Supplementary Figure 1 A B C Supplementary Figure
Supplementary Figure 1: A B C Supplementary Figure 1: Chimerism and hematopoietic cell reconstitution. Three months after bone marrow transplantation, the chimerism and hematopoietic cell reconstitution of BMC-transplanted irradiated mice was monitored in spleen and PP by flow cytometry. (A) Percentages of CD 45. 1+ and CD 45. 2+ positive cells. (B and C) Percentages of CD 3+, CD 19+ and CD 11 c+ cells. At least n=6 per group from 3 independent experiments; mean±s. e. m.
s 0 f 2 10 D K 0. 20 0. 15 0. 10 0. 05 0. 00 0 f s 0. 0 2 10 0. 5 D 1. 0 O 1. 5 103 N 1. 5 102 GFP 2 wt 101 D Phosphatase alkaline G 2 O G 1 N G or 100 ct G 3 ve D pt y 104 Fold increase (2 -ΔΔct) NOD 2 WT Em s 0 f 103 2 10 D O N 2 wt D 0. 0 O 102 GFP O 2. 0 N 0. 5 G 2 N Sucrase isomaltase 2 wt or ct ve 101 D y G 1 O 0. 0 pt C N 0. 5 Em F or Zo-1 100 ct 104 ve 1. 0 Fold increase (2 -ΔΔct) 103 y G 3 pt 1. 0 Akaline Phosphatase Activity (U/ml/min) s 0 f G 2 2 10 102 GFP D O N 2 wt Empty Vector Em s 0 f Occludin 1. 5 2 10 J D 0. 5 x 106 D 1. 0 x 106 O 1. 5 O NOD 21007 fs G 1 N Empty vector N 7 or E 101 2 wt 0. 0 ct 0 0 10 D 0. 5 500 400 O I 6 ve B N pt y 3 4 5 Time (days) or NOD 2 wt Fold increase (2 -ΔΔct) 0 ct Em 200 ve 400 y 600 Number of cell 1000 800 pt 1. 0 Fold increase (2 -ΔΔct) s 0 f D 2 10 O D 2 wt ct or ve O N pt y N Em Fold increase (2 -ΔΔct) A Em s 2 0 f 1 2 10 1. 5 x 106 D 2. 0 x 106 O Number of Caco-2/TC 7 cells 2. 5 x 106 N 2 wt D 1. 5 0 O or ct ve Fold increase (2 -ΔΔct) 0 N y pt Em Supplementary Figure 2: NOD 21007 fs G 3 300 200 104 H Mlck 1. 0 0. 5 0. 0
% of positive B 220+ cells for Annexin V 4 3 2 1 0 20 10 G 6 4 2 0 30 6 0 8 4 2 0 H 6 4 2 0 60 40 20 0 I 1. 5 1. 0 0. 5 0 % of positive CD 8+ cells D % of positive CD 8+ cells for Caspase 3 5 8 % of positive CD 4+ cells C % of positive CD 4+ cells for Caspase 3 F 40 % of positive CD 11 c+ cells B % of positive CD 3+ cells for Caspase 3 80 70 60 50 40 30 20 10 0 % of positive CD 3+ cells A % of positive CD 3+ cells for Annexin V % of positive B 220+ cells Supplementary Figure 3: E 40 2. 0 8 7 6 5 4 3 2 1 0 WT KO 30 20 10 0 J WT KO
Supplementary Figure 4: WT KO
Supplementary Figure 5: 1. 0 Percentage of CD 8+ positive for TNF-α Percentage of CD 8+ positive for IFN-γ 0. 8 WT KO 0. 6 0. 4 0. 2 WT KO 0. 8 0. 6 0. 4 0. 2 0 0 Basal Stimulated
Supplementary Figure 6: Graphical abstract MDP Nod 2 MLCK - + TNF-R 2 Nod 23020 ins. C RICK TAK 1 MLCK +Nod 2 + TNF-R 2 TNF-R 1 TNF-α CD 4+ T cells IFN-γ
Supplementary Table 1: Peyer’s patches homeostasis. Nod 2+/+ Nod 2 -/- Nod 2+/+ CD 4 - Nod 2 -/CD 4 - Nod 2+/+ IFNγ- Nod 2 -/IFNγ- Mlck 1. 0± 0. 1 2. 7± 0. 2** 1. 1± 0. 2 0. 9± 0. 3φφφ 0. 9± 0. 2 0. 9± 0. 3φφφ Claudin 2 1. 0± 0. 2 2. 6± 0. 3*** 1. 1± 0. 1 0. 9± 0. 2φφφ 1. 1± 0. 1 1. 3± 0. 2φφφ Claudin 5 1. 0± 0. 2 1. 1± 0. 3 0. 9± 0. 2 1. 0± 0. 1 0. 9± 0. 2 1. 1± 0. 3 Claudin 8 1. 1± 0. 2 1. 0± 0. 1 1. 1± 0. 1 0. 9± 0. 2 0. 9± 0. 3 1. 1± 0. 1 Occludin 1. 0± 0. 1 1. 1± 0. 3 1. 0± 0. 2 1. 0± 0. 1 1. 1± 0. 3 1. 3± 0. 4 Zo-1 1. 1± 0. 1 0. 7± 0. 1** 1. 0± 0. 2 1. 0± 0. 1φφ 1. 1± 0. 1 1. 2± 0. 2φφ Zo-2 1. 0± 0. 2 0. 6± 0. 1** 1. 1± 0. 2 1. 2± 0. 1φφ 1. 2± 0. 4 0. 9± 0. 3φφ Tnf-r 1 1. 0± 0. 2 1. 2± 0. 2 0. 9± 0. 3 1. 3± 0. 2 1. 3± 0. 4 0. 9± 0. 2 Tnf-r 2 1. 1± 0. 1 2. 6± 0. 2** 0. 9± 0. 1 0. 9± 0. 2φφ 1. 2± 0. 3 1. 1± 0. 2φφ
Supplementary Table 2: Ileal mucosa homeostasis. Nod 2+/+ Nod 2 -/- Nod 2+/+ CD 4 - Nod 2 -/CD 4 - Nod 2+/+ IFNγ- Nod 2 -/IFNγ- Mlck 1. 2± 0. 2 3. 3± 0. 4** 1. 0± 0. 2 1. 1± 0. 1φφ 0. 9± 0. 3 1. 3± 0. 4φφ Claudin 2 1. 1± 0. 2 1. 2± 0. 1 1. 0± 0. 2 1. 2± 0. 1 1. 1± 0. 2 1. 3± 0. 3 Claudin 5 1. 0± 0. 2 0. 9± 0. 1 1. 1± 0. 2 0. 9± 0. 2 1. 1± 0. 3 1. 2± 0. 4 Claudin 8 1. 1± 0. 2 1. 0± 0. 1 0. 9± 0. 2 0. 8± 0. 3 Occludin 1. 1± 0. 2 0. 6± 0. 2* 1. 2± 0. 3 1. 1± 0. 2φ 1. 1± 0. 3 0. 9± 0. 2φφ Zo-1 1. 1± 0. 1 0. 8± 0. 2 0. 8± 0. 1 0. 9± 0. 2 1. 2± 0. 2 1. 1± 0. 2 Zo-2 1. 0± 0. 1 0. 5± 0. 2* 0. 9± 0. 2 1. 2± 0. 2φφ 0. 9± 0. 2 0. 9± 0. 1φ Tnf-r 1 0. 9± 0. 3 1. 1± 0. 2 1. 0± 0. 1 1. 2± 0. 3 1. 0± 0. 2 0. 9± 0. 2 Tnf-r 2 1. 1± 0. 2 2. 8± 0. 3* 0. 9± 0. 2 1. 2± 0. 2φφ 0. 9± 0. 3φφ
Supplementary Table 3: Summary of the in vivo experiments. Permeability Cytokine expression Nod 2 expression in intestinal epithelium Impact of ML-7 treatment Impact of MDP treatment Nod 2 is expressed in both compartments (WT→WT) Normal No change Nod 2 is absent in both compartments (KO→KO) Increased IFNγ, TNFα & IL-12 are increased Not expressed Normalized permeability and cytokine expression (versus KO→KO) No change Increased IFNγ, TNFα & IL-12 are increased Increased Normalized permeability and cytokine expression (versus KO→KO) Normalized permeability and cytokine expression are (versus KO→KO) Normal permeability (similar to WT→WT & restored in comparison to KO→KO) Restored a normal IFNγ, TNFα & IL-12 expression similar to WT→WT chimera Not expressed No change Nod 2 is only expressed in the non-hematopoietic compartment (KO→WT) Nod 2 is only expressed in the hematopoietic compartment (WT→KO)
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