Indoleamine 2,3-Dioxygenase Overexpression Causes Kynurenine-modification of Proteins, Fiber Cell Apoptosis and Cataract Formation in the Mouse Lens

Maneesh Mailankot; Magdalena M. Staniszewska; Heather Butler; Moonkyung H. Caprara; Scott Howell; Benlian Wang; Catherine Doller; Lixing W. Reneker; Ram H. Nagaraj

Disclosures

Lab Invest. 2009;89(5):498-512. 

In This Article

Materials and Methods

Establishment of Transgenic Mouse Lines Expressing hIDO

The studies conformed to the ARVO Statement on the Use of Animals in Ophthalmic and Vision Research and were approved by the Case Western Reserve University Institutional Animal Care and Use Committee. Two independent transgenic mouse lines were produced by the standard pronuclear microinjection technique. The details of the transgene DNA construct are illustrated in Figure 1. The human IDO (hIDO) gene was inserted between EcoR1 sites of a minigene construct, which contained a chick δ1-crystallin lens enhancer upstream of the αA-crystallin promoter and a rabbit ß-globin intron. Human growth hormone polyA was inserted downstream from the αA-crystallin promoter. At 2-3 weeks after birth, tail biopsies were obtained, and genomic DNA was screened for the transgene integration using PCR with forward primer 5'-TCTGAGAGCCTCTGCTGCTC-3' and reverse primer 5'-GGTCCATGGTGATACAAGGGAC-3'. Identified founders were crossed with wild-type (Wt) C57BL/6 to establish a hemizygous transgenic (hemTg) line. Homozygous transgenic (homTg) mouse lines were established by breeding the hemizygous mice within the same line.

Figure 1.

Transgene. Schematic representation of the hIDO transgenic construct. The chick δ1-crystallin enhancer (δ1) was fused to the mouse αA-crystallin promoter (αA-P) to make the chimeric promoter, δenαA. The hIDO cDNA was inserted between the ß-globin intron (ß-glo int) and human growth hormone polyA signal (hGH pA, 660 bp).

Morphological Changes

Mice were killed by CO2 asphyxiation. Following dissection, lenses were immediately placed on an electron microscopy grid to assess opacity. Lenses were then examined in bright field using a x 4 objective on an Olympus BX-60 upright microscope (Olympus, Tokyo, Japan). Images were captured in color using a SPOT RT Slider camera (Diagnostic Instruments, Sterling Heights, MI, USA). Lens diameter measurements were carried out using Spot software version 3.5.5.

Hematoxylin and Eosin Staining

Immediately after dissection, eyes were fixed in 10% neutral-buffered formalin, embedded in paraffin, cut into 5-µm sections, and stained with hematoxylin and eosin using a standard procedure.

Electron Microscopy

Mouse eyeballs were fixed with triple aldehyde-DMSO fixative for 2 h at room temperature. The specimens were thoroughly rinsed in 0.1 M phosphate buffer (pH 7.4), and then fixed for 2 h in a buffered 1:1 mixture of 2% osmium tetroxide and 3% potassium ferricyanide. After rinsing in water, the specimens were soaked overnight in an acidified solution of 0.25% uranyl acetate. After another rinse in distilled water, they were dehydrated in ascending concentrations of ethanol, passed through propylene oxide, and embedded in an Epon mixture. Sections (80 nm) were cut with an RMC MT 6000-XL ultramicrotome. The sections were sequentially stained with acidified methanolic uranyl acetate and lead tartrate and examined with a JEOL 1200EX transmission electron microscope.

Slit Lamp Imaging

Animals were anesthetized by 2,2,2-tribromoethanol in 2-methyl-2-butabol (both from Sigma-Aldrich) (i.p. injection at 0.2 ml per 10 g). Pupils were dilated with topically applied 0.5% mydriacyl and examined using a fixed slit lamp with an adapted Nikon D2Xs digital SLR camera. Images were taken using axial light with full illumination. Slit beams were offset by 10°; minimum beam size was used and additional fill light was provided with a fiber optic source.

IDO Activity

Lenses were homogenized on ice in 200 µl PBS and centrifuged (14 000 g, 4°C, 15 min). Supernatants containing water-soluble proteins were used for the IDO assay. The reaction mixture contained 50 mM sodium phosphate buffer (pH 6.5), 20 mM ascorbic acid sodium salt, 200 µg/ml bovine liver catalase (Sigma-Aldrich), 10 µM methylene blue, and 400 µM L-tryptophan. The reaction was carried out at 37°C for 1 h and was stopped with 40 µl 30% (w/v) trichloroacetic acid (TCA). The samples were incubated at 65°C for 15 min to convert NFK to KYN and then centrifuged (14 000 g, 4°C, 15 min). Controls were prepared in the same way except that the water-soluble protein was incubated with an IDO inhibitor, 20 µM 1-methyl-D,L-tryptophan (Sigma-Aldrich). KYN content was estimated by reverse-phase HPLC along with standards ranging from 0.2 to 5.0 nM L-KYN (Sigma-Aldrich) using a GraceVydac C18 column (250 x 4.6 mm, 5.0 µm) with ammonium acetate (10 mM) as solvent A and 10% methanol in ammonium acetate as solvent B. The percentage of solvent B in the gradient was 0% (10 min), 0-100% (10 min), and 100-0% (15 min). The flow rate was 0.8 ml/min. The eluant was monitored at 360 nm (Jasco UV-970 UV-VIS detector), and the peak areas were determined using PowerChrom software. IDO activity was expressed as nanomoles of KYN formed per milligram protein per minute. Protein concentration was determined using the Bio-Rad reagent.

Immunostaining of IDO

After rehydration in xylene and ethanol series, paraffin-embedded eye sections were treated with citrate buffer (pH 6.0) for 20 min at 70°C, cooled, and then incubated in 3.0% hydrogen peroxide to block endogenous peroxidase. The sections were incubated with streptavidin D and biotin blocking solution for 15 min each at room temperature and then in mouse-on-mouse (M.O.M; Vector Laboratories, CA, USA) Ig blocking solution for 1 h at room temperature. After washing in PBS, sections were incubated in M.O.M. diluent, followed by incubation in mouse anti-IDO mAb (Chemicon International, CA, USA) diluted to 24 µg/ml in PBS, overnight at 4°C. After thorough washing in PBS, the slides were incubated with M.O.M. biotinylated anti-mouse IgG reagent and rinsed thoroughly in PBS. The slides were then incubated in ABC Vectastain Elite Peroxidase (Vector Laboratories) and rinsed in PBS. Sections were stained by incubating in 3,3'-diaminobenzidine substrate, rinsing thoroughly in deionized water, and counterstained with hematoxylin. The negative control was processed similarly, but without the primary antibody. The slides were viewed with an Olympus BX-60 upright microscope (Olympus). Images were captured in color using a SPOT RT Slider camera (Diagnostic Instruments) connected to a Macintosh computer using Spot software version 3.5.5.

Quantification of hIDO mRNA

RNA was extracted from lenses using TRIZOL reagent, and RNA quality was verified by agarose gel electrophoresis. cDNA was generated using the Super Script First Strand Synthesis System for RT-PCR (Invitrogen). Total RNA, 2.5 µg, was denatured, then reverse-transcribed using random primers in a reaction mixture containing RT buffer, 10 mM dNTP, RNase inhibitor, reverse transcriptase, and DTT at 42°C for 1.5 h in a Bio-Rad iCycler. Amplification was verified by electrophoresis.

The hIDO cDNA was first amplified with gene-specific primers: forward 5'-CACTTTGCTAAAGGCGCTGTTGGA-3' and reverse 5'-GGTTGCCTTTCCAGCCAGACAAAT-3' using the Invitrogen PCR Reagent System. The PCR product was 140 bp and the amplification specificity was verified by electrophoresis. The cDNA concentration was determined and standards ranging from 1 x 109 to 1 x 100 cDNA molecules were prepared. Real-time PCR was performed on duplicate samples and the standards using SYBR Green PCR Master Mix (Applied Biosystems). The PCR protocol comprised an initial denaturation step at 95°C for 10 min followed by 40 cycles of denaturation (95°C, 15 s each) and annealing/extension (60°C, 1 min). In preliminary experiments, the PCR products were visualized by agarose gel electrophoresis to verify that a single product was amplified. Real-time PCR data were analyzed with an ABI PRISM 7700 detector using ABI PRISM 7700 dissociation curve software (Applied Biosystems).

Kynurenine Content

Lenses were weighed and homogenized in 100 µl ethanol. The homogenate was stored at -20°C for 1 h and then centrifuged (14 000 g, 10°C, 15 min). The supernatant was removed, kept at -20°C whereas the pellet was reextracted with 100 µl, 80% ethanol. The homogenate was kept at -20°C for 1 h, centrifuged as before, and the supernatants were combined and dried in a SpeedVac concentrator. Samples and standards (0.2-5.0 nmol of KYN) were analyzed by reverse-phase HPLC as described above. Sodium acetate (20 mM)/acetic acid buffer (pH 4.5) used as solvent A and 20% methanol as solvent B. The percentage of solvent B in the gradient was 0% (30 min), 0-50% (2 min), 50-100% (8 min), and 100-0% (6 min). The flow rate was 0.6 ml/min. KYN was expressed as nmol/mg lens.

ELISA for Kynurenine-Mediated Lens Protein Modification

Microplate wells were coated with water-soluble lens proteins in 0.05 M carbonate buffer (pH 9.7) at a concentration of 1 µg per 50 µl, incubated for 1 h at 4°C, and then washed three times with PBST. The wells then were blocked with 5% nonfat dry milk in PBST and washed three times with PBST. The primary antibody, mouse anti-KYN mAb [29] (1:1000 diluted in PBS), was added, the plates were incubated for 1 h at 37°C, washed with PBST, and then incubated with HRP-conjugated goat anti-mouse IgG secondary antibody (Promega; 1:15 000 in PBST). Substrate was added (3,3',5,5'-tetramethylbenzidine (Sigma-Aldrich) with H2O2), the reaction stopped, and absorbance was measured at 450 nm. Primary and secondary antibody specificity was verified by preincubating the primary antibody with KYN-modified RNase A and incubating coated wells with secondary antibody only.

Immunostaining for Kynurenine Modification

After rehydration, high-temperature antigen retrieval, and incubation in 3.0% H2O2, paraffin-embedded sections were blocked with goat serum. The sections were rinsed briefly in PBS and incubated with anti-KYN mAb (1:500 diluted in PBS) or anti-KYN mAb incubated with KYN-modified RNase A. Sections were then incubated with goat anti-mouse IgG Texas Red (1:400 diluted in PBS; Molecular Probes), rinsed, incubated with DAPI/Vectashield, and then permanently mounted. The negative control was processed similarly but without the primary antibody. Images of lenses were acquired on a Leica DMI 6000 B inverted microscope using a x 20 objective and a Retiga EXI camera (QImaging, Vancouver, BC, USA). To obtain 'stitched' images, Metamorph software (Molecular Devices Corp., Downingtown, PA, USA) was used.

Incubation of Lens Homogenate with L-Tryptophan

Water-soluble lens protein was incubated with or without 1 mM L-tryptophan for 12 h at 37°C, after which the samples were mixed with an equal volume of 20% TCA and centrifuged at 14 000 g, 10°C, 15 min. The supernatant was assayed for KYN by reverse-phase HPLC as described above. KYN concentrations were expressed as pmol/mg protein.

Organ Culture with L-Tryptophan

Each lens was carefully dissected by a posterior approach and placed in the well of a 12-well culture plate containing 2 ml M199 medium (pH 7.4, 300 ± 5 mOsm; Sigma-Aldrich) and incubated for 6 h in a CO2 incubator. Only transparent lenses were selected for further assay. M199 medium containing 1 mM L-tryptophan (stock made in 2 N NaOH) was prepared (pH 7.4 and 300 ± 5 mOsm). The medium was changed every 24 h for 3 days, after which KYN was assayed by reverse-phase HPLC as described above and was expressed as nmol/mg lens.

Measurement of Glutathione

Glutathione (GSH) was determined according to the method of Cui and Lou.[31] Lens homogenate was prepared as described above for the incubation with L-tryptophan, and 10 µl was mixed with 10 µl 5,5'-dithiobis(2-nitrobenzoic acid) (2.0 mg per 2.5 ml methanol). The volume was adjusted to 200 µl with 1.0 M Tris-HCl buffer (pH 8.2), containing 0.02 M EDTA. Absorbance of the reaction product was measured at 412 nm.

Apoptosis

After rehydration and antigen retrieval by microwave irradiation in 0.1 M citrate buffer (pH 6.0), paraffin-embedded sections were stained for apoptosis in situ using a kit (In situ Cell Death Detection Kit; Roche) as per the manufacturer's instructions. The sections were counterstained with DAPI. For the negative control, sections were incubated without terminal transferase.

Measurement of Caspase-3 and Caspase-9 Activity

Freshly dissected whole lenses were homogenized in 50 mM Tris-buffered saline. An equal volume of fluorogenic substrate solution (2 x reaction buffer: 10 mM DTT, and 50 µM Ac-DEVD-AFC (for caspase-3) or Ac-LEHD-AFC (for caspase-9)) was added to each lysate. Lysates were incubated for 2 h at 37°C in the dark. Samples were read in a spectrofluorometer (FluoroMax-4; HORIBA Jobin Yvon, USA) at excitation/emission wavelengths of 400/505 nm. Recombinant human caspases (Calbiochem) were used as positive controls.

Identification of KYN-Modified Proteins

Lens homogenate from Wt or homTg lenses corresponding to 200 µg protein in PBS was incubated with 2 µg mouse anti-KYN mAb or nonimmune IgG for 1 h at room temperature followed by addition of protein G-sepharose. The mixture was again incubated on a shaker for 1 h at room temperature. After centrifugation the pellet was washed five times with PBS. To do SDS-PAGE, the pellet was boiled with SDS sample buffer for 5 min at 95°C, centrifuged and the supernatant was run on 15% Tris-HCl buffer. The gel were stained with Bio-Safe staining solution (Bio-Rad) and destained in water. A major protein band (indicated by an arrow in Figure 10c) was cut out from the gel, minced, and subjected to in-gel digestion with trypsin and the peptides were analyzed on an LTQ linear ion trap mass spectrometer (Thermo Fisher Scientific) coupled with an Ettan MDLC system (GE Healthcare). The spectra were acquired by data-dependent methods: one full scan (m/z of 300-2000) followed by MS/MS on the five most abundant precursor ions at the 30% normalized collision energy. The dynamic exclusion was set as follows: repeat count, 1; repeat duration, 45 s; and exclusion duration 180 s. The obtained data were submitted to Mascot by searching Swiss-Prot (sprot 50.3) mouse database, which includes 228670 sequences, 83849098 residues.

Figure 10.

Caspase activity in the lens. (a) Caspase-3 activity in the homTg lens was significantly higher than in Wt and hemTg lenses. (b) Caspase-9 activity was significantly higher in homTg lens compared with Wt and hemTg lenses. Results shown are mean ± s.d. of five independent experiments of five lenses from five mice. *P < 0.0001.

Statistics

The results were analyzed using one-way analysis of variance, followed by the Fisher's protected least significant difference test (using Statview 5.0 software, SAS Institute Inc.). The level of significance was set at less than 0.05.

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