Lung Interstitial Macrophages Redefined: It Is Not That Simple Anymore.

Tissue resident macrophages (TRMFs) are required for embryonic development and postnatal tissue homeostasis, as well as for reinstating steady-state functionality after tissue injury (1). TRMFs function by secreting various growth factors and cytokines that promote cell regeneration and survival. In addition, TRMFs clear invading pathogens, surround dead cells (through a process known as efferocytosis), and clear extraneous cellular debris and fluids (2). Dysregulation of TRMF function has been associated with various acute and chronic diseases, including inflammatory lung injury (2, 3). Thus, it is crucial to understand the complexity of TRMF function to devise novel strategies for suppressing inflammation while accelerating tissue regeneration. In the lung, two major populations of TRMFs were previously thought to exist: alveolar (AMFs) and interstitial (IMFs) macrophages. AMFs reside within the alveolus, whereas IMFs localize to the interstitial space of the alveolar septum or alveolar corners (4) (Figure 1). Although AMFs have been the principal focus of study because of their salient localization and the feasibility of tracking their behavior during lung injury and repair, the identity of other subsets of TRMFs has remained largely elusive. AMFs act as lung sentinels by sensing pathogenic insults and rapidly triggering leukocyte recruitment to mount host defense (5). Recently, subsets of nonsessile and sessile AMFs have been described (6). Nonsessile AMFs patrol the alveolar space and initiate an inflammatory response after sensing inhaled pathogens or toxicants, whereas sessile AMFs remain attached to the alveolar epithelium and communicate with those cells to dampen the inflammatory response (6). In contrast, IMFs appear to be functionally and phenotypically distinct from AMFs (7, 8). IMFs are proportionally less abundant (z2–3-fold), have low phagocytic potential, and respond to pathogenic or inflammatory stimuli differently than do AMFs (7–9). IMFs have been shown to play an important role in blunting unwarranted immune reactions elicited by dendritic cells in steady-state and in response to pathologic stimuli (7). Strategies for depleting IMFs or AMFs using clodronate or F4/80 antibody (a pan monocytic/macrophage marker), respectively, have produced varied results. For example, depletion of IMFs, but not AMFs, exaggerated endotoxin/antigeninduced airway allergic responses (7), whereas depletion of sessile AMFs augmented LPS-induced lung injury (6). Thus, a more thorough molecular characterization of IMFs and AMFs and a better characterization of their exact functions is required for understanding the complexity of MF populations in the lung under normal conditions and in disease states. In the July issue of the Journal, Gibbings and colleagues (pp. 66–76) describe studies using complementary in vivo and ex vivo approaches to provide convincing evidence detailing the lack of IMFs in the alveolar–capillary lung interstitium during normal healthy steady-state conditions (10). These authors demonstrate that IMFs in fact reside within the bronchial interstitium in close proximity to lymphatic vessels, cells we herein refer to as bronchial IMFs (BIMFs). The authors classified them as BIMF1 (CD11c1MHCII), BIMF2 (CD11c1MHCII), and BIMF3 (CD11c1MHCII) subsets. The BIMFs have phagocytic potential similar to AMFs both in vivo and ex vivo, but express distinct yet overlapping MF-specific cell surface phenotype markers compared with AMFs. Gene expression analysis of these BIMFs clearly revealed the presence of monocyte-specific markers (e.g., CD14, CD163, and Csfr1) and the absence of AMF-specific expression markers (e.g., Marco, Siglec-F, and Csfr2), supporting the notion that BIMFs probably originate from circulating monocytes. Consistent with this view, increased expression of inflammatory mediators and receptors are noted in BIMFs compared with AMFs under steady-state. Using BrdU labeling and parabiotic mice, the authors show that within the three BIMF subsets, BIMF3 turnover rate is faster and they exhibit less phagocytic potential in vivo than do other two subtypes. They also report that three BIMFs subpopulations exist in other organs such as the skin, gut, and heart and express similar gene expression patterns, suggesting a ubiquitous nature. The existence of macrophages in the bronchial interstitium was noted previously in the lungs of the rhesus monkey (7, 8), further supporting the authors’ findings and implicating their potential roles in human lung disease or diseases. Previous studies have shown that MFs exist in the interstitium of alveoli and capillary. For example, immuno-colocalization studies in lung tissue sections with anti-CD11c and anti-F4/F80 or CD206 antibodies revealed coexistence of IMFs and AMFs in the alveoli (7, 8), which we refer to as alveolar IMFs (AIMFs). However, in the current study, the investigators used c-mer protooncogene tyrosine kinase (MerTK)-positive staining as the bona fide marker for detecting MFs (11) in the alveolar region of CX3CR1 GFP1 reporter mice. It is noteworthy that the authors detected CX3CR1 GFP1 cells within and around alveoli that were negative for MerTK immunostaining. It is possible that these cells either expressed low-to-undetectable levels of MerTK or represent another subset or subsets of AIMFs. Also, the authors detected MerTKGFP cells in the bronchial interstitium, but their identity remains unclear. The findings of Gibbings and colleagues (10) no doubt represent an important step toward defining the role of BIMFs in maintaining bronchial homeostasis. Further, their findings suggest