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Transduction of salivary glands with antimicrobial peptide genes has great potential for oral infection control. Our ultimate goal is to introduce antimicrobial peptide genes into salivary glands that secrete these peptides into saliva to control bacterial/fungal infection in the oral cavity. However, an animal study model to test this potential has not been established. Therefore, we determined to test (i) whether the potent antimicrobial peptide human β-defensin-2 (hBD-2) can be overexpressed in saliva after transduction of salivary glands and (ii) whether oral fungal infection can be developed in a NOD/SCID murine model. Lentiviral vector SIN18cPPTRhMLV bearing hBD-2 cDNA was introduced into SCID mouse submandibular glands via cannulation. Reverse transcription polymerase chain reaction (RT-PCR), immunohistochemistry or enzyme-linked immunosorbent assay (ELISA) were performed to detect hBD-2 expression in glands or in saliva. Candida albicans 613p was inoculated orally into SCID mice to establish oral candidiasis. Whilst expression of hBD-2 was detected in mouse salivary glands by RT-PCR and immunohistochemistry 1 day or 1 week following delivery of lentivirus, hBD-2 was not detected in saliva. There was recoverable C. albicans from the oral cavity and gastrointestinal tract 4 days to 4 weeks after infection, but there was no establishment of observable oral candidiasis in SCID mice under a stereomicroscope. Our data indicate that lentiviral vectors transduce mouse salivary glands, but not at a sufficient level to allow hBD-2 detection in saliva. Other vectors for gene transduction and additional treatment of SCID mice to establish oral candidiasis are needed in order to utilise mouse salivary glands to test antimicrobial gene therapy.
Since the discovery of natural antimicrobial peptides/proteins, researchers have tried not just to understand how these peptides/proteins work and applied them in topical use for infection control, but have also explored the possibility of linking their use to gene-based therapeutics, i.e. transducing cells with these peptide genes to augment the innate resistance of tissues/organs against infection [1–5]. Tracheal antimicrobial peptide (TAP), a member of the β-defensin family of antibiotic peptides found in the tracheal mucosa of cows, was used to generate transgenic animals to produce TAP in milk that is antimicrobial . Among a long list of naturally occurring antimicrobial peptides, some well characterised ones are encoded by single genes and their products do not need post-translational processing, making them good candidates for antimicrobial gene therapy. Human β-defensins (hBDs or DEFB) have drawn considerable attention for this purpose as they have potent antimicrobial abilities and some also exert inhibitory effects on viral infections [6–13]. Although only four hBDs (hBD-1–4) have been well characterised, up to 28 hBDs may exist based on genomic surveys using a computational search strategy [14,15]. Fourteen hBD or DEFB gene transcripts (DEFB-1, -4 and -103–114) were investigated using reverse transcription polymerase chain reaction (RT-PCR) to detect their expression in gingival keratinocytes . Transduction of epithelial cells in skin or oral mucosa with hBDs has been tested for its potential to enhance infection control [8,12,17]. Primary fibroblasts have also been proposed as target cells to express hBDs, but the amount of antimicrobial peptide secreted may be insufficient to exert an effect [17,18].
Salivary glands have been considered an excellent target organ for gene-based therapeutics compared with other tissues or organs (e.g. liver and skeletal muscle) both for systemic and upper gastrointestinal tract gene therapeutic applications. Salivary glands are well encapsulated, which limits undesired extraglandular vector dissemination. In addition to being an exocrine secretion organ, physiological existence of endocrine secretory pathways in these tissues has been reported [19–21]. In 1996, O’Connell et al.  first utilised salivary glands as a target organ for antimicrobial gene-based therapy to control oral infection. The gene encoding the anticandidal protein histatin 3 was transferred to rat salivary glands using an adenovirus-directed approach, and up to 1 mg/mL of histatin exerting a strong candicidal effect was detected in saliva. However, the study did not test whether the secreted histatin 3 in saliva controls oral fungal infection in vivo.
hBDs are expressed in salivary glands and are detected in saliva [22–25]. They are presumably among a plethora of defence mechanisms in the oral cavity to combat infections. hBD-2 is a potent antimicrobial peptide effective against a broad spectrum of bacteria, including Gram-positive bacteria, Gram-negative bacteria and fungi. Among these microbes, Streptococcus mutans and Lactobacillus acidophilus, which are associated with dental caries, and certain strains of periodontal bacteria such as Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis have been shown to be sensitive to hBD-2 [18,26]. hBD2 is chemotactic for T-cells, immature dendritic cells, mast cells and tumour necrosis factor-alpha (TNF-α)-primed neutrophils [10,27,28]. Its expression in saliva may enhance not only the innate but also adaptive immunity of the oral mucosa. Together with its ability to inhibit human immunodeficiency virus (HIV) infectibility [11,13], these characteristics make hBD-2 an ideal candidate for a study model of antimicrobial gene therapy to control oral infection.
Transduction of salivary glands with antimicrobial peptide genes has great potential for oral infection control. Our ultimate goal is to introduce antimicrobial peptide genes into salivary glands, which secrete these peptides into saliva to control bacterial/fungal/viral infection in the oral cavity. However, an animal study model to test this potential has not yet been established. In the present study, our objective was to address two questions before establishing this model: (i) whether the potent hBD-2 can be expressed in saliva after transduction of salivary glands using lentiviral vectors; and (ii) whether oral fungal infection can be developed in a NOD/SCID murine model. Our findings provide an insightful understanding on the technical issues and concerns for future investigation regarding salivary gland antimicrobial gene therapy for oral infection control.
The antimicrobial activity of recombinant (r)hBD-2 was assayed as previously described [6,18]. The following microorganisms were used for the assays: Candida albicans 613p (from Dr C. Haidaris, University of Rochester, Rochester, NY) ; P. gingivalis ATCC 33277 (from Dr S.K. Haake, UCLA, Los Angeles, CA); and Escherichia coli ML-35p (from Dr T. Ganz, UCLA) . Candida albicans 613p was grown in yeast extract–peptone–dextrose (YEPD) broth, P. gingivalis was grown in mycoplasma broth base (Becton Dickinson, Franklin Lakes, NJ) supplemented with 0.5 μg/mL hemin SS (Sigma, St Louis, MO) and 0.5 μg/mL menadione (Sigma) and E. coli ML-35p was grown in Lauria–Bertani broth. Microorganisms were cultured at 37 °C in aerobic conditions (except P. gingivalis, which used anaerobic conditions), grown to exponential phase, washed with phosphate-buffered saline (PBS) twice and re-suspended in low or high salt buffer (10 mM Na phosphate, pH 7.4 or 100 mM Na phosphate, pH 7.4, respectively, each containing 0.03% trypticase soy broth) or in filter-sterilised (0.2 μm) human saliva from healthy donors. The input concentration was adjusted to 2 × 106 cells/mL and the suspension was incubated with high-performance liquid chromatography (HPLC)-purified rhBD-2 (from Dr T. Ganz) with final hBD-2 concentrations of 0, 0.1, 1 and 10 μM. Mixtures of microbes and hBD-2 were then incubated under aerobic or anaerobic (P. gingivalis) conditions for 1 h at 37 °C. Subsequently, the mixtures were serially diluted and plated onto appropriate agar plates to quantitate surviving microbes for colony-forming unit (CFU) analysis.
Whole saliva from healthy mouse or human donors was collected. Mouse saliva was collected by administering 0.5 mg/kg pilocarpine via subcutaneous injection to induce saliva secretion. Saliva was filtered (0.2 μm) and stored at −80 °C until use. Forty-five microlitres of saliva was mixed with 5 μL of hBD-2 (16 ng/mL) and incubated at 37 °C for 0, 10, 30 and 60 min. At each time point, samples were snap frozen in liquid N2 until enzyme-linked immunosorbent assay (ELISA).
HT-1080 (human fibrosarcoma cell line, ATCC CCL-121) was grown in Alpha-Minimum Essential Medium (Life Technologies/GIBCO BRL, Gaithersburg, MD) with 10% fetal bovine serum supplemented with 100 units/mL penicillin G, 100 μg/mL streptomycin and 0.25 μg/mL fungizone (Gemini Bio-Products, Inc., Woodland, CA).
A lentiviral vector SIN18cPPTRhMLV-E (SIN18-E), the packaging plasmid pCMVR8.2DVPR and the vesicular stomatitis virus G protein expression plasmid (pHCMV-G) were obtained from Dr I.S.Y. Chen, UCLA . The SIN18-E vector containing the enhanced green fluorescent protein (EGFP) reporter cDNA was released and inserted with hBD-2 cDNA yielding SIN18-hBD-2 as described previously .
Packaging cells 293T (2 × 107) were co-transfected with 12.5 μg of SIN18-hBD-2 or SIN18-E, 12.5 μg of pCMVR8.2DVPR and 5 μg of pHCMV-G plasmids using a calcium phosphate precipitation method . The viral supernatant was collected on Days 2 and 3 after transfection, filtered (0.45 μm) and concentrated ca. 100-fold by ultracentrifugation as described previously . Viral concentration was measured against known concentrations of viral protein p24 and virus titration was performed using 293T cells with various dilutions of the viral stock and analysed for EGFP expression by fluorescence microscopy and flow cytometry on Day 3 post infection. Vector titres were routinely 108 infectious units/mL. Expression of hBD-2 was analysed by ELISA as described below.
The amount of secreted hBD-2 was determined by ELISA as described previously [9,17] using monoclonal anti-human hBD-2 antibodies (1:5000 dilution) as anchoring antibodies, polyclonal rabbit anti-human hBD-2 antibodies (1:2000 dilution) as detecting antibodies and horseradish peroxidase (HRPO)-labelled polyclonal goat anti-rabbit immunoglobulin G (Pierce, Rockford, IL) as a second-step antibody. Bound HRPO was visualised with fresh developing buffer containing a substrate of optimal concentrations of o-phenylenediamine dihydrochloride (Sigma), 0.01% H2O2 and 20 mM sodium citrate pH 4.0. The developing reaction was stopped with the addition of 2.5 M sulphuric acid. Absorbance was determined at 490 nm with a microplate reader (Bio-Tek Instrument, Inc., Laguna Hills, CA) and the concentrations were determined with Delta Soft III software. rhBD-2 produced in baculovirus-transfected insect cells was used as a standard. All antibodies were kindly provided by Dr T. Ganz (UCLA).
Virus from the packaging cell line 293T was used to infect HT-1080 cells. Cells (5 × 105) were exposed to 400 ng of virus at 37 °C in the presence of 5 μg/mL polybrene. After overnight incubation, cell cultures were replaced with fresh medium and incubated for 3 days. Secreted hBD-2 was detected by ELISA. To select clones with the highest hBD-2 expression, transduced cells were subsequently diluted to 1 cell/well in 96-well plates for subcloning. Single cell colonies were selected and hBD-2 expression was detected by ELISA. Colonies expressing high levels of hBD-2 were further subcultured for subsequent use.
Male 6–8-week-old NOD/SCID mice (SCID, NOD.CB17-Prkdc-scid/J; Jackson Laboratory, Bar Harbor, ME) were anaesthetised via intraperitoneal injection with a mixture of 80 mg/kg ketamine and 8 mg/kg xylazine and mounted on a custom-made operating board. Mouse jaws were held open with a custom-made device for cannulation under a stereomicroscope. Extended polyethylene tubes (PE-10; Intramedic, Sparks, MD) were inserted into the openings of glandular ducts and 50 μL of SIN18-hBD-2 or SIN18-E viral vector was administered into each submandibular gland by retrograde ductal instillation. Virus with an average HIV-1 p24 concentration of 12 × 103 ng/mL was used. NOD/SCID mice were euthanised 24 h or 1 week after gene transfer. Submandibular glands were removed from mice at the time of sacrifice and subjected to hBD-2 detection. Mice were maintained in the animal care centre at the UCLA Division of Laboratory Animal Medicine. All procedures involving experimentation and handling of mice described herein followed the protocols approved by the UCLA Animal Research Committee.
Tumours were generated by transcutaneous injection of 107 HT-1080 cells transduced with pBabe-hBD-2 as described previously [9,17] or SIN18-hBD-2 in 200 μL of Hank’s balanced salt solution, using a 26 gauge needle, into the back of NOD/SCID mice (Jackson Laboratory). Tumours were grown until they reached ca. 1.5 cm × 1.5 cm in size. Mice were than euthanised and tumours were resected for subsequent hBD-2 detection by RT-PCR and immunostaining.
Total RNA was extracted from resected submandibular glands or tumours from NOD/SCID mice using TRIzol reagent (Invitrogen, Carlsbad, CA) and cDNA was synthesised using Advantage RT-for-PCR kit (BD Biosciences Clontech, Palo Alto, CA). The appropriate amounts of cDNA and specific primers (final concentration 0.4 μM each) for hBD-2 in the reaction mixture plus Taq DNA polymerase were then used for PCR. Amplification was performed at 95 °C for 1 min followed by 35 cycles of 94 °C for 45 s, 55 °C for 30 s, 72 °C for 1.5 min and 72 °C for 7 min as the final step. Sequences of the primer set for hBD-2 were 5′-CCAGCCATCAGCCATGAGGGT-3′ (sense) and 5′-GGAGCCCTTTCTGAATCCGCA-3′ (antisense) with an expected target size of 255 bp. PCR products were analysed by 1% agarose gel electrophoresis with ethidium bromide staining. Human placenta RNA and human glyceraldehyde-3-phosphate dehydrogenase primers provided in the RT kit were used to ensure the accuracy of the RT-PCR procedures and to determine the quality of the sample RNA.
Resected submandibular glands and tumours were divided into several fragments and some were snap frozen in tissue freezing medium (Triangle Biomedical Sciences, Durham, NC) and stored at −80 °C. Cryostatic sections (10 μm) were fixed in cold acetone and blocked in serum followed by overnight incubation with primary antibodies (polyclonal rabbit anti-human hBD-2 (1:500)) at 4 °C. Sections were then incubated with fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit antibodies (Sigma) for 2 h at room temperature, washed, cover-slipped with VECTASHIELD Mounting Medium with 4′,6-diamidino-2-phenylindole (DAPI) according to the manufacturer’s instructions (Vector Laboratories, Burlingame, CA) and examined under a fluorescence microscope.
The oral mucosal surfaces of male NOD/SCID mice (8–10 weeks old; Jackson Laboratory) or normal wild-type C57BL/6 mice were swabbed using cotton-tipped applicators soaked with C. albicans (107 blastoconidia/mL) grown to exponential phase in YEPD broth. Visual observation under a stereomicroscope was performed at different time points to determine the establishment of oral candidiasis (white patches of plaque) on the tongue and cheek, followed by an oral swab with PBS-wetted cotton-tip applicators. The cotton tips were then placed in 5 mL of YEPD broth containing 100 units/mL penicillin G and 100 μg/mL streptomycin and incubated overnight at 37 °C. Some mice were euthanised and the tongue and intestine were resected. One-half of the resected tissues were homogenised and added to 5 mL of YEPD broth with antibiotics as above for overnight incubation at 37 °C. The other half of the tissues were fixed in 10% formalin for standard haematoxylin and eosin (H&E) staining for histological observation of fungal colonisation of the mucosa.
Candida albicans 613p is known to establish oral candidal infection in rats after surgical hyposalivation . It was therefore chosen as the infectious agent to establish oral fungal infection for the study model. We first tested its sensitivity to recombinant hBD-2 by an in vitro antimicrobial assay. The potency of hBD-2 was verified by performing parallel experiments using E. coli and P. gingivalis ATCC 33277 as positive controls, which are reported to be sensitive to hBD-2 at various concentrations [6,32].
Microorganisms grown to exponential phase were incubated with HPLC-purified hBD-2 under low or high salt conditions. After 1 h at 37 °C, the mixtures were plated on agar plates to quantitate surviving microbes (CFU analysis). The findings for E. coli susceptibility to hBD-2 (Fig. 1A) are in accordance with a report by Liu et al. , which showed that E. coli is only sensitive to hBD-2 under low salt conditions. Porphyromonas gingivalis ATCC 33277 was also sensitive to hBD-2 but at a higher concentration, as reported by Joly et al. , although it was more resistant than E. coli. Under low salt conditions, growth of P. gingivalis at 10 μM hBD-2 was completely inhibited (Fig. 1B), similar to the findings in a report by Mineshiba et al. . Growth of C. albicans 613p was inhibited by 1 μM hBD-2 by 87% or 97% under high and low salt conditions, respectively (Fig. 1C), suggesting that this C. albicans 613p strain appears to be a suitable candidate for the proposed study model.
In order for hBD-2 secreted into saliva to exert its function in the oral cavity, it is important that the secreted hBD-2 is not degraded in saliva, which contains many enzymes and proteases. To verify this possibility, whole saliva collected from NOD/SCID mice or a human donor was incubated with rhBD-2 at 37 °C for various time intervals. The samples were then subjected to ELISA to detect hBD-2. The results shown in Fig. 2 indicate no degradation of hBD-2 by mouse saliva after 60 min of incubation, whereas intact hBD-2 was reduced to ca. 60% in human saliva after 30–60 min of incubation, suggesting that hBD-2 is resistant to mouse salivary enzyme but is sensitive to human salivary enzyme degradation to some extent. It should be noted that mouse saliva was collected by stimulation of salivation with pilocarpine.
One day after hBD-2 gene transfer into submandibular glands of SCID mice, glands were removed and RNA was isolated to detect hBD-2 mRNA expression using RT-PCR. A positive control experiment was performed in parallel to ensure that in vivo hBD-2 expression can be detected. Selected clones of HT-1080 transduced with either pBabe-hBD-2 or SIN18-hBD-2 that secrete hBD-2 in vitro were injected into SCID mice subcutaneously to form tumours. RNA was isolated from the resected tumours and underwent the same procedures as for mouse submandibular glands. hBD-2 was detected via RT-PCR in tumours (Fig. 3A) and in submandibular glands (Fig. 3B).
To verify the presence of hBD-2 peptides in submandibular glands 1 week after gene transfer, immunohistochemistry staining was performed. hBD-2 was observed in submandibular glands as well as in HT-1080 tumours (Fig. 4). The data confirmed successful hBD-2 gene integration into salivary gland acinar cells following salivary duct cannulation. In addition, salivary gland staining (Fig. 5) verified the positive expression of hBD-2 in gland cells. However, hBD-2 was not detected by ELISA in saliva collected (after pilocarpine stimulation) from mice transduced with hBD-2 in the submandibular glands after 1 day or 1 week, nor was it detected from mice transduced after 2–3 months.
On Day 0 (before candidal infection), no fungus was detected either in NOD/SCID or normal C57BL/6 mice. On Days 4 and 12 after oral inoculation, no C. albicans was detected from the oral cavities of C57BL/6 mice, whereas a large number of C. albicans was recovered from the oral cavity swab samples and from homogenised tissues (tongue and intestine) of NOD/SCID mice after overnight incubation in YEPD broth. However, no oral manifestation of oral candidiasis was observed in any of the tested mice under stereomicroscopic examination, nor was there detection of any fungal colonisation or invasion into the epithelial or subjacent tissues of oral and intestinal mucosa using H&E analysis. Similar findings were noted 4 weeks after fungal infection of NOD/SCID mice, although some mice appeared ill and died after several weeks of infection.
The ultimate goal of this study was to utilise salivary glands as a target organ for antimicrobial gene therapy against oral infections, especially for immunocompromised individuals. Although there are available animal models and a potent hBD-2 ready to test this potentially applicable clinical treatment modality, several questions need to be addressed before a workable animal study model may be established. We stepwise tested the antimicrobial activity against C. albicans 613p that was to be used as part of the study model and verified its sensitivity to hBD-2, which has been shown to be a potent antimicrobial agent against some strains of C. albicans [6,32,33]. The effective concentrations of hBD-2 against C. albicans are 1–14 μM based on published reports, which coincides with our finding using C. albicans 613p.
The presence of hBD-2 in human saliva was detected by Mathews et al.  with an estimated concentration of 150 ng/mL, which is below the level required for effective antimicrobial function (1 μM hBD-2 = 4.3 μg/mL). The exact source of hBD-2 in saliva remains elusive. It was speculated to be from oral keratinocytes (an abundant source of this peptide) and from submandibular glands and minor salivary glands in which hBD-2 mRNA, not its peptide, was detected in a few human samples [25,34]. hBD-2 was not detected in human parotid glands [24,34]. In contrast, hBD-1 mRNA is expressed in all human salivary glands and its peptides in duct cells of minor salivary glands as well as in saliva [34,35]. Since hBD-2 is more potent than hBD-1, overexpressing hBD-2 in human saliva appears to be a logical approach for oral infection control. A study has shown that individuals who develop oral candidiasis have lower hBD expression in saliva, including hBD-2 . Together with our finding of some loss of hBD-2 integrity (down to 60%) in human saliva, these facts further support the approach of overexpressing hBD-2 in saliva via gene transfer of salivary glands to compensate for this loss.
O’Connell et al.  used pilocarpine to induce rat saliva secretion after adenoviral vector-mediated histatin 3 gene transduction of submandibular glands. In the collected rat saliva, they found >1 mg/mL of histatin 3 expressed. Although adenoviral vectors offer a sufficient level of peptide expression in salivary glands and saliva, its inherited transient effect and elicitation of strong immune reactions preclude its usability for this purpose. Our attempt to use the lentiviral vector to transduce mouse salivary glands indicated that although hBD-2 was detected both at mRNA and protein levels in glands, it was not detectable in saliva. This finding answers our first question regarding whether hBD-2 can be expressed in mouse saliva after transduction by a lentiviral vector. Although the lentiviral vector is powerful in transducing non-dividing cells in vitro and in vivo, we found that it does not appear to be potent in producing a high level of transgene expression in mouse cells . This is in agreement with the findings of Shai et al.  who compared a number of vector systems for transducing mouse salivary glands. Lentiviral vector transduction was found among others to have lower transgene expression levels. None the less, the research group found that feline immunodeficiency virus-based lentiviral vectors appear to provide satisfactory long-term expression of the transgene in mouse salivary glands , suggesting a potential future use of this vector for gene transduction of mouse salivary glands.
Rodents, especially mice, have been used to establish oropharyngeal candidiasis or more extensive fungal infections as a study model to test antifungal agents for treatment or for clinical monitoring of infection [29,39–44]. SCID mice have been used by many investigators as a model to study mucosal candidiasis [40,45]. However, a clear establishment of oral candidal infection was only reported by Takakura et al. [39,42] using prednisolone to immunosuppress the mice and they were able to observe macroscopically typical lesions consisting of white patches on the tongues. Candida albicans 613p, although showing establishment of oral candidiasis in the rat after surgical hyposalivation by ligation of the parotid salivary ducts plus surgical removal of the submandibular and sublingual salivary glands, based on our finding it did not establish oral candidiasis in NOD/SCID mice. Hyposalivation to induce formation of candidiasis after fungal infection is a contraindication for our purpose. Whilst oral candidiasis was not established in NOD/SCID mice, recovery of live fungus after incubation in broth indicates that there was some degree of fungal colonisation in the mouse oral cavity and gastrointestinal tract. This may be sufficient to serve as a study model for antifungal gene therapy. Nevertheless, the main issue is the level of hBD-2 expression in saliva. In conclusion, our studies addressed a technical barrier that needs to be overcome before an optimal study model can be established to test salivary gland antimicrobial gene therapy for oral infection.
The authors wish to acknowledge Dr T. Ganz for providing E. coli ML-35p, rhBD-2 and antibodies; Dr I.S.Y. Chen for the lentiviral vectors; Dr C. Haidaris for C. albicans 613p; Dr S.K. Haake for P. gingivalis ATCC 33277; and Drs B. Baum and J. Wang at NIH for providing technical instructions on mouse salivary duct cannulation. This study was supported in part by an NIH/NIDCR Grant R21 DE14585 (G.T.-J.H.).
This work was performed at UCLA School of Dentistry.