Date Published: April 3, 2014
Publisher: Public Library of Science
Author(s): Christoph Schneider, Samuel P. Nobs, Alex K. Heer, Michael Kurrer, Glynis Klinke, Nico van Rooijen, Johannes Vogel, Manfred Kopf, Andrew Pekosz.
Alveolar macrophages (AM) are critical for defense against bacterial and fungal infections. However, a definitive role of AM in viral infections remains unclear. We here report that AM play a key role in survival to influenza and vaccinia virus infection by maintaining lung function and thereby protecting from asphyxiation. Absence of AM in GM-CSF-deficient (Csf2−/−) mice or selective AM depletion in wild-type mice resulted in impaired gas exchange and fatal hypoxia associated with severe morbidity to influenza virus infection, while viral clearance was affected moderately. Virus-induced morbidity was far more severe in Csf2−/− mice lacking AM, as compared to Batf3-deficient mice lacking CD8α+ and CD103+ DCs. Csf2−/− mice showed intact anti-viral CD8+ T cell responses despite slightly impaired CD103+ DC development. Importantly, selective reconstitution of AM development in Csf2rb−/− mice by neonatal transfer of wild-type AM progenitors prevented severe morbidity and mortality, demonstrating that absence of AM alone is responsible for disease severity in mice lacking GM-CSF or its receptor. In addition, CD11c-Cre/Ppargfl/fl mice with a defect in AM but normal adaptive immunity showed increased morbidity and lung failure to influenza virus. Taken together, our results suggest a superior role of AM compared to CD103+ DCs in protection from acute influenza and vaccinia virus infection-induced morbidity and mortality.
Alveolar macrophages (AM) are lung-resident macrophages important for the maintenance of surfactant homeostasis in the alveolar space . Their importance for lung physiology becomes evident in a rare human syndrome termed “pulmonary alveolar proteinosis” (PAP), which is characterized by the accumulation of surfactant material and a varying degree of respiratory insufficiency . PAP patients have a higher risk for pulmonary infections with opportunistic pathogens . PAP typically occurs in patients that spontaneously develop GM-CSF autoantibodies  or carrying mutations in the GM-CSF receptor α chain  associated with impaired function and/or reduced numbers of AM. Similarly, mice lacking GM-CSF (Csf2−/−) or the receptor β chain (Csf2rb−/−) develop PAP , , ,  and display increased susceptibility to a range of bacterial and fungal infections, which is associated with impaired innate functions of AM , , , , . High phagocytic activity and expression of pattern recognition receptors provides them with the capacity to respond to bacterial and fungal pathogens. However, AM were also described to have anti-inflammatory properties based on direct inhibition of the antigen-presenting function of lung DCs  and production of immunosuppressive mediators such as IL-10 and nitric oxide . In addition, AM were proposed to sequester pulmonary antigen and thereby interfere with efficient priming of immune responses by DCs . In contrast to the well-described functions of AM in bacterial and fungal infections, their precise role during viral infections is poorly understood. AM have the capacity to endocytose adenovirus particles, which was impaired in cells isolated from Csf2-deficient mice . Furthermore, AM are potent producers of type I IFNs upon pulmonary virus infection , . A beneficial role of AM in influenza infection has been proposed based on AM depletion experiments , ,  and treatment of mice with GM-CSF that increased numbers of AM , . However, besides the described role of GM-CSF for AM function under steady-state conditions, GM-CSF also influences DCs as demonstrated by its positive effects on the homeostasis of CD103+ DCs  or the upregulation of CD103 on CD11b− DCs . Indeed, a recent report proposed that GM-CSF protects from lethal influenza virus infection by enhancing CD103+ DC mediated anti-viral T cell responses .
In this study, we revisited the role of GM-CSF in AM homeostasis and function of this cell population in respiratory viral infection. According to the current understanding, Csf2- and Csf2rb-deficient mice develop pulmonary alveolar proteinosis (PAP) due to a defect in terminal maturation of AM involving impaired lipid catabolism. However, using 8-parameter flow cytometry in combination with dead cell exclusion, we found that Csf2−/− mice were completely devoid of AM and presented a massive accumulation of dead CD45-negative cells, which are presumably epithelial cells, in the BAL and lung consistent with an important role of AM in removal of dead cells (efferocytosis). Indeed, usage of a viability dye in combination with CD45 staining was inevitable for the exclusion of highly auto-fluorescent dead cells and avoidance of misidentification as AM. We also observed increased numbers of neutrophils in the BAL of naive Csf2−/− mice supporting an association of impaired efferocytosis and chronic inflammatory lung disease . Using mixed bone marrow chimeras we demonstrated that AM exclusively differentiated from WT and not from Csf2rb−/− BM. The absence of Csf2rb−/− AM in mixed BM chimeras also excludes the possibility that these cells have developed and died subsequently due to a functional defect in surfactant metabolism and accumulation of cellular debris, as the mixed chimeras did not develop PAP pathology. These results demonstrate a cell intrinsic requirement of GM-CSFR signaling for licensing development of AM from a precursor cell. Our results are in line with a recent report describing highly reduced numbers of AM in Csf2−/− mice and a requirement of GM-CSF signaling for the reconstitution of AM post-irradiation . The license is provided by GM-CSF secretion of radio-resistant lung cells, as shown by our 4-way BM chimeras. Whether during homeostasis GM-CSF also influences the functionality of AM needs to be determined using inducible knock out strategies or antibody-mediated neutralization.