumonia. Moreover, we are able to resolve the immune dynamics throughout this entire process in real-time at high spatiotemporal resolution. Moving forward, we are investigating the systemic contributions to immune-experience to compare with pulmonary contributions, mechanistic depletion studies for cells-of-interest in systemic vs. pulmonary compartments, and characterizing differential immune cell dynamics throughout pneumonia pathogenesis.

Acknowledgements (Optional): References
[1] Banerji et al., Nature Methods, 2023
[2] Shenoy et al., Mucosal Immunology, 2020