Differential Susceptibility of Transgenic Mice Expressing Human Surfactant Protein B Genetic Variants to Pseudomonas Aeruginosa Induced Pneumonia

Biochemical and Biophysical Research Communications 469 (2016) 171e175

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Biochemical and Biophysical Research Communications

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Differential susceptibility of transgenic mice expressing human surfactant protein B genetic variants to Pseudomonas aeruginosa induced pneumonia

Lin Ge a, b, Xinyu Liu a, Rimei Chen c, Yongan Xu a,YiY.Zuoc, Robert N. Cooney a, * Guirong Wang a, a Department of Surgery, SUNY Upstate Medical University, Syracuse, NY, 13210, USA b Department of Biochemistry and Molecular Biology, Tianjin Medical University, Tianjin, 300070, China c Department of Mechanical Engineering, University of Hawaii at Manoa, Honolulu, HI, 96822, USA article info abstract

Article history: Surfactant protein B (SP-B) is essential for lung function. Previous studies have indicated that a SP-B Received 4 November 2015 1580C/T polymorphism (SNP rs1130866) was associated with lung diseases including pneumonia. The Accepted 20 November 2015 SNP causes an altered N-linked glycosylation modification at Asn129 of proSP-B, e.g. the C allele with this Available online 24 November 2015 glycosylation site but not in the T allele. This study aimed to generate humanized SP-B transgenic mice carrying either SP-B C or T allele without a mouse SP-B background and then examine functional sus- Keywords: ceptibility to bacterial pneumonia in vivo. A total of 18 transgenic mouse founders were generated by the Allele DNA microinjection method. These founders were back-crossed with SP-B KO mice to eliminate mouse Humanized transgenic mice Pneumonia SP-B background. Four founder lines expressing similar SP-B levels to human lung were chosen for Psedomonas aeruginosa further investigation. After intratracheal infection with 50 mlofPseudomonas aeruginosa solution 6 Surfactant protein B (1 10 CFU/mouse) or saline in SP-B-C, SP-B-T mice the mice were sacrificed 24 h post-infection and Surface tension tissues were harvested. Analysis of surfactant activity revealed differential susceptibility between SP-B-C and SP-B-T mice to bacterial infection, e.g. higher minimum surface tension in infected SP-B-C versus infected SP-B-T mice. These results demonstrate for the first time that human SP-B C allele is more susceptible to bacterial pneumonia than SP-B T allele in vivo. © 2015 Elsevier Inc. All rights reserved.

1. Introduction like respiratory distress syndrome in pre-term neonates (RDS), congenital alveolar protein deposition disease (CAP), broncho- Surfactant protein B (SP-B), a hydrophobic protein, is essential pulmonary dysplasia (BPD) [6], and the interstitial lung disease [7]. for normal lung function, which is expressed by alveolar type II Critical mutations of hSP-B gene result in SP-B deficiency which is epithelial cells in the lung. SP-B protein is critical for the formation lethal for newborn infants [8]. For example, a two-base-insertion in of the pulmonary surfactant film at the surface of alveoli that codon 121 of hSP-B cDNA causes SP-B deficiency and neonatal lowers the surface tension and prevents the collapse of alveoli in alveolar proteinosis [9] and a 1-bp deletion (1553delT) in exon 4 the lung [1]. Human SP-B (hSP-B) is encoded by sftpb gene causes a reading frame shift and the premature translational (approximately 9.5 kb containing 11 exons) on chromosome 2 [2,3]. termination in exon 6. Infants carrying these homozygous mutants The mature SP-B product is an 8 kDa protein (79 residues) which is died shortly after birth because of a lack of mature SP-B protein derived from a SP-B precursor (pro-SP-B) by a complex protein [10]. processing pathway [4,5]. A common SP-B single nucleotide polymorphism, SP-B 1580C/T hSP-B genetic variation is associated with various lung diseases, (SNP, rs1130866), causes a change in the amino acid residue from Threonine (Thr) for the C allele to Isoleucine (Ile) for the T allele at position 131 of SP-B precursor. This altered residue located at a glycosylation recognition sequence results in the C allele containing * Corresponding author. Department of Surgery, UH Room 8715, SUNY Upstate fi Medical University, 750 E Adams St, Syracuse, NY, 13210, USA. a glycosylation modi cation at Asn129 which is not present in the T E-mail address: [email protected] (G. Wang). allele [11]. Patients-based genotyping studies demonstrate that this http://dx.doi.org/10.1016/j.bbrc.2015.11.089 0006-291X/© 2015 Elsevier Inc. All rights reserved. 172 L. Ge et al. / Biochemical and Biophysical Research Communications 469 (2016) 171e175

SP-B SNP (rs1130866 C/T) is associated with several pulmonary 2.2. Constructs diseases including pneumonia [12] and pneumonia-induced ARDS [13], but its effects on functional susceptibility to pulmonary A 5.4-kb DNA fragment (Fig. 1) used for DNA microinjection was pathogens induced pneumonia have never been tested. We hy- excised from a recombinant plasmid by restriction enzymes Nde I pothesized that the change of glycosylation status at the Asn129 in and Not I. The DNA fragment consisted of a human SP-C promoter SP-B precursor has an impact on SP-B processing and physiological/ (3.7-kb), a human SP-B cDNA (1.3-kb), and a SV40 small t-intron pathophysiological function. poly (A) sequence (0.4-kb). The basic 3.7-hSP-C/SV40 vector was In the current study, to test our hypotheses we generated hu- kindly provided by Drs. Jeffrey A. Whitsett and Stephan W. Glasser manized SP-B transgenic (hTG) mice carrying either SP-B C or T (Cincinnati Children's Research Foundation, Cincinnati, OH) [14]. allele but without a mouse SP-B (mSP-B) gene background, and The recombinant DNA processes were performed using standard then examine the functional susceptibility of SP-B variants on methods of molecular cloning. The cDNA of human SP-B was cloned surfactant activity in response to Pseudomonas aeruginosa pneu- into a basic 3.7-hSP-C/SV40 vector and recombinant construct was monia in the hTG SP-B-C and SP-B-T mice. veriﬁed by DNA sequencing. A 5.4 kb DNA fragment was microinjected into fertilized FVB/N oocytes from WT mice.

2.3. Generation of hTG SP-B mice 2. Materials and methods Human SP-B positive transgenic founders carrying either hSP-B 2.1. Mice C or T allele (hSP-Bþ, mSP-B þ/þ) were bred with conditional SP-B KO mice (mSP-B /) for several generations to eliminate mSP-B Wild type (WT) FVB/N mice used in the present study were gene background. Then homozygous hTG SP-B-C and SP-B-T mice purchased from the Jackson laboratory and maintained in the an- (hSP-B þ/þ, mSP-B /) were generated by the self-breeding of imal core facility at SUNY Upstate Medical University. The hSP-B hemizygous mice (hSP-B þ/, mSP-B /). All mice were geno- transgenic mice carrying either hSP-B C or T allele without a typed using DNA from tail samples by PCR genotyping with Primer mouse SP-B gene background were generated in this study. Mice pair 1458/189 for hSP-B and primer pair 75/76 for mSP-B. To verify were housed in pathogen-free conditions and the animal protocols hSP-B in hTG mice, primer pair 1458/1401 was used to amplify the (IACUC# 236 and 380) in this study were approved by Institutional whole fragment of hSP-B gene and the PCR products were analyzed Animal Care and Use Committee at SUNY Upstate Medical Univer- by DNA sequencing. sity, they also meet the National Institutes of Health and ARRIVE guidelines on the use of laboratory animals. 2.4. Western blotting analysis

To examine the expression of hSP-B protein level in hTG SP-B mice, Bronchoalveolar lavage ﬂuid (BALF) from hTG mice was analyzed by Western blotting with anti-SP-B antibody (Hycult Biotech, Plymouth Meeting, PA) at 1:200 dilutions as previously described [15,16].

2.5. Analysis of histology and immunohistochemistry (IHC)

Mouse lungs from hTG mice (8e12 weeks) were ﬁxed by 10% formalin solution at about 25 cm of water pressure for at least 24 h, and then processed into parafﬁn blocks. The sections of lung tissue were approximately 5 mm in thickness. Lung sections were stained with hematoxylin and eosin, or used for IHC analysis using the ABC kit (Vector Laboratories, Burlingame, CA) as described previously [15,16].

2.6. Pseudomonas aeruginosa infection

hTG SP-B-C and SP-B-T mice were infected by intratracheal inoculation of 50 mlofP. aeruginosa PA01 (1 106 CFU/mouse) or sterile saline (sham control). Mice were sacrificed 24 h post infec- Fig. 1. Recombinant construct and genotyping analysis of hSP-B transgenic mouse tion. Tissues were harvested and prepared as described previously offspring. A diagram of recombinant DNA construct is shown in Panel A. A DNA frag- [15,16]. ment consisting of a human SP-C promoter, a human SP-B cDNA and a SV40 small t- intron poly A sequence was cloned into the basic 3.7-hSP-C/SV40 vector by restriction enzymes Nde I and Not I (A). The transgenic mice were genotyped by PCR amplification 2.7. Electron microscopy analysis of both human SP-B and mouse SP-B fragment. hSP-B was amplified with primers which locate on hSP-C promoter and hSP-B cDNA (þ421 bp), and the PCR product is fi 0 Lung tissues were prepared as previously described [17]. The 577 bp. mSP-B was ampli ed with primers which locate on the 3 end of mSP-B gene fi intron 3 and 50 end of intron 4. The WT mSP-B product is 279 bp, but the SP-B fragment samples were xed by 4% glutaraldehyde, 2.5% paraformaldehyde in SP-B KO mice could not be amplified with the used PCR conditions due to a neo- for 24 h, then stained with osmium tetroxide, 1.5% potassium mycine resistance gene (1.8 kb) in mouse SP-B gene exon 4 (Panel B). Panel C depicts ferrocyanide and embedded in Embed812 resin (Electron micro- the results of genotyping analysis of mouse offspring: the mice containing only hSP-B scopy science). The tissues were cut into ultrathin sections (90 nm) gene were defined as F2 (lanes 1, 2, 4e6, 8); the mice with both hSP-B and mSP-B were and stained with lead citrate and 2% aqueous uranyl acetate before defined as F1 (lane 7), the mice carrying only mSP-B were defined as WT (lane 9, 10). The recombinant plasmid was used as positive control and the KO mouse genomic electron microscopy analysis. Each sample was examined at the DNA as negative control. magnification varied from 10, 000 to 40, 000, and at least 100 L. Ge et al. / Biochemical and Biophysical Research Communications 469 (2016) 171e175 173

fields were examined from several sections. Furthermore, hSP-B expression was analyzed in the lung by IHC method (Fig. 2B) and western blotting (data shown in 3.4). The 2.8. Surfactant large and small aggregates results indicated that only alveolar type II epithelial cells showed positive staining for SP-B expression in the lung tissue of both hTG Mouse lungs were lavaged for 3 times each with 0.7 ml saline SP-B-T and SP-B-C mice (Fig. 2B). solution. BALFs from 6 mice of each group were prepared and centrifuged at 150 g,4C for 10 min and the supernatant was 3.3. Bacterial pneumonia then centrifuged for 15 min at 40,000 g,4C. After centrifugation, the pellet containing surfactant large aggregates was resuspended To examine the functional variation of hSP-B genetic variants in 0.3 ml of saline for surface tension study as previous description under infectious conditions, a bacterial pneumonia model was [18,19]. The phospholipid concentration of the large aggregates was developed and applied for the assessment of functional effects of determined using phosphate assays to be around 1 mg/mL. hSP-B genetic variants in vivo. hTG mice were infected intratracheally by 50 mlofP. aeruginosa PA01 (106 CFU/mouse) or same 2.9. Analysis of surfactant activity amount of saline solution (Sham). The infected mice were sick but survival for 24 h. The lung pathohistology displayed pneumonia Surface activity of the mouse surfactants was determined with a pathogenic characteristics and lung injury in the infected mice but constrained drop surfactometer (CDS; BioSurface Instruments, HI) not in Sham control (Fig. 3A). A large amount of neutrophils were [20]. A droplet of the mouse surfactant of ~10 mL was dispensed observed in the BALF of infected mice but not in the control. Ul- onto the CDS drop holder. After the equilibrium surface tension was trastructural analysis revealed significant decrease of lamellar established by rapid adsorption, the surfactant film was com- bodies in the type II cells and remarkable less microvilli on the pressed and expanded at a rate of 3 s per cycle with a compression surface of type II cells in infected mice compared to Sham (Fig. 3B, ratio controlled to be less than 40% of the initial surface area. At p < 0.05), suggesting bacterial infection decreased activation of least five compression-expansion cycles were studied for each type II cells and surfactant/protein expression. droplet. Cycles were quantified with the minimum surface tension (gmin) at the end of compression and the maximum surface tension 3.4. Differential surfactant activity between SP-B-C and SP-B-T in (gmin) at the end of expansion. Drop images were taken at 10 frames response to bacterial infection per second. The surface tension and surface area were determined with Axisymmetric Drop Shape Analysis (ADSA) [21]. Surfactant activity of large aggregates of BALF from infected and sham mice was analyzed by the Constrained Drop Surfactometer 2.10. Statistical analysis (CDS) [18e20]. The results indicated that uninfected hSP-B-T and hSP-B-C mice (sham) had similar surface activity, i.e., minimum All the experiments were repeated at least 3 times in this study. surface tension (gmin) about 1.8e2.2 (mN/m) (Fig. 4A). After infec- Western blot bands were quantified by software Quantity One tion, the minimum surface tension in infected SP-B-C mice (version 4.6.1). Statistics were performed by SigmaStat version 3.5 increased significantly by two times compared to uninfected con- software. Significant difference in statistics among groups was trol (Fig. 4A, p < 0.05), suggesting that surfactant activation has considered when p < 0.05 by t-test or ANOVA. been inhibited in the infected SP-B-C mice. But the minimum surface tension of infected SP-B-T mice (hSP-B-T) showed less change 3. Results compared to uninfected SP-B-T mice (Fig. 4A). These data indicate that the SP-B-T mice had less susceptibility on surfactant activity to 3.1. Generation of hTG SP-B-C and SP-B-T mice bacterial infection when compared to SP-B-C mice (p < 0.05) (Fig. 4A). Analysis of Western blotting demonstrated decreased SP- To generate hTG SP-B-C and SP-B-T mice, the cDNA of hSP-B B levels of both infected SP-B-C and SP-B-T mice compared to their gene was cloned into a basic 3.7-hSP-C/SV40 vector (Fig. 1A) and uninfected mice (Fig. 4B, p < 0.01). Infected SP-B-C mice had lower A 5.4 kb DNA fragment was microinjected into fertilized FVB/N SP-B tendency vs. infected SP-B-T mice. oocytes from WT mice. A total of 18 hSP-B positive founders were identified by PCR. To eliminate the mSP-B background, the condi- 4. Discussion tional SP-B knockout (KO) mice by inserting Neomycin resistant gene into the gene (Fig. 1B) were used to breed with hSP-B positive Pulmonary surfactant consists of about 90% phospholipids and founders. The hSP-B positive F1 were bred with conditional SP-B KO 10% surfactant-associated proteins, including SP-B, an essential mice to generate F2 hSP-B positive mice (hSP-B þ/mSP-B /) protein for normal lung function [1]. The SP-B precursor is about (Fig. 1C). Finally, four homozygous hTG SP-B-C and SP-B-T mouse 42 kDa peptide containing three sapsin-like protein domains, e.g. lines, e.g. (hSP-B þ/þ, mSP-B /), were obtained by the self- domain N, M, and C. Mature 8-kDa SP-B protein is derived from breeding of hemizygous mice (hSP-B þ/, mSP-B /) and sapsin-like domain M, which plays a key role in lowering surface showed healthy status suggesting that hSP-B protein functions well tension in the alveolar space [22]. Decreased levels of mature SP-B in the transgenic mice. significantly influence lung function and oxygenation [23] and cause disease exacerbation [24]. The SP-B SNP (rs1130866 C/T) was 3.2. Expression of transgene SP-B-T or SP-B-C in hTG mice identified to be associated with several pulmonary diseases including pneumonia [12], in which the individuals carrying the C The histology of lung in the hTG SP-B-c and SP-B-T mice from allele of SP-B are more susceptible than those with the T allele [13]. four founders was assessed using H/E staining sections (Fig. 2A). However, the detailed mechanisms are unclear. The humanized SP- The results demonstrated hTG SP-B-C founder line and SP-B-T B transgenic mouse model provides a powerful in vivo tool for founder lines displayed normal alveolar structure and septal studying the mechanistic role of human SP-B genetic variants in the thickness. The chord length (mean linear intercepts: Lm) as a pathogenesis of pulmonary diseases [25]. measure of the acinar air space complex was measured, no signif- Previous studies found that the production of mature SP-B icant difference was found between hTG SP-B-C and SP-B-T mice. protein is completed by complex SP-B precursor processing and 174 L. Ge et al. / Biochemical and Biophysical Research Communications 469 (2016) 171e175

Fig. 2. Histology and hSP-B expression in the lung of hTG SP-B-C and SP-B-T mice. Lung tissue was ﬁxed by formalin for 24 h and then processed into parafﬁn blocks. About 5-mm- thick sections were prepared and slides were stained using hematoxylin and eosin (H/E) method. hTG SP-B-C and SP-B-T mice exhibited normal lung histology (A). To identify SP-B expression in the lung the sections of lung were analyzed by IHC with anti-SP-B antibody. The results showed positive in the type II alveolar cells (pointed by arrows) (B).

Fig. 4. Different susceptibility of surfactant activity in hTG SP-B-C and SP-B-T mice to bacterial infection. Surfactant large aggregates were prepared from the BALF samples

as described in the methods. Minimum surface tension (gmin) was measured. The results showed that there are similar minimum surface tension (gmin) in the uninfected SP-B-C and SP-B-T mice, but the level of minimum surface tension (gmin) was increased significantly in infected SP-B-C mice compared to infected SP-B-T mice, as well as uninfected SP-B-C mice (Panel A). Western blot analysis showed that the hSP-B expression in BALF 20 mg of total protein from the BALF of sham or infection group samples was subjected to 12% SDS-PAGE under non-reducing condition. Human BALF was used as a positive control. The hSP-B protein was detected by a rabbit anti-pig SP-B Fig. 3. Lung injury caused by bacterial pneumonia. Mice were infected intratracheally antibody (at 1:200X). The dimer (16 kDa) of hSP-B was detected on the blot. The data with 50 ml of bacterial solution (1 106 CFU/mouse) or saline (sham) and sacrificed show that samples from infection group express a decreased level of hSP-B protein by 24 h after infection. Lung tissues were fixed with 10% Formalin solution. The results comparing with sham group (B). There is a significant difference between sham and showed that infected mice had severe lung injury with a large amount of inflammatory infection groups (p < 0.01) (C). cells e.g. PMN and macrophages in the alveoli and pathogenic changes of lung tissues but not in uninfected mice (A). For analysis of lung ultrastructure after infection lung fi tissues from infection and sham group of hSP-B-T and hSP-B-C mice were xed stained observed in the healthy individuals with either the C or T allele, and embedded, and then analyzed by electron microscopy as described in the fi methods. The results showed decreased lamellar bodies in the type II alveolar cells indicating the additional Asn129 glycosylational modi cation in from infected mice compared with sham mice (B). The hSP-B-T and hSP-B-C mice SP-B precursor may not significantly influence physiological func- showed a normal ultrastructure of type II alveolar cells in health mice. tion under healthy conditions. The similar levels of the SP-B expression in the hTG SP-B-C and SP-B-T mice in this study also confirmed the observation in human beings. However, it is un- fi traf cking [26]. SP-B was secreted to alveoli or stored in the known whether the additional glycosylation site at the As129 in the lamellar bodies in the alveolar type II cells of lung [26]. Several SP-B-C variant has an negative impact on pro-SP-B processing and proteases are involved in the protein processing, including protease trafficking under disease conditions, like bacterial pneumonia. Napsin and Cathepsin H [26]. Differentially posttranslational Previous studies demonstrate that there are decreased expres- fi modi cations of SP-B precursor caused by genetic variation may sions of SP-B after a variety of infectious conditions [28]. In the fi fi have an impact on the ef ciency of SP-B processing and traf cking present study we observed a significant decrease of SP-B level in in the type II cells. The C allele of human SP-B has an additional the BALF of infected mice, and SP-B level in infected SP-B-C mice protein glycosylation site at the residue Asn129 compared to the T was lower than infected SP-B-T mice, suggesting that altered allele [11,27]. SP-B C and T alleles are two common genetic alleles in glycosylation modification at the Asn129 in the SP-B-C mice human population and no obviously abnormal SP-B expression was L. 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