Sessile alveolar macrophages communicate with alveolar epithelium to modulate immunity

Abstract

The tissue-resident macrophages of barrier organs constitute the first line of defence against pathogens at the systemic interface with the ambient environment. In the lung, resident alveolar macrophages (AMs) provide a sentinel function against inhaled pathogens. Bacterial constituents ligate Toll-like receptors (TLRs) on AMs, causing AMs to secrete proinflammatory cytokines that activate alveolar epithelial receptors, leading to recruitment of neutrophils that engulf pathogens. Because the AM-induced response could itself cause tissue injury, it is unclear how AMs modulate the response to prevent injury. Here, using real-time alveolar imaging in situ, we show that a subset of AMs attached to the alveolar wall form connexin 43 (Cx43)-containing gap junction channels with the epithelium. During lipopolysaccharide-induced inflammation, the AMs remained sessile and attached to the alveoli, and they established intercommunication through synchronized Ca(2+) waves, using the epithelium as the conducting pathway. The intercommunication was immunosuppressive, involving Ca(2+)-dependent activation of Akt, because AM-specific knockout of Cx43 enhanced alveolar neutrophil recruitment and secretion of proinflammatory cytokines in the bronchoalveolar lavage. A picture emerges of a novel immunomodulatory process in which a subset of alveolus-attached AMs intercommunicates immunosuppressive signals to reduce endotoxin-induced lung inflammation.

Figure 1. Live confocal microscopy of AMs in situ

(a) Interstitial (arrowhead) and luminal (arrow) YFP⁺ cells (yellow-green) in autofluorescent alveoli (red). Sketch of imaged field. n = 15. (b) Immunofluorescence and quantification of alveolar (top) and interstitial (bottom) YFP⁺ cells. n = 4 or 5. (c) Dye (green) injection in alveolus (Alv). Interstitial (Int) MHC-II⁺ DC (red, arrowhead) and luminal AM (arrow). n = 3. (d) Merge of S. aureus (green, arrow in inset) with AMs (red; arrowhead in inset). n = 4 (e) Ca²⁺ uncaging in epithelium (dotted circle) spreads Ca²⁺ (arrow) to AM (arrowhead). Bars show GAP 26/27 effect. n = 4 or 5. (f) Alveoli with Cx43 (red) and calcein (green) show Cx43-low (arrowhead) and Cx43-high AMs (arrow) expressing YFP (yellow-green). High power images show calcein in AMs before (pre) and after (post) photobleaching. Line drawn by linear regression. n = 23 AMs, 4 lungs. (g) Cx43 (red), CD11c (green) and nuclei (blue) in AMs from BAL (B) or lung tissue (T). Bars show protein (left) and mRNA (right) expressions in AMs. n = 4. Scale bars, 15 μm. mean±SEM. *P<0.05.

Figure 2. Communicating Ca²⁺ spikes in AMs

(a) Fluorescent LPS (green), AM (red) and DC (blue) in interstitium (Int) and alveolar lumen (Alv). n = 3 (b) YFP-expressing AMs (yellow; topmost panel), pseudocolored sequential images show increased fluo-4 fluorescence (arrowheads), 24 h after LPS. Dashes sketch spike path between AMs. Tracings show synchronous AM responses (arrows). n = 4 (c) Ca²⁺ spikes normalized for 10 AMs per imaging field. Total, all spikes; Sync, Synchronous spikes; BL, baseline (n = 8); 1h, n = 6; 4h, n = 4; 24h, n = 12 (d) Ca²⁺ oscillations and spikes (arrows) in AMs and adjoining alveolar epithelium (AE) 24 h after LPS or PBS. GAP, GAP26/27. n = 4 (e) Ca²⁺ spikes are 24h after LPS, normalized for 10 AMs per imaging field.; Ctl, LPS alone (n = 12); PP, PPADS (n = 4); XeC, xestospongin C (n = 4); Ca⁰, Ca²⁺ depletion (n = 3). GAP, n = 5. Scale bars, 20 μm. Bars, mean±SEM. *P<0.05 versus Ctl or corresponding BL.

Figure 3. AM-MyD88 in inflammatory signaling

(a-c) Synchronized (Syn) and non-synchronized (Non) Ca²⁺ spikes (arrows) and oscillations in wild type (WT, n = 12), CD11cMyD88^−/− (My^−/−, n = 5) and CD11cCx43^−/− (Cx^−/−, n = 5) mice. (d) Alveoli (green), and Ly6G⁺ (red) and CD11b⁺ neutrophils (blue) 24 h after LPS. Scale bar, 30 μm. n = 4 (e) Responses are 24 h after treatments. LPS WT: n = 9, others: n = 4. Bars, mean±SEM. *P<0.05.

Figure 4. AM-epithelial signaling

(a) BAL-cytokine ELISA in CD11cCx43^−/− (Cx^−/−) and littermate control (LC) mice. nd, not detectable. n = 3 or 4. (b) Kaplan-Meier plots. n = 16 LC, 17 Cx43^−/−. (c,d) Data are 24 h after treatment. IP, immunoprecipitation. n = 4 (e) In situ immunofluorescence (red) of alveolar epithelium and YFP⁺ AMs (yellow-green) 24 h after LPS. n = 4 (f,g) Lung lysate Western blots and BAL leukocyte counts 24 h after LPS in lungs given scrambled (Sc) or CAMKKα-specific (Si) siRNA. n = 4 for PBS+si, n = 5 for LPS+si, others: n = 6. All blots (n = 3) from same sample set. CAMKKα and Actin processed on different gel. Bars, mean±SEM. *P<0.05.