Abstract
BackgroundIndividuals with deficiencies of pulmonary surfactant protein C (SP-C) develop interstitial lung disease (ILD) that is exacerbated by viral infections including respiratory syncytial virus (RSV). SP-C gene targeted mice (Sftpc -/-) lack SP-C, develop an ILD-like disease and are susceptible to infection with RSV.MethodsIn order to determine requirements for correction of RSV induced injury we have generated compound transgenic mice where SP-C expression can be induced on the Sftpc -/- background (SP-C/Sftpc -/-) by the administration of doxycycline (dox). The pattern of induced SP-C expression was determined by immunohistochemistry and processing by Western blot analysis. Tissue and cellular inflammation was measured following RSV infection and the RSV-induced cytokine response of isolated Sftpc +/+ and -/- type II cells determined.ResultsAfter 5 days of dox administration transgene SP-C mRNA expression was detected by RT-PCR in the lungs of two independent lines of bitransgenic SP-C/Sftpc -/- mice (lines 55.3 and 54.2). ProSP-C was expressed in the lung, and mature SP-C was detected by Western blot analysis of the lavage fluid from both lines of SP-C/Sftpc -/- mice. Induced SP-C expression was localized to alveolar type II cells by immunostaining with an antibody to proSP-C. Line 55.3 SP-C/Sftpc -/- mice were maintained on or off dox for 7 days and infected with 2.6x107 RSV pfu. On day 3 post RSV infection total inflammatory cell counts were reduced in the lavage of dox treated 55.3 SP-C/Sftpc -/- mice (p = 0.004). The percentage of neutrophils was reduced (p = 0.05). The viral titers of lung homogenates from dox treated 55.3 SP-C/Sftpc -/- mice were decreased relative to 55.3 SP-C/Sftpc -/- mice without dox (p = 0.01). The cytokine response of Sftpc -/- type II cells to RSV was increased over that of Sftpc +/+ cells.ConclusionsTransgenic restoration of SP-C reduced inflammation and improved viral clearance in the lungs of SP-C deficient mice. The loss of SP-C in alveolar type II cells compromises their response to infection. These findings show that the restoration of SP-C in Sftpc -/- mice in response to RSV infection is a useful model to determine parameters for therapeutic intervention.
Highlights
surfactant protein C (SP-C) is an abundant surfactant associated lipoprotein expressed in alveolar type II epithelial cells that synthesize and secrete pulmonary surfactant into the airspace
Because the Clara cell secretory protein (CCSP)-rtTA transgenic line was tightly regulated by dox administration and the transgene was expressed in the SP-C producing type II cells the CCSP-rtTA transactivator line was used to breed and establish the model of inducible SP-C replacement
Four of the founder tetO7CMV-SP-C mice were successfully bred to Sftpc -/- mice on the 129S6 background and crossed with CCSP-rtTA/Sftpc -/mice in order to test F1 offspring for inducible transgene SP-C expression to selectively replace SP-C in the lungs of the SP-C deficient, Sftpc -/- mice
Summary
SP-C is an abundant surfactant associated lipoprotein expressed in alveolar type II epithelial cells that synthesize and secrete pulmonary surfactant into the airspace. Individuals lacking SP-C survive but develop a variety of interstitial or fibrotic lung disease outcomes [1]. The SFTPC mutations have distinct consequences, altering mRNA splicing, proprotein structure, protein expression, or processing These mutation-derived defects induce both a cellular stress injury due to altered structure and processing of the SP-C proprotein and a decrease in the amount of functional mature SP-C lipoprotein released into the airspace. On the 129S6 background Sftpc -/- mice develop parenchymal lung injury with age that is similar to the interstitial lung disease (ILD) reported for individuals with deficiencies of SP-C or SFTPC mutations [7,8]. Individuals with deficiencies of pulmonary surfactant protein C (SP-C) develop interstitial lung disease (ILD) that is exacerbated by viral infections including respiratory syncytial virus (RSV). SP-C gene targeted mice (Sftpc -/-) lack SP-C, develop an ILD-like disease and are susceptible to infection with RSV
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