SP-C deficient mice have increased inflammation and prolonged recovery from injury following infection with RSV . A genetic model of regulated SP-C restoration was generated wherein proSP-C was expressed, processed to mature SP-C, and secreted in the airspace. When SP-C expression was induced in the lungs of Sftpc -/- mice prior to RSV infection, the pulmonary infiltration, viral titers, and morphologic indicators of injury were reduced. The increased susceptibility of Sftpc -/- mice to RSV and partial rescue by short-term alveolar expression supports the concept that SP-C contributes to innate alveolar defense against viral infection.
Reduced expression of SP-C can compromise lung defense by multiple mechanisms
Pulmonary surfactant is essential for normal respiration and for protection from inspired microorganisms. Rare forms of familial ILD have been linked to decreased SP-C expression and indicate that SP-C is required for lung homeostasis. The age of onset and severity of disease can vary significantly between affected family members ranging from an immediate neonatal RDS to childhood or adult onset ILD/IPF . This variable progression suggests the activity of additional disease modifiers.
The complex underlying molecular pathology of SP-C related ILD results from decreased SFTPC gene expression through mutations that alter the structure and abundance of either mRNA or protein. In addition, familial SP-C deficient ILD has been reported without any structural SFTPC mutation [4, 5]. The affected individuals appear to be a true null phenotype. A recent report identifies individuals with SP-C deficiency due to clustered mutations in a distal upstream promoter region and implicates this region as an essential regulatory element for SFTPC gene expression . Thus, the simple absence of SP-C can produce neonatal and/or childhood ILD.
The majority of individuals with SP-C related disease have mutations in the SFTPC gene. Mutations are primarily dispersed throughout the region downstream of the mature peptide altering mRNA splicing, proprotein processing, and proprotein structure and folding [3, 22, 23]. Mutations that alter processing or structure can induce both a cellular stress response as well as produce a net decrease in the amount of the secretory form of the protein. These distinct genetic alterations and resulting protein abnormalities may account for the reported pleiotropic phenotype. The Sftpc -/- mice have been used to explore the impact of the deficiency separate from deleterious effects of aberrant forms of proSP-C.
SP-C abnormalities increase the severity of RSV lung disease
Viral infections are a risk factor for preexisting conditions that include immunosuppression, cystic fibrosis, or prematurity . In a similar pattern, the pulmonary disease in young SFTPC patients can be complicated by influenza A/B virus or RSV infection. RSV infection is a common cause of respiratory infections in children that can vary from mild upper airway disease to severe bronchiolitis that requires hospitalization. RSV bronchiolitis during infancy also increases the incidence of chronic respiratory conditions such as wheezing, asthma, and allergy later into childhood [11, 24]. RSV related exacerbation has occurred in affected individuals either with or without a defined SFTPC mutation [2, 9, 10]. In a separate study, expression of an SFTPC mutation in vitro rendered the cells more susceptible to RSV infection . In support of the human clinical findings, a sub-strain of cattle with hereditary SP-C deficiency has an increased incidence of newborn respiratory distress. These cattle had increased fatal lung disease following an attempt to vaccinate against bovine RSV . The findings from human case histories, as well as bovine and mouse model of SP-C deficiency indicate that SP-C is an important factor in both the control of RSV infection and recovery from RSV infection.
In the current study Sftpc -/- type II cells had increased basal levels of key cytokines and expressed increased cytokine levels when challenged with either synthetic dsRNA or upon RSV infection in vitro. Thus the absence of SP-C is sufficient to predispose the type II cell to aberrant inflammation upon viral infection. These findings indicate that type II cell function can contribute to alveolar defense during severe RSV infection that in part relies upon the immune-protective activity of SP-C.
Restoration of SP-C in type II cells improves the outcome of RSV infection
RSV infection of Sftpc +/+ and -/- mice produced increased tissue and cellular inflammation in the lungs of the Sftpc -/- mice . Recovery from RSV related disease was incomplete in the lungs of Sftpc -/- mice thirty days after initial infection. As a first step to determine how SP-C confers alveolar protection, we have transiently replaced SP-C in a cell correct manner in vivo to assess reduction of inflammation and viral clearance. The SP-C proprotein was expressed and processed to the mature peptide that was secreted and detected in the BALF. SP-C expression was only detected following dox administration consistent with tight regulation of the SP-C transgene. Tissue inflammation and inflammatory cell infiltration was decreased on day 3 post-infection in the line 55.3/Sftpc -/- mice on doxycycline while more severe histopathology was apparent in the same mice without the induced SP-C expression. The incomplete reversal of RSV-induced injury may reflect the pattern of induced transgenic SP-C expression in a subset of type II cells when compared to wild type mice. The presence of the CCSP-rtTA transgene has been reported to alter alveolar size in mice on a different genetic background than the mice in this study and by inbreeding of lines of mice . Based upon histological examination of three sections from three representative mice in each group there were no indications of differences in morphology between either the CCSP-rtTA or tetO7-SP-C transgenic mice or in two independent lines of the double transgenic-Sftpc -/- mice (lines 55.3 and 54.2) on the defined 129S6 genetic background.
Other surfactant components modify the outcome of RSV infection
Additional protein and lipid components of pulmonary surfactant have been reported to influence the pulmonary response to RSV infection. Previous studies have shown that SP-A and SP-D also reduce injury from RSV infection. Both SP-A and SP-D (Sftpa-/-, Sftpd -/-) deficient mice were more susceptible to RSV infection. Co-administration of isolated SP-A with virus at the time of instillation reduced the cellular inflammation and increased viral clearance from the lungs of Sftpa -/- mice [28, 29]. However neither SP-A nor D levels were altered in the BALF from lungs of RSV infected Sftpc -/- mice relative to Sftpc +/+ mice . Thus neither SP-A nor SP-D influenced RSV infection in the SP-C deficient lung.
In the present study, vesicles generated with the major representative surfactant phospholipid species (DPPC and POPC) did not reduce the viral infection in vitro. The DPPC:POPC vesicles complexed with SP-C decreased RSV infectivity indicating that SP-C associated with major surfactant phospholipids confers a protective role during RSV infection. The phospatidylglycerol (PG) derivative palmitoyloleoyl-phosphatidylglycerol (POPG) is found in pulmonary surfactant and was shown to independently suppress RSV infectivity . The activity of pure POPG was noted at concentrations eight fold above phospholipid levels tested in this study. It is unknown if POPG in surfactant-like phospholipid mixtures would confer antiviral activity on the order of our SP-C dependent findings. While not directly comparable, the POPG activity could exert a synergistic affect with SP-C to modify the outcome of RSV infection. The surfactant phospholipid composition of Sftpc -/- mice was unchanged from that of Sftpc +/+ mice inferring that there is no change in potential PG derived phospholipid antiviral activity between the two Sftpc genotypes .
Immuno-protection by SP-C extends to other lung pathogens
The function of SP-C as a key protective component of surfactant was identified in a model of influenza A infection. The commercial surfactant extract Surfacten® acted as an adjuvant increasing the effective nasal mucosal immunity upon viral challenge . Surfacten lacks SP-A and SP-D and is enriched in SP-C. Representative surfactant phospholipid and protein mixtures were assessed to identify active components that confer the viral immune response. Synthetic phospholipid mixtures similar to the commercial Surfacten preparation alone or in combination with SP-B did not induce a protective antiviral response. However addition of SP-C to the phospholipid preparations produced a nasal IgA response equivalent to the Surfacten-influenza mixture . These findings on SP-C mediated influenza protection along with the current SP-C regulation of RSV injury directly infer an immune-protective role for SP-C independent of representative surfactant phospholipids alone.
Some SFTPC patient histories document recurrent infections. These findings are supported by a bacterial infection study in the Sftpc -/- mice demonstrating reduced clearance of the respiratory pathogen Pseudomonas aeruginosa. Taken together these reports identify a protective role for SP-C during both bacterial and viral infections.
The Toll-like receptor TLR3 influences the type II cell response in SP-C deficient mice
While the epithelial response to RSV is complex, cytokine activation and the activity of the Toll-like innate immune receptors have been shown to be integral to the inflammatory and epithelial protective reaction to RSV [34, 35]. Because SP-C is synthesized and secreted in the alveolus we hypothesized that the increased susceptibility to infection resulted from compromised type II cells. Sftpc -/- type II cells had increased basal levels of key cytokines and expressed increased cytokine levels when challenged in vitro with either the TLR3 specific ligand poly I:C, or RSV infection. TLR3 has previously been shown to mediate immune response to RSV infection and SP-C blocked TLR3 signaling in vitro[14, 36]. In addition Sftpc -/- mice had increased lung inflammation when challenged in vivo with poly I:C. Our findings indicate that the absence of SP-C is sufficient to predispose the type II cell to viral infection and stimulate TLR3 mediated inflammatory responses. TLR3 is associated with intracellular signaling vesicles and SP-C is an obligate membrane or phospholipid associated lipopeptide. The increased intrinsic expression of inflammatory cytokines by SP-C deficient type II cells suggest the interpretation that under normal conditions there is intracellular vesicle-to-vesicle interaction where SP-C restrains unwarranted or non-specific activation of TLR3 inflammatory signaling. Collectively our results support the concept that type II cells contribute to alveolar defense during severe RSV infection that in part relies upon the immune-protective activity of SP-C.