The alveolar macrophage toponome of female SP-A knockout mice differs from that of males before and after SP-A1 rescue

Abstract

Using the Toponome Imaging System (TIS), a serial immunostainer, we studied the patterns of expression of multiple markers in alveolar macrophages (AM) from female mice lacking surfactant protein A (SP-A knockouts; KO) after “rescue” with exogenous SP-A1. We also used a 7-marker subset to compare with AM from males. AM were harvested 18 h after intrapharyngeal SP-A1 or vehicle, attached to slides, and subjected to serial immunostaining for 12 markers. Expression of the markers in each pixel of the image was analyzed both in the whole image and in individual selected cells. The marker combination in each pixel is referred to as a combinatorial molecular phenotype (CMP). A subset of antibodies was used to compare AM from male mice to the females. We found: (a) extensive AM heterogeneity in females by CMP analysis and by clustering analysis of CMPs in single cells; (b) AM from female KO mice respond to exogenous SP-A1 by increasing CMP phenotypic diversity and perhaps enhancing their potential innate immune capabilities; and (c) comparison of male and female AM responses to SP-A1 revealed that males respond more vigorously than females and clustering analysis was more effective in distinguishing males from females rather than treated from control.

Comparison of the Toponomes of Alveolar Macrophages From Wild Type and Surfactant Protein A Knockout Mice and Their Response to Infection

“…TIS was performed as described previously ( 14 – 16 ). As discussed in previous studies, the choice of antibodies was limited by the presence of artifacts in fluorescent images from some samples that may prevent the use of some of these images/markers for study.…”

“…Table 1 lists the nine antibodies or reagents we used in the image analysis, their gene names (where appropriate), their Uniprot accession numbers, the commercial source, and the catalog number of each antibody. Calibration and optimization of antibodies for the TIS procedure was done as previously described ( 14 – 16 ). Briefly, commercially available antibodies were tested at several dilutions.…”

“…The process employed to generate TIS data from a collection of immunofluorescent images has been described in detail in our previous publications ( 14 – 16 ) and is summarized in Figure 2 . Briefly, a sample slide is subjected to repeated cycles of immunostaining with fluorescein-labeled antibodies, an image captured, the fluorescence bleached out, and the cycle repeated with the next antibody.…”

“…In a number of previous reports we have studied the effect of SP-A on the function of the alveolar macrophage (AM) ( 1 ). These studies have examined the expression of inflammatory mediators ( 2 – 5 ), the AM proteome ( 6 – 9 ), miRNome ( 10 – 12 ), and AM gene expression ( 1 , 13 ), and more recently the AM toponome ( 14 – 16 ), i.e. the organization of proteins within the cells.…”

“…The technology used in the present study, the Toponome Imaging System (TIS), is an ideal tool with which one may study these events because it looks at combinations of proteins, rather than individual proteins. TIS, also known as Imaging Cycler Microscopy, or Multi-epitope ligand cartography (MELC), allows one to study the toponome or the expression and/or the presence of multiple markers in their spatial networks in intact AM after different experimental manipulations ( 14 – 16 , 37 – 39 ).…”