Hata et al.
Online Supplement
Critical Role of Th17 Cells in Inflammation and Neovascularization After Ischemia
Hata et al.
Expanded Materials and Methods
Histology and Immunohistochemistry
The mice were euthanized, and their adductor muscles were then excised; the
muscles were embedded in the OCT compound (Tissue-Tek; Miles Laboratories, Elkhart, IN)
and frozen in liquid nitrogen, and each specimen was then cut into 10-m sections. The sections
were stained with hematoxylin and eosin (HE). For immunohistochemical analysis, the sections
were stained with antibodies against F4/80 (clone A3-1; RDI, Flanders, NJ), Gr-1 (clone
RB6-8c5; eBioscience, San Diego, CA), CD4 (clone H129.19, BD Biosciences, San Jose, CA),
and CD31 (BD Biosciences). This was followed by incubation with biotin-conjugated
secondary antibodies. Next, the sections were washed and treated with avidin-peroxidase (ABC
kit; Vector Laboratories, Burlingame, CA). The reaction was developed using the DAB
substrate kit (Vector Laboratories). The sections were then counterstained with hematoxylin. No
signals were detected when irrelevant IgG (Vector Laboratories) was used instead of the
primary antibody as a negative control.
Flow cytometric analysis
Flow cytometric analysis was performed as described previously1. Circulating cells
were identified using the nucleated cell fraction. The nucleated cells were labeled with the
following antibodies: anti-CD4 (BD Biosciences), anti-CD34 (clone RAM34; BD Biosciences),
anti-Flk-1 (BD Biosciences), anti-IL-17 (eBioscience, San Diego, CA), anti-CD45 (BD
Biosciences), anti-Gr-1 (BD Biosciences), and F4/80 (Abcam, Cambridge, MA). To identify
IL-17 expression, cells were permeabilized with an intracellular antigen detection kit
(Cytofix/Cytoperm, BD Biosciences) according to the manufacturer’s instructions. The cells
1
Hata et al.
were examined by flow cytometer (FACSCalibur, BD Biosciences) and analyzed using
CellQUEST software ver.3.3 (BD Biosciences).
Real-time reverse transcription-polymerase chain reaction (RT-PCR)
Total RNA was prepared from the adductor muscles or cultured CD4 T+ cells using
ISOGEN (Nippon Gene Co., Ltd., Toyama, Japan), according to the manufacturer’s instructions.
Real-time RT-PCR analysis was performed using the Takara TP-800 PCR Thermal Cycler Dice
Detection System (Takara Bio Inc, Shiga, Japan) to detect the mRNA expression of IL-17,
RORt, FoxP3, IL-22, VEGF-A, IL-1, IL-4, IL-6, IFN-, MCP-1, and -actin. The expression
levels of each target gene were normalized by subtracting the corresponding -actin threshold
cycle (CT) values; normalization was carried out using the  CT comparative method.
Bone marrow transplantation
Bone marrow-transplanted mice were developed as described previously1, 2. Whole
bone marrow cells from the WT and IL-17–/– mice were harvested by flushing their femurs with
phosphate-buffered saline (PBS). Red blood cells were lysed with ammonium chloride
potassium (ACK) buffer (150 mM NH4Cl, 10 mM KHCO3, 0.1 mM ethylenediaminetetraacetic
acid [EDTA]; pH 7.2) at 4°C for 20 min. They were washed 3 times with PBS and resuspended
in 0.5 mL PBS. Recipient mice (WT and IL-17–/– mice, 6–8 weeks old) were lethally irradiated
with a total dose of 9 Gy (MBR-155R2, Hitachi, Japan) and injected with bone marrow cells
through the tail vein. To verify the reconstitution of bone marrow after transplantation by this
protocol, we used green fluorescent protein (GFP) transgenic mice (kindly provided by
Professor M. Okabe, Osaka, Japan) as donors. Flow cytometry analysis revealed that at 8 weeks
after transplantation, peripheral blood cells consisted of more than 90% GFP-positive cells. By
using this protocol, we produced 3 types of bone marrow-transplanted mice: WT to WT
(BMTWT→WT) mice, WT to IL-17–/– (BMTWT→IL-17–/–) mice, and IL-17–/– to WT (BMTIL-17–/–→WT)
mice.
2
Hata et al.
In vitro experiments
Bone marrow cells were isolated from the femurs of WT mice. The cells were incubated at 95%
air, 5% CO2, in a humidified incubator at 37°C in Dulbecco’s modified Eagle’s medium
(DMEM; Sigma, St. Louis, MO) supplemented with 10% fetal calf serum (FCS: Hyclone,
Logan, UT) for 6 h in the presence or absence of murine recombinant IL-17 (R&D Systems).
The expression of VEGF-A, IL-1, IL-6, and MCP-1 was assessed by real-time RT-PCR
analysis.
References
[1]
Shiba Y, Takahashi M, Yoshioka T, Yajima N, Morimoto H, Izawa A et al. M-CSF
accelerates neointimal formation in the early phase after vascular injury in mice: the critical role
of the SDF-1-CXCR4 system. Arterioscler Thromb Vasc Biol 2007;27:283-289.
[2]
Yajima N, Takahashi M, Morimoto H, Shiba Y, Takahashi Y, Masumoto J et al.
Critical role of bone marrow apoptosis-associated speck-like protein, an inflammasome adaptor
molecule,
in
neointimal
formation
after
vascular
injury
in
mice.
Circulation
2008;117:3079-3087.
Supplemental Figure Legends
Supplemental figure I. Expression of IL-6, IL-17, RORt, FoxP3, and IL-22 after hindlimb
ischemia
(A) IL-6 protein levels were assessed in the adductor muscles of contralateral and ischemic
limbs at 24 h after ischemia induction. Data are expressed as mean ± SEM (n = 4). **p < 0.01
vs. contalateral. (B–D) The adductor muscles of sham-operated, contralateral, and ischemic
limbs were excised at 24 h after ischemia induction and digested using collagenase. The isolated
cells were treated with CD3/CD28 antibodies for 6 h. The mRNA expression of (B) IL-17, (C)
RORt, (D) FoxP3, and (E) IL-22 was analyzed by real-time RT-PCR. Splenocytes were used as
3
Hata et al.
an internal control. Data are expressed as mean ± SEM (n = 4).
Supplemental figure II. Capillary density in the ischemic hindlimbs of CD4-depleted mice
Mice were treated intraperitoneally with a neutralizing antibody against CD4. The adductor
muscles were excised from vehicle (PBS)-treated or anti-CD4-antibody-treated (CD4) mice at
21 days after ischemia induction and immunohistochemically stained with an antibody against
CD31. Representative photographs of capillary density, determined by staining CD31+
endothelial cells, are shown (n = 3).
Supplemental figure III. Circulating CD34+/Flk-1+ cells after hindlimb ischemia
Hindlimb ischemia was produced in WT and IL-17–/– mice. (A) The percentage of CD34+ and
Flk-1+ cells in peripheral circulation was analyzed by flow cytometry. (B) Quantitative analysis
of CD34+/Flk-1+ cells was performed. Data are expressed as mean ± SEM (n = 4). **p < 0.01;
ns, no significance.
Supplemental figure IV. Infiltration of monocytes, neutrophils, and CD4+ T cells
Hindlimb ischemia was produced in WT and IL-17–/– mice. The adductor muscles of the
ischemic limbs were excised and digested using collagenase. The percentages of CD45 +, Gr-1+,
F4/80+ cells, and CD4+ cells were assessed by flow cytometric analysis. Representative results
were shown (n = 3).
Supplemental figure V. Production of HGF and bFGF in the ischemic limbs
Hindlimb ischemia was produced in WT and IL-17–/– mice. The adductor muscles of the
ischemic and contralateral limbs were excised at 2 days after ischemia induction. The protein
levels of HGF and bFGF were measured. Data are expressed as mean ± SEM (n = 5).
4
Hata et al.
Supplemental figure VI. Production of IL-1 in the ischemic limbs
Hindlimb ischemia was produced in WT and IL-17–/– mice. The adductor muscles of the
ischemic and contralateral limbs were excised at 5 days after ischemia induction. The protein
levels of IL-1 were measured. Data are expressed as mean ± SEM (n = 3–6). *p < 0.05 and
**p < 0.01.
Supplemental figure VII. Effect of IL-17 on the expression of VEGF-A, IL-1, IL-6, and MCP-1
Bone marrow cells were isolated from WT mice and incubated for 6 h in the presence or
absence of murine recombinant IL-17. Real-time RT-PCR was performed to evaluate the
expression of VEGF-A, IL-1, IL-6, and MCP-1. Data are expressed as mean ± SEM (n = 4)
Supplemental figure VIII. Proposed mechanisms
Ischemia induces accumulation of inflammatory cells, such as monocytes/macrophages and
neutrophils, that produce inflammatory cytokines. Among these cytokines, IL-6 can promote
differentiation from CD4+ T cells into Th17 cells. IL-17 produced by Th17 cells may stimulate
the production of IL-1b and VEGF-A, and induce the subsequent angiogenic responses.
5