Surfactant protein B (SP-B), a key component of surfactant, is essential for normal lung respiratory physiology. It plays critical roles in lowering surface tension to prevent alveoli from collapse and host defense against bacterial infection (1, 2). Human SP-B gene (sftpb) contains several genetic variations, including a critical single nucleotide polymorphism (SNP rs1130866), which has a nucleotide T to C transition. The nucleotide transition of the SNP alters the N-linked glycosylation status of the Asn129 site in pro-SP-B protein, i.e., the SP-B C allele with the additional N-linked glycosylation site but not in the T allele (3, 4). This altered glycosylation may have an implication on SP-B protein processing and function under disease or stress conditions, although both C and T alleles are common SP-B genetic variation in the general population (5, 6). Genotyping–association studies of patients demonstrated that patients with C allele are more susceptible than those with T allele to pneumonia and pneumonia-induced acute respiratory distress syndrome (ARDS) (7–9). However, the mechanisms underlying differential susceptibility of human SP-B genetic variants to pneumonia-induced acute lung injury (ALI) and ARDS are unclear yet (10, 11).
SP-B gene belongs to a member of Saposin-like protein (SAPLIN) family, which is involved in multiple functions such as lipid interaction and antimicrobial activity (2, 12). The SP-B precursor contains three SAPLIN domains: the first two domains produced two mature proteins, i.e., SP-BN and SP-BM, which are involved in host defense and surface tension, respectively (2). However, the function of the other SAPLIIN domain, which locates in C-terminal (SP-BC) region, is not known yet (2). SP-B expression is restricted to the alveolar type II cells and Club cells in the respiratory epithelium. The processing of pro-SP-B precursor to mature SP-B in type II cells in the lung requires proteolytic cleavages and a series of post-translational modifications, including glycosylation. The mature SP-B protein should be transported into the lamellar body, which consists of surfactant-specific phospholipid and other proteins (13, 14).
Humanized mouse models have proven to be powerful tools in the study of many human biological processes and excellent models for preclinical testing. Humanized transgenic (hTG) mouse model has helped overcome the limitation in the study of human biology in vivo. The physiological and pathological functions of human gene/variants (alleles) can be investigated in the complex biological or pathological processes using hTG mouse method (15, 16). In the last several years, we have generated and characterized hTG SP-B mice, i.e., SP-B-C and SP-B-T mice, which expressed either human SP-B C or T allele without mouse SP-B gene (17, 18). These hTG mice have provided us an excellent tool to elucidate the cellular and molecular mechanisms of the regulatory roles of human SP-B genetic variants on the differential susceptibility in response to bacterial-induced ALI/ARDS.
Pseudomonas aeruginosa, a gram-negative bacteria, is a clinically relevant pathogen, especially in the patients with ventilator-associated infection and in cystic fibrosis. Previous studies demonstrated that the FVB/N strain of mice is sensitive to this pathogen, although one common laboratory strain PA01 has moderate toxicity to mice (19, 20). To establish a severe septic mouse model and to monitor in vivo bacterial growth, we have used one P. aeruginosa strain Xen5 with high toxicity and expressing bioluminescence in this study. Using the pneumonia-induced ARDS model, we tested our hypothesis that SP-B genetic variants have differentially regulatory roles in the pathogenesis of ALI/ARDS. We examined dynamic bacterial growth in vivo, cellular and molecular changes, and surfactant dysfunction in the lung of the three types of mice after intratracheally bacterial infection by various cellular and molecular methodology. The findings from the present study demonstrated that human SP-B genetic variants differentially regulate susceptibility to pneumonia-induced ALI/ARDS through influencing bacterial growth and surfactant activation, modulating host cell death, inflammation, and inflammatory NF-κB signaling pathway.
MATERIALS AND METHODS
We used hTG SP-B-C and SP-B-T (FVB/N strain background) and wild-type FVB/N in this study. hTG SP-B-C and SP-B-T, carrying either human SP-B C or T allele without mouse SP-B gene, were generated in our laboratory; the genotypes and human SP-B expression were confirmed by PCR and Western blot as described previously (17). Original WT FVB/N mice were purchased from the Jackson Laboratory (Bar Harbor, Maine) and were bred for experimental use. All the animals were maintained under the pathogen-free condition in the animal core facility at SUNY Upstate Medical University, with a standard 12 h/12 h light/dark time cycle, the stable-controlled temperature at 24°C, and regular mouse chow and water ad libitum. Both male and female mice of each type (8–12 weeks old, about 25 g bw) were used for the experiments. In this study, each genetic variant (SP-B-C, SP-B-T and WT) of mice was divided into two groups: the pneumonia group (infected with P. aeruginosa Xen5 strain) and the control group (the sham group with same surgery and the same volume of the sterile vehicle given). All the related animal protocols were strictly adhered to according to with National Institutes of Health and ARRIVE guidelines and approved by the Institutional Animal Care and Use Committee of SUNY Upstate Medical University (IACUC#380).
Pseudomonas aeruginosa-induced mouse sepsis
The pneumonia-induced sepsis model was established intratracheally by injecting 50 μL of bioluminescent P. aeruginosa Xen5 solution (about 4 × 104 CFU/mouse) based on several pilot experiments. The optimal dose of P. aeruginosa Xen5 used in this study is appropriate for in vivo imaging analysis and induced ARDS and about 50% of survival rate 48 h postinfection. In brief, a single colony of P. aeruginosa Xen5 was cultured in 5 mL of LB liquid medium at 37°C, 250 RPM incubator overnight. Bacterial pellets were obtained from a subculture of the bacteria. Then pellets were resuspended with cold saline buffer and adjusted to OD600 value to 0.6. The bacterial solution was diluted 500 times for animal infection. Each mouse was injected 50 μL of the bacterial solution for the infection group or the same size of sterile saline with the same surgery for the control group. The procedures of anesthesia, surgery, and intratracheal inoculation of bacteria were performed as described previously (18, 21).
In vivo imaging analysis
Bacterial dynamic changes were detected by monitoring the bioluminescence signaling at 0, 12, 24, 36, and 48 h postinfection by in vivo imaging system (Caliper Life Science, Hopkinton, Mass) as described previously (18, 22). All the measurement was performed following the same imaging setting mode: i.e., exposure time, 3 min; binning, large; f/stop, 1.
Mouse specimen collection
Mice were anesthetized with ketamine/xylazine cocktail (90 mg/kg ketamine, 10 mg/kg xylazine) by intraperitoneal injection and be killed by exsanguination. Blood was collected from the inferior vena cava using a 1-mL syringe. Lung tissue and bronchoalveolar lavage fluid (BALF) were harvested as described previously (18, 23). To obtain BALF, each lung was lavaged with 0.5 mL of cold saline and repeated 3 times. About 1.2 mL of BALF per mouse was obtained as described previously (18, 22, 23).
Lung tissues were fixed for 24 h and then were embedded into paraffin. Sections (about 5 μm) of lung tissues were prepared and stained with hematoxylin and eosin (H&E). Lung injury score was blindly determined by two experienced investigators (22, 23).
Ultrastructural analysis by transmission electron microscopy
Analysis of the ultrastructural properties of lung cells was performed as described previously (23). In brief, lung tissues were fixed by 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) and further fixed in 1% osmium tetroxide in 0.1 M phosphate buffer (pH 7.4) for 1 h. Samples dehydrated and embedded in LX-112 (Ladd Research Industries, Vermont, Va). Thin sections (70 nm) were cut and stained with uranyl acetate and lead citrate. Samples were viewed in a JEOL JEM1400 TEM (JEOL USA Inc., Peabody, Mass). Each sample was examined at various magnifications from 6,000× to 35,000×.
BALF of each mouse was obtained as described above. To determine bacterial numbers in the BALF, fresh BALF (100 μL) from each mouse was diluted at 1,000 times, and then 100 μL of diluted BALF was plated on LB-Agar plates. Experiments were performed by triplicates for each mouse, and the CFUs for each mouse were calculated by an average of CFU counts from three plates. Plate cultures were incubated overnight at 37°C, and colony counts were then determined. Total CFUs were calculated and expressed CFUs per lung (21).
Inflammatory cell analysis in BALF
Quantification of inflammatory cells from BALFs was performed as described previously (21–23). In brief, BALF sample was prepared from individual animals (n = 8 mice per group). About 10 μL of each fresh BALF was used for total cell counting with a hemocytometer. The remaining BALF samples were centrifuged for 10 min at 250 x g at 4°C. The cell pellet was resuspended with 1 mL of cold sterile saline. The cells in 200 μL resuspended cell solutions were mounted on a slide by cytospin centrifugation at 1,000 rpm for 3 min. Cells were stained using the Protocol HEMA-3 cell staining kit (Fisher, Pittsburg, Pa). Macrophages/monocytes and polymorphonuclear neutrophils (PMNs) on the slides were identified and quantified by Nikon 2000 research light microscope (×200 magnification).
Analysis of the surface activity of pulmonary surfactants
The cell-free supernatant from the first time centrifugation was followed by a second-time high-speed centrifugation for 15 min at 40,000 x g at 4°C. The large aggregates (LAs) were resuspended in 0.1 mL buffer (140 mM NaCl, 2.5 mM CaCl2, and 10 mM HEPES and at pH 6.9), and they were adjusted to a phospholipid concentration of 1 mg/mL (17, 24). The surface activity of the LAs was determined as previously described, using constrained drop surfactometry (CDS) (25, 26). In CDS, the adsorbed pulmonary surfactant film is accommodated at the air–water surface of a sessile droplet (∼4 mm in diameter, ∼16 μL in volume, and ∼0.25 cm2 in surface area), which is “constrained” on a carefully machined pedestal. The pedestal has a knife-sharp edge that prevents film leakage and keeps the droplet integrity even at near-zero surface tensions. The surfactant film can be compressed and expanded periodically via regulating the surface area of the droplet by controlling liquid flow into and out of the droplet with a motorized syringe. The droplet is small enough to be enclosed in an environmental control chamber that permits simulations of physiologically relevant intra-alveolar conditions, i.e., the core body temperature of 37°C and relative humidity close to 100%. Specifically, a 16 μL surfactant droplet was dispensed onto the CDS pedestal via a pipette. The surface tension decrease during film adsorption was recorded until an equilibrium value of around 23 mN/m was reached. The adsorbed surfactant film was then compressed and expanded at a rate of three seconds per cycle to simulate normal tidal breathing. The surface tension and surface area of the surfactant film were determined simultaneously using closed-loop axisymmetric drop shape analysis (CL-ADSA) (26). The minimum surface tension (γmin) during dynamic cycling was reported as an indication of surface activity.
Cytokine analysis by ELISA
Mouse lungs were harvested 24 h postinfection, and then lung tissues were homogenated in cold saline and centrifuged at 12,000 x g at 4°C for 20 min by Eppendorf centrifuge. The supernatant was used for enzyme-linked immunosorbent assay (ELISA) assay. The ELISA analysis was performed with a Muti-Analyte ELISArray Kit (QIAGEN, Germantown, Md), which can analyze a panel of 12 proinflammatory cytokines simultaneously using a conventional ELISA protocol. The cytokines analyzed in this study include interleukin (IL)-1α, IL-1β, IL-2, IL-4, IL-6, IL-10, IL-12, IL-17A, interferon-gamma (IFN-γ), tumor necrosis factor alpha (TNFα), granulocyte colony-stimulating factor (G-CSF), and granulocyte-macrophage colony-stimulating factor (GM-CSF).
Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay of the sections of lung tissues was performed with the TUNEL kit (Roche, Indianapolis, Ind) following the protocol, as described previously (22). Apoptotic cells were quantified by counting TUNEL-positive cells in 20 random fields of light microscopy at 200× magnification.
To detect the protein SP-B, NLRP3, and pNF-κB p65 expression in the cells of lung tissue, immunofluorescence (IF) staining was performed with the paraffin-embedded lung sections on slides and anti-SP-B antibody (1:100; Hycult Biotech, Plymouth Meeting, Pa), NLRP3 antibody (1:50; ThermoFisher Scientific, Rockford, Ill), p-NF-κB p65 (1:300; CST, Boston, Mass) as described previously (22).
Western blotting analysis
The Western blotting analysis was performed following our previous description (23). In brief, protein samples were prepared from lung tissues with RIPA buffer containing a cocktail of protease and phosphatase inhibitors. Total protein concentrations of samples were determined by the micro-BCA method. Protein samples (50 μg of protein per lane) were separated by 12% SDS-polyacrylamide gel electrophoresis under reducing (for phosphorated NF-κB p65, i.e., pNF-κB p65, Caspase-3) or nonreducing (for SP-B) condition. The PVDF membrane (Bio-Rad, Hercules, Calif) was blocked with 5% nonfat milk of Tris-buffered saline, and then followed by primary antibody, Caspase-3 (1:1,000; CST), pNF-κB p65 (1:1,000; CST), anti-SP-B antibody (1:2,000; Hycult Biotech), the secondary HRP-conjugated antibody (Bio-Rad). After finishing incubation and washing, the signal of protein was detected with Pierce ECL Western Blotting detection reagent (Thermo Scientific, Rockford, Ill), and then exposed to X-film in a darkroom. For the internal control, the blot was stripped and reprobed with β-actin antibody (1:2,000; Abcam, Cambridge, Mass). The band on the film was quantified by scanning the film with the Quantity One software (Bio-Rad).
Data were expressed as means ± SEM in this study. All statistics were performed with SigmaStat Software (version 3.5). The comparisons among the three groups of mice were analyzed by one-way ANOVA, and then the pairwise comparison was performed using Student t test. The survival analysis of animals was performed with Kaplan–Meyer survival curves and examined statistically by the log-rank test. There are significant differences when P < 0.05.
Bacterial dynamic changes in vivo in the lung of infected mice
To examine dynamic bacterial growth in vivo P. aeruginosa Xen5 expressing with bioluminescence was used for lung infection of three types of mice, i.e., hTG SP-B-C, SP-B-T, and WT mice. Total bioluminescence flux detected by the in vivo imaging system represents the bacterial burden in the lung of infected mice. The total flux of three types of infected mice was monitored at 0, 12, 24, 36, and 48 h postinfection of three types of infected mice. The representative images of several time points of three types of infected mice were shown in Figure 1A. Bacterial burden increased dramatically starting 12 h postinfection in all three types of mice; the level of bacterial burden was the highest in SP-B-C mice compared with SP-B-T and WT mice at 12, 24, 36, and 48 h postinfection (P < 0.01) (Fig. 1B). Furthermore, the CFUs of bacteria were determined in the BALF of mice at 24 h postinfection; the results showed similar pattern observed in the in vivo imaging method; the CFUs in the lung of SP-B-C were the highest among three types of mice (i.e., SP-B > SP-B-T > WT) (P < 0.01); as expected, no bacteria were detected in controls (Fig. 1C). In addition, we also analyzed the survival rate in this study; SP-B-C mice had higher mortality rate compared with SP-B-T (P < 0.05) and WT mice (P < 0.01), respectively; no mortality of sham mice was found (Fig. 1D), suggesting differential susceptibility of three types of mice in response to pneumonia-induced sepsis.
Inflammatory cells in BALF
To assess the inflammatory cell filtration into lung alveoli after infection, total cell counts were assessed in the BALF of infected mice and controls. Inflammatory cells (macrophages and neutrophils) were quantified in the sections prepared by cytospin centrifugation. As expected, only macrophages were observed in the BALF from controls, but more than 90% of inflammatory cells were neutrophils in the BALF from infected mice (Fig. 2A). Total cell counts in BALF increased significantly after infection compared with controls (P < 0.01), and the SP-B-C mice had the most significant number of total cell counts among three types of infected mice, i.e., SP-B-C > SP-B-T > WT (P < 0.01) (Fig. 2B).
Histopathological analysis of lung injury
Lung histopathologic changes were assessed in the H&E staining sections of infected mice and controls; lung injury scores of three types of mice were calculated as described previously (18, 22, 23). We have observed remarkable lung injury in infected mice but not in controls; the injurious lung showed large amounts of inflammatory cells in alveolar spaces and interstitials, proteinaceous debris accumulation, and the thicker alveolar wall (Fig. 2C). The injury score of each type of infected mice was significantly higher than that of each control group (P < 0.01), and infected SP-B-C mice had higher injury score compared with infected SP-B-T (P < 0.05) and WT mice (P < 0.01) (Fig. 2D).
Ultrastructural analysis of lung injury
To examine the ultrastructural changes of cells in the lung after bacterial infection, the ultrastructural properties of the lung cells were assessed by transmission electron microscopy at various magnifications from 6,000× to 35,000×. There were many normal lamellar bodies and healthy mitochondria in the alveolar epithelial type II cells in all controls, suggesting human SP-B genetic variants are functional in mice (control panel of Fig. 3A). But, it was observed increased autophagosomes, and damaged lamellar bodies and abnormal mitochondria as well as decreased microvilli in type II cells in infected mice (pneumonia panel of Fig. 3A). The quantitative analysis indicated that there is a significantly reduced number of lamellar bodies in type II cells of infected mice compared to the controls (P < 0.01). The number of lamellar bodies of infected SP-C was less than that of infected SP-B-T and WT mice (P < 0.01) (Fig. 3B).
Apoptosis in the lung of infected mice
We examined apoptosis in the lung of three types of infected mice using TUNEL assay and Western blotting analysis. As shown in Figure 4, remarkable apoptotic cells showed in red color were detected in the images of TUNEL assay in the lung of infected mice but not in the lung of controls (Fig. 4A). The data from Western blotting analysis with anti-caspase-3 showed an increased level of activated caspase-3 (a band with 17 kDa) in all infected mice (Fig. 4B). The quantitative analyses indicated that there is a significant increase of apoptotic cells in the lung of infected mice compared to the controls (P < 0.01), and there are differences among three types of infected mice, i.e., SP-B-C > SP-B-T > WT (P < 0.01) (Fig. 4C). The level of activated caspase-3 in infected SP-B-C was higher than that of SP-B-T and WT mice (P < 0.01) (Fig. 4D).
Decreased SP-B expression in the lung of infected mice
SP-B protein is essential for life through lowering surface tension and host defense in the alveolar space of the lung. We examined the SP-B expression and its level in the lung by IF and Western blotting analyses. As shown in Figure 5, many IF positive cells were detected in the lung tissues of hTG SP-B-C, SP-B-T, WT mice, but there were decreased IF positive cells in the lung of infected mice (Fig. 5A); the results from Western blotting with anti-SP-B antibody further confirmed decreased SP-B level in all three types of infected mice compared with the controls (Fig. 5B). The quantitative analyses of IF images indicated a significant decrease in IF positive cell number per HPF in the infected mice compared with the controls (P < 0.01) and there were differences among three types of infected mice, i.e., SP-B-C < SP-B-T < WT (P < 0.01) (Fig. 5C). Furthermore, the level of SP-B protein from quantitative analysis of the Western blot indicated a significant decrease in the infected mice compared with the controls (P < 0.01) and there were differences among three types of infected mice (P < 0.05) but not in controls (Fig. 5D).
Compromised surface activity of pulmonary surfactants of infected mice
SP-B is a critical component of pulmonary surfactant that helps lower alveolar surface tension, thus preventing lung collapse and closure of small airways in the distal lung. We examined the surface activity of large aggregates (LAs) extracted from the BALF of infected mice and the controls using CDS (see “Materials and Methods”). Panel A of Figure 6 showed the dynamic cycling results in which the surface tension changes as a function of rapidly decreased and increased surface area that simulates tidal breathing. Panel B of Figure 6 showed the statistical analysis of the minimum surface tension (γmin) obtained at the end of film compression during the dynamic cycling process. It was found that the γmin of the infected mice increased significantly compared with the control mice. In addition, we found that the γmin of the three infection groups ranks in the order of SP-B-C > SP-B-T > WT (P < 0.05), which suggests differential regulation of hSP-B genetic variants to the alveolar surface tension under infectious conditions.
Multiple cytokine analysis in the lung of infected mice
Inflammatory cytokine expression is a remarkable character in pneumonia and pneumonia-induced sepsis. We examined a total of 12 cytokines, which are in response to bacteria-induced lung inflammation and injury, with lung homogenates of infected (24 h posttreatment) mice and controls by a Muti-Analyte ELISArray Kit. Out of 12 cytokines, 9, i.e., TNF-α, IL-6, IL-1α, IL-1ß, G-CSF, GM-CSF, IFN-γ, IL-4, and IL-12, increased significantly in infected mice when compared with the controls (P < 0.01) (Fig. 7). Furthermore, the levels of TNF-α, IL-6, IL-1α, and IL-1ß of infected SP-B-C mice were higher than those of infected SP-B-T and WT mice (P < 0.05 or P < 0.01) (Fig. 7), indicating differential regulation of proinflammatory cytokines between human SP-B genetic variants. By contrast, there was no difference of G-CSF, GM-CSF, IFN-γ, IL-4, and IL-12 among three types of infected mice. We also found no significant changes in the levels of IL-2, IL-10, and IL-17A between infected mice and the controls (data not shown).
Inflammatory NF-κB signaling activation in the lung of infected mice
Previous studies have demonstrated that the activation of the NF-κB signaling pathway plays a critical role in the host defense and inflammatory response to the bacterial infection and sepsis (18, 27). We examined the NF-κB activation by IF and phosphorated NF-κB p65 (pNF-κB p65), and the critical component of the NF-κB signaling pathway by Western blotting analysis. As shown in Figure 8, both IF and Western blotting analyses showed increased NF-κB activation in infected mice compared with the controls. Furthermore, different levels of NF-κB activation were observed among three types of infected mice, indicating that infected SP-B-C mice showed higher level compared with infected SP-B-T (P < 0.05) and infected WT mice (P
< 0.01), respectively.
Increased NLRP3 inflammasome activation in the lung of infected mice
We examined the NLRP3 inflammasome activation in the lung of infected mice and the controls by IF. As shown in Figure 9, increased NLRP3 IF positive cells were detected in the infected mice compared with the controls, and different levels of NLRP3 inflammasome activation were observed among three types of infected mice, i.e., infected SP-B-C mice had more positive cells compared with infected SP-B-T and infected WT mice (P < 0.01). The results of NLRP3 inflammasome activation analysis were consistent with the observation of NF-κB activation in this study.
Pneumonia-induced sepsis is a major cause of ARDS and multiple organ dysfunction, which have very high associated mortality (40%–60%). Some individuals are more susceptible to bacterial pneumonia-induced sepsis compared with others, but the underlying mechanisms are unclear. Human genomic variation, including human SP-B genetic variants, should contribute to the susceptibility and disease severity of ARDS and sepsis because SP-B protein is essential for maintaining formal surfactant activity and host defense in the lung. Patients-based genotyping studies have demonstrated the association of hSP-B polymorphisms with pneumonia-induced ALI/ARDS, and the decreased level of SP-B protein was used as an essential biomarker of ARDS progression (10, 11). In the present study, we investigated the regulatory roles of hSP-B genetic variants on the lung injury and surface activity of pulmonary surfactant in the P. aeruginosa-induced mouse septic model. We found differentially regulatory effects of hTG SP-B C and T alleles in response to bacteremia through influencing in vivo bacterial growth, regulating inflammatory cells and molecular processes, as well as surfactant activation in the lung.
SP-B, an essential protein of pulmonary surfactant, causes lethal respiratory failure when an acute reduction by about 60% of normal levels occurs in animals (28) or ARDS patients (10). The SP-B precursor can produce two proteins, i.e., SP-BM and SP-BN, which are involved in lowering surface tension and innate host defense, respectively (2). Several hSP-B polymorphisms and mutations have been identified, including the SNP (rs1130866), one of the most important SNPs (9, 29). This SNP is associated with several pulmonary diseases, including pneumonia and pneumonia-induced ARDS (7–9), neonatal respiratory distress syndrome (RDS) (30). Our findings from this study demonstrated the C allele of hSP-B exhibited decreased host defense and increased compromised surface activity of pulmonary surfactant in the infected mice compared with the T allele. These in vivo results from our septic hTG mouse model are consistent with the observations of patients-based genotyping analysis (6), indicating the regulatory roles of the hSP-B genetic variants due to the SNP rs1130866 in the pathogenesis of septic ALI/ARDS.
hTG mice have provided an ideal tool to explore biological function and variation of human gene/allele in the pathophysiological processes, which is able to overcome the limitation of human beings, for example, to test in vivo capacity and the mechanisms of human gene/allele in response to various infectious conditions (15, 31). In this study, we found no difference of the surface activity of pulmonary surfactants (minimum surface tension) and lung structural properties among three types of mice, i.e., SP-B-C, SP-B-T, and WT mice in normal conditions (control groups), indicating that human SP-B variants function well in the transgenic mice. Furthermore, higher bacterial load, increased inflammatory cells, and proinflammatory cytokine expression were observed in the lungs of infected SP-B-C mice when compared with infected SP-B-T and WT mice in the present study, suggesting the decreased innate immune ability of SP-B C allele in comparison with the T allele, as well as WT mice.
The previous studies demonstrated that the process of producing mature SP-B protein is involved in complex SP-B precursor processing and differentially complicated posttranslational modification (32). Other surfactant lipids and proteins, including mature SP-B, are packaged into lamellar bodies from which surfactants are secreted into the air space by exocytosis (3). The SNP (rs1130866) can result in the alteration of N-linked glycosylation of SP-B, thus may influence the level of mature SP-B by affecting the transfer of SP-B to lamellar bodies (32) and the efficiency of pro-SP-B cleavage under infectious or stress conditions (30). Lung ultrastructural analysis by electron microscopy revealed abnormal type II cells and many damaged lamellar bodies in the infected mice. However, it is necessary to investigate detailed molecular mechanisms further how the N-linked glycosylation in the C allele influences SP-B precursor processing and trafficking in the type II cells under infectious conditions.
SP-B plays a role in the host defense and surfactant physiology (33). The protein product (SP-BN) produced from SP-B precursor recognized as an antimicrobial peptide, directly killed bacteria and indirectly promoted uptake of bacteria by macrophages. In vivo, the ability of bacterial killing was enhanced in overexpressing SP-BN mice (34). In vitro, the growth of bacteria P. aeruginosa Xen5 was significantly inhibited when co-incubated with mice BALF, whereas the effect reversed when SP-BN IgG was added (2). The results from this study demonstrated that P. aeruginosa infection could cause cell death and induce proinflammatory cytokine expression by NF-κB-dependent transcriptional upregulation with increased NLRP3 and Caspase-3 activation. The data about NF-κB signaling activation and a panel of inflammatory cytokine expression in response to P. aeruginosa infection further revealed differential regulation of SP-B genetic variants on the lung injury. NF-κB is usually composed of the p50/p65 heterodimers and its effector form, phosphorylated NF-κB-p65, dissociates from NF-κB complex, and then translocates into the nucleus upon stimulation. The protein of 17-kDa Caspase-3 is the active form of Caspase-3, recognized as an indicator of apoptosis. In this study, we found that, compared with SP-B-T and WT mice, SP-B-C mice induced more pNF-κB p65 and NLRP3 activation, a higher level of activated Caspase-3, thus causing more apoptosis in the lung of this model.
An accumulating body of evidence showed cytokines such as IL-4 and IL-10, served as anti-inflammatory chemokines, whereas TNF-α, IL-1α, IL-1β, IL-6, IFN-γ as proinflammatory cytokines (35). In the present study, at the time point of 24 h postinfection, cytokines of TNF-α, IL-1α, IL-1β, and IL-6 significantly increased compared with the control group; meanwhile, the SP-B-C mice demonstrated higher levels than SP-B-T and WT mice. The cytokines IL-4, IL-12, IFN-γ, G-CSF, and GM-CSF increased significantly after the infection, but no significant difference among the two SP-B genetic variants and WT. By contrast, no difference was observed in IL-2, IL-10 and IL-17A between the control and infection groups. The differential response time to infection may be one of the reasons. For example, IL-17A, as an early proinflammatory cytokine, responded to the bacteria infection transiently and returned to the baseline at 24 h postinfection (34). The previous study indicated levels of TNF-α and IL-6 rose sharply and reached a peak at 24 h after LPS exposure, whereas IL-10 and IFN-γ elevated gradually from 24 h and peaked at 96 h. However, as to the most-recognized proinflammatory cytokines TNF-α, IL-1β, and IL-6, their significant differences were observed between infected SP-B-C and SP-B-T mice in this study.
In summary, we have used hTG SP-B-C, SP-B-T, and WT mice to study the regulatory roles of hSP-B genetic variants on the lung injury and the surface activity of pulmonary surfactant in pneumonia-induced sepsis. We found that SP-B genetic variants differentially influenced lung inflammation and tissue injury, as well as surfactant dysfunction in this P. aeruginosa-induced sepsis model. The underlying mechanisms of the differential regulation of SP-B T and C variant involved in influencing of bacterial growth and lung cell death, modulating cytokine expression and inflammatory NF-κB signaling activation, as well as regulating surface activity in the lung of infected mice.
The authors thank Dr. Jennifer F. Moffat of the Department of Microbiology and Immunology, SUNY Upstate Medical University, Syracuse, NY, for kindly providing the bioluminescent bacterial strain of P. aeruginosa Xen5 and in vivo imaging system. They also thank all members of Profs. R.N. Cooney and G. Nieman's laboratories for their kind support to this project.
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