Supplementary information Disrupting ceramide-cd3f interaction prevents septic peritonitis by stimulating neutrophil recruitment Kumi Izawa 1,2, Akie Maehara 1,2, Masamichi Isobe 1,2, Yuka Yasuda 3, Makoto Urai 4, Yasutaka Hoshino 4, Keigo Ueno 4, Toshihiro Matsukawa 2,5, Mariko Takahashi 2, Ayako Kaitani 1,2, Emiko Shiba 1,6, Ayako Takamori 1, Shino Uchida 1,7, Koichiro Uchida 1, Keiko Maeda 1, Nobuhiro Nakano 1, Yoshinori Yamanishi 2,8, Toshihiko Oki 2, David Voehringer 9, Axel Roers 1, Susumu Nakae 11, Junko Ishikawa 3, Yuki Kinjo 4, Toshiaki Shimizu 1,6, Hideoki Ogawa 1, Ko Okumura 1, Toshio Kitamura 2 & Jiro Kitaura 1,2
Supplementary figure legends S1-S9 Figure S1. Concentrations of all and indicated ceramide species in plasma. Concentrations of all and indicated ceramide species in plasma before (n =3) or 4 h after a sham operation (n = 4) or CLP (n = 4) in WT mice. The data are expressed as mean ± SD. Figure S2. Disruption of ITIMs and ITSM of CD3f attenuated the inhibition of E. coli-stimulated mast cell activation. (a) Expression of c-kit, FcεRIα, or CD3f in CD3f -/- BMMCs transduced with CD3f WT, CD3f-Y241F/Y289F/Y325F mutant, or mock. (b,c) These BMMC transfectants were stimulated with 5 x 1 7 CFU/ml heat-killed E. coli on plates coated with ceramide, PC, or vehicle. Production of (b) the release of β-hexosaminidase or (c) KC. The data are representative of three independent experiments and are expressed as mean ± SD. *P <.1 (Student s t-test). Figure S3. Expression of CD11b, Gr-1, and CD3f in WT or CD3f -/- neutrophils. Neutrophils purified from WT or CD3f -/- BM were stained with an anti-cd11b Ab, anti-gr-1 Ab, or anti-cd3f Ab. The data are representative of three independent experiments. Figure S4. Neither CD3f expression nor ceramide-cd3f interaction significantly influenced bactericidal activity of neutrophils or BMMCs against E. coli. (a,b) Neutrophils (a) or (b) BMMCs were incubated with E. coli (4 x 1 5 cells per 1 6 CFU/ml) for 6 min on plates coated with the indicated lipids or vehicle. Numbers
of viable E. coli cells were estimated by colony-counting methods. The data are representative of three independent experiments and are expressed as mean ± SD. Figure S5. Transfusion of 1 7 WT or CD3f -/- neutrophils equally improved the survival of CLP-operated mice. CLP-operated WT mice were intraperitoneally injected with 1 7 WT or CD3f -/- neutrophils or PBS as a control (n = 6 per group) and were monitored regarding survival. *p <.1 compared to CLP-operated control mice; n.s.: not significant (long-rank test). Figure S6. Kit W-sh/W-sh mice reconstituted intraperitoneally with WT or CD3f -/- BMMCs had equivalent numbers of peritoneal mast cells. (a) Peritoneal lavage cells from Kit W-sh/W-sh mice that received a transplant of WT or CD3f -/- BMMCs were stained for FcεRI and c-kit to determine the percentage of mast cells among peritoneal lavage cells (upper). Surface expression levels of CD3f in FcεRI + c-kit + mast cell populations of peritoneal lavage cells (lower). (b) Numbers of mast cells among peritoneal lavage cells from Kit W-sh/W-sh mice that received a transplant of WT or CD3f -/- BMMCs (each, n = 7). The data are representative of two independent experiments. The data are expressed as mean ± SD. Figure S7. Mcpt5-Cre/R-DTA mice reconstituted intraperitoneally with WT or CD3f -/- BMMCs had equivalent numbers of peritoneal mast cells. Numbers of mast cells among peritoneal lavage cells from Mcpt5-Cre/R-DTA mice with a transplant of WT or CD3f -/- BMMCs (each, n = 5). The data are representative of two independent experiments and are expressed as mean and ± SD.
Figure S8. Reconstitution with CD3f -/- BMMCs increased the number of neutrophils recruited to the peritoneal cavity and improved survival in CLP-operated Kit W-sh/W-sh CD3f -/- mice. (a) Kit W-sh/W-sh CD3f -/- mice that had received an intraperitoneal transplant of WT or CD3f -/- BMMCs (n = 11 per genotype) or were injected with PBS (n = 3) were subjected to CLP and monitored regarding survival; *p <.1 compared to the mice with a transplant of CD3f -/- BMMCs. (b) Numbers of neutrophils recruited into the peritoneal cavity (n = 4 per group). The data are expressed as mean ± SD; *p <.1 (Student s t-test). The data are representative of two independent experiments. (c) Numbers of mast cells among peritoneal lavage cells from Kit W-sh/W-sh CD3f -/- mice with a transplant of WT or CD3f -/- BMMCs (each, n = 6). The data are representative of two independent experiments. The data are expressed as mean ± SD. Figure S9. Transfusion of WT or CD3f -/- BM-derived macrophages equally improved the survival of macrophage-depleted WT mice after CLP. Macrophage-depleted WT mice were intravenously injected with 1 6 WT or CD3f -/- or PBS as a control (n = 7 per group) 6 h before CLP induction and were monitored regarding survival. *p <.1 compared to CLP-operated control mice; n.s.: not significant (long-rank test).
Supplementary Fig. S1 Total ceramide (ng/ml) Total ceramide (ng/ml) 2 15 1 5 Plasma normal Sham-4 sham-4hrh CLP-4hr h Ceramide (ng/ml) 1 8 6 4 2 d18:1/16: d18:1/18: d18:1/2: normal Sham-4 sham-4hrh CLP-4hr h d18:1/22: d18:1/24: d18:1/24:1 Plasma
Supplementary Fig. S2 A mock CD3f-WT CD3f-YF B β-hexosaminidase release (%) c-kit FcεRI CD3f 15 1 5 * KO-MC (-) Cer PC mock Mock CD3f-WT LMIR3(WT) CD3f-YF LMIR3(YF) C KC (pg/ml) 35 3 * 25 2 15 1 5 vehicle ceramide PC vehicle (-) ceramide Cer PC KO-MC Mock mock LMIR3(WT) CD3f-WT LMIR3(YF) CD3f-YF
Supplementary Fig. S3 WT neutrophils KO neutrophils 1 4 1 4 Gr-1 1 3 1 2 1 3 1 2 1 1 1 1 1 1 1 1 1 1 2 1 3 1 4 1 1 1 1 2 1 3 1 4 CD11b 1 1 8 8 6 6 4 4 2 2 1 2 3 4 1 1 1 1 1 1 1 1 1 2 1 3 1 4 CD3f
Supplementary Fig. S4 A PBS WT-neutrophils KO-neutrophils 1 B 1 PBS WT-MC KO-MC KO-MC 1 CFU CFU 1 1 CFU CFU 1 1 1 1 MetOH Cer PC PS MetOH Cer PC PS Vehicle ceramide PC PS vehicle ceramide PC PS
Supplementary Fig. S5 Percent Survival survival (%) 1 8 6 4 2 *p <.1 *p <.1 WT PBS+ PBS (n = 6) WT Cd3lf + WT +/+ - neutrophils (1 7 ) (n = 6) WT Cd3lf + KO-neutrophils -/- (1 7 ) (n = 6) n.s. 1 2 3 4 5 6 7 Days after CLP
Supplementary Fig. S6 A c-kit 1 4 1 3 1 2 1 1 1 Kit Wsh/Wsh % 1 1 1 1 2 1 3 1 4 1 4 1 3 1 2 1 1 1 1 8 Kit W-sh/W-sh + WT-MC 1 1 1 1 2 1 3 1 4 1 4 1 3 1 2 1 1 1 FcεRI 1 8 Kit W-sh/W-sh + KO-MC 1 1 1 1 2 1 3 1 4 B Percentage of c-kit + FcεRI + cells (/peritoneal cavity) 1 8 6 4 2 Kit WT W-sh/W-sh + WT-MC Kit KO W-sh/W-sh + KO-MC 6 6 4 4 2 2 1 1 1 1 2 1 3 1 4 1 1 1 1 2 1 3 1 4 CD3f
Supplementary Fig. S7 Percentage of c-kit + FcεRI + cells (/peritoneal cavity) 6 4 2 WT-MC KO-MC Mcpt5-Cre/R-DTA
Supplementary Fig. S8 A Survival (%) (%) 1 8 6 4 2 Kit W-sh/W-sh CD3f -/- (n = 3) Kit W-sh/W-sh CD3f -/- + WT-MC (n = 11) Kit W-sh/W-sh CD3f -/- + KO-MC (n = 11) *p <.1 B CD11b + Gr-1 high cells (x1 6 cells/peritoneal cavity) 15 1 5 * 1 2 3 4 5 6 7 Days Days after after CLP CLP PBS WT-MC KO-MC Kit W-sh/W-sh CD3f -/- C Percentage of c-kit + FcεRI + cells (/peritoneal cavity) 8 6 4 2 WT-MC KO-MC Kit W-sh/W-sh CD3f -/-
Supplementary Fig. S9 Survival (%) 1 8 6 4 2 clodronate ΜΦ-depleted WT (n = 7) clodronate+wt-mf ΜΦ-depleted + WT-ΜΦ (n = 7) clodronate+ko-mf ΜΦ-depleted WT + KO-ΜΦ (n = 7) *p <.1 *p <.1 n.s. 1 2 3 4 5 6 7 Days after mild CLP