In vivo rescue of alveolar macrophages from SP-A knockout mice with exogenous SP-A nearly restores a wild type intracellular proteome; actin involvement

Center for Host defense, Inflammation, and Lung Disease (CHILD) Research and Department of Pediatrics, The Pennsylvania State University College of Medicine, Hershey, PA 17033, USA. .
Proteome Science (Impact Factor: 1.73). 10/2011; 9(1):67. DOI: 10.1186/1477-5956-9-67
Source: PubMed


Mice lacking surfactant protein-A (SP-A-/-; knockout; KO) exhibit increased vulnerability to infection and injury. Although many bronchoalveolar lavage (BAL) protein differences between KO and wild-type (WT) are rapidly reversed in KO after infection, their clinical course is still compromised. We studied the impact of SP-A on the alveolar macrophage (AM) proteome under basal conditions. Male SP-A KO mice were SP-A-treated (5 micrograms/mouse) and sacrificed in 6 or 18 hr. The AM proteomes of KO, SP-A-treated KO, and WT mice were studied by 2D-DIGE coupled with MALDI-ToF/ToF and AM actin distribution was examined by phalloidon staining.
We observed: a) significant differences from KO in WT or exogenous SP-A-treated in 45 of 76 identified proteins (both increases and decreases). These included actin-related/cytoskeletal proteins (involved in motility, phagocytosis, endocytosis), proteins of intracellular signaling, cell differentiation/regulation, regulation of inflammation, protease/chaperone function, and proteins related to Nrf2-mediated oxidative stress response pathway; b) SP-A-induced changes causing the AM proteome of the KO to resemble that of WT; and c) that SP-A treatment altered cell size and F-actin distribution.
These differences are likely to enhance AM function. The observations show for the first time that acute in vivo SP-A treatment of KO mice, under basal or unstimulated conditions, affects the expression of multiple AM proteins, alters F-actin distribution, and can restore much of the WT phenotype. We postulate that the SP-A-mediated expression profile of the AM places it in a state of "readiness" to successfully conduct its innate immune functions and ensure lung health.

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Available from: Omar Quintero, Oct 04, 2015
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    • "This manifested itself in several ways. When the total numbers of significantly changing protein spots and identified whole proteins were examined, in females there were many fewer changes in the 18 hr SP-A treatment and WT groups when each was compared to KO as opposed to what we observed for the males (Figure 2 and Table 2 versus our previously published study in males (see Additional file 2 in [29])). Furthermore, the principal component analysis that compared male and female WT and KO mice showed a much greater separation between the male and female WT groups than the KO groups, indicating that SP-A may play a role in the observed sex differences. "
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    ABSTRACT: Background Male wild type (WT) C57BL/6 mice are less capable of clearing bacteria and surviving from bacterial pneumonia than females. However, if an oxidative stress (acute ozone exposure) occurs before infection, the advantage shifts to males who then survive at higher rates than females. We have previously demonstrated that survival in surfactant protein-A (SP-A) knockout (KO) mice compared to WT was significantly reduced. Because the alveolar macrophage (AM) is pivotal in host defense we hypothesized that SP-A and circulating sex hormones are responsible for these sex differences. We used 2D-DIGE to examine the relationship of sex and SP-A on the AM proteome. The role of SP-A was investigated by treating SP-A KO mice with exogenous SP-A for 6 and 18 hr and studying its effects on the AM proteome. Results We found: 1) less variance between KO males and females than between the WT counterparts by principal component analysis, indicating that SP-A plays a role in sex differences; 2) fewer changes in females when the total numbers of significantly changing protein spots or identified whole proteins in WT or 18 hr SP-A-treated males or females were compared to their respective KO groups; 3) more proteins with functions related to chaperones or protease balance and Nrf2-regulated proteins changed in response to SP-A in females than in males; and 4) the overall pattern of SP-A induced changes in actin-related proteins were similar in both sexes, although males had more significant changes. Conclusions Although there seems to be an interaction between sex and the effect of SP-A, it is unclear what the responsible mechanisms are. However, we found that several of the proteins that were expressed at significantly higher levels in females than in males in WT and/or in KO mice are known to interact with the estrogen receptor and may thus play a role in the SP-A/sex interaction. These include major vault protein, chaperonin subunit 2 (beta) (CCT2), and Rho GDP alpha dissociation inhibitor. We conclude that sex differences exist in the proteome of AM derived from male and female mice and that SP-A contributes to these sex differences.
    Proteome Science 07/2012; 10(1):44. DOI:10.1186/1477-5956-10-44 · 1.73 Impact Factor
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    ABSTRACT: The key feature of respiratory distress syndrome (RDS) is the insufficient production of surfactant in the lungs of preterm infants. As a result, researchers have looked into the possibility of surfactant replacement therapy as a means of preventing and treating RDS. We sought to identify the role of surfactant in the prevention and management of RDS, comparing the various types, doses, and modes of administration, and the recent development. A PubMed search was carried out up to March 2012 using phrases: surfactant, respiratory distress syndrome, protein-containing surfactant, protein-free surfactant, natural surfactant, animal-derived surfactant, synthetic surfactant, lucinactant, surfaxin, surfactant protein-B, surfactant protein-C. Natural, or animal-derived, surfactant is currently the surfactant of choice in comparison to protein-free synthetic surfactant. However, it is hoped that the development of protein-containing synthetic surfactant, such as lucinactant, will rival the efficacy of natural surfactants, but without the risks of their possible side effects. Administration techniques have also been developed with nasal continuous positive airway pressure (nCPAP) and selective surfactant administration now recommended; multiple surfactant doses have also reported better outcomes. An aerosolised form of surfactant is being trialled in the hope that surfactant can be administered in a non-invasive way. Overall, the advancement, concerning the structure of surfactant and its mode of administration, offers an encouraging future in the management of RDS.
    The Open Respiratory Medicine Journal 07/2012; 6(1):44-53. DOI:10.2174/1874306401206010044
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    ABSTRACT: Human surfactant protein A, an innate immunity molecule is encoded by two genes SFTPA1 (SP-A1) and SFTPA2 (SP-A2). The 5' untranslated (5'UTR) splice variant of SP-A2 (ABD), but not of SP-A1 (AD), contains exon B (eB), which is an enhancer for transcription and translation. We investigated whether eB contains cis regulatory elements that bind trans-acting factors in a sequence-specific manner as well as the role of the eB mRNA secondary structure. Binding of cytoplasmic NCI-H441proteins to wild type eB-, eB mutants-, AD-, and ABD- 5'UTR mRNAs were studied by RNA electromobility shift assays (REMSAs). The bound proteins were identified by mass spectroscopy and specific antibodies (Abs). We found i) proteins bind eB mRNA in a sequence-specific manner, with two cis elements identified within eB to be important; ii) eB secondary structure is necessary for binding; iii) mass spectroscopy and specific Abs in REMSAs identified 14-3-3 proteins to bind (directly or indirectly) eB and the natural SP-A2 (ABD) splice variant but not the SP-A1 (AD) splice variant; iv) other ribosomal and cytoskeletal proteins, and translation factors are also present in the eB mRNA:protein complex; v) knock-down of 14-3-3 β/α isoform resulted in a downregulation of SP-A2 expression. In conclusion, proteins including the 14-3-3 family bind two cis elements within eB of hSP-A2 mRNA in a sequence- and secondary structure- specific manner. Differential regulation of SP-A1 and SP-A2 is mediated by the 14-3-3 protein family as well as by a number of other proteins that bind UTRs with or without eB mRNA.
    AJP Lung Cellular and Molecular Physiology 03/2013; 304(11). DOI:10.1152/ajplung.00324.2012 · 4.08 Impact Factor
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