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Biochemical and Biophysical Research Communications 469 (2016) 171e175

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Biochemical and Biophysical Research Communications

journal homepage: www.elsevier .com/locate/ybbrc

Differential susceptibility of transgenic mice expressing humansurfactant protein B genetic variants to Pseudomonas aeruginosainduced pneumonia

Lin Ge a, b, Xinyu Liu a, Rimei Chen c, Yongan Xu a, Yi Y. Zuo c, Robert N. Cooney a,Guirong Wang a, *

a Department of Surgery, SUNY Upstate Medical University, Syracuse, NY, 13210, USAb Department of Biochemistry and Molecular Biology, Tianjin Medical University, Tianjin, 300070, Chinac Department of Mechanical Engineering, University of Hawaii at Manoa, Honolulu, HI, 96822, USA

a r t i c l e i n f o

Article history:Received 4 November 2015Accepted 20 November 2015Available online 24 November 2015

Keywords:AlleleHumanized transgenic micePneumoniaPsedomonas aeruginosaSurfactant protein BSurface tension

* Corresponding author. Department of Surgery, UMedical University, 750 E Adams St, Syracuse, NY, 132

E-mail address: wangg@upstate.edu (G. Wang).

http://dx.doi.org/10.1016/j.bbrc.2015.11.0890006-291X/© 2015 Elsevier Inc. All rights reserved.

a b s t r a c t

Surfactant protein B (SP-B) is essential for lung function. Previous studies have indicated that a SP-B1580C/T polymorphism (SNP rs1130866) was associated with lung diseases including pneumonia. TheSNP causes an altered N-linked glycosylation modification at Asn129 of proSP-B, e.g. the C allele with thisglycosylation site but not in the T allele. This study aimed to generate humanized SP-B transgenic micecarrying either SP-B C or T allele without a mouse SP-B background and then examine functional sus-ceptibility to bacterial pneumonia in vivo. A total of 18 transgenic mouse founders were generated by theDNA microinjection method. These founders were back-crossed with SP-B KO mice to eliminate mouseSP-B background. Four founder lines expressing similar SP-B levels to human lung were chosen forfurther investigation. After intratracheal infection with 50 ml of Pseudomonas aeruginosa solution(1 � 106 CFU/mouse) or saline in SP-B-C, SP-B-T mice the mice were sacrificed 24 h post-infection andtissues were harvested. Analysis of surfactant activity revealed differential susceptibility between SP-B-Cand SP-B-T mice to bacterial infection, e.g. higher minimum surface tension in infected SP-B-C versusinfected SP-B-T mice. These results demonstrate for the first time that human SP-B C allele is moresusceptible to bacterial pneumonia than SP-B T allele in vivo.

© 2015 Elsevier Inc. All rights reserved.

1. Introduction

Surfactant protein B (SP-B), a hydrophobic protein, is essentialfor normal lung function, which is expressed by alveolar type IIepithelial cells in the lung. SP-B protein is critical for the formationof the pulmonary surfactant film at the surface of alveoli thatlowers the surface tension and prevents the collapse of alveoli inthe lung [1]. Human SP-B (hSP-B) is encoded by sftpb gene(approximately 9.5 kb containing 11 exons) on chromosome 2 [2,3].The mature SP-B product is an 8 kDa protein (79 residues) which isderived from a SP-B precursor (pro-SP-B) by a complex proteinprocessing pathway [4,5].

hSP-B genetic variation is associated with various lung diseases,

H Room 8715, SUNY Upstate10, USA.

like respiratory distress syndrome in pre-term neonates (RDS),congenital alveolar protein deposition disease (CAP), broncho-pulmonary dysplasia (BPD) [6], and the interstitial lung disease [7].Critical mutations of hSP-B gene result in SP-B deficiency which islethal for newborn infants [8]. For example, a two-base-insertion incodon 121 of hSP-B cDNA causes SP-B deficiency and neonatalalveolar proteinosis [9] and a 1-bp deletion (1553delT) in exon 4causes a reading frame shift and the premature translationaltermination in exon 6. Infants carrying these homozygous mutantsdied shortly after birth because of a lack of mature SP-B protein[10].

A common SP-B single nucleotide polymorphism, SP-B 1580C/T(SNP, rs1130866), causes a change in the amino acid residue fromThreonine (Thr) for the C allele to Isoleucine (Ile) for the T allele atposition 131 of SP-B precursor. This altered residue located at aglycosylation recognition sequence results in the C allele containinga glycosylation modification at Asn129 which is not present in the Tallele [11]. Patients-based genotyping studies demonstrate that this

L. Ge et al. / Biochemical and Biophysical Research Communications 469 (2016) 171e175172

SP-B SNP (rs1130866 C/T) is associated with several pulmonarydiseases including pneumonia [12] and pneumonia-induced ARDS[13], but its effects on functional susceptibility to pulmonarypathogens induced pneumonia have never been tested. We hy-pothesized that the change of glycosylation status at the Asn129 inSP-B precursor has an impact on SP-B processing and physiological/pathophysiological function.

In the current study, to test our hypotheses we generated hu-manized SP-B transgenic (hTG) mice carrying either SP-B C or Tallele but without a mouse SP-B (mSP-B) gene background, andthen examine the functional susceptibility of SP-B variants onsurfactant activity in response to Pseudomonas aeruginosa pneu-monia in the hTG SP-B-C and SP-B-T mice.

2. Materials and methods

2.1. Mice

Wild type (WT) FVB/N mice used in the present study werepurchased from the Jackson laboratory and maintained in the an-imal core facility at SUNY Upstate Medical University. The hSP-Btransgenic mice carrying either hSP-B C or T allele without amouse SP-B gene background were generated in this study. Micewere housed in pathogen-free conditions and the animal protocols(IACUC# 236 and 380) in this study were approved by InstitutionalAnimal Care and Use Committee at SUNY Upstate Medical Univer-sity, they also meet the National Institutes of Health and ARRIVEguidelines on the use of laboratory animals.

Fig. 1. Recombinant construct and genotyping analysis of hSP-B transgenic mouseoffspring. A diagram of recombinant DNA construct is shown in Panel A. A DNA frag-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 restrictionenzymes Nde I and Not I (A). The transgenic mice were genotyped by PCR amplificationof both human SP-B and mouse SP-B fragment. hSP-B was amplified with primerswhich locate on hSP-C promoter and hSP-B cDNA (þ421 bp), and the PCR product is577 bp. mSP-B was amplified with primers which locate on the 30 end of mSP-B geneintron 3 and 50 end of intron 4. The WT mSP-B product is 279 bp, but the SP-B fragmentin SP-B KO mice could not be amplified with the used PCR conditions due to a neo-mycine resistance gene (1.8 kb) in mouse SP-B gene exon 4 (Panel B). Panel C depictsthe results of genotyping analysis of mouse offspring: the mice containing only hSP-Bgene were defined as F2 (lanes 1, 2, 4e6, 8); the mice with both hSP-B and mSP-B weredefined 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 genomicDNA as negative control.

2.2. Constructs

A 5.4-kb DNA fragment (Fig. 1) used for DNA microinjectionwasexcised from a recombinant plasmid by restriction enzymes Nde Iand Not I. The DNA fragment consisted of a human SP-C promoter(3.7-kb), a human SP-B cDNA (1.3-kb), and a SV40 small t-intronpoly (A) sequence (0.4-kb). The basic 3.7-hSP-C/SV40 vector waskindly provided by Drs. Jeffrey A. Whitsett and Stephan W. Glasser(Cincinnati Children's Research Foundation, Cincinnati, OH) [14].The recombinant DNA processes were performed using standardmethods of molecular cloning. The cDNA of human SP-B was clonedinto a basic 3.7-hSP-C/SV40 vector and recombinant construct wasverified by DNA sequencing. A 5.4 kb DNA fragment was micro-injected into fertilized FVB/N oocytes from WT mice.

2.3. Generation of hTG SP-B mice

Human SP-B positive transgenic founders carrying either hSP-BC or T allele (hSP-Bþ, mSP-B þ/þ) were bred with conditional SP-BKO mice (mSP-B �/�) for several generations to eliminate mSP-Bgene background. Then homozygous hTG SP-B-C and SP-B-T mice(hSP-B þ/þ, mSP-B �/�) were generated by the self-breeding ofhemizygous mice (hSP-B þ/�, mSP-B �/�). All mice were geno-typed using DNA from tail samples by PCR genotyping with Primerpair 1458/189 for hSP-B and primer pair 75/76 for mSP-B. To verifyhSP-B in hTG mice, primer pair 1458/1401 was used to amplify thewhole fragment of hSP-B gene and the PCR products were analyzedby DNA sequencing.

2.4. Western blotting analysis

To examine the expression of hSP-B protein level in hTG SP-Bmice, Bronchoalveolar lavage fluid (BALF) from hTG mice wasanalyzed by Western blotting with anti-SP-B antibody (HycultBiotech, Plymouth Meeting, PA) at 1:200 dilutions as previouslydescribed [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 tissuewere approximately 5 mm in thickness. Lung sections were stainedwith hematoxylin and eosin, or used for IHC analysis using the ABCkit (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 intratrachealinoculation of 50 ml of P. aeruginosa PA01 (1 � 106 CFU/mouse) orsterile saline (sham control). Mice were sacrificed 24 h post infec-tion. Tissues were harvested and prepared as described previously[15,16].

2.7. Electron microscopy analysis

Lung tissues were prepared as previously described [17]. Thesamples were fixed by 4% glutaraldehyde, 2.5% paraformaldehydefor 24 h, then stained with osmium tetroxide, 1.5% potassiumferrocyanide and embedded in Embed812 resin (Electron micro-scopy science). The tissues were cut into ultrathin sections (90 nm)and stained with lead citrate and 2% aqueous uranyl acetate beforeelectron microscopy analysis. Each sample was examined at themagnification 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.

2.8. Surfactant large and small aggregates

Mouse lungs were lavaged for 3 times each with 0.7 ml salinesolution. BALFs from 6 mice of each group were prepared andcentrifuged at 150 � g, 4 �C for 10 min and the supernatant wasthen centrifuged for 15min at 40,000� g, 4 �C. After centrifugation,the pellet containing surfactant large aggregates was resuspendedin 0.3 ml of saline for surface tension study as previous description[18,19]. The phospholipid concentration of the large aggregates wasdetermined using phosphate assays to be around 1 mg/mL.

2.9. Analysis of surfactant activity

Surface activity of the mouse surfactants was determined with aconstrained drop surfactometer (CDS; BioSurface Instruments, HI)[20]. A droplet of the mouse surfactant of ~10 mL was dispensedonto the CDS drop holder. After the equilibrium surface tensionwasestablished by rapid adsorption, the surfactant film was com-pressed and expanded at a rate of 3 s per cycle with a compressionratio controlled to be less than 40% of the initial surface area. Atleast five compression-expansion cycles were studied for eachdroplet. Cycles were quantified with the minimum surface tension(gmin) at the end of compression and the maximum surface tension(gmin) at the end of expansion. Drop images were taken at 10 framesper second. The surface tension and surface area were determinedwith Axisymmetric Drop Shape Analysis (ADSA) [21].

2.10. Statistical analysis

All the experiments were repeated at least 3 times in this study.Western blot bands were quantified by software Quantity One(version 4.6.1). Statistics were performed by SigmaStat version 3.5software. Significant difference in statistics among groups wasconsidered when p < 0.05 by t-test or ANOVA.

3. Results

3.1. Generation of hTG SP-B-C and SP-B-T mice

To generate hTG SP-B-C and SP-B-T mice, the cDNA of hSP-Bgene was cloned into a basic 3.7-hSP-C/SV40 vector (Fig. 1A) andA 5.4 kb DNA fragment was microinjected into fertilized FVB/Noocytes from WT mice. A total of 18 hSP-B positive founders wereidentified by PCR. To eliminate the mSP-B background, the condi-tional SP-B knockout (KO) mice by inserting Neomycin resistantgene into the gene (Fig. 1B) were used to breed with hSP-B positivefounders. The hSP-B positive F1were bredwith conditional SP-B KOmice to generate F2 hSP-B positive mice (hSP-B þ/mSP-B �/�)(Fig. 1C). Finally, four homozygous hTG SP-B-C and SP-B-T mouselines, e.g. (hSP-B þ/þ, mSP-B �/�), were obtained by the self-breeding of hemizygous mice (hSP-B þ/�, mSP-B �/�) andshowed healthy status suggesting that hSP-B protein functions wellin the transgenic mice.

3.2. Expression of transgene SP-B-T or SP-B-C in hTG mice

The histology of lung in the hTG SP-B-c and SP-B-T mice fromfour founders was assessed using H/E staining sections (Fig. 2A).The results demonstrated hTG SP-B-C founder line and SP-B-Tfounder lines displayed normal alveolar structure and septalthickness. The chord length (mean linear intercepts: Lm) as ameasure of the acinar air space complex was measured, no signif-icant difference was found between hTG SP-B-C and SP-B-T mice.

Furthermore, hSP-B expression was analyzed in the lung by IHCmethod (Fig. 2B) and western blotting (data shown in 3.4). Theresults indicated that only alveolar type II epithelial cells showedpositive staining for SP-B expression in the lung tissue of both hTGSP-B-T and SP-B-C mice (Fig. 2B).

3.3. Bacterial pneumonia

To examine the functional variation of hSP-B genetic variantsunder infectious conditions, a bacterial pneumonia model wasdeveloped and applied for the assessment of functional effects ofhSP-B genetic variants in vivo. hTG mice were infected intra-tracheally by 50 ml of P. aeruginosa PA01 (106 CFU/mouse) or sameamount of saline solution (Sham). The infected mice were sick butsurvival for 24 h. The lung pathohistology displayed pneumoniapathogenic characteristics and lung injury in the infected mice butnot in Sham control (Fig. 3A). A large amount of neutrophils wereobserved in the BALF of infected mice but not in the control. Ul-trastructural analysis revealed significant decrease of lamellarbodies in the type II cells and remarkable less microvilli on thesurface of type II cells in infected mice compared to Sham (Fig. 3B,p < 0.05), suggesting bacterial infection decreased activation oftype II cells and surfactant/protein expression.

3.4. Differential surfactant activity between SP-B-C and SP-B-T inresponse to bacterial infection

Surfactant activity of large aggregates of BALF from infected andsham mice was analyzed by the Constrained Drop Surfactometer(CDS) [18e20]. The results indicated that uninfected hSP-B-T andhSP-B-C mice (sham) had similar surface activity, i.e., minimumsurface tension (gmin) about 1.8e2.2 (mN/m) (Fig. 4A). After infec-tion, the minimum surface tension in infected SP-B-C miceincreased significantly by two times compared to uninfected con-trol (Fig. 4A, p < 0.05), suggesting that surfactant activation hasbeen inhibited in the infected SP-B-C mice. But the minimum sur-face tension of infected SP-B-T mice (hSP-B-T) showed less changecompared to uninfected SP-B-T mice (Fig. 4A). These data indicatethat the SP-B-T mice had less susceptibility on surfactant activity tobacterial infection when compared to SP-B-C mice (p < 0.05)(Fig. 4A). Analysis of Western blotting demonstrated decreased SP-B levels of both infected SP-B-C and SP-B-T mice compared to theiruninfected mice (Fig. 4B, p < 0.01). Infected SP-B-C mice had lowerSP-B tendency vs. infected SP-B-T mice.

4. Discussion

Pulmonary surfactant consists of about 90% phospholipids and10% surfactant-associated proteins, including SP-B, an essentialprotein for normal lung function [1]. The SP-B precursor is about42 kDa peptide containing three sapsin-like protein domains, e.g.domain N, M, and C. Mature 8-kDa SP-B protein is derived fromsapsin-like domain M, which plays a key role in lowering surfacetension in the alveolar space [22]. Decreased levels of mature SP-Bsignificantly influence lung function and oxygenation [23] andcause disease exacerbation [24]. The SP-B SNP (rs1130866 C/T) wasidentified to be associated with several pulmonary diseasesincluding pneumonia [12], in which the individuals carrying the Callele of SP-B are more susceptible than those with the T allele [13].However, the detailed mechanisms are unclear. The humanized SP-B transgenic mouse model provides a powerful in vivo tool forstudying themechanistic role of human SP-B genetic variants in thepathogenesis of pulmonary diseases [25].

Previous studies found that the production of mature SP-Bprotein is completed by complex SP-B precursor processing and

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-Bexpression 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. 3. Lung injury caused by bacterial pneumonia. Mice were infected intratracheallywith 50 ml of bacterial solution (1 � 106 CFU/mouse) or saline (sham) and sacrificed24 h after infection. Lung tissues were fixed with 10% Formalin solution. The resultsshowed that infected mice had severe lung injury with a large amount of inflammatorycells e.g. PMN and macrophages in the alveoli and pathogenic changes of lung tissuesbut not in uninfected mice (A). For analysis of lung ultrastructure after infection lungtissues from infection and sham group of hSP-B-T and hSP-B-C mice were fixed stainedand embedded, and then analyzed by electron microscopy as described in themethods. The results showed decreased lamellar bodies in the type II alveolar cellsfrom infected mice compared with sham mice (B). The hSP-B-T and hSP-B-C miceshowed a normal ultrastructure of type II alveolar cells in health mice.

Fig. 4. Different susceptibility of surfactant activity in hTG SP-B-C and SP-B-T mice tobacterial infection. Surfactant large aggregates were prepared from the BALF samplesas described in the methods. Minimum surface tension (gmin) was measured. The re-sults showed that there are similar minimum surface tension (gmin) in the uninfectedSP-B-C and SP-B-T mice, but the level of minimum surface tension (gmin) was increasedsignificantly in infected SP-B-C mice compared to infected SP-B-T mice, as well asuninfected SP-B-C mice (Panel A). Western blot analysis showed that the hSP-Bexpression in BALF 20 mg of total protein from the BALF of sham or infection groupsamples was subjected to 12% SDS-PAGE under non-reducing condition. Human BALFwas used as a positive control. The hSP-B protein was detected by a rabbit anti-pig SP-Bantibody (at 1:200X). The dimer (16 kDa) of hSP-B was detected on the blot. The datashow that samples from infection group express a decreased level of hSP-B protein bycomparing with sham group (B). There is a significant difference between sham andinfection groups (p < 0.01) (C).

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trafficking [26]. SP-B was secreted to alveoli or stored in thelamellar bodies in the alveolar type II cells of lung [26]. Severalproteases are involved in the protein processing, including proteaseNapsin and Cathepsin H [26]. Differentially posttranslationalmodifications of SP-B precursor caused by genetic variation mayhave an impact on the efficiency of SP-B processing and traffickingin the type II cells. The C allele of human SP-B has an additionalprotein glycosylation site at the residue Asn129 compared to the Tallele [11,27]. SP-B C and T alleles are two common genetic alleles inhuman population and no obviously abnormal SP-B expressionwas

observed in the healthy individuals with either the C or T allele,indicating the additional Asn129 glycosylational modification inSP-B precursor may not significantly influence physiological func-tion under healthy conditions. The similar levels of the SP-Bexpression in the hTG SP-B-C and SP-B-T mice in this study alsoconfirmed the observation in human beings. However, it is un-knownwhether the additional glycosylation site at the As129 in theSP-B-C variant has an negative impact on pro-SP-B processing andtrafficking under disease conditions, like bacterial pneumonia.

Previous studies demonstrate that there are decreased expres-sions of SP-B after a variety of infectious conditions [28]. In thepresent study we observed a significant decrease of SP-B level inthe BALF of infected mice, and SP-B level in infected SP-B-C micewas lower than infected SP-B-T mice, suggesting that alteredglycosylation modification at the Asn129 in the SP-B-C mice

L. Ge et al. / Biochemical and Biophysical Research Communications 469 (2016) 171e175 175

influenced SP-B processing and trafficking under bacterial pneu-monia. Although decreased levels of other surfactant proteins (SP-A, SP-C and SP-D) in the BALF were also observed in the infectedmice compared to uninfected mice (data not shown), but no dif-ferences of these protein expressions were detected in infected SP-B-C and SP-B-T mice in the infectious condition. Furthermore, ul-trastructural analysis of the lung tissues indicated lower density ofLB in type II cells suggesting decreased activation of alveolar type IIcells in infected mice. These pathological changes in the cellularand molecular levels in the lung of infected SP-B-C and SP-B-T micemight cause lung dysfunction in mouse pneumonia.

The SNP (rs1130866 C/T) studied in the present work has beenfound to be associated with several pulmonary diseases in severalindependent groups [13] [7,12]. For instance, individual with C/Cgenotype was found more susceptible to RDS [29] while individualwith T/T genotype was found to protect the patients with SSCagainst the development of ILD [7]. The individuals carrying one ormore C allele had more severe lung injury and required MVcompared to those with T/T genotype [30]. These patients-basedgenotyping analyses demonstrate that the C allele of SP-B is moresusceptible to various pulmonary diseases. This hTG model mayprovide a unique tool to study the mechanisms of human SP-Bgenetic variants on functional variation in various pulmonary dis-eases. Indeed, the results observed in this work revealed differen-tial susceptibility of human SP-B genetic variants in response tobacterial pneumonia. Decreased surfactant activity of BALF in thelung might lead to worse respiratory function and disease severityin pneumonia. However, it is warrant to explore detailed mecha-nisms how the altered glycosylational modification at the Asn129influence pro-SP-B processing, folding and trafficking in the alve-olar type II cells in vivo in various diseases in the future.

Disclosure

All the authors declared no competing interests.

Acknowledgments

We would like to thank Drs. Jeffrey A. Whitsett and Stephan W.Glasser (Cincinnati Children's Research Foundation, Cincinnati, OH)for kindly providing SP-B KO mice and the basic 3.7-hSP-C/SV40vector. This work was supported in part by NIH grant HL096007,Michael Connolly Endowment Fund and Hendricks Foundation.

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