Sessile alveolar macrophages communicate with alveolar epithelium to modulate immunity

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 alveolarmacrophages (AMs)providea sentinel functionagainst inhaledpathogens.Bacterial constituents ligate Toll-like receptors (TLRs) onAMs, causingAMs 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 howAMsmodulate the response toprevent injury.Here, using real-time alveolar imaging in situ, we show that a subset ofAMsattached to the alveolarwall formconnexin43 (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 Cawaves, using the epithelium as the conducting pathway. The intercommunication was immunosuppressive, involvingCa-dependent activationofAkt, becauseAM-specific knockout of Cx43 enhanced alveolar neutrophil recruitment and secretionofproinflammatorycytokines in thebronchoalveolar lavage. Apicture emerges of a novel immunomodulatory process inwhich a subset of alveolus-attached AMs intercommunicates immunosuppressive signals to reduce endotoxin-induced lung inflammation. As most of our understanding of AMs is based on studies in which AMswere isolatedbybronchoalveolar lavage (BAL)and studied in vitro, or inwhich theywere depleted from the lungs, we lack an understanding of the dynamic interactions between AMs and the alveolar epithelium that might critically modulate the lung’s inflammatory response. To determine these interactions in situ, we took advantage of the fact that AMs express CD11c and crossed Cd11c-cre mice with Rosa26LSL-eYFP mice to obtain mice expressing enhanced yellow fluorescence protein (eYFP) in CD11c-expressing cells. Live confocal microscopy of isolated, blood-perfused mouse lungs derived from CD11c/eYFP mice revealed YFP cells in the subpleural interstitium and the alveolar lumen (Fig. 1a). To distinguishAMs from lung dendritic cells that also express CD11c, we gave mice intraalveolar microinjections of the AM marker Siglec F (ref. 13) and an antibody for the major histocompatibility complex class II (MHC-II), whichmarks lung dendritic cells. Thesemicroinjections revealed that luminal YFP cells were AMs, because they stained strongly for Siglec F but weakly forMHC-II (Fig. 1b), whereas interstitial YFP cells that we stainedby topical antibodyapplications to the saponin-permeabilized pleura were MHC-II/Siglec F dendritic cells (Fig. 1b). The alveolar epithelium inhibits transepithelial transit of reagents injected in the alveolar lumen.Hence, alveolarmicroinjections of dye resulted in fluorescence uptake in AMs, but not in dendritic cells (Fig. 1c), suggesting that dendritic cells did not communicate with the alveolar lumen. In further affirmation that luminal YFP cells were AMs, YFP-MHCII cells recovered in the BAL failed to induce T-cell proliferation in antigen-presentation assays (Extended Data Fig. 1a). Together, these findings indicate that YFP AMs localized to the alveolar lumen, whereas YFP dendritic cells were compartmentalized in the perialveolar interstitium. Wedetecteda singleAMforapproximately every three alveoli (Extended Data Fig. 1b), suggesting that AMs carry out pathogen surveillance by patrolling the alveolar surface. However, AMs remained stationary at fixed alveolar locations for the duration of our imaging studies, which lasted up to 4 h (ExtendedData Fig. 1c). Our attempts to dislodge AMs byBALor by direct alveolarmicroinjection of bufferwere unsuccessful (ExtendedData Fig. 1c). To determine whether AMsmight be induced tomigrate towards bacteria, wemicroinjected Staphylococcus aureus in the alveolar lumen. Although AMs rapidly ingested the S. aureus that lay within one AMdiameter, they did notmigrate towards the bacteria (Fig.1d and ExtendedData Fig. 1c). The alveolar liquid flow appeared to wash bacteria towards the AMs (data not shown). Thus, contrary to expectations, our findings indicate that AMs were sessile. Macrophages express Cx43 (ref. 16), potentially enabling AMs to form gap junction channels (GJCs) with the alveolar epithelium. To determine thepresence ofGJCs,we appliedphotolytic uncaging to induce cell-specific increases in cytosolic Ca (ref. 17), and fluorescence recovery after photobleaching (FRAP) toquantify intercellulardyediffusion. In 40% of AMs, uncaging-induced Ca waves travelled from the epithelium toAMs (Fig. 1e) and in the opposite direction (data not shown). Cx43 expression in AMs correlated directly with FRAP (Fig. 1f). A 1 h treatment with GAP26 and 27, inhibitors of Cx43-based GJCs and hemichannels, blocked uncaging-induced Ca waves (Fig. 1e) as well as FRAP (data not shown) between AMs and the epithelium. In CD11cMHC-II AMs, which we obtained by BAL and by extraction from lung tissue after BAL, respectively (ExtendedData Fig. 1a, d), Cx43 protein andmessenger RNAexpressionwerehigher in tissue than in BAL AMs (Fig. 1g), suggesting that Cx43 was higher in alveolusadherent than alveolus-non-adherent AMs. In mice with CD11cspecific Cx43 knockout (CD11cCx43) (Extended Data Fig. 2a), AMs remained immobile even after alveolar microinjections of bacteria or PBS buffer (Extended Data Fig. 2b, c). Hence, Cx43 was not responsible for AM immobility. In lungs given intranasal Escherichiacoli-derived lipopolysaccharide (LPS) instillation, neutrophils entered and migrated freely on the alveolar surface (Supplementary Fig. 1 and Supplementary Video 1), ruling out non-specific physical factors in causing AM immobility. Taken together, our findings reveal the presence of Cx43 and Cx43 AM populations in the lung, in which Cx43 AMs formed GJCs with the alveolar epithelium. Next, we gave mice intranasal LPS to induce lung injury or PBS as a control. We removed lungs 1, 4 or 24 h after the instillations to establish isolated perfused lungs for imaging studies. Instillation of fluorescent LPS confirmed LPS entry in AMs (Fig. 2a). Fluorescent LPS did not enter interstitial dendritic cells, indicating that in alveoli LPS ligated AMs, not dendritic cells. Fluorescent LPS uptake wasmarkedly greater in BAL-derived dendritic cells than in those recovered from the tissue