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

Abstract

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 SPA 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 SPA treatment. Future studies may identify key protein(s) responsible for CMP integrity and consequently for a given function, and target it for therapeutic purposes.

Differences in the alveolar macrophage toponome in humanized SP-A1 and SP-A2 transgenic mice

“…The mean values of 2-of-4 + 1-of-4 totals were compared with a Kruskal-Wallis test and differed significantly (p=0.007). As shown previously (8), AM are heterogeneous and while conditions in the alveolus, such as SP-A levels, may favor certain phenotypes, it is likely that most phenotypes are found in more than one group, although their relative abundance may vary.…”

“…AF varies from cell to cell and is a useful characteristic for analyzing myeloid cells ( 32 ). AF tends to be localized in intracytoplasmic organelles that may contain NAD(P)H, flavins, ceroid/lipofuscin, bilirubin, and porphyrins, among others ( 32 ), and is often punctate or granular in nature ( 8 ) ( Supplemental Figure 1 ; supplemental material available online with this article; https://doi.org/10.1172/jci.insight.141410DS1 ). The positive organelles may be related to AM bactericidal capacity.…”

“…However, none of the exclusive phenotypes were from SP-A2 AM. A strategy described previously (8) was also used to identify cell subgroups that were almost exclusive for each group, although some overlap with other groups was common. First, for each biomarker the range of its presence in all cells of the data set was determined in order to define high and low levels of that marker (Table 8) .…”

“…What is not well investigated is whether the “resting” phenotype is based on a single phenotype or a variety of phenotypes. Previous studies ( 7 ), including one employing the technology used here ( 8 ), showed that alveolar macrophages (AMs) in the “resting” or unstimulated state are heterogeneous and that various insults or stimulatory protocols may result in heterogeneous cell populations.…”

“…Treating SP-A–KO mice acutely with SP-A variants produces more pronounced effects ( 19 ) than those seen in hTG mice with chronic or constitutive exposure to SP-A ( 18 ). Building on previous studies, we investigated the effects of rescue with a single dose of exogenous SP-A1 on the AM toponome by investigating combinatorial molecular phenotypes (CMPs) of proteins ( 8 ) to study their spatial network within intact cells. CMP is a designation describing the presence or absence of all markers in a given pixel, rather than simply variations in expression of a single marker.…”