Pleural Cavity-resident B Cells and Macrophages Form Immune Scaffolds on the Lung Surface in Response to Bacterial Stimuli

Abstract Rationale: Infectious and inflammatory pulmonary diseases can involve the pleural cavity, a unique immune niche that contains various leukocytes in mice and humans. The role of these cells in the response to thoracic infection remains largely unexplored. Herein, we test the hypothesis that patrolling pleural cavity immune cells are recruited to the pleural surface in response to bacteria or their components, contributing to infection resolution and concomitant lung inflammation. Methods: Using an intercostal approach, we administered vehicle (PBS, n=5), LPS (0.05 μg/μL, n=7), or pHRhodo green+ E.coli bioparticles (n=4) into the pleural cavities of WT B6 mice and LPS into B cell-deficient muMT mice (n=6). We also performed intratracheal administration of LPS (n=2 WT B6 mice). Naïve WT B6 (n=10) received no intervention. Pleural cavity lavage and chest walls with intact pleura were collected to quantify free-floating and tissue-adherent leukocytes, respectively. Human pleural tissue from healthy donor lung (n=4) and diseased lung explants (n=3), and pleural fluid from lung transplant recipients (n=9) were also collected for analysis by flow cytometry and whole-mount tissue confocal imaging. Results: Compared to naïve controls, pleural lavage F4/80hi CD11b+ resident macrophages and CD19+ B cells were significantly reduced and Ly6G+ neutrophils were increased in LPS- but not PBS-treated mice. Accordingly, we found numerous resident macrophage-B cell immune scaffolds adherent to the pleura 4 hours post-intrapleural or intratracheal administration of LPS, absent in naïve mice (Fig 1, top left). Reminiscent of bronchus-associated lymphoid tissue found in inflamed lungs, these adherent pleural leukocytes form remarkably circumscribed and organized structures, with B cells located centrally (Fig 1, top right). Strikingly, immune scaffolds were absent in B cell-deficient muMT mice post-intrapleural LPS challenge, indicating a pivotal role for B cells in organizing macrophages and other immune cells on the pleural surface. After administration of pH-sensitive E.coli bioparticles which fluoresce only in the acidic environment of phagolysosomes, immune scaffolds were highly pHRhodo green+, suggesting they function to phagocytose and clear bacteria during infection. We observed adherent resident macrophages and neutrophils on human pleural tissue from diseased explants (Fig 1, bottom right) but not healthy controls (Fig 1, bottom left) and identified CD19+ B cells, CD163+ CD11b+ resident macrophages and CD66+ CD11b+ neutrophils in human pleural fluid. Conclusions: Our data suggest that B cell-dependent formation of immune scaffolds on the pleural surface functions to clear pulmonary bacterial infection, implicating this phenomenon as an essential component of the host response to infection.

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