In the present study, we aimed to dissect the impact of chronic lung disease on the alveolar macrophage phenotype. To this end, we utilized the SPC-HAxTCR-HA transgenic mouse model for T cell-mediated lung inflammation. Despite different etiologies, a participation of lymphocyte immune responses has been suggested for the pathogenesis of several lung disorders such as COPD [
14] or sarcoidosis [
15] and SPC-HAxTCR-HA lungs phenocopy [
7] histopathological manifestations of human CLD, e.g., airway remodeling and lymphocytic infiltrations.
By using fluorescence-based cytometric assays, we detected several important morphological changes of alveolar macrophages in our in vivo model. In detail, we show that their size, structural, and intrinsic fluorescence properties are significantly affected within an inflammatory environment. Interestingly, alterations in airway macrophage SSC and autofluorescence have also been reported in another study in SHIP1 (an inositol polyphosphate-5-phosphatase)-deficient mice, that spontaneously develop severe lung inflammation associated with a pathologic M2-macrophage phenotype [
16]. These findings are of general interest for two reasons. First, FACS technology provides a valuable and commonly utilized tool to characterize inflammatory processes on airway mucosal surfaces. Yet the massive autofluorescence signals of airway macrophages within, e.g., FITC, PE, and PerCp detectors require well-considered panel design and the utilization of additional control stainings to correctly separate large intrinsic (false) from antibody-derived (true) fluorescence signals. Since we and others have proven fundamental differences of AM fluorescence properties in healthy versus pathologic conditions, it is likely to observe similar effects in other non-infectious and infectious respiratory disease settings. In practice, this would adversely affect accuracy of, e.g., cell surface molecule quantitation. Thus, for similar studies using comparative flow cytometric investigations on airway macrophages, the aforementioned effects should definitely be considered. Second, information on the mentioned parameters offers a simple additive tool to characterize the alveolar macrophage phenotype in more detail and to detect morphologic alterations that possibly hint to molecular and functional cellular adaptations. Accordingly, we could provide a link between affected fluorescent properties and activation, polarization, and functional alterations in alveolar macrophages in CLD. Here, the blunted AM-dependent TNF-
α response toward Gram-positive bacterial ligands in SPC-HAxTCR-HA airways suggests a functional impairment of these cells during infection with airborne pathogens. Of note, TNF-
α was shown to exert protective efficacy in several models of respiratory infection [
17‐
20]. In detail, this cytokine acts on macrophages as well as neutrophils by enhancing phagocytosis of opsonized bacteria and antibody-dependent cytotoxicity [
21,
22]. On the other hand, it was previously demonstrated that lung-specific overexpression of TNF-
α in mice was linked to a disease phenotype mirroring hallmark features of emphysema and pulmonary fibrosis [
23]. In this context, macrophages from pre-diseased human and murine lungs were found to mount blunted cytokine responses upon stimulation with bacterial ligands [
24,
25]. It is speculated that this airway hyporesponsiveness provides an advantage to the host by evading PAMP-induced immunopathology that would further compromise tissue integrity in the pre-inflamed lung. However, given the two faces of TNF-
α (and other pro-inflammatory molecules) in alveolar macrophage-mediated immune responses, further studies will be needed to explore their role on the shape of antibacterial host defense in the pre-diseased lung.