Optical Methods for Determining the Phagocytic Activity Profile of CD206-Positive Macrophages Extracted from Bronchoalveolar Lavage by Specific Mannosylated Polymeric Ligands

Macrophage (Mph) polarization and functional activity play an important role in the development of inflammatory lung conditions. The previously widely used bimodal classification of Mph into M1 and M2 does not adequately reflect the full range of changes in polarization and functional diversity observed in Mph in response to various stimuli and disease states. Here, we have developed a model for the direct assessment of Mph from bronchial alveolar lavage fluid (BALF) functional alterations, in terms of phagocytosis activity, depending on external stimuli, such as exposure to a range of bacteria (<i>E. coli</i>, <i>B. subtilis</i> and <i>L. fermentum</i>). We have employed polymeric mannosylated ligands (the "trapping ligand") specifically targeting the CD206 receptor to selectively isolate activated Mph from the BALF of patients with pulmonary inflammatory conditions: primary ciliary dyskinesia (PCD), pneumonia and bronchial asthma. An "imaging ligand" allows for the subsequent visualization of the isolated cells using a sandwich technique. Five model strains of <i>E. coli</i>, MH-1, JM109, BL21, W3110 and ATCC25922, as well as <i>B. subtilis</i> and <i>L. fermentum</i> strains, each exhibiting distinct properties and expressing red fluorescent protein (RFP), were used as a phagocytosis substrate. Fluorometric, FTIR- and confocal laser scanning microscopy (CLSM) assessments of the phagocytic response of Mph to these bacterial cells were performed. Mph absorbed different strains of <i>E. coli</i> with different activities due to the difference in the surface villosity of bacterial cells (pili and fimbriae, as well as signal patterns). In the presence of other competitor cells (like those of <i>Lactobacilli</i>), the phagocytic activity of Mph is changed between two and five times and strongly dependent on the bacterial strain. The relative phagocytic activity indexes obtained for BALF-Mph in comparison with that obtained for model human CD206+ Mph in the M1 polarization state (derived from THP-1 monocyte cultures) were considered as a set of parameters to define the Mph polarization profile from the BALF of patients. Mannan as a marker determining the selectivity of the binding to the CD 206 mannose receptor of Mph significantly inhibited the phagocytosis of <i>E. coli</i> and <i>B. subtilis</i> in cases of pneumonia, suggesting an important role of CD206 overexpression in acute inflammation. Conversely, <i>L. fermentum</i> binding was enhanced in PCD, possibly reflecting altered macrophage responsiveness in chronic lung diseases. Our approach based on the profiling of Mph from patient BALF samples in terms of phagocytosis for a range of model bacterial strains is important for the subsequent detailed study of the factors determining dangerous conditions and resistance to existing therapeutic options.