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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 ( = 3) was treated with exogenous human surfactant protein A1 (hSP-A1) and the other with vehicle ( = 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

We compared identical CMPs in KO and SP-A1 cells and found them to differ significantly ( = 0.0007). Similarities between pairs of markers in the two populations also differed significantly ( < 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.

BACKGROUND

METHODS

RESULTS

We compared identical CMPs in KO and SP-A1 cells and found them to differ significantly ( = 0.0007). Similarities between pairs of markers in the two populations also differed significantly ( < 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

背景

我们使用拓扑基因组成像系统（TIS）来识别“标志物表达模式”，在此称为肺泡巨噬细胞（AM）中对固有免疫分子SP-A1产生反应的组合分子表型（CMP）。

方法

我们比较了来自雄性SP-A缺陷小鼠的114个肺泡巨噬细胞。一组（n = 3）用外源性人表面活性蛋白A1（hSP-A1）处理，另一组用赋形剂处理（n = 3）。通过支气管肺泡灌洗获得的肺泡巨噬细胞被铺在载玻片上，并使用TIS进行分析，以研究肺泡巨噬细胞拓扑基因组，即完整细胞内蛋白质的空间网络。使用TIS时，每张载玻片依次用多种异硫氰酸荧光素（FITC）偶联抗体进行免疫染色。对图像进行逐像素分析，识别每个像素内的所有蛋白质，然后将其指定为CMP。CMP代表推测有助于特定功能的有组织的蛋白质簇。

结果

1）我们比较了敲除（KO）细胞和SP-A1细胞中相同的CMP，发现它们有显著差异（P = 0.0007）。两个群体中各对标志物之间的相似性也有显著差异（P < 0.0001）。2）针对每个细胞中最丰富的20种CMP，我们开发了一种方法来生成表征不同细胞组的CMP“特征”。表型被定义为表现出相似CMP特征的细胞。i）肺泡巨噬细胞极其多样，每个组都包含具有多种表型的细胞。ii）在分析的114个肺泡巨噬细胞中，没有两个细胞是相同的。iii）然而，CMP特征可以区分组内和组间的细胞亚群。iv）一些细胞群体在SP-A1处理后富集，一些在没有SP-A1时更常见，还有一些似乎不受SP-A1存在的影响。v）我们还发现，与用赋形剂处理的小鼠相比，用SP-A1处理的小鼠中的肺泡巨噬细胞更多样化。