Download Supplementary Materials and Methods

Supplementary Materials and Methods
Cell preparation. Lungs were digested for 1 h at 37 °C in RPMI medium containing 0.4
mg/ml collagenase Type IV (Sigma-Aldrich), 25 mM HEPES (Gibco), and 0.1 mg/ml
DNase (Sigma-Aldrich). The cell suspension was filtered through a 70 m cell strainer,
resuspended in 40% Percoll (GE Healthcare) and layered onto 70% Percoll solution, then
centrifuged at 1150 g for 30 min. The middle leukocyte fraction of the gradient was
collected for further analysis.
Endothelial cell isolation. Isolation for flow cytometry analysis: lungs were digested for
1 h at 37 °C in RPMI medium containing 0.4 mg/ml collagenase Type IV, 2.4 mg/ml
dispase (Roche Diagnostics), 25 mM HEPES, and 0.1 mg/ml DNase. Cell suspension was
filtered through a 100 m cell strainer, pelleted and resuspended in RPMI medium
supplemented with 20% FCS and 1% sodium citrate (Sigma-Aldrich), layered onto
Ficoll-Paque PLUS (1.077 +/- 0.001 g/ml, GE-Healthcare) and then centrifuged at 400 g
for 20 min. The interphase was collected for further analysis. Isolation for transendothelial migration assay and electrical resistance measurements: lungs were digested
for 1 h at 37 °C in Hank’s Balanced Salt Solution (Gibco) containing 1 mg/ml
collagenase type IV. Cell suspension was filtered through 100 m cell strainer, pelleted
and stained with anti CD31-biotin antibody (MEC 13.3, BD Biosciences) followed by
staining with magnetically labeled streptavidin- MicroBeads (Miltenyi Biotec). Cells
were separated using MACS separation system (Miltenyi Biotec). CD31 positive cells
were collected and cultured in endothelial cell basal medium-2 EBM-2 (Lonza).
Trans-endothelial migration assay. Primary lung endothelial cells were grown to
confluence on 8 m pore size gelatinized polyester (PET) transwell inserts (Falcon).
Cells were stimulated with 100 ng/ml of IL17A (PeproTech) for 10 h. Media was
changed and membrane labeled red fluorescent (PKH26, Sigma-Aldrich) B16F10 cells
were added to the upper chamber for 16 h. Next, cells from the upper face of the
membrane were scraped and the number of tumor cells transmigrated to the lower face of
the insert was calculated using fluorescence microscopy (Slidescanner, Axio Scan.Z1,
Carl Zeiss Microscopy).
Flow cytometry. For surface and intracellular staining following antibodies were used:
CD45 (30-F11, BD Biosciences), CD3 (17A2, eBioscience), CD4 (RM 4-5, BioLegend),
NK1.1 (PK136, BD Biosciences), CD11b (M1/70, BD Biosciences), CD5 (53-7.3, BD
Biosciences), TCR (GL3, eBioscience), CD11c (HL3, BD Biosciences), B220 (RA36B2, BD Biosciences), IL17A (TC11-18H10, BD Biosciences), CD31 (390, BioLegend),
CD34 (RAM34, BD Biosciences). Cells were incubated for 5 min at 4 °C with 1 g of
anti CD16/CD32 (93, eBioscience) antibodies, followed by 25 min surface staining at 4
°C. For intracellular staining, cells were stimulated with PMA (Applichem) and
ionomycin (Invitrogen) in the presence of Golgi Plug (BD Biosciences) for 4 h. After
surface staining cells were fixed and permeabilized according to the manufacturer’s (BD
Biosciences) recommendations and next stained intracellularly. Samples were analyzed
with a FACS LSRII Fortessa (BD Biosciences). Post-acquisition analysis was done with
FlowJo (Tree Star) software.
ELISA assay. Serum from naïve VE-cadherin-Cre+IL17Aind/+ or IL17Aind/+ mice was
collected and analyzed for IL17A expression using an ELISA assay. Antibodies used for
the assay were purchased from: coating antibody anti IL17A (BD Biosciences), detection
antibody biotin anti IL17A (BD Biosciences), streptavidin-HRP (BD Biosciences).
RNA extraction and Real-Time Quantitative PCR. Total RNA was isolated from the
whole mouse lung tissue using Qiagen RNeasy Plus kit (Qiagen GmbH). cDNA was
prepared using M-MLV reverse transcriptase (Invitrogen). Mouse Eselectin, Vcam1,
Il17ra, Il17rc and Polr2a expression was measured by real- time quantitative PCR
analysis using the CFX 384 Real-Time detection system (Bio-Rad) with SYBR Green
Supermix (Bio-Rad). Sequences for PCR primers were: Polr2a, left primer 5’-CTG GTC
CTT CGA ATC CGC ATC, right primer 5’-GCT CGA TAC CCT GCA GGG TCA;
Eselectin, left primer 5’- CGC CAG AAC AAC AAT TCC AC, right primer 5’- ACT
GGA GGC ATT GTA GTA CC; Vcam1, left primer 5’- TCT TAC CTG TGC GCT GTG
AC, right primer 5’- ACT GGA TCT TCA GGG AAT GAG T; Il17ra, left primer 5’TGG GAT CTG TCA TCG TGC T, right primer 5’- ATC ACC ATG TTT CTC TTG
ATC G; Il17rc, left primer 5’- CCA GAA AGA GCT CAA CCT CAC, right primer 5’GCT CCT CAG AGA CAT CCA GTG. The expression level of the genes was
normalized to the mouse Polr2a house-keeping gene and represented as 2-∆CT (∆CT = CT
gene of interest - CT house-keeping gene).
Statistics. Differences between more than three groups were evaluated with One Way
ANOVA with Bonferroni post test, differences between two sets of data were evaluated
using unpaired two tailed t-test
*
P < 0.05, ** P < 0.01, *** P < 0.001, n.s. (not significant).
Data were analyzed using Prism software (GraphPad Software, Inc.).