Supplementary Materials for - Science Translational Medicine€¦ · the plasmid pEasy Flirt in...

www.sciencetranslationalmedicine.org/cgi/content/full/5/196/196ra100/DC1

Supplementary Materials for

Transforming Growth Factor–ββββ Signaling in T Cells Promotes Stabilization of Atherosclerotic Plaques Through an Interleukin-17–

Dependent Pathway

Anton Gisterå, Anna-Karin L. Robertson, John Andersson, Daniel F. J. Ketelhuth, Olga Ovchinnikova, Stefan K. Nilsson, Anna M. Lundberg, Ming O. Li, Richard A. Flavell,

Göran K. Hansson*

*Corresponding author. E-mail: [email protected]

Published 31 July 2013, Sci. Transl. Med. 5, 196ra100 (2013) DOI: 10.1126/scitranslmed.3006133

The PDF file includes:

Material and Methods Fig. S1. Smad7 deletion. Fig. S2. CD4Cre+ Smad7fl/fl phenotype. Fig. S3. CD4Cre+ Smad7fl/fl/Ldlr−/− phenotype. Fig. S4. Plasma lipid analyses. Fig. S5. Lipase activity. Fig. S6. IL-6 immunoblot. Fig. S7. Immunohistochemical evaluation of lesions after neutralizing IL-17A treatment. Table S1. Inflammation-associated mRNA expression. Table S2. Basal characteristics of CD4Cre+ Smad7fl/fl/Ldlr−/− . Table S3. Basal characteristics in the neutralizing anti–IL-17A experiment. Table S4. Metabolism-associated mRNA expression.

Gisterå, Robertson et al.

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Supplemental data

Detailed Material and Methods

Generation of Smad7 deficient mice

The Smad7fl/fl mouse strain was developed using standard gene targeting techniques. The

construct with Smad7’s proximal promoter, exon 1 and exon 2 being floxed was created using

the plasmid pEasy Flirt in conjunction with p-Bluescript and PSL1180. The mice were made

using TC1 ES cells (129S6 background). Correctly targeted Smad7fl/+.clones were injected

into d3.5 C57BL/6 blastocysts. The Neomycin resistance gene was removed by crossing the

germ-line mouse with a Flipase-expressing mouse (Jackson Laboratories Strain #3800). The

Smad7fl/+ was backcrossed to C57Bl/6NCr for 6 generations and then crossed with CD4Cre+/+

(from Taconic, strain 4196), backcrossed to C57Bl/6NTac for 9 generations (47). The

offspring was thereafter intercrossed in heterozygous breeding, using CD4Cre+ Smad7fl/fl in

the study group and their littermates, CD4Cre+ Smad7fl/+, as controls. Genotyping was

performed by PCR using Taq DNA polymerase (Invitrogen) according to manufacturer’s

instructions. CD4Cre was detected using forward primer 5'-

TAGTTTGGCAGGACCTTTGG-3' and reverse primer 5'-GCAAACGGACAGAAGCATTT-

3' and heterozygosity for the floxed smad7 gene was determined with forward primer 5'-

TGGCATGGTGATCCAACCTGAC-3' and reverse primer 5'-

TTCCTTCCACAACGTAGGGCTC-3'.

Bone marrow transplantation and experimental atherosclerosis

Bone marrow from femur and tibia was harvested from 14 weeks old male donor mice, and

injected intravenously into 14 weeks old lethally irradiated (14 Gy) male Ldlr-/- (Jackson

Laboratories) recipients, 7.5 million cells/recipient. After 3 weeks on antibiotics (Sulfadiazine

and Trimethoprim) and totally 6 weeks of recovery, high fat diet was fed to the animals

(D12108C 1.25 % cholesterol, 0% cholic acid; Research Diets). Following 8 weeks of

treatment, mice were anaesthetized by CO2 asphyxiation and organs were harvested. The

experiment was repeated twice comparing CD4Cre+ Smad7fl/+ and CD4Cre+ Smad7fl/fl.

During a third experiment only CD4Cre+ Smad7fl/fl bone marrow was transplanted and the

mice were treated with weekly intraperitoneal injections of 50 µg neutralizing anti-IL-17A rat

IgG2A (R&D systems) (48) or unspecific rat IgG2A throughout the eight weeks on high fat


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diet. In a separate study, non-bone marrow transplanted male Ldlr-/-, 20 weeks of age, were

subjected to 8 weeks of high fat diet or standard chow. All animal experiments were approved

by the Stockholm Regional Board for Animal Ethics.

Tissue processing, immunohistochemistry and lesion analysis

Blood from sacrificed mice was collected by cardiac puncture and vascular perfusion

was performed with sterile RNase-free PBS. Aorta, one third of the spleen, liver, inguinal

lymph nodes and para-aortic lymph nodes were dissected and snap-frozen for later RNA

isolation. Hearts were dissected and preserved for immunohistochemistry and lesion analysis.

Lesion analysis was performed as previously described (49). Briefly, hearts were serially

sectioned from the proximal 1 mm of the aortic root on a cryostat. Sections stained with

hematoxylin and oil red O were used to evaluate lesion size. The lesion size was determined

by measuring 8 sections, collected at every 100 μm over a 1 mm segment of the aortic root.

For each section, images were captured, and the surface areas of the lesions and of the entire

vessel were measured. Primary antibodies to CD4, CD8, CD68 and VCAM-1 (all rat anti-

mouse from BD Biosciences) were applied to acetone-fixed cryosections followed by

biotinylated rabbit anti-rat IgG. The primary antibody for I-Ab (mouse anti-mouse, BD

Biosciences) was directly biotin-conjugated. Anti-α-smooth muscle actin (mouse anti-mouse,

Sigma-Aldrich) was detected using M.O.M. kit (Vector Laboratories), and mouse IgG2a (BD

Biosciences) was used as isotype control. Staining was visualized with VECTASTAIN ABC

kit and diaminobenzidine (Vector Laboratories). Plaque size was measured using Image J

software (NIH). Quantification of immunohistochemical stainings were documented as ratio

of thresholded chromogen area using Leica Qwin Standard Y 2.8 computerized analysis

(Leica Microsystem Imaging Solutions) or calculated as number of stained cells divided by

the intimal lesion area. For comparative analysis of lesion content of CD4, CD8, CD68,

VCAM-1, I-Ab and collagen, lesions were size-matched between groups. Collagen content

was assessed by analyzing formaldehyde fixed sections stained 1 hour in saturated picric acid

containing 0.1 % Picrosirius Red (Direct Red 80; Fluka Buchs) and visualized under linear

polarized light as previously described (11).

RNA isolation, cDNA synthesis, and real-time PCR

RNA was isolated from the indicated tissues or cells using RNeasy kit (Qiagen). Total RNA

quality was analyzed on a BioAnalyzer instrument (Agilent Technologies) and quantified with


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260 nm absorbance measurement using a Nanodrop 1000 spectrophotometer (Thermo Fisher

Scientific). Reverse transcription was performed with Superscript-II and random hexamers

(both from Invitrogen). Amplification was performed by real time-PCR using TaqMan

Universal Master Mix and pre-manufactured primers and probes for the genes of interest and

hypoxanthine guanidine ribonucleosyl transferase (HPRT) (Assay-on-demand) in an ABI

7900HT Fast Real-Time PCR System (all Applied Biosystems). Data was analyzed on the

basis of the relative expression method with the formula 2-ΔΔCt, where ΔΔCt = ΔCt (sample) –

ΔCt (calibrator = mean Ct values of the controls), and ΔCt is the Ct of the housekeeping gene

(HPRT) subtracted from the Ct of the target gene.

Western blot

Total proteins were isolated from tissue homogenate by AllPrep DNA/RNA/Protein kit

(Qiagen). For Western blot analysis of IL-6, samples were diluted with 2x Laemmli sample

buffer (Bio-Rad) and boiled. Samples were separated by 4-15 % Mini-PROTEAN TGX

Precast Gels (Bio-Rad) and transferred to a PVDF membrane (GE Healthcare). IL-6 was

detected using a rabbit polyclonal anti-IL6 antibody (Abcam), followed by a goat anti-rabbit

IgG-HRP (Dako), and visualized using ECL system (GE Healthcare). Rabbit polyclonal α-

tubulin antibody (Abcam) was used as a loading control.

FACS analysis and in vitro differentiation

Flow cytometry was performed on leukocytes isolated from spleen. Primary labeled

antibodies were employed for extracellular staining against CD3e (BioLegend), CD4, CD44

and CD62L (BD Biosciences). Intracellular staining was performed using the anti-mouse/rat

Foxp3 staining Set (eBioscience) and anti-Foxp3 (eBioscience). Samples were analyzed on a

Dako CyAn (Beckman Coulter) flow cytometer and data were analyzed using FlowJo

software (Tree Star).

Spleens were harvested from 14 weeks old CD4Cre+Smad7fl/fl and CD4Cre+Smad7fl/+ mice,

single cell suspension from 2 mice were pooled and naïve CD4+ T cells were prepared by

magnetic-activated cell separation purification using CD4+ CD62L+ T Cell Isolation Kit II

(Miltenyi Biotec). Dynabeads Mouse CD3/CD28 T cell expander beads (Invitrogen) was

added to all cultures together with different combinations of antibodies and cytokines: for Th1

conditions, murine IL-12 (10 ng/ml), anti-IL-4 (3 μg/ml); for Treg conditions, human TGF-β1


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(2 ng/ml), murine IL-2 (100 U/ml); for Th17 conditions, human TGF-β1 (2 ng/ml), murine

IL-6 (20 ng/ml), anti-IFN-γ (3 μg/ml), anti-IL-4 (3 μg/ml); and as a control condition, no

cytokines. All cytokines and antibodies were from PeproTech except IL-2 and TGF-β1,

purchased from R&D systems. Cells were cultured in a humid 5% CO2 atmosphere in serum-

free RPMI 1640 media, containing ITS + Premix (BD Bioscience), 0.1 % bovine serum

albumin, non-essential amino acids, L-glutamin, 1mM Sodium pyruvate and 50µM β-ME or

IMDM, serum-free as above or with 10 % FCS, 1 mM Sodium Pyruvate, 50 µM β-ME and

antibiotics. After three days of incubation GolgiPlug (BD Bioscience), 50 ng/ml Phorbol 12-

myristate 13-acetate and 1 µg/ml Ionomycin was added for 4 hours. Thereafter intracellular

staining was performed using anti-Foxp3 (eBioscience), anti-IFN-γ (BioLegend) and anti-

IL-17A (BioLegend) antibodies.

Stimulation of smooth muscle cells (SMCs) with human recombinant IL-17

Human aortic SMCs from 3 different individuals (provided by Drs. Piotr Religa and Nina

Wolmer-Solberg, Karolinska Institutet) and commercially available primary culture of

coronary SMCs (Cascade Biologics) were maintained in smooth muscle basal medium

(Cambrex Bio Science). After 48 hours starvation in medium supplemented with 1% FCS, the

following incubations were performed; medium only, IL-17A (50 ng/ml) or TGF-β1 (10

ng/ml) (PeproTech) as a positive control for 48 and 72 hours. Incubation with IL-17A and 75

µM PD-98509 (ERK-inhibitor, Calbiochem) was also performed. Experiments were repeated

at least twice.

RNA was isolated as previously described. Assay-on-demand for procollagen I α1-chain

(COL1A1) and cyclophilin A (PPIA) were used for real time-PCR. Data were analyzed with

the relative expression method with the formula 2-∆∆Ct, with PPIA as the housekeeping gene

and the ∆Ct of cells treated with cytokine-free medium as calibrator. The relative expression

upon IL-17 or TGF-β stimulation was calculated for each individual SMC line separately.

Collagen analysis in cultured SMCs

After stimulation, SMC layers were extensively washed with 1xPBS, fixed with 2 %

paraformaldehyde for 10 minutes and washed in running tap water for 15 minutes. The culture

dishes were then air-dried before incubation with 1 ml Picrosirius Red dye for 1 hour under

constant mild shaking. Excess dye solution was removed by suction and the cell layers were


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washed extensively with 0.01 N hydrochloric acid to remove non-bound dye. Cell

morphology was photo documented under microscope. The stained material was dissolved in

0.2 ml 0.1 N sodium hydroxide using a microplate shaker for 30 minutes at room temperature.

The dye solution was transferred to Nunc microtiter plates and the optical density (OD) was

measured at 550 nm against 0.1 N sodium hydroxide as a blank (50).

Total protein isolation and western blot were performed as described above. Protein extracts

from SMCs were separated by 12 % SDS-PAGE. Collagen type I was detected using a rabbit

polyclonal anti-human collagen I antibody (AbD Serotec). Mouse anti-human α-tubulin

antibody (Sigma) was used as a loading control.

Blood and lipid analyses

Blood from sacrificed mice was collected by cardiac puncture or through tail vein bleeding at

other time-points. Whole blood (EDTA) was analyzed on a Scil Vet abc hemocounter (Scil

animal care company). Plasma was separated by 1500g centrifugation for 15 minutes. Plasma

cholesterol and triglycerides were analyzed using enzymatic colorimetric kits (Randox

Laboratories) according to the manufacturer’s protocol. Plasma lipoprotein profiles were

analyzed using a Superose 6 10/300 GL column (GE Healthcare) coupled to Prominence

UFLC system (Shimadzu) and equilibrated with Tris-buffered saline, pH 7.4. Fractions of

200µl were collected using Foxy Jr. fraction collector (Teldyne Isco) and subsequent

detection of cholesterol and triglycerides using above mentioned enzymatic kits.

For lipoprotein lipase and hepatic lipase activity measurement, the lipases were released to

plasma with heparin injections (100 U/kg) and post-heparin plasma was collected after 15 min

to heparin coated tubes. Lipoprotein lipase activity was assessed in a system using [3H]-

labelled triolein in an emulsion with Intralipid 10 % composition as enzyme substrate. Rat

serum was added as donor of apoCII (53). Hepatic lipase was inhibited using antibodies

against rat hepatic lipase (54). Specific hepatic lipase activity was measured using a gum

Arabic-stabilized emulsion (55). Post-heparin plasma lipase activity was determined by

triplicate readings and the mean was used. Lipase activity is expressed as mU/ml where one

mU corresponds to one nmol of fatty acid released per minute at 25°C (56).

The concentrations of IFN-γ, TNF, IL-2, IL-4, IL-5 and IL-17A in plasma were determined

by cytofluorometric bead assay (BD Bioscience) using a Dako CyAn (Beckman Coulter)


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flow cytometer as described by the manufacturer. Concentration of Serum Amyloid A was

determined with PHASE Murine Serum Amyloid A Enzyme Immunoassay (Invitrogen).

For blood glucose measurement a drop of fresh tail vein blood were applied to a test stick for

immediate analysis using a FreeStyle Lite blood glucose monitoring system (Abbott Diabetes

Care).


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Supplemental figures

Supplemental figure S1. Smad7 deletion

A. To verify the deletion of Smad7 in CD4Cre+ Smad7fl/fl, T cells were isolated from both CD4Cre+ Smad7fl/fl and CD4Cre+ Smad7fl/+ splenocytes by magnetic microbead separation using Pan T Cell Isolation Kit (Miltenyi Biotec). Smad7 mRNA was then analyzed in the isolated T cells and compared to that in T cell depleted splenocyte preparations. The bar graph shows that Smad7 mRNA expression is abrogated in CD4 and CD8 T cells in CD4Cre+ Smad7fl/fl mice but preserved in non-T cells and in CD4Cre+ Smad7fl/+ mice (n=3).

B. Phosphorylated Smad2/3 assessed in splenocytes with BD phosflow (BD Biosciences) according to the manufacturer’s instruction. Histograms showing CD3+CD4+ cells, the dashed line representing unstimulated control and the black lines cells stimulated with 3 ng/ml TGF-β1 for 15 minutes, CD4Cre+ Smad7fl/+ vs. CD4Cre+ Smad7fl/fl, the latter tinted in grey (n=2).

C. Western blot of protein extracts from T cells, isolated from lymph node single cell suspensions with Dynal Mouse CD4 Negative Isolation kit (Invitrogen), and stimulated with 3 ng/ml recombinant TGF-β1. Phosphorylated Smad2 (Phospho-Smad2, polyclonal rabbit antibody, Cell Signaling) was analyzed as an indicator of TGF-β signaling. Smad2 (rabbit monoclonal antibody, Cell Signaling) and α-tubulin (rabbit polyclonal antibody, Abcam) were loading controls. (n=4, Student’s t-test, P=0.0034)


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Supplemental figure S2. CD4Cre+ Smad7fl/fl phenotype

A. Spleen cell composition regarding CD3+CD4+CD62L/CD44 (naïve/effector-memory T cells) comparing CD4Cre+ Smad7fl/fl to CD4Cre+ Smad7fl/+mice. 2 mice were pooled per group for immunostaining and FACS analysis (n=1).

B. CD3+CD4+Foxp3+ cell populations in spleen (n=4). C. T cells were separated as described in supplemental fig 1A, cultured for 48 hours with

Dynabeads Mouse CD3/CD28 T cell expander with addition of PMA/Ionomycin/GolgiPlug for the last 4 hours, and then stained for intracellular IL-17A (n=3).


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Supplemental figure S3. CD4Cre+ Smad7fl/fl /Ldlr-/- phenotype

A. Dissected aortic arches were stained with Sudan IV en face. Representative stained samples from each group are shown.

B. Quantification of en face prepared aortas. The percentage lesion areas of the total aortic arch area are shown (n=4-5, Student’s t-test, P=0.096).

C. The percentage lesion areas in the innominate artery are shown (n=3-5, Student’s t-test, P=0.20).

D. No difference in CD3+CD4+FOXP3+ T regulatory cell populations in the spleen of CD4Cre+ Smad7 /Ldlr-/- chimeras was detected after the high fat diet period of eight weeks (n=3-4, Student’s t-test, P=0.98).


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Supplemental figure S4. Plasma lipid analyses

A. Plasma cholesterol levels were measured throughout the high fat diet treatment period. Non-fasted and fasted samples are plotted on separate y-axes for clarity, on the left y-axis showing non-fasted samples (n=5 in each group) and on the right fasted samples. Week 1, 3 and 6, plasma was collected after 4 hours fasting. Week 9 plasma was collected after 8 hours fasting (n=4 in each group). Data presented as relative to the mean of the controls.

B. Triglycerides measured in plasma as in A. Data are presented as relative to the mean of the fasted control group.

C. Total plasma cholesterol in non-fasting (fed) (n=9-15, Student’s t-test, P=0.013) and fasted animals (n=3-4, P=0.24).

D. Total plasma triglycerides in non-fasting (fed) (n=9-15, Student’s t-test , P=0.0007) and fasted animals (n=3-4, P=0.16)

E. Cholesterol lipoprotein profile shows significantly elevated VLDL/CR peak in CD4Cre+ Smad7fl/fl /Ldlr-/- mice (n=5, P=0.032). Student’s t-test P value for the LDL-peak = 0.22.

F. Triglyceride lipoprotein profile shows significantly elevated VLDL/CR peak (n=5, Student’s t-test P=0.0031).


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Supplemental figure S5. Lipase activity

A. LPL activity measured in fed-state plasma collected 15 minutes after heparin injection (100 U/kg) (n=4-5, Student’s t-test, P=0.90).

B. LPL activity measured in soleus muscle lysate (n=4-5, Student’s t-test, P=0.71). C. LPL activity measured in gonadal white adipose tissue lysate (n=4-5, Student’s t-test,

P=0.44). D. Hepatic lipase activity measured in post-heparin plasma as in A (n=4-5, Student’s t-

test, P=0.20).


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Supplemental figure S6. IL-6 immunoblot Immunoblots of para-aortic lymph node tissue stained for IL-6 and α-tubulin. The left lines show samples from four Ldlr-/- mice fed standard chow and the four lines to the right, samples from high fat diet fed Ldlr-/-. Quantification shown in fig 3D.


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Supplemental figure S7. Immunohistochemical evaluation of lesions after neutralizing IL-17A treatment

A. CD68 staining of size-matched lesions in the aortic root (n=7, Student’s t-test, P=0.81). Arrows indicate CD68+ area in the atherosclerotic lesions.

B. CD4 staining (n=7-8, Student’s t-test, P=0.37). Arrows indicate CD4+ cells in the atherosclerotic lesions.

C. CD8 staining (n=7-8, Student’s t-test, P=0.092). Arrows indicate CD8+ cells in the atherosclerotic lesions.

D. I-Ab staining (n=6, Student’s t-test, P=0.35). Arrows indicate IAb+ cells in the atherosclerotic lesions.

E. VCAM-1 staining (n=7, Student’s t-test, P=0.096). Arrows indicate VCAM-1+ area in the atherosclerotic lesions.


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Supplemental table S1. Inflammation associated mRNA expression -mRNA levels evaluated in para-aortic lymph nodes, aorta, inguinal lymph nodes, spleen, and liver. Calculated as relative expression using the 2-ΔΔCt formula, normalized to the house-keeping gene HPRT.

Organ Gene name

CD4Cre+ Smad7fl/+ /Ldlr-/-

CD4Cre+ Smad7fl/fl /Ldlr-/-

Significance (Mann-Whitney test)

Spleen Tbx21 1.24 ± 0.30 N=7 1.39 ± 0.49 N=9 n.s. Gata3 1.22 ± 0.29 N=7 1.42 ± 0.41 N=9 n.s. Foxp3 1.19 ± 0.25 N=7 1.23 ± 0.30 N=9 n.s. Ror-γ 1.22 ± 0.28 N=7 1.16 ± 0.26 N=9 n.s. IL-17A 2.69 ± 1.62 N=7 2.41 ± 1.14 N=9 n.s. IL-6 1.07 ± 0.24 N=4 0.93 ± 0.10 N=5 n.s. TGF-β1 1,19 ± 0,43 N=4

0,87 ± 0,21 N=5 n.s.

Inguinal lymph nodes

Tbx21 1.03 ± 0.11 N=7 1.09 ± 0.12 N=8 n.s.

Gata3 1.02± 0.08 N=7 1.30 ± 0.13 N=8 n.s. Foxp3 1.01 ± 0.07 N=7 1.26 ± 0.05 N=8 P=0.014 Ror-γ 1.03 ± 0.12 N=7 0.92 ± 0.07 N=8 n.s. IL-17A 1.84 ± 0.78 N=7 1.37 ± 0.26 N=8 n.s. IL-6 1.03 ± 0.13 N=4 1.15 ± 0.08 N=5 n.s. TGF-β1 1,02 ± 0,11 N=4 1,03 ± 0,05 N=5 n.s. Para-aortic lymph nodes

IL-4 1.16 ± 0.35 N=5 0.70 ± 0.10 N=10 n.s.

IL-6 1.08 ± 0.22 N=5 1.27 ± 0.15 N=10 n.s. IL-10 1.15 ± 0.33 N=5 0.66 ± 0.12 N=10 n.s. IFN-γ 1.05 ± 0.18 N=5 0.92 ± 0.12 N=10 n.s. TGF-β1 1.02 ± 0.11 N=5 1.43 ± 0.15 N=10 n.s. Tnf 1.02 ± 0.12 N=5 0.85 ± 0.03 N=10 n.s. Ccl2 1.04 ± 0.13 N=5 1.06 ± 0.18 N=10 n.s. Ccl5 1.02 ± 0.10 N=5 1.28 ± 0.13 N=10 n.s. Helios 1.05 ± 0.15 N=5 1.07 ± 0.14 N=10 n.s. Aorta Tbx21 1.09 ± 0.18 N=8 1.34 ± 0.28 N=14 n.s. Gata3 1.09 ± 0.17 N=8 1.16 ± 0.13 N=14 n.s. Foxp3 1.24 ± 0.32 N=8 1.42 ± 0.39 N=14 n.s. Ror-γ 1.19 ± 0.25 N=8 1.20 ± 0.20 N=14 n.s. IL-17A Not detected Not detected - IFN-γ 1.32 ± 0.37 N=8 1.38 ± 0.34 N=14 n.s. TGF-β1 1,02 ± 0,10 N=4 0,73 ± 0,09 N=5 n.s. Liver TGF-β1 1,02 ± 0,10 N=4 0,96 ± 0,18 N=5 n.s.


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Supplemental table S2. Basal characteristics of CD4Cre+ Smad7fl/fl /Ldlr-/-

Phenotype of the Ldlr-/- chimeras after the high fat diet period of eight weeks.



Significance (Student’s t-test)

Body weight (g)

26.1 ± 0.7 N=7 26.0 ± 0.6 N=9 n.s.

B-glucose (mmol/l)

7.35 ± 0.45 N=4 7.84 ± 0.43 N=5 n.s.

Lymphocytes (109/L)

3.01 ± 0.64 N=7 4.89 ± 0.31 N=14 P=0.0075

Monocytes (109/L)

0.36 ± 0.10 N=7 0.46 ± 0.08 N=14 n.s.

Granulocytes (109/L)

2.63 ± 0.79 N=7 2.54 ± 0.4058 N=14 n.s.

Plasma IFN-γ (pg/ml)

12.0 ± 5.1 N=8 15.6 ± 1.8 N=14 n.s.

Plasma TNF (pg/ml)

27.9 ± 10.7 N=8 35.3 ± 4.4 N=14 n.s.

Plasma IL-2 (pg/ml)

7.6 ± 2.4 N=8 9.3 ± 0.9 N=14 n.s.

Plasma IL-4 (pg/ml)

6.0 ± 2.7 N=8 8.6 ± 1.0 N=14 n.s.

Plasma IL-6 (pg/ml)

38.3 ± 15.9 N=8 29.7 ± 13.8 N=14 n.s.

Plasma IL-10 (pg/ml)

140.0± 66.28 N=8 193.3 ± 47.0 N=14 n.s.

Plasma IL-17A (pg/ml)

22.5 ± 4.7 N=8 25.3 ± 3.0 N=14 n.s.

Plasma Serum Amyloid A (µg/ml)

79.2 ± 54.3 N=3 96.6 ± 32.8 N=4 n.s.


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Supplemental table S3. Basal characteristics in the neutralizing anti-IL 17A experiment -

of the phenotype of the CD4Cre+ Smad7fl/fl /Ldlr-/- treated with control IgG och neutralizing anti-IL-17A treatments.

Control IgG2A CD4Cre+ Smad7fl/fl /Ldlr-/-

Anti-IL-17A CD4Cre+ Smad7fl/fl /Ldlr-/-

Significance (Student’s t-test)

Body weight (g)

26.7 ± 0.8 N=7 29.3 ± 0.9 N=8 n.s.

Plasma Cholesterol (mmol/l)

31.7 ± 1.6 N=7 35.6 ± 1.3 N=8 n.s.

Plasma Triglycerides (mmol/l)

3.7 ± 0.3 N=7 3.7 ± 0.6 N=8 n.s.

B-glucose (mmol/l) 5.27 ± 0.50 N=7 7.56 ± 0.44 N=8 P=0.0039 Lymphocytes (109/L) 5.82 ± 1.18 N=5 6.22 ± 1.12 N=5 n.s. Monocytes (109/L) 0.52 ± 0.10 N=5 0.52 ± 0.06 N=5 n.s. Granulocytes (109/L) 2.30 ± 0.41 N=5 2.34 ± 0.38 N=5 n.s.


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Supplemental table S4. Metabolism associated mRNA expression -

mRNA expression of genes associated with metabolism in liver, quadriceps femoris muscle, gonadal white adipose tissue, and proximal small intestine. Measured with real-time quantitative PCR and calculated as relative expression using the 2-ΔΔCt formula, normalized to the house-keeping gene HPRT.

Organ Gene name



Significance (Mann-Whitney test)

Liver Apoa5 1.04 ± 0.15 N=4 0.98 ± 0.19 N=5 n.s. Apob 1.00 ± 0.04 N=4 1.07 ± 0.22 N=5 n.s. Apoc1 1.02 ± 0.10 N=4 0.94 ± 0.16 N=5 n.s. Apoc2 1.04 ± 0.19 N=4 0.67 ± 0.14 N=5 n.s. Apoc3 1.10 ± 0.25 N=4 0.72 ± 0.14 N=5 n.s. Apoe 1.02 ± 0.12 N=4 1.11 ± 0.21 N=5 n.s. Abca1 1.05 ± 0.15 N=5 1.01 ± 0.12 N=10 n.s. Abcg1 1.00 ± 0.04 N=4 0.95 ± 0.13 N=5 n.s. Abcg5 1.02 ± 0.10 N=5 1.22 ± 0.15 N=10 n.s. Abcg8 1.02 ± 0.09 N=5 1.28 ± 0.16 N=10 n.s. Htgl 1.07 ± 0.20 N=4 0.92 ± 0.20 N=5 n.s. Sdc1 1.01 ± 0.10 N=4 0.86 ± 0.16 N=5 n.s. Dgat1 1.00 ± 0.02 N=5 1.09 ± 0.03 N=10 n.s. LTβR 1.01 ± 0.06 N=5 0.98 ± 0.04 N=10 n.s. Mttp 1.00 ± 0.02 N=5 0.98 ± 0.05 N=10 n.s. Acat2 1.01 ± 0.06 N=5 0.92 ± 0.03 N=10 n.s. Lrp1 1.03 ± 0.14 N=4 1.20 ± 0.20 N=5 n.s. Quadriceps femoris muscle

Lpl 1.01 ± 0.09 N=4 1.12 ± 0.05 N=5 n.s.

Lrp1 1.09 ± 0.25 N=4 1.29 ± 0.18 N=5 n.s. Vldlr 1.02 ± 0.12 N=4 1.12 ± 0.06 N=5 n.s. Abcg1 1.01 ± 0.08 N=4 1.50 ± 0.20 N=5 n.s. White adipose tissue

Lpl 1.02 ± 0.11 N=4 0.88 ± 0.04 N=5 n.s.

Apoc1 1.05 ± 0.18 N=4 1.05 ± 0.08 N=5 n.s. Apoe 1.00 ± 0.04 N=4 1.16 ± 0.10 N=5 n.s. Hsl 1.00 ± 0.04 N=4 0.95 ± 0.08 N=5 n.s. Lrp1 1.10 ± 0.29 N=4 1.20 ± 0.17 N=5 n.s. Vldlr 1.00 ± 0.05 N=4 0.95 ± 0.15 N=5 n.s. Abcg1 1.02 ± 0.14 N=4 1.37 ± 0.09 N=5 n.s. Proximal small intestine

Npc1l1 1.01 ± 0.09 N=4 1.05 ± 0.09 N=4 n.s.

Sdc1 1.02 ± 0.13 N=4 0.83 ± 0.08 N=4 n.s. Apob 1.18 ± 0.35 N=4 1.84 ± 0.21 N=4 n.s. Apoc2 1.11 ± 0.27 N=4 2.03 ± 0.12 N=4 P=0.020 Apoc3 1.13 ± 0.28 N=4 1.75 ± 0.13 N=4 n.s. Hsl 1.00 ± 0.04 N=4 1.24 ± 0.14 N=4 n.s.

Supplementary Materials for - Science Translational Medicine€¦ · the plasmid pEasy Flirt in...

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