Nod2-mediated recognition of the microbiota is critical for mucosal adjuvant activity of cholera toxin

Transcription

1 Supplementry informtion Nod2-medited recognition of the microiot is criticl for mucosl djuvnt ctivity of choler toxin Donghyun Kim 1,2, Yun-Gi Kim 1,2, Sng-Uk Seo 1,2, Dong-Je Kim 1,2, Nouhiko Kmd 3, Dve Prescott 4, Dn J Philpott 4, Philip Rosenstiel 5, Nohiro Inohr 1,2 & Griel Núñez 1,2 1 Deprtment of Pthology, University of Michign Medicl School, Ann Aror, Michign, USA. 2 Comprehensive Cncer Center, University of Michign Medicl School, Ann Aror, Michign, USA. 3 Deprtment of Internl Medicine, Division of Gstroenterology, University of Michign Medicl School, Ann Aror, Michign, USA. 4 Deprtment of Immunology, University of Toronto, Toronto, Ontrio, Cnd. 5 Institute of Clinicl Moleculr Biology, University of Kiel, Kiel, Germny. Correspondence should e ddressed to Y.-G.K. (yungikim77@gmil.com) or G.N. (griel.nunez@umich.edu). This file includes Supplementry figures 1 to 13 Nture Medicine: doi:1.138/nm.475

2 HSA-specific IgG (OD 45 ) µg/mouse.1 µg/mouse.2 µg/mouse.4 µg/mouse , 8, Supplementry Figure 1. Antigen-specific IgG production induced y vrious mounts of choler toxin. Mice were intrnslly immunized with 3 μg of HSA nd vrious mounts of CT. The mounts of HSA-specific IgG were nlyzed in plsm on dy 14 post-immuniztion. Dt re shown s men ± s.e.m. Nture Medicine: doi:1.138/nm.475

3 HSA-specific IgG (OD 45 ) IL-5 (ng/ml) IFN-γ (ng/ml) Untreted Ax , 4, HSA + + Untreted Ax HSA n.d. n.d. + + Untreted Ax Supplementry Figure 2. Symiotic cteri re criticl for orl immuniztion with ntigen nd choler toxin. () The mounts of HSA-specific IgG were nlyzed in plsm otined from ntiiotic (Ax)-treted mice (n = 5) nd untreted control mice (n = 4) on dy 14 fter orl immuniztion with 1 mg of HSA nd 1 μg of CT. () Splenocytes were isolted from Ax-treted mice nd control mice on dy 14 post-immuniztion, nd then restimulted with or without 5 μg/ml of HSA in triplicte cultures for 4 dys. The production of IFN-γ nd IL-5 ws exmined in superntnts of restimulted splenocytes y ELISA. The results re representtive of t lest two independent experiments. Vlues represent men ± s.e.m. () or men of three technicl replictes ± s.d. (). P <.5 nd P <.1 y Mnn-Whitney test () nd y Student s t-test (). n.d., not detected. Nture Medicine: doi:1.138/nm.475

4 HSA-specific IgG (OD 45 ) HSA-specific IgG (OD 45 ) 1.. µg/mouse.1 µg/mouse 1. µg/mouse 5. µg/mouse 2. Untreted Ax , 8, 25 1, 4, 16, Supplementry Figure 3. Symiotic cteri re dispensle for ntigen-specific IgG response induced y intrperitonel immuniztion with ntigen nd choler toxin. Mice were intrperitonelly immunized with 1 μl of PBS contining 1 μg of HSA nd vrious mounts of CT () or 1 μg of CT (). (, ) The mounts of HSAspecific IgG were nlyzed in plsm on dy 14 post-immuniztion. () Antiiotic-treted mice (n = 5) were given ntiiotic cocktil in the drinking wter d liitum from 2 weeks prior to immuniztion while control group (n = 5) ws provided with norml wter. Vlues represent men ± s.e.m. Nture Medicine: doi:1.138/nm.475 4

5 HSA-specific IgG (OD 45 ) HSA-specific IgG (OD 45 ) Untreted Ax 1. Untreted Ax , 4, 16, 25 1, 4, 16, Supplementry Figure 4. Symiotic cteri enhnce ntigen-specific IgG production fter nsl immuniztion with CpG, ut not MALP-2. The mounts of HSA-specific IgG in plsm were nlyzed in Ax-treted nd untreted mice on dy 14 fter intrnsl immuniztion with 3 μg of HSA nd 1 μg of CpG (, n = 4 or 5 per group) or.5 μg of MALP-2 (, n = 5 or 7 per group). Vlues represent men ± s.e.m. P <.5 y Mnn-Whitney test. Nture Medicine: doi:1.138/nm.475 5

6 HSA-specific IgG 1 (OD 45 ) HSA-specific IgG 2 (OD 45 ) HSA-specific IgM (OD 45 ) c WT WT Nod2 / Nod2 / WT Nod2 / , 8, Supplementry Figure 5. Antigen-specific IgG 1, IgG 2, nd IgM induced y nsl immuniztion with HSA nd choler toxin re decresed in Nod2 deficient mice. The mounts of HSA-specific IgG 1, IgG 2, nd IgM were mesured in plsm from Nod2 / nd WT mice (n = 5 per group) on dy 14 fter intrnsl immuniztion. Dt shown represent men ± s.e.m. P <.5 nd P <.1 y Mnn-Whitney test. Nture Medicine: doi:1.138/nm.475

7 HSA-specific IgG (OD 45 ) HSA-specific IgA (OD 45 ) 1. WT Nod2 / WT Nod2 / Reciprocl plsm dilution (1 x 1 ) Reciprocl NALF dilution (x 1 ) Supplementry Figure 6. Nod2 is importnt for systemic production of IgG nd locl production of IgA induced y nsl immuniztion with high dose of choler toxin. WT nd Nod2 / mice (n = 4 per group) were intrnslly immunized with 1 μg of HSA nd 1 μg of CT. HSA-specific IgG in plsm () nd HSA-specific IgA in NALF () were mesured on dy 14 post-immuniztion. Dt represent men ± s.e.m (, ) P <.5 y Mnn-Whitney test. Nture Medicine: doi:1.138/nm.475

8 HSA-specific IgG (OD 45 ) HSA-specific IgG (OD 45 ) WT 2.5 WT 1. Ripk2 / 2. Nod2 / , 4, , 4, Supplementry Figure 7. Ripk2 nd Nod2 re dispensle for intrperitonel immuniztion with choler toxin or lum. WT nd Ripk2 / () or Nod2 / () mice (n = 3 ~ 5 per group) were intrperitonelly immunized with 1 μl of PBS contining 1 μg of HSA nd 1 μg of CT () or 3 μg of HSA nd 1 μl of lum (), respectively. HSAspecific IgG ws mesured in plsm on dy 14 post-immuniztion. Dt represent men ± s.e.m. Nture Medicine: doi:1.138/nm.475

9 Normlized reltive mrna mount (Nod2/GAPDH; fold) Totl cells BMDM HEK293 CD11c NALT CD CD45 + CD11c + CD45 + CD11c - CD45 - CD11c - Supplementry Figure 8. Nod2 expression in NALT cell popultions. () Cells isolted from NALT were sorted y flow cytometry s indicted. () Anlysis of Nod2 expression in mrna from totl cells nd sorted NALT cells ws performed y rel-time qpcr. Bone mrrow-derived mcrophges (BMDM) nd HEK293 cells re shown s positive nd negtive controls, respectively. Gpdh gene expression ws used for dt normliztion. Nture Medicine: doi:1.138/nm.475

10 Nod2 18s Nod2 fl/fl CD11c Cre ;Nod2 fl/fl Nod2 / Nod2 fl/fl CD11c Cre ;Nod2 fl/fl Nod2 / CD11c + cells CD11c cells Supplementry Figure 9. Verifiction of CD11c positive cell-specific knockout of Nod2. Totl splenocytes were isolted from CD11c Cre ;Nod2 fl/fl, their littermte Nod2 fl/fl nd whole-ody Nod2 / mice. CD11c-positive nd CD11c-negtive cells were isolted y mgnetic sorting kit nd RT-PCR using primers spnning exons 2 nd 3 of the Nod2 trnscript ws performed on totl RNA isolted from purified cells. Nture Medicine: doi:1.138/nm.475 1

11 HSA-specific IgG (OD 45 ) IFN-γ (pg/ml) IL-5 (pg/ml) Nod2 fl/fl Krt14-Cre;Nod2 fl/fl 8 n.s , 8, HSA + + Nod2 fl/fl Krt14-Cre;Nod2 fl/fl HSA + + Nod2 fl/fl Krt14-Cre;Nod2 fl/fl Supplementry Figure 1. Nod2 in epithelil cells is dispensle for djuvnt ctivity of choler toxin. () HSA-specific IgG ws mesured in plsm from immunized Krt14-Cre;Nod2 fl/fl nd their littermte Nod2 fl/fl mice (n = 4 per group) on dy 14 post-immuniztion.() Splenocytes were isolted from Krt14-Cre;Nod2 fl/fl nd Nod2 fl/fl mice on dy 14 post-immuniztion, nd then restimulted with or without HSA for 4 dys. Concentrtions of IFN-γ nd IL-5 were determined in the superntnts of restimulted splenocytes y ELISA. Results re representtive of t lest two independent experiments. Dt re shown s men ± s.e.m. () or ± s.d. (). P <.1 nd P <.1 y Mnn-Whitney test () nd y Student s t-test () n.s.; not significnt. Nture Medicine: doi:1.138/nm.475

12 Normlized reltive mrna mount (Nod2/Gpdh) Normlized reltive mrna mount (Nod2/Gpdh) Time fter CT tretment (hours) Time fter camp tretment (hours) Supplementry Figure 11. Choler toxin nd camp induce the expression of Nod2 mrna in DCs. BMDCs were treted with.5 μg/ml of CT () nd 1 μm of camp () in triplicte cultures nd totl RNA were prepred t vrious time-points, followed y reltime qpcr nlysis using specific primer sets for Nod2 nd Gpdh. Gpdh ws used s for dt normliztion. Results re representtive of two independent experiments. Dt shown represent men ± s.d. Nture Medicine: doi:1.138/nm.475

13 Reltive Nod2 ctivity in NALF (fold) Reltive totl cteril mount in NALF (fold) Bcteril numer in NALF n.s. n.s HSA+CT HSA+CT+S.s. HSA+CT+S.g S.sciuri S.gllinum S.sciuri S.gllinum HSA + CT c 6 n.s S.sciuri S.gllinum HSA + CT Supplementry Figure 12. Bcteril coloniztion nd Nod2-stimultory ctivity in the nsl cvity of mice stimulted intrnslly with HSA, choler toxin nd indicted cteri. SPF mice (ech group n = 5) were intrnslly immunized with HSA nd CT together with c.f.u./mouse of live S. sciuri or S. gllinrum nd, 2 weeks lter, NALFs were collected from ech mouse. () Totl cteril coloniztion ws mesured y rel-time qpcr nlysis using totl cteril DNA otined from NALF nd set of universl 16S rdna sequence primers () The numers of S. sciuri nd S. gllinrum in the NALF were clculted sed on distinct colony morphology fter culturing NALF cteri in BHI gr pltes. The identity of selected colonies ws verified y 16S rdna sequencing of V3-V4 regions. (c) Nod2-stimultory ctivity in NALF from SPF, S. sciuri-, or S. gllinrum-treted mice. Ech dot represents n individul mouse nd the men vlue is displyed y line. P <.5 nd P <.1 y Mnn-Whitney test. n.s.; not significnt. 13 Nture Medicine: doi:1.138/nm.475

14 HSA-specific IgG (OD 45 ) S.s. E.c , 4, Supplementry Figure 13. Antigen-specific IgG responses in germ-free mice stimulted with UV-inctivted cteri. GF mice (n = 3 or 4 per group) were intrnslly immunized with HSA nd CT together with c.f.u./mouse of UVinctivted S. sciuri or E. coli. The mounts of HSA-specific IgG were nlyzed in plsm on dy 14 post-immuniztion. Dt re shown s men ± s.e.m. P <.5 y Mnn- Whitney. Nture Medicine: doi:1.138/nm.475