Using toponomics to characterize phenotypic diversity in alveolar macrophages from male mice treated with exogenous SP-A1

BACKGROUND: We used the Toponome Imaging System (TIS) to identify “patterns of marker expression”, referred to here as combinatorial molecular phenotypes (CMPs) in alveolar macrophages (AM) in response to the innate immune molecule, SP-A1. METHODS: We compared 114 AM from male SP-A deficient mice. One group (n = 3) was treated with exogenous human surfactant protein A1 (hSP-A1) and the other with vehicle (n = 3). AM obtained by bronchoalveolar lavage were plated onto slides and analyzed using TIS to study the AM toponome, the spatial network of proteins within intact cells. With TIS, each slide is sequentially immunostained with multiple FITC-conjugated antibodies. Images are analyzed pixel-by-pixel identifying all of the proteins within each pixel, which are then designated as CMPs. CMPs represent organized protein clusters postulated to contribute to specific functions. RESULTS: 1) We compared identical CMPs in KO and SP-A1 cells and found them to differ significantly (p = 0.0007). Similarities between pairs of markers in the two populations also differed significantly (p < 0.0001). 2) Focusing on the 20 most abundant CMPs for each cell, we developed a method to generate CMP “signatures” that characterized various groups of cells. Phenotypes were defined as cells exhibiting similar signatures of CMPs. i) AM were extremely diverse and each group contained cells with multiple phenotypes. ii) Among the 114 AM analyzed, no two cells were identical. iii) However, CMP signatures could distinguish among cell subpopulations within and between groups. iv) Some cell populations were enriched with SP-A1 treatment, some were more common without SP-A1, and some seemed not to be influenced by the presence of SP-A1. v) We also found that AM were more diverse in mice treated with SP-A1 compared to those treated with vehicle. CONCLUSIONS: AM diversity is far more extensive than originally thought. The increased diversity of SP-A1-treated mice points to the possibility that SP-A1 enhances or activates several pathways in the AM to better prepare it for its innate immune functions and other functions shown previously to be affected by SP-A treatment. Future studies may identify key protein(s) responsible for CMP integrity and consequently for a given function, and target it for therapeutic purposes.