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Supplemental Information
Methods
Mice
A conditional targeting vector for the DNase II gene was designed to delete exons 3 to 5, as
shown in Supplementary Figure S1. In brief, a neo-loxP cassette carrying the PGK
promoter-driven neo gene flanked by lox sequences (provided by Dr. J. Takeda) was
inserted into a Hind III site in intron 5 of the DNase II gene. A 0.44 kb Sma I fragment
containing exons 1 and 2 was replaced with a DNA fragment carrying the corresponding
sequences and a loxP sequence. The diphtheria toxin A-fragment (DT-A) driven by the tk
promoter was inserted at the 3’ end of the vector.
To establish mice carrying the floxed allele of the DNase II gene, the linearized
targeting vector was introduced into R1 ES cells by electroporation. ES clones resistant to
G418 (150 g/ml) were screened for homologous recombination by PCR. Positive clones
were aggregated with BDF1 morula embryos to generate DNase II3lox/+ mice. To remove
the Neo cassette, DNase II3lox/+ mice were crossed with E2a-Cre transgenic mice, and the
resultant DNase IIflox/+E2a-CreT (mosaic) mice were crossed with DNase II+/- mice to
establish DNase IIflox/+ and DNase IIflox/- mice. The Mx1-Cre transgene was then introduced
into the DNase IIflox/+ or DNase IIflox/- mice. DNase IIflox/-Mx1-CreT mice at the age of 4-6
weeks were injected i.p. with 1.5 g/weight (g) of poly(I:C)(Sigma) 3 times at 1-day
interval to activate the IFN gene.
Most of the mice were housed in a specific pathogen-free facility at Osaka University
Medical School and Oriental Bioservices. Some conditional knock-out mice were kept in a
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conventional facility at Osaka University Medical School. DNase II-/-IFN-IR-/- mice had the
C57BL6 background. The conditional DNase II targeting mice carried a mixed background
of the 129 and C57BL6 strains. All animal experiments were carried out in accordance with
protocols approved by the Osaka University Medical School Animal Care and Use
Committee.
Primers used for genotyping
The following primers were used to genotype the respective wild-type and mutant alleles.
DNase II: wild-type sense, 5’-GCCCATCTAGACTAACTTTC-3’
mutant sense, 5’-GATTCGCAGCGCATCGCCTT-3’
common antisense, 5’-GAGTCTTAGTCCTTTGCTCCG-3’
IFN-IR: wild-type antisense, 5’-AAGATGTGCTGTTCCCTTCCTCTGCTCTGA-3’
mutant antisense, 5’-CCTGCGTGCAATCCATCTTG-3’
common sense, 5’-ATTATTAAAAGAAAAGACGAGGCGAAGTGG-3’
TLR-9: wild-type antisense, 5’-GAGTCTTAGTCCTTTGCTCCG-3’
mutant antisense, 5’-ATCGCCTTCTATCGCCTTCTTGACGAG-3’
common sense, 5’-GCAATGGAAAGGACTGTCCACTTTGTG-3’
Mx1-Cre: sense, 5’-GTGGGCACAGCAAGCTCAGG-3’
antisense, 5’-TCCATGAGTGAACGAACCT-3’
flox allele: sense, 5’-CGCACGTCTAAGAAACCATT-3’
antisense, 5’-CAGTACTAGTGAACCTCTTC-3’
Treatment with anti-TNF mAb
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A rat hybridoma MP6-XT22 producing a neutralizing anti-TNF mAb was obtained
from Dr. K. Okumura. The hybridomas were cultured in GIT medium (Nihon Seiyaku), and
the mAb was purified by fractionation with (NH4)2SO3. The mAb (20 g/g body weight)
was administered twice a week by i.p. injection, and clinical parameters were assessed three
days after the last injection.
MRI and radiography
For MRI, the mice were anesthetized by i.p. injection of pentobarbital sodium (Dainippon
Pharmaceutical). 1H-MRI was performed at 3.0 T using a Signa Horizon LX 3T (General
Electric), and MR images were obtained using T1, T2, and fat-suppressed T2-weighted
sequences with a coil designed for small animals. Pixel sizes were 0.12 x 0.12 x 1.50 mm3
(1.50-mm thickness). Radiography of limbs was performed using the MX-20 Specimen
Radiography system (Faxitron X-ray), and imaged on high-speed radiographic film (Fuji
Photo Film).
Histology
To prepare paraffin sections, tissues were fixed with 4% paraformaldehyde in 0.1 M
sodium phosphate buffer (pH 7.2) containing 4% sucrose, and embedded in paraffin. Joints
were incubated at room temperature for 24 h in Morse’s solution (10% sodium citrate and
22.5% formic acid) for decalcification before paraffinization. The blocks were sectioned at
4 m, deparaffinized, and subjected to staining with hematoxylin and eosin, or DAPI
(Dojindo).
For frozen sections, the paraformaldehyde-fixed tissues were placed in PBS
containing 20% sucrose, embedded in OCT compound (SAKURA), quickly frozen,
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sectioned at 6 m, and mounted onto MAS-coated glass slides (Matsunami). The joints
were decalcified by incubation at 4°C for 11 days in 10% EDTA (pH 7.4) after fixation.
TRAP staining was performed using an Acid Phosphatase Leukocyte kit (Sigma
Diagnostics). For immunohistochemical staining, the sections were post-fixed with cold
acetone and treated for 10 min with Peroxidase Blocking Reagent (Dako). After blocking
non-specific binding sites with 5% goat serum and 1% BSA in PBS, the sections were
stained with biotinylated mAb. The sections were further incubated with
streptavidin-conjugated horseradish peroxidase (HRP) (Roche) with 3,3’-diaminobenzidine
tetrachloride (DAB) (DAB Kit, Zymed) as the substrate, and counterstained with Mayer’s
hematoxylin (WAKO). The mAbs used were anti-CD68 (FA-11; Serotec), and anti-CD4
(H129.19; Beckton Dickinson).
For the histochemical staining of TNF, sections were post-fixed in cold acetone, and
washed with HEPES-buffered saline containing 0.1% saponin (HBSS). After blocking the
non-specific binding sites with 5% rabbit serum and 1% BSA in HBSS, the sections were
incubated at 4°C overnight with goat anti-TNF (L-19; Santa Cruz). The signals were
detected with biotin-labelled rabbit anti-goat IgG (Vector), followed by incubation with
Cy3-labelled streptavidin (Sigma). The sections were mounted with Fluorsave containing
0.5 g/ml DAPI, and observed by fluorescence microscopy (Olympus).
Real-time PCR
Limbs were removed from just above the ankle joint and homogenized with a polytron
homogenizer. Total RNA was prepared using an ISOGEN kit (Nippon Gene), treated with
RNase-free DNase I (Qiagen), and purified using an RNeasy kit (Qiagen). RNA (2 g) was
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reverse-transcribed in a total volume of 20 l using Superscript III (Invitrogen) with
oligo(dT) as primer, and Real-Time PCR was carried out using a LightCycler (Roche
Diagnostics). The amount of specific mRNA was quantified at the point where the
LightCycler System detected the upstroke of the exponential phase of PCR accumulation,
referred to the standard curve, and was normalized to glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) or -actin mRNA in each individual sample.
Primers for Real-time PCR
The primers used for Real-Time PCR were as follows:
TNF- sense primer, 5’-CACAGAAAGCATGATCCGCGACGT-3’
antisense primer, 5’-CGGCAGAGAGGAGGTTGACTTTCT-3’
IFN-,
sense primer, 5’-CCACCACAGCCCTCTCCATCAACTAT-3’
antisense primer, 5’-CAAGTGGAGAGCAGTTGAGGACATC-3’
IFN-,
sense primer, 5’-CTTTGCAGCTCTTCCTCATGGCTGTTTCTG-3’
antisense primer, 5’-TGACGCTTATGTTGTTGCTGATGGCCTG-3’
TGF-
sense primer, 5’-GACCGCAACAACGCCATCTA-3’
antisense primer, 5’-GGCGTATCAGTGGGGGTCAG-3’
IL-1,
sense primer, 5’-CCAGCTTCAAATCTCACAGCAG-3’
antisense, 5’-CTTCTTTGGGTATTGCTTGGGATC-3’
IL-6,
sense primer, 5’-ACAACGATGATGCACTTGCAGA-3’
antisense primer, 5’-GATGAATTGGATGGTCTTGGTC-3’
IL-10,
sense primer, 5’- TGGCCCAGAAATCAAGGAGC -3’
antisense primer, 5’-CAGCAGACTCAATACACACT-3’
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IL-18,
sense primer, 5’-ACTGTACAACCGCAGTAATACGG-3’
antisense primer, 5’-AGTGAACATTACAGATTTATCCC-3’
DNase II, sense primer, 5’-GAGACGGTGATCAAGAACCAA-3’
antisense primer, 5’-AATTTTGCCAGAACTGGACCT-3’
MMP-3, sense primer, 5’-ACTCTACCACTCAGCCAAGG-3’
antisense primer, 5’-TCCAGAGAGTTAGACTTGGTGG-3’
-actin, sense primer 5’-TGTGATGGTGGGAATGGGTCAG-3’
antisense primer 5’-TTTGATGTCACGCACGATTTCC-3’
GAPDH, sense primer 5’-AACGACCCCTTCATTGAC-3’
antisense primer 5’-TCCACGACATACTCAGCAC-3’
Analysis of DNA in serum
To quantify the DNA in serum, sera were diluted 4-fold with lysis buffer (100 mM
Tris-HCl buffer [pH 8.5] containing 5 mM EDTA, 0.2% SDS, 200 mM NaCl, and 100
g/ml Proteinase K), and incubated at 55°C for 4 h. The DNA concentration was then
determined using a PicoGreen kit (Molecular Probes) and a SPECTRAmax GEMINI XS
(Molecular Devices) with an excitation wavelength of 480 nm and emission wavelength of
520 nm. Each sample was treated with DNase I, and the value obtained after DNase Itreatment was subtracted as background.