C57BL/6J mice were purchased from CLEA Japan (Kanagawa, Japan) and maintained

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1 Supplementary Materials and Methods Mouse model of lymphedema C57B/6J mice were purchased from CEA Japan (Kanagawa, Japan) and maintained on a standard mouse chow diet. Rag2 -/-, Cd4 -/- and Cd8a -/- mice were purchased from Taconic (Germantown, NY). Eight-week-old mice were anesthetized and a longitudinal skin incision was made in the middle of the right side of the abdomen, after which transverse skin incisions were made at the two ends of the first incision to form a rectangular skin flap (Supplementary Figure S1). The skin flap was then peeled from the epifascial layer of the rectus abdominis, external oblique and pectoralis major muscles to expose the subdermal tissues. ymphatic vessels were visualized by injecting dye into the subcutaneous tissues around the inguinal lymph node. Note that during this operation dye was not directly injected into the inguinal lymph node to avoid additional insult to the lymphatic system. The superficial axillary lymph node, which was revealed by the inguinal dye injection, was dissected out and several major collector lymphatic vessels were ligated approximately 5 mm caudal from the superficial axillary lymph node, where the first major branch from the vein running parallel to the lymphatic vessels was situated. The skin flap was then returned to its original position and sewn back together. All experiments were approved by the University of Tokyo Ethics Committee for Animal Experiments and strictly adhered to the guidelines for animal experiments of the University of Tokyo. Histopathologic assessment of lymphedema Skin tissue (dermal and subcutaneous) along with the major collector lymphatic vessels extending from immediate cranial of the inguinal lymph node to the lymphatic vessel 1

2 ligation site was collected as a strip approximately 10 mm in width. The morphologic characteristics of skin sections were then assessed under a light microscope using paraffin-embedded sections (5 μm thick) stained with hematoxylin-eosin and sirius red. The skin thickness was measured as the distance from the base of the epidermis to the subdermis, and was analyzed at 3 standardized points in each section from an area 5 mm cranial of the inguinal lymph node (see Figure S1c). The mean of the thickness at the 3 points was used as the skin thickness. In addition, the wet-to-dry skin weight ratio was used as an index of skin fluid accumulation after lymphatic obstruction. To calculate the ratio, skin samples were excised from the same area identified above and rapidly weighted. The samples were then dried in an oven for 5 days at 60C, as previously reported (Xie et al., 2013), after which they were weighed to obtain the dry weight. ymphangiography and venography For lymphangiography, which was used to analyze lymphatic vessels after the lymphatic obstruction operation, either 10 l of Evans blue dye (1% in PBS, Sigma-Aldrich Japan, Tokyo, Japan) or indocyanine green (0.25% in distilled water, Daiichi Sankyo, Tokyo, Japan) was injected into the inguinal lymph node. ymphatic vessels were then assessed using a stereoscopic microscope and a digital camera. For venography, 100 l of Evans blue dye was injected into the tail vein. Immunohistochemical analysis Paraffin-embedded sections (5 μm thick) were deparaffinized and blocked with 2% BSA, after which they were incubated with primary antibodies (anti-mouse F4/80 and anti-mouse CD45 (BD Biosciences, San Diego, CA) and anti-mouse podoplanin (AngioBio, Del Mar, CA) and the corresponding biotinylated secondary antibodies 2

3 (anti-rat from Dako, Glostrup, Denmark and anti-hamster from Santa Cruz, Dallas, Texas). The sections were then developed using an ABC-alkaline phosphatase kit and Vector Red substrate (Vector aboratories, Bunlingame, CA) and counterstained with hematoxylin. To detect VEGF-C producing cells, skin tissues were fixed in methanol and stained in whole-mount with goat anti-mouse VEGFC antibody (Santa Cruz) and rat anti-mouse CD11b antibody (BD), after which and secondary anti-goat antibody was labeled with Alexa-488 (BD) and the anti-rat antibody was labeled with Alexa 555 (BD). To identify lymphatics and blood vessels, the sections were stained with rabbit anti-mouse YVE-1 antibody (AngioBio, Del Mar, CA), hamster anti-mouse podoplanin antibody (mentioned) and rat anti-mouse CD31 antibody (BD), as well as the appropriate secondary antibodies. Hoechst (Invitrogen) was used to stain nucleic acids in whole-mount, and the stained samples were observed using a confocal microscope (SM510 Meta, Zeiss) as described (Eguchi et al., 2012). Miles assay To assess vascular permeability, 1.0% Evans blue dye in saline (100 l) was injected into the tail vein. Fifteen minutes later, mice were perfused with saline via the right ventricle after venting the left atrium. The skin extending from the area immediately cranial to the inguinal lymph node to the lymphatic vessel ligation site was then collected as a strip approximately 10 mm in width, and the Evans blue dye was extracted from the samples in formamide (Invitrogen) overnight at 60 C. The dye collected was then quantified based on the absorbance at 600 nm, as previously described, and the absorbance was first normalized to the weight of tissue and then further normalized to the level at day 0 (Whitehead et al., 2009). 3

4 Inhibition of lymphangiogenesis in vivo To inhibit lymphangiogenesis, we used a recombinant mouse VEGFR3-Fc chimeric protein (R & D Systems, Minneapolis, MN)(Cursiefen et al., 2004). VEGFR3-Fc (6 g) was injected into the tail vein one day prior to the lymphatic obstruction operation. Then an additional 6 g were administered subcutaneously into the region adjacent to the epigastric vein and major collector lymphatic vessels during the operation. Human IgG1-Fc (R & D Systems) was used as a control. Flow cytometric analysis and cell sorting Skin tissue (dermal and subcutaneous) along the major collector lymphatic vessels extending from a point immediately cranial of the inguinal lymph node to the lymphatic vessel ligation site were collected as a strip approximately 10 mm wide (Figure S1C). Dermal tissue samples from the edematous areas of at least two mice in the same experimental group were pooled into one sample for flow cytometric analysis. The skin strips were cut into fine pieces in FACS buffer (PBS supplemented with 5% FBS) on ice, and then dissociated into individual cells by incubation in Hank s balanced salt solution containing 400 U/m collagenase type II (Worthington abs, San Francisco, CA) and 60 U/m DNase I (Roche, Mannheim, Germany) at 37C for 20 min with shaking. After dissociation, the cells were passed through a 100 m Cell Strainer (BD Biosciences) and centrifuged at 1500 rpm for 5 min at 4C. The resultant cell pellets were resuspended in ice-cold FACS buffer and then washed three times with FACS buffer before flow cytometric analysis. The antibodies used for analysis were anti-cd11b-fitc or Pacific Blue (clone M1/70) and anti-i17r-pe (clone PAJ-17R) from ebioscience; anti-f4/80-apc or PE/Cy7 (BM8), anti-y6c-apc/cy7 or FITC (HK1.4), and anti-y-6g-pe/cy7 or Pacific Blue (1A8) from Bioegend. 4

5 Corresponding isotype controls for each antibody were also used. PI + dead cells were gated out, and the remaining cells were subjected to further analyses and sorting using a FACSAria III (BD Biosciences). For intracellular cytokine staining, cells were stimulated for 4 h with PMA (50 ng/ml) and ionomycin (500 ng/ml) (Sigma-Aldrich) in PBS in the presence of Golgistop (BD Biosciences). Surface staining was performed in the presence of Fc-blocking Abs (2.4G2), followed by intracellular staining for cytokines using a fixation and permeabilization kit (BD Biosciences) according to the manufacturer s instructions. Anti-IFN--PE, -I-4-PE, -I-17A-PE, -CD3-APC and -CD4-FITC antibodies were from Biolegend (San Diego, CA). Nonviable cells were excluded using Zombie Aqua dye (Biolegend). Quantitative real-time PCR analysis Total RNA was purified from tissue and cultured cell samples using RNeasy kits (Qiagen) or from the sorted cells using an RNeasy Plus Micro Kit (Qiagen). Complementary DNA was synthesized using a SuperScript III First-Strand Synthesis System (Invitrogen). Quantitative real-time PCR analyses were conducted using a ightcycler system (Roche). The results were first normalized to 18s levels and then to the levels of controls (the levels on day 0 in Figures 2a, 3a, and 6d, those in samples of control IgG treatment in Figures 5b and 5e, and those in untreated cells in Figure 5c). Primer sequences for the analyzed genes were: 18s, 5 -GCAATTATTCCCCATGAACG-3 and 5 -GGGACTTAATCAACGCAAGC-3 ; Vegfc, 5 -CAGACAAGTTCATTCAATTATTAGACG-3 and 5 -CATGTCTTGTTAGCTGCCTGA-3 ; Il17a, 5 -GAAGCTCAGTGCCGCCA -3 and 5 -TTCATGTGGTGGTCCAGCTTT -3 ; Ifng, 5

6 5 -GGAGGAACTGGCAAAAGGAT-3 5 -TTCAAGACTTCAAAGAGTCTGAGG-3 ; and Tbx21, 5 -TTCCCATTCCTGTCCTTCAC-3 and 5 -CCACATCCACAAACATCCTG-3 ; Gata3, 5 -GGAAACTCCGTCAGGGCTA -3 and 5 -AGAGATCCGTGCAGCAGAG-3 ; Rorc (RORt), 5 -GCCTCCTGCCACCTTGAGT-3 and 5 -TCTGCCTTCAGCTTTGCCTC-3. Tube formation assay Matrigel (growth factor reduced, BD Biosciences) that had been thawed on ice was plated in the bottom wells of 24-well Boyden chambers (Corning Incorporated, Corning, NY). These plates were then incubated at 37 C for 30 min to allow the Matrigel to polymerize, after which HDMECs ( cells/well) were seeded onto the Matrigel. In some wells, lesional CD4 + cells ( cells/well) and/or CD11b + cells ( cells/well), isolated from the edematous tissues 4 days after the lymphatic obstruction surgery using CD4 + T cell isolation kit II and CD11b microbeads (Milteny Biotec, Tokyo, Japan), were cultured on polycarbonate membrane filter inserts (0.4 m pore size, Thermo Scientific, Waltham, MA) for 48 h at 37 C. Three separate fields were chosen at random in each well and photographed. Three or four wells were analyzed per condition. The lengths of the tubes were measured using ImageJ software (National Institutes of Health). Culture of lesional CD11b + and CD4 + cells CD4 + cells and CD11b + cells were isolated from edematous tissue on postoperative day 4. CD11b + cells (5x10 5 cells/well) were plated in the bottom wells of 24-well Boyden chambers and starved for 24 h in RPMI 1640 supplemented with 0.5% FBS prior to 6

7 coculture. After activation of the CD4 + by incubation in 5% PBS containing PMA (50 ng/ml) and ionomycin (500 ng/ml) from Sigma for 4 h prior to coculture, the cells were placed in the chamber inserts (5x10 5 cells/insert). In some wells, anti-i17 antibody (10 g/ml, Biolegends), anti-ifn- antibody (10 g/ml, Biolegends), recombinant I-17 (10 ng/ml, R&D), recombinant IFN- (20 ng/ml, R&D) or atorvastatin (10 mol/) were added to the culture medium. For mrna analysis, the cells were collected after 24 h of coculture. For measurement of VEGF-C in the media, the cells were cocultured for 48 h. VEGF-C concentrations were then measured using mouse enzyme-linked immunosorbent assay (EISA) kits (WS, Hubei, China). Absorbance at 450 nm was determined using a multimode plate reader (Perkin Elmer, Waltham, MA). Monocyte/macrophage depletion To deplete monocytes/macrophages, we used Itgam-HBEGF transgenic mice and clodronate liposomes. Itgam-HBEGF transgenic mice (Jackson aboratory, Bar Harbor, ME) were treated with either intraperitoneal injection of vehicle or diphtheria toxin (25 ng/g body weight) 3 days prior to the operation and 3 days after the operation. In addition, vehicle or the same dose of diphtheria toxin was intraoperatively injected subcutaneously into the area adjacent to the epigastric vein and collector lymphatic vessels. Alternatively, clodronate-filled liposomes (0.01 mg/g body weight) were administered via the tail vein 3 days prior to operation and 3 days after operation, and were administered intraoperatively into the subcutaneous tissues adjacent to the superficial epigastric vein and major collector lymphatic vessels. Control mice were administered empty liposomes. Effects of inhibiting IFN- and I-17 on lymphangiogenesis 7

8 To inhibit IFN- and I-17 we used a neutralizing antibody composed of a mixture of anti-ifn- (Clone MIB-5E9.1, Biolegend) and anti-i-17 (Clone TC11-18H10.1, Biolegend) antibodies. The neutralizing antibodies (200 g, 100 g each) were administered via the tail vein 1 day prior to lymphatic obstruction, and an additional subcutaneous injection was made on the day of the lymphatic obstruction. After the procedure, the same amount of antibody was intravenously injected weekly for one month. Control mice were given isotype IgG instead (Biolegend). Statin treatment Atorvastatin (10 mg/kg/day) or vehicle (0.5% carboxymethylcellulose) was administered by oral gavage daily beginning from 7 days prior to the lymphatic obstruction and continued for 1 month after the operation. For cell culture, lesional CD11b + and CD4 + cells were isolated from edematous tissues and respectively cultured for 30 h in the bottom wells and inserts of Boyden chambers with vehicle or ATV (10 M). Human subjects Written informed consent was obtained from each subject after a full explanation of the study, which was approved by the Ethics Committee of the University of Tokyo Hospital. After obtaining informed consent, we acquired lymphatic vessels from lymphedema patients. The patient from whom the presented histological section (Figure S4) was obtained suffered from stage 2 lymphedema. Paraffin-embedded sections (5 μm thick) were deparaffinized and blocked with 2% BSA. For immunohistochemical staining, D2-40 (Dako) was used with the avidin-biotin complex technique and Vector Red substrate (Vector aboratories, Burlingame, CA). Sections were counterstained 8

9 with hematoxylin. Statistic analysis Comparisons between two groups were made using Student s t test (2-tailed). Differences among more than two groups were analyzed using one-way ANOVA followed by post hoc Bonferroni (3 groups) or Tukey-Kramer (>3 groups) tests. Values of P less than 0.05 were considered significant. Error bars represent S.E. 9

10 Supplementary References Cursiefen C, Chen, Borges P, et al. (2004) VEGF-A stimulates lymphangiogenesis and hemangiogenesis in inflammatory neovascularization via macrophage recruitment. J Clin Invest 113: Eguchi K, Manabe I, Oishi-Tanaka Y, et al. (2012) Saturated Fatty Acid and TR Signaling ink Cell Dysfunction and Islet Inflammation. Cell Metab 15: Whitehead KJ, Chan AC, Navankasattusas S, et al. (2009) The cerebral cavernous malformation signaling pathway promotes vascular integrity via Rho GTPases. Nat Med 15: Xie W, Wang H, Wang, et al. (2013) Resolvin D1 reduces deterioration of tight junction proteins by upregulating HO-1 in PS-induced mice. ab Invest 93:

11 Supplementary Figure egends Supplementary Figure S1 The mouse abdominal wall lymphedema model (a) View of the lymphatic system in the anterior abdominal wall of a mouse using Evans blue lymphangiography. (b) Schematic of the procedure in the lymphatic obstruction operation. (c) Sample preparation methods for histology, gene expression analysis, flow cytometry and wet-to-dry skin weight ratio. Supplementary Figure S2 Wet-to-dry edematous skin weight ratios evels of skin edema were evaluated based on the wet-to-dry weight ratios of the skin at the indicated times after lymphatic obstruction. n=4 in each group. Supplementary Figure S3 Immunofluorescent staining of lymphatic vessels Shown are images of immunofluorescent staining for YVE-1 (green), podoplanin (red) and CD31 (pink) on day 7. YVE-1 + podoplanin + CD31 + vessels are lymphatic vessels (), while YVE-1 podoplanin CD31 + vessels are blood vessels (B). Scale bars, 100 m. Supplementary Figure S4 Blood and lymphatic vessels in lymphedematous tissues (a) Quantitative analysis of blood and lymphatic vessels. Blood vessels were identified as CD31 + podoplanin vessels and lymphatics as CD31 + podoplanin + vessels. To quantify the numbers of blood and lymphatic vessels, the vessels were counted in three 0.2-mm 2 areas of edematous skin from each mouse. n=3 mice in each group. P<0.05 vs. Day 0. (b) Quantification of Evans blue dye extravasation using the Miles assay. Shown are 11

12 relative absorbance levels, which were normalized to the levels on day 0. n=3 in each group. (c) Blood vessels visualized using Evans blue 7 days after lymphatic obstruction. Shown is a representative image that was taken immediately after dye injection into the tail vein. Scale bar, 5 mm. Supplementary Figure S5 ymphatic vessels in lymphedematous tissues (a) Sections of dermal tissues under basal conditions and 28 days and 3 months after lymphatic obstruction were stained for podoplanin. B: major blood vessels, C: major collector lymphatic vessels. Podoplain + lymphatic vessels. Podoplanin lymphatic vessels. Scale bars, 100 m. Note that the antibody nonspecifically stains hair roots. (b) Evans blue lymphangiography 3 months after lymphatic obstruction. The lymphangiogram of an age-matched normal mouse is also shown. Scale bar, 5 mm. Supplementary Figure S6 ymphatic vessels in human lymphedematous tissue Section of lymphedema tissue from the ankle of a stage 2 lymphedema patient. The section was immunostained for podoplanin (red). A major collector lymphatic (C) and smaller lymphatic vessels () are marked. Note that the collector lymphatic is negative for podoplanin. Scale bar, 100 m. Supplementary Figure S7 Macrophages express VEGF-C in edematous tissue (a, b) Representative confocal images of a section of day 7 edematous lesions stained for CD11b and VEGF-C. In b, higher magnification views are shown. For the merged image, cross sectional images generated from Z-stack images are also shown. Scale bars, 10 m. (c) Representative images of serial sections of day 7 edematous lesions stained for podoplanin and F4/80 (red). Note that F4/80 + cells are located in vicinity of newly 12

13 formed, small, podoplanin + lymphatic vessels., lymphatic vessels. Scale bar, 10 m. Supplementary Figure S8 Macrophage depletion (a) CD11b + F4/80 + y6g cell fractions among total living cells in edematous tissues collected on day 7 from clodronate liposome-treated (left) and Itgam-HBEGF transgenic mice (right). n=4 in each group. P<0.05 vs. control. (b) Effects of clodronate liposomes on lesional Vegfc expression. Vegfc expression in the edematous tissues of mice administered empty or clodronate-filled liposomes, as described in Figure 2f. evels of mrna expression were normalized first to those of 18s rrna and then to the levels in empty-liposome treated mice on day 0. n=6 in each. P<0.05 vs. day 0. P<0.05. Supplementary Figure S9 CD4 + and CD8 + T cells in edematous tissues Shown are representative plots of CD4 and CD8 expression on CD3 + cells from edematous tissues isolated at the indicated times after lymphatic obstruction. Mean CD3 + CD4 + and CD3 + CD8 + cell fractions among CD3 + cells are also shown. n=4 for each time point. Supplementary Figure S10 CD4 + T cell polarization in edematous tissues (a) Cell fractions expressing the signature cytokines of Th1 (IFN-), Th2 (I-4) and Th17 (I-17) cells among total living cells. n=3. P<0.05 vs. day 0 of the same cell-type. P<0.05 vs. day 4 of the same cell-type. (b) Expression levels of Tbx21 (T-bet), Gata3 and T cell-specific isoform of Rorc (RORt), markers for Th1, Th2 and Th17 cells, respectively, were analyzed in CD4 + T cells isolated from edematous tissues. evels of mrna expression were normalized first to those of 18s rrna and then to the 13

14 levels on day 0 in each cell-type. n=3. P<0.05 vs. Day 0. Supplementary Figure S11 I-17RA expression of macrophages Representative plot of I-17 receptor A (I-17RA) expression in CD11b + F4/80 + y6g - macrophages in edematous tissues. The gray line is the isotype control. Supplementary Figure S12 Effects of inhibiting IFN- and I-17 signaling in lymphatic vessel obstruction-induced lymphedema model Shown are representative picrosirius red-stained sections from mice treated with control IgG and those treated with anti-ifn-and anti-i-17 antibodies. Scale bars, 500 m (upper panels of each group) or 100 m (lower panels). 14

15 a Axillary lymph node ymphatic vessels Blood vessels Inguinal lymph node b Axillary lymph node ymphe node dissection and lymphoatic vessel ligation ymphatic vessels Blood vessels ymphatic flow Inguinal lymph node c RNA, flow cytometry Histology 0.5 mm 5 mm Skin thickness Supplementary Figure S1

16 9 Wet/dry weight ratio Day 0 Day 2 Day 4 Day 7 Day 14 Supplementary Figure S2

17 Podoplanin YVE-1 Hoechst CD31 B Merge Supplementary Figure S3

18 a Blood vessel ymphatic vessel Vessel number / 0.2 mm Vessel number / 0.2 mm 2 0 Day 0 Day 7 Day 14 Day 0 Day 7 Day b c Day 7 Relative absorbance Day 0 Day 7 Day 14 Head Tail Supplementary Figure S4

19 a Normal Day 28 C B C B B B B b Normal Month 3 Supplementary Figure S5 C B B C B B B Month 3

20 C Supplementary Figure S6

21 a CD11b VEGFC Hoechst Merge b CD11b VEGFC Hoechst Merge c Podoplanin F4/80 Supplemetnary Figure S7

22 a 9 9 % in live cells 6 3 % in live cells Control Clodronate 0 Control DT b 12 Relative Vegfc level 8 4 Control Clodronate 0 Day 0 Day 7 Supplementary Figure S8

23 CD4 63.5% Day 0 Day 7 Day 14 Day % 47.9% 32.2% 22.4% 39.5% 40.8% 43.3% CD8 Supplementary Figure S9

24 a (%) 12 Fraction of cytokine + cells Day 0 Day 4 Day 7 IFN I4 I17 b Relative transcript level Tbx21 Gata3 Rorc Day 0 Day 7 Day 0 Day 7 Day 0 Day 7 Supplementary Figure S10

25 Count I-17RA Supplementary Figure S11

26 Day 14 Day 28 anti-ifn-j/i-17 IgG Day 7 Supplementary Figure S12 Month 3