Differences in the alveolar macrophage proteome in transgenic mice expressing human SPA 1 and SPA 2

Surfactant protein A (SP-A) plays a number of roles in lung host defense and innate immunity. There are two human genes, SFTPA1 and SFTPA2, and evidence indicates that the function of SP-A1 and SP-A2 proteins differ in several respects. To investigate the impact of SP-A1 and SP-A2 on the alveolar macrophage (AM) phenotype, we generated humanized transgenic (hTG) mice on the SP-A knockout (KO) background, each expressing human SP-A1 or SP-A2. Using two-dimensional difference gel electrophoresis (2D-DIGE) we studied the AM cellular proteome. We compared mouse lines expressing high levels of SPA1, high levels of SP-A2, low levels of SP-A1, and low levels of SP-A2, with wild type (WT) and SP-A KO mice. AM from mice expressing high levels of SP-A2 were the most similar to WT mice, particularly for proteins related to actin and the cytoskeleton, as well as proteins regulated by Nrf2. The expression patterns from mouse lines expressing higher levels of the transgenes were almost the inverse of one another - the most highly expressed proteins in SP-A2 exhibited the lowest levels in the SP-A1 mice and vice versa. The mouse lines where each expressed low levels of SP-A1 or SP-A2 transgene had very similar protein expression patterns suggesting that responses to low levels of SP-A are independent of SP-A genotype, whereas the responses to higher amounts of SP-A are genotype-dependent. Together these observations indicate that in vivo exposure to SP-A1 or SP-A2 differentially affects the proteomic expression of AMs, with SP-A2 being more similar to WT.


INTRODUCTION
1 min. PCR products were resolved on 1% agarose and confirmed by sequencing.

SP-A assessment
Initial characterization of BAL fluid was done to confirm the presence of the expected SP-A protein.
Blots were prepared as described subsequently and immunostained with either an antibody specific to SP-A1 [48] or an antibody specific for SP-A2 (Aviva Systems Biology, San Diego, CA). Because these two antibodies differed considerably in their relative affinities, these were used for positive identification of SP-A1 and SP-A2 transgenes and not for quantitation.
To determine the amount of secreted hSP-A in the lungs of hTG mice, lungs were lavaged and lavage samples (25 µl of BAL/sample) were subjected to gel electrophoresis (12.5% SDS-PAGE) under reducing conditions. Human SP-A in the BAL fluid was detected by Western blot analysis using a specific SP-A antibody that recognizes both SP-A1 and SP-A2, as described previously [40]. Briefly, proteins were then transferred to nitrocellulose and SP-A detected using a rabbit antibody (IgG) to human SP-A (1:20,000) and goat anti-

Supplementary Table 1. Primers used in this study
Supplementary Table 1 secondary antibody. Following enhanced chemiluminescent detection, blots were exposed to Xray film and densitometry performed on the films using a Bio-Rad GS-800 Densitometer and Quantity One Software.

Proteomics
For the proteomic study we used humanized transgenic mice expressing SP-A1 and SP-A2 at high levels and two mouse lines that expressed SP-A1 and SP -A2 at low levels, in addition to KO mice and WT mice.
The SP-A1 mouse lines were 6A 2 T1 (subsequently referred to as 6A 2 ) that expressed high levels of SP-A1, and a mouse expressing low levels of SP-A1 that is

Generation of heat maps
Heat maps were generated in Excel (Microsoft).

SP-A hTG mice and transgene copy number
First, we confirmed the presence of the transgene by Southern Blot (Figure 1)     and 6A 2 lines (Figure 2)) did not have a major influence on the differences between these mouse lines and that these differences were largely variant-specific rather than dose-specific. As in the lower expressing mouse lines, there were some proteins (n=10) that were significantly different from KO in both lines. However, of these ten, while five were similarly regulated in both mouse lines (actin-related protein 2/3 complex, subunit 5; heat shock protein 5 precursor; keratin complex 2, basic, gene 8; prolyl 4-hydroxylase, beta polypeptide precursor; protein synthesis initiation factor 4A), there were five significantly changed proteins that were increased relative to KO mice in one mouse line and decreased in the other (ferritin heavy chain; gelsolin precursor; high mobility group 1 protein; protein disulfide isomerase associated 6; Rab GDP dissociation inhibitor beta). This implied that although the expression of the nine proteins was human SP-A-

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effects and the consequences of the above-described proteomic changes in vivo is an important undertaking for future studies. However, based on the distinct proteomic differences observed in the present study, we speculate that significant differences will be found in vivo between SP-A1 and SP-A2 in terms of their ability to regulate inflammatory processes, responses to oxidative stress, and other functions.

Low expressors:
It was interesting to note that although there were many differences in protein expression between AM from mice with higher levels of SP-A1 and SP-A2, a high degree of similarity was observed between mice expressing low levels of SP-A2    Table 2A) and each of these two low expressing hTG mouse lines.  It should be noted that despite the fact that the 6A 2 mice had lower levels of SP-A than 1A 0 mice did, they exhibited almost 50% more significant changes vs KO, further reinforcing that the different response patterns reflect primarily functional differences between 1A 0 and 6A 2 rather than differences between absolute amounts of SP-A. Further evaluation of the expression data for individual proteins in this study did not show that differences in specific protein levels between the 1A 0 and 6A 2 mice were due to levels of SP-A, but rather due to the specific variant present. This was also supported by the very different response patterns elicited by 1A 0 and

Summary
In the present study we compared the AM proteome of hTG mice to KO mice and were able to gain insight into changes in protein expression resulting from the presence of the SP-A transgenes. We studied the expression of 76 AM proteins using a 2D-DIGE experimental design. We found that even when SP-A was expressed at low levels the AM proteome differed markedly from that of SP-A KO mice. While there were relatively few significant differences from KO, the similarity in these responses led us to speculate that these low amounts of SP-A might be affecting the AM by acting through a high affinity receptor that was not variant-specific.
On the other hand, when we studied the mice expressing higher levels of SP-A there were more than twice as many significant changes, with greater numbers of changes occurring in the SP-A1 (6A 2 ) mice. The other difference between these mouse lines that became apparent with the study of heat maps was that these two   This study was supported in part by R01 ES009882 from the National Institute of Environmental Health Sciences.

SUPPLEMENTARY MATERIALS
Supplementary