Single-cell analysis reveals differential regulation of the alveolar macrophage actin cytoskeleton by surfactant proteins A1 and A2: implications of sex and aging

Abstract

BackgroundSurfactant protein A (SP-A) contributes to lung immunity by regulating inflammation and responses to microorganisms invading the lung. The huge genetic variability of SP-A in humans implies that this protein is highly important in tightly regulating the lung immune response. Proteomic studies have demonstrated that there are differential responses of the macrophages to SP-A1 and SP-A2 and that there are sex differences implicated in these responses.MethodsPurified SP-A variants were used for administration to alveolar macrophages from SP-A knockout (KO) mice for in vitro studies, and alveolar macrophages from humanized SP-A transgenic mice were isolated for ex vivo studies. The actin cytoskeleton was examined by fluorescence and confocal microscopy, and the macrophages were categorized according to the distribution of polymerized actin.ResultsIn accordance with previous data, we report that there are sex differences in the response of alveolar macrophages to SP-A1 and SP-A2. The cell size and F-actin content of the alveolar macrophages are sex- and age-dependent. Importantly, there are different subpopulations of cells with differential distribution of polymerized actin. In vitro, SP-A2 destabilizes actin in female, but not male, mice, and the same tendency is observed by SP-A1 in cells from male mice. Similarly, there are differences in the distribution of AM subpopulations isolated from SP-A transgenic mice depending on sex and age.ConclusionsThere are marked sex- and age-related differences in the alveolar macrophage phenotype as illustrated by F-actin staining between SP-A1 and SP-A2. Importantly, the phenotypic switch caused by the different SP-A variants is subtle, and pertains to the frequency of the observed subpopulations, demonstrating the need for single-cell analysis approaches. The differential responses of alveolar macrophages to SP-A1 and SP-A2 highlight the importance of genotype in immune regulation and the susceptibility to lung disease and the need for development of individualized treatment options.

Biological sex influences susceptibility to Acinetobacter baumannii pneumonia in mice

“…In this report we showed that alveolar macrophage depletion in female mice resulted in a decrease of the eukaryotic initiation factor 2 initiation machinery, possibly resulting in decreased protein translation as well as severe alterations in mitochondria, which led to metabolic dysfunctions. A recent report raises the hypothesis of sex-dependent actin cytoskeleton regulation in alveolar macrophages (33), which has the potential to affect several processes, such as phagocytosis.…”

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

“…B) As one moves from the most abundant to the less abundant CMPs the diversity of the CMP (i.e. the number of markers contributing to the particular CMP) increases (Panels A and B) shows the order [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] of the top 20 CMPs from most abundant to least abundant. Then this is followed (Panels A and B) by a column giving the CMP number (5-27 CMP#; from data in Fig.…”

“…Its many functions are subject to complex regulation through autocrine [1] and paracrine mechanisms, as well as environmental factors [2], and other cell types, such as type II alveolar epithelial cells [3], which are not only responsible for the production of surfactant, a complex of proteins and lipids which is essential for lung function, but also produce a number of immunoregulatory molecules [4]. There is a large body of evidence demonstrating that the surfactant constituent, surfactant protein A (SP-A) has a profound regulatory effect on the AM, including regulating proinflammatory cytokine production, enhancing phagocytosis, and influencing actin metabolism [5][6][7]. This multi-faceted regulatory pattern may be one of the reasons for the well-documented heterogeneity of AM in the lung [7][8][9][10].…”

“…There is a large body of evidence demonstrating that the surfactant constituent, surfactant protein A (SP-A) has a profound regulatory effect on the AM, including regulating proinflammatory cytokine production, enhancing phagocytosis, and influencing actin metabolism [5][6][7]. This multi-faceted regulatory pattern may be one of the reasons for the well-documented heterogeneity of AM in the lung [7][8][9][10]. A model formulated some years ago described macrophages as being either classically (M1) or alternatively (M2) activated [11].…”

“…In humans, however, unlike in rodents, there are two genes, Sftpa1 and Sftpa2, encoding SP-A1 and SP-A2, respectively, and these two gene products have been shown to have a differential impact on several AM functions. These functions include bacterial phagocytosis and cytokine production by AM [18,24,25], actin polymerization in the AM [7], and effects on the AM proteome and miRNome [26][27][28]. Moreover, the effects of SP-A variants on the regulation of the AM proteome and miRNome, survival, and pulmonary mechanics after infection, vary with sex [26][27][28][29][30].…”