Human Surfactant Proteins (SP-) A1 and SP-A2: to Include or Not in Surfactant Replacement Therapy, and If Yes, Both or Which One?

Based on a large number of animal studies, surfactant protein A plays important roles in lung innate immunity under basal conditions and in response to various insults such as infection and oxidative stress. SP-A interacts with the Alveolar Macrophage (AM), the sentinel cell of innate immunity, and regulates many of its functions. These include the ability of the AM to produce cytokines and to carry out phagocytosis of various pathogens. Moreover, the AM proteomic expression profile has been shown to be significantly affected by SP-A. In a study where SP-A -/mice were treated (or rescued) in vivo with a single dose of human SP-A, the treatment nearly restored the AM proteomic expression to that of the wild type [1], supporting a role of SP-A on AM protein expression. Among the protein groups affected were actin-related/cytoskeletal proteins, which was the major group, proteins regulating inflammatory processes, and proteins related to Nrf2-mediated oxidative stress. Of interest, earlier studies have also indicated a role for SP-A in actin filament dynamics via its ability to enhance cell migration and AM chemotaxis [2], phagocytosis [3], other actin-dependent processes [4-6] and F-actin assembly [7]. The actin cytoskeleton is highly regulated and dynamic, with globular (G) and filamentous (F) actin, under the influence of many actin binding proteins, constantly changing by polymerization, depolymerization, branching, and remodeling, modulating inflammatory response and oxidative stress (Figure 1).


As shown in
signaling via NF-kB [8] and Nrf2 [9] can both depend on cytoskeletal changes, indicating that SP-A-mediated modulation of the actin cytoskeleton could have significant effects on AM responses. Moreover, the SP-A -/-rescue experiment [1] points to the possibility and feasibility of using SP-A as a therapeutic intervention in lung diseases influenced by innate immunity. Furthermore, based on animal studies [10] where small amounts of SP-A elicited significant differences in the AM proteome, it is possible that the SP-A amount needed may not be high, although, further experimentation will be needed to determine optimal amount and efficacy in the clinical setting.
However, humans, (unlike rodents) have two genes, SFTPA1 (SP-A1) and SFTPA2 (SP-A2) with several genetic variants identified for each SP-A1 and SP-A2 gene. These variants have been associated with many lung diseases including neonatal diseases, such as Respiratory Distress Syndrome (RDS) and Bronchopulmonary Dysplasia (BPD) [11]. The SP-A1 and SP-A2 gene products have been shown to differ in several functions related to innate immunity [12] and in surfactantrelated functions [13,14]. These differences have been reviewed previously [12] and include differences in their oligomerization status [13,15,16], their ability to form phospholipid monolayers [17], their ability to bind sugars [18], and their ability to enhance the phagocytic activity and cytokine production by the alveolar macrophages and macrophage-like cell lines, respectively [3,[19][20][21][22], as well inhibit surfactant secretion by Type II cells [13]. In most of these cases SP-A2 appeared to exhibit higher functional activity. However, in preliminary studies [23], we observed that SP-A1 produced a higher reduction in surface tension than SP-A2, with no significant differences observed between SP-A1 and SP-A that contained both SP-A1 and SP-A2. Furthermore, in the presence of oxidative stress the activity of SP-A2 to enhance phagocytosis may be reduced more than that of SP-A1 [3], and the SP-A2 resistance to trypsin is lower than that observed in SP-A1 and SP-A1/SP-A2 [15]. Collectively, these observations indicate that SP-A1 and SP-A2 may each be better for different functions in the lung i.e. either for surfactant-related functions and/or innate immunity. Thus, the addition of SP-A1 and/or SP-A2 in surfactant replacement therapies may be advantageous to both groups of functions, to innate immunity and surfactant, and in some cases and under certain conditions one may be better than the other. SP-A humanized Transgenic (hTG) mice, generated on the SP-A -/background where each hTG mouse carries a different human SP-A variant provide models to study the in vivo functional activity of each variant. With these mice we observed differences in the oligomerization pattern of SP-A1 and SP-A2 confirming in vitro findings and also observed that both SP-A1 and SP-A2 may be needed for an extracellular form of surfactant [14]. In addition, we have shown differences in the proteomic profile of AM derived from SP-A1 and SP-A2 hTG mice [24]. In other words, AM constitutively exposed to different human SP-A variants in vivo exhibit different expression profiles, and presumably this reflects different stages of functional activity either in one or more AM functions. In fact, the protein differences observed span several functional protein groups. Among these there were actin-related and cytoskeletal proteins, which was the major group of differentially regulated proteins by SP-A1 and SP-A2, as well as proteins involved in the regulation of inflammation, and proteins related to the Nrf2-mediated oxidative stress response [24]. As shown in Figure, remodeling of the actin cytoskeleton is central to several AM functions. Therefore, SP-A1 and SP-A2 are likely to differentially regulate several AM functions.
In the prematurely born infant, apart from potential exposure of the lung to varied levels of oxidative stress, one of the major complications is infection. Although innate immunity and the surfactant proteins could play a significant role in this process, none of the surfactant replacement therapies include the surfactant proteins SP-A1 and/or SP-A2, or SP-D [25]. Of interest, surfactant treatment of babies with RDS defined as "simple" or with pneumonia indicated that in the latter group, surfactant treatment was less effective [26]. In addition, after surfactant therapy, neonates with respiratory failure and Group B streptococcal infection, although most of them showed an improvement in gas-exchange, this improvement was slower compared to RDS [27]. Whether the outcome in RDS with or without infection, or in other diseases beyond RDS [28] would or could be better if SP-A is included in the surfactant preparations or given alone remains to be determined. Of interest, the efficacy of surfactant in its current form on bacterial pneumonia in preterm infants, remains an open question. An attempt to assess randomized clinical trials for efficacy of surfactant in late term and preterm infants with bacteria pneumonia could not reach any conclusions one way or another at the present time [29]. However, based on the available literature, and the role of human SP-A1 and SP-A2 on AM functions, it is imperative that considerations for including the surfactant proteins in existing surfactant preparations or using them individually in the absence of surfactant be investigated. Moreover, as noted above, SP-A1 and SP-A2 may also contribute to surfactant-related functions and therefore its potential therapeutic use may benefit these functions as well. Furthermore, considerations as to whether both or which genetic variant(s) for a given condition be used must not be overlooked. These questions must be investigated with further experimentation.  [1] show that SP-A has significant impact on the AM expression profile and specifically on the cytoskeletal/actin-related proteins. Based on the literature cytoskeletal changes may affect several signaling processes including NF-kB [8] and Nrf2 [9]. These processes in turn via transcriptional regulation modulate the inflammatory response and the response to oxidative stress. In addition cytoskeletal changes are key in AM activities such as phagocytosis, motility, and cell shape. Thus, SP-A, via its ability to alter expression of cytoskeletal/actin-related proteins in the AM, may affect several of the AM functions or responses [i.e. inflammation, oxidative stress, phagocytosis, and other]. For example, human SP-A variants have been shown to differentially enhance the phagocytic activity of AM [3] and mice that lacked SP-A exhibited a lower phagocytic activity and survival [10].
Because low levels of SP-A have been associated with neonatal disease (RDS, BPD) risk, as well as with risk in adult disease (IPF) or condition (lung trauma) [11], enhancing the expression of SP-A1, SP-A2, or both depending on the particular functional need may be another way to increase their levels in neonatal life. Although individuals with lung disease have been shown to have altered levels of SP-A, it is not known whether these reflect change in both gene products, or just one, and if the latter, which one. Given the functional differences between the two gene products and eventually functional differences among the variants of each gene, it is important to have a good understanding of the SP-A1 and SP-A2 gene-specific regulatory mechanisms in order to guide therapeutic decision making. Although differences in the regulation between SP-A1 and SP-A2 have been observed in organ cultures and in cell cultures, the details of the mechanisms are not fully understood [12]. Recent studies have implicated regulatory protein factors [30] and miRNAs [31] in the differential regulation of SP-A1 and SP-A2 expression. For example, some isoforms of the 14-3-3 family of proteins are specific for the translation of SP-A2, but not SP-A1 [30]. Whether these or other factors can in the future be used therapeutically to differentially regulate specific SP-A1 and SP-A2 expression remains to be determined.
In summary, serious consideration should be given to the question of whether SP-A1 and SP-A2, as well as SP-D (not discussed here in detail), should be included in surfactant replacement preparations or be given as an individual treatment. The mouse rescue experiment [1] discussed above for SP-A indicates feasibility and potentially a functional benefit to the AM as shown by changes in the cytoskeletal/ actin-related proteins. The cytoskeletal changes, as depicted in Figure, are central to several AM functions. Innate immunity, being the first line of defense, provides a logical target for therapy, because deranged innate immunity most likely leads to dire downstream consequences. Thus, ensuring a well-functioning AM is essential to lung health. SP-A1 and SP-A2 appear to be important regulators of AM protein expression and function, and using these therapeutically may help ameliorate disease severity in several conditions. Moreover, preliminary evidence indicates that SP-A1 and SP-A2 treatment may have a positive impact beyond innate immunity by benefiting surfactant-related functions, and perhaps clinical cases beyond RDS. Alternatively, understanding the regulatory mechanisms of SP-A1 and SP-A2 could help modulate SP-A1 and SP-A2 levels and therefore this line of investigation should be a research priority. In specific, such knowledge can help identify points of therapeutic intervention to enhance individual expression of SP-A1 and/or SP-A2 under certain conditions.