Aquaporin-9-expressing neutrophils are required for the establishment of contact hypersensitivity
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1 Supplemental Material quaporin-9-expressing neutrophils are required for the establishment of contact hypersensitivity atharina Sagita Moniaga 1, Sachiko Watanabe 1, Tetsuya Honda 1, 2, Søren Nielsen 3, Mariko Hara-hikuma 1* 1 enter for Innovation in Immunoregulative Technology and Therapeutics, 2 Department of Dermatology, Graduate School of Medicine, Kyoto University, Kyoto , Japan, 3 Department of Health Science and Technology, alborg University, alborg 922, Denmark ddress correspondence to: Mariko Hara-hikuma, Ph.D. enter for Innovation in Immunoregulative Technology and Therapeutics Graduate School of Medicine, Kyoto University Yoshida Konoe-cho, Sakyo-ku, Kyoto , JPN Phone: ; Fax: haramari@kuhp.kyoto-u.ac.jp
2 Ear swelling (um) Supplementary Fig. S1 QP9-/ PM (x2) (x1) (x2) QP9-/- (x2) QP9-/- (x2) Supplementary Fig. S1. () Ear thickness in and QP9-/- mice with single topical application of.5% dinitrofluorobenzene () or.2 mg/ml phorbol myristate acetate (PM) at 2 h as a model of irritant contact dermatitis. oth of ear swellings were comparable (n=3-). (, ) Mice were sensitized with, and challenged 5 d later on the ear. The ears were collected at 2 h after challenge. representative image of neutrophils infiltration (arrow head) with Giemsa staining (), and mast cell infiltration (arrow head) with Toluidine blue staining () in the -challenged ear of (left) and QP9-/- (right) mice. ar, 1 µm.
3 Supplementary Fig. S2 > QP9 -/- QP9 -/- > 91.7% D % D5.2 Total cell no. (x1 7 ) QP9 -/- D + cell no. (x1 6 ) D D8 + cell no. (x1 6 ) E Neutrophil cell no. (x1 3 ) ** Supplementary Fig. S2. () Mice ( or QP9 -/- ) were gamma-irradiated with two doses of 6rad, 3 hours apart. The irradiated mice received intravenous injection of bone marrow from or QP9 -/- mice. The chimerism was checked using 57L/6 D5.1 congenic mice. Two months after bone marrow transfer, blood cells were stained with D5.1-pacific blue and D5.2-P, and analyzed by flow cytometry. More than 9% cells of the recipient cells were replaced by donor cells. (-E) The compositions of skin dlns cells after sensitization with.5%. Five days after sensitization, the total cell numbers (), D + cells (), and D8 + cells (D) were comparable in the skin dlns between and QP9 -/- mice; while the neutrophil numbers (E) were increased in compare to QP9 -/- mice (n= per group; **, p<.1).
4 Supplementary Fig. S3 1.83% SS 13.5% nti-ly6g D11b Spleen ontrol D D SS- SS- 22 nti-ly6g D8 D8 Lymph node ontrol nti-ly6g 22 D8 Lymph node nti-ly6g ontrol SS- D 22 Supplementary Fig. S3. Effects of neutrophil depletion during HS. () Representative FS analysis of SShigh D11b+ neutrophils in the blood of mice injected with anti-ly6g mb at day Representative FS analysis of D+, D8+, and 22+ cells in spleen and/or skin dlns of mice in the steady state () and after sensitization () with or without anti-ly6g injection.
5 Supplementary Fig. S IL-17 + cell no.(x1 3 ) D8 + Neutrophil 18 h Day 5 Neutrophils percentage of total dlns cell hr hr 18 hr 2 d 3 d 5 d Neutrophils cell no. (x1 5 /ml) M % of neutrophils lood QP9 -/- Supplementary Fig. S. () Skin dlns were collected at 18 h and 5 d after sensitization in mice for intracellular IL-17 staining by flow cytometry analysis. LN cells were cultured for 2 h with PM (5ng/ml) and ionomycin (1µM) in the presence of GolgiStop (n=3 per group). ().5% was applied to shaved abdomen skin of mice at indicated times, and the percentage of neutrophil (Gr1 + D11b + ) infiltration in skin dlns was calculated by flow cytometry. () one Marrow (M) and blood cells were collected at 18 h after application. Neutrophils (Gr1 + D11b + ) cell numbers in M (left) and the ratio in blood (right) were comparable between and QP9 -/- mice as analyzed by flow cytometry (n= per group).
6 Supplementary Fig. S5 2 h 2 72 h QP9 -/- ell no. (x1 ) ell no. (x1 ) R-D M-D R-D M-D Supplementary Fig. S5. FIT-induced cutaneous dendritic cell (D) migration assay. Mice were painted on the shaved abdomen with 2 µl of 1% FIT in 1:1 (v/v) acetone/dibutyl phalate mixture. The number of D11c + MH class II + FIT + cutaneous Ds that migrated into the skin dlns was enumerated by flow cytometry. The migrated cell numbers were comparable between and QP9 -/- mice, either at 2 h (left) and 72 h (right) after the application of FIT (n=-5 per group). R-D = resident D; M-D = migrated D. Supplementary Fig. S6 % of Max, QP9 -/- (-), (-), (+) QP9 -/-, (+) isotype R7 Supplementary Fig.S6. R7 expression on neutrophils from and QP9 -/- mice with or without sensitization. R7 expressions were similar between each group.
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Citation The Journal of Dermatology, 37(8), available at
NAOSITE: Nagasaki University's Ac Title Two cases of blaschkitis with promi Author(s) Utani, Atsushi Citation The Journal of Dermatology, 37(8), Issue Date 2010-08 URL Right http://hdl.handle.net/10069/25634