Animals, husbandry, and experimental group design
The Institutional Animal Care and Use Committee of Allegheny Health Network Research Institute approved all animal experimentation described herein. The C57BL/6.NOD-Aec1 Aec2 mouse line [9] was acquired as a kind gift from Dr Ammon Peck (University of Florida, Gainesville, FL, USA) and mice were genotyped to confirm their identity using the probes: D3Mit151-F: 5′-GGTAAAATATTTTCTGGGCAAGC, D3Mit151-R: 5′-TTGTTAATTGTAATTCTGTTTCTGTCG. All gene transfer was performed on animals at 8 weeks of age.
A total of 48 mice were utilized in this study. One cohort of Aec1/Aec2 mice (n = 33) was assigned for determination of relative levels of IL-17R:Fc transcript and local and systemic levels of IL-17 protein. These animals received the following treatments: naïve (n = 2), UAGT/IL17R:Fc (n = 4), AdIL17R:Fc 1 × 107 viral particles (vp) (n = 4), AdIL17R:Fc 1 × 108 vp (n = 5), and AdIL17R:Fc 1 × 109 vp (n = 5) and were sacrificed 48 hours after gene transfer. A second cohort (n = 15) was utilized for proteomic profiling of submandibular glands following gene transfer. These Aec1/Aec2 animals received the following treatments: UAGT/Luc (n = 5) and UAGT/IL17R:Fc (n = 5). A third group in this cohort (n = 5) were C57/BL6 background strain animals who received sham gene transfer consisting of anesthesia and ductal cannulation.
Vector design and preparation
The plasmid vectors pCMV-GL3 (Luc) have been previously described by our group [10] and were used to generate the pCMV-IL17R:Fc vector, ensuring both vectors were isogenic with respect to the backbone. The adenoviral vector expressing IL-17R:Fc (AdIL17R:Fc) was acquired as a kind gift from Dr Jay Kolls (University of Pittsburgh, Pittsburgh, PA, USA) and was upscaled and purified using standard methods previously described by our group [11]. The cDNA for the IL-17R:Fc fusion protein was generated from adenoviral genomic DNA, sequence verified, and cloned into pCMV-GL3, replacing the GL3 sequence with the IL-17R:Fc sequence. Plasmid vectors were upscaled by growing in DH5α-competent cells (Life Technologies, Carlsbad, CA, USA) and vectors were purified using CsCl gradient ultracentrifugation.
Salivary gland gene transfer
At 8 weeks of age, Aec1/Aec2 animals or background C57/BL6 animals received gene transfer to the submandibular glands bilaterally via UAGT or adenovirus. UAGT was performed as we have previously described [10]. Briefly, animals were anesthetized with a mixture of ketamine and xylazine and the submandibular duct was cannulated bilaterally, then 50 μl of solution containing either the adenoviral vector or 15 % v/v Definity microbubbles and 1 μg/μl of plasmid vector in normal saline was infused. Bubbles were destroyed by four 30-s bursts from a Sonigene device (Visualsonics, Inc., Toronto, ON, Canada) set for 1 MHz, 50 % duty cycle and 2 W/cm2, with 10 s between pulses. Following the four pulses, the transducer was withdrawn and the animal was rested for 10 minutes before the catheter was removed.
IL-17R:Fc transcript and IL-17 protein detection in submandibular glands
At 48 hours after gene transfer, animals were sacrificed by cervical dislocation and submandibular glands removed and homogenized in RNALater. Total RNA was isolated from homogenized tissue using the RNEasy protocol (Qiagen, Venlo, Limburg Netherlands) and cDNA synthesized using a first strand synthesis kit (Roche Life Sciences, Indianapolis, Indiana USA). IL-17R:Fc transcript levels were determined by CYBR green quantitative real-time reverse transcription PCR using mouse glyceraldehyde 3-phosphate dehydrogenase (GAPDH) transcription level as an internal control on a Roche LightCycler® 480. The following primers were used: IF-17R:Fc-F: 5′-ATGGCTGCTTCTGCTGCT, IL-17R:Fc-R: 5′- CTTGACTCTGCAGCTCAGCC -3′ GAPDH-F: 5′- CGTCCCGTAGACAAAATGGT, GAPDH-R: 5′- TTGATGGCAACAATCTCCAC. The PCR program used was: 1) 95 °C, 5 minutes and 2) 95 °C 10 s, 60 °C 10 s, 72 °C 10 s, with 45 cycles.
An aliquot of these samples was analyzed by western blot. Protein concentrations were evaluated using the Bradford method and adjusting protein concentration to 1 μg/μl: 20 μg of each individual sample was separated by 15 % SDS-PAGE and transferred onto nitrocellulose membrane. The membranes were blocked with 5 % milk and probed with anti-IL-17 (1:2,000) (Abcam, Inc. Cambridge, MA, USA) and visualized using a horseradish-peroxidase-linked secondary antibody and Amersham ECL prime Western blotting Detection Reagent (GE Healthcare, Pittsburgh, PA, USA). GAPDH (Abcam) was used as a loading control.
Serum IL-17 measurement
At 48 hours after gene transfer animals were sacrificed and blood collected by cardiac puncture. Serum IL-17 concentrations were determined by the Bio-Plex Pro™ Assay (Bio-Rad, Hercules, CA, USA).
Proteomic profiling of salivary glands
Animals were sacrificed 21 days after gene transfer (to mimic the optimal functional improvements observed by Nguyen et al. [7]) by cervical dislocation and submandibular glands removed and homogenized on ice using 2D lysis buffer (7 M Urea, 2 M Thiourea, 2 % CHAPS). Samples were cleaned using a 2-D Quant kit (GE Healthcare) and resuspended in 2D gel rehydration buffer (7 M Urea, 2 M Thiourea, 2 % CHAPS) containing 50 mM Tris pH8.5. Protein concentrations were determined using the Bradford method and adjusted to 1 μg/μl. Profiling was performed initially on pooled samples (n = animals/group): 1 μl of Cy2, Cy3 or Cy5 were added to 50 μg of each pooled group sample respectively. Three gels were performed with dye swapping, to ensure that each pooled group was labeled with each Cy dye. After addition of dye, samples were incubated on ice for 30 minutes in the dark. After 30 minutes, 1 μl of 10 mM lysine was used to quench the reaction by incubating on ice for 10 minutes in the dark.
Isoelectric focusing (IEF) and SDS electrophoresis were performed as we have previously described [12]. Briefly, 15 μg of each labeled sample was diluted in rehydration buffer to 450 μl for IEF. A 24-cm strip, pH3-10NL was rehydrated by adding 2 % DTT, 0.5 % IPG buffer and 0.002 % bromophenol blue room temperature for 8 hours. Samples were applied to rehydrated strips and loaded onto an Ettan IPGphor 3. IEF was performed overnight for 60,000 volt-hours (vhr). The next day, the strips were equilibrated with 1 % DTT followed by 2.5 % iodoacetamide for 15 minutes each and separated in a 13.5 % SDS gel. Finished gels were scanned on a Typhoon Trio (GE Healthcare). The pictures were edited using ImageQuant TL 7.0 software (GE Healthcare). Differential in-gel analysis (DIA) and biological variation analysis (BVA) of the two-dimensional differential gel electrophoresis (2D DIGE) results were performed using DeCyder 2D 7.0 Software (GE Healthcare).
Pairwise comparisons
In order to confirm the statistical validity of spots of interest identified by DIA analysis, pairwise comparisons of the various groups were made to allow BVA. Pairwise comparisons were performed on randomly paired individual samples from each group labeled respectively with Cy3 or Cy5 or vice versa and using a mixed sample labeled using Cy2 as an internal standard.
Phosophoprotein staining
In order to determine the phosphorylation of spots 4 and 5, 2-D gels were run (as described above) with a single sample, either UAGT/Luc and UAGT/IL-17R:Fc, and stained with Pro-Q Diamond phosphoprotein gel stain reagent for phosphorylated protein. The locations of spots 4 and 5 were identified using landmarks, and the gels were washed and stained with Sypro Ruby for total protein. Spots 4 and 5 were then cut from the gel and protein ID performed to confirm their identity. Both reagents were obtained from Thermo Fisher Scientific Inc. Waltham, MA USA.
Protein identification
Protein identification was performed as previously described [13]. Briefly, protein spots of interest were excised from the 2-D gel, reduced by DTT, alkylated with iodoacetamide, digested with trypsin and desalted with C18 ZipTips (Millipore Corporation, Billerica, MA, USA). Both mass spectrometry (MS) and tandem mass spectrometry (MS/MS) analyses of the digested peptides were performed on the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF)/TOF tandem MS (Bruker UltrafleXtreme MALDI TOF/TOF Mass Spectrometer, Bruker Daltonics Inc. Billerica, MA, USA). The database search and analysis were performed using FlexAnalysis and BioTools software (Bruker Daltonics Inc.) against mouse Swiss-Prot protein database using a local Mascot search engine.
Statistical analysis
Group comparisons in Figs. 1a and 2 were made using the Mann-Whitney U test. Analysis of proteomic profiles was performed within the DeCyder software, with 2-fold changes as the threshold. DIA was used for pooled gels and BVA was used for pairwise comparisons. In all cases, statistical significance was considered to be p <0.05.