1. A B C Supplementary Figure 1:
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 CD45.1+ and CD45.2+ positive cells. (B and C) Percentages of CD3+, CD19+ and CD11c+ cells. At least n=6 per group from 3 independent experiments; mean±s.e.m.

2. Akaline Phosphatase Activity (U/ml/min)
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 CD45.1+ and CD45.2+ positive cells. (B and C) Percentages of CD3+, CD19+ and CD11c+ cells. At least n=6 per group from 3 independent experiments; mean±s.e.m.
Supplementary Figure 2: Transfection and selection of stably transfected Caco-2/TC7 cells.
(A) After transfection with the empty control vector or the vector including the NOD2WT or NOD21007fs, cDNA, mRNA expression of NOD2WT and NOD21007fs was investigated by RT-PCR (at least n=4 per group; mean±s.e.m). (B-D) 48h after transfection, selection of positively transfected cells was achieved by addition of G418 to the culture medium. Three consecutive cycles of selection were performed. Then, the level of GFP fluorescence was assessed by FACS and cells with the highest fluorescence (gate G3) were selected. (E) Cellular growth was monitored each day for six days. (at least n=8 per group; 3 independent experiments; mean±s.e.m). (F-K) Stably transfected Caco-2/TC7 cells were cultivated in Transwell chambers for 14 days and the level of cell differentiation was monitored by mRNA expression levels of (F) sucrase-isomaltase, (G) alkalin phosphatase, (H) Mlck, (I) occludin and (J) Zo-1 (at least n=4 per group; mean±s.e.m). (K) Alkaline phosphatase activity was monitored using a commercial kit from Biovision (n=4 per group; mean±s.e.m).
Supplementary Figure 3: Characterization of Peyer’s patch immune cell composition and apoptotic cells after seven days of culture.
(A-E) The immune cell composition of PP from WT and KO mice was investigated by flow cytometry. Relative proportions of PP (A) CD45+B220+, (B) CD3+, (C) CD11c+, (D) CD3+CD4+ and (E) CD3+CD8+. Data represent the means ± SEM of six mice per group. (**p<0.01 vs. WT mice). (F-J) After seven days of culture, the relative proportions of PP (F) CD45+B220+, (G) CD3+ positive for annexin V and the percentage of (H) CD3+, (I) CD3+CD4+ and (J) CD3+CD8+ positive for capase-3 have been investigated. At least n=6 per group; 3 independent experiments; mean±s.e.m.
Supplementary Figure 4: Characterization of CD4+ depletion in WT and KO mice.
In vivo depletion of CD4+ T-cells in WT and KO mice was obtained by intraperitoneal injection of 100 mg purified GK1.5 (anti-L3T4 (CD4+) monoclonal antibody 96 and 24 hours before experimentation. The relative proportion of CD3+CD4+ cells in PP were assessed by flow cytometry. At least n=8 per group; 3 independent experiments; mean±s.e.m; ***p<0.001 vs. indicated group.
Supplementary Figure 5: Percentage of IFN- or TNF-α secreting CD8+ T cells in PP of WT and Nod2KO mice.
Cells from PP of WT and KO mice were cultured with or without anti-CD3 and anti-CD28 for four hours. After surface (CD3/CD8) and intracellular (IFN- or TNF-α) staining, cells were gated as CD3+CD8+ and the percentage of cells positive for IFN- or TNF-α were determined using Cell Quest 3.3 software. At least n=6 per group; 3 independent experiments; mean±s.e.m; *p<0.05 vs. control group.
Supplementary Figure 6: Graphical abstract.
Nod2 is a key player in the control of the homeostasis of the gut mucosa by regulating the crosstalk between immune cells and the epithelium. IFN-γ secretion by CD4+ T cells up-regulates the expression of TNF-R2 by enterocytes. The interaction between TNF-α and TNF-R2 promotes the expression and activity of MLCK, which alters the structure of tight junctions. By contrast, TNF-α interaction with TNF-R1 strongly increases Nod2 expression. When activated with MDP, epithelial Nod2 inhibits the expression of MLCK, thereby inhibiting tight junction opening via recruitment of the TAK1/RICK complex resulting in a reinforced epithelial barrier. In case of Nod2 deletion or loss of function mutation, the protective role of Nod2 for the control of the epithelial barrier function is lost.
Supplementary Figure 2: A B C D
Fold increase (2-ΔΔct)
Empty vector
NOD2wt
NOD2100fs
400
600
800
1000
200
Empty Vector
100
101
102
103
104
GFP G1 G2 G3
200
400
500
Number of cell
300
NOD2WT G1 G2 G3
100
101
102
103
104
GFP
GFP
100
101
102
103
104
NOD21007fs G1 G2 G3 E F G H
2.5x106
NOD21007fs
Empty vector
NOD2wt
Empty vector
NOD2wt
NOD2100fs
Fold increase (2-ΔΔct)
0.0
0.5
1.0
1.5
Sucrase isomaltase
Empty vector
NOD2wt
NOD2100fs
Fold increase (2-ΔΔct)
0.0
0.5
1.0
1.5
Phosphatase alkaline
2.0
Empty vector
NOD2wt
NOD2100fs
Fold increase (2-ΔΔct)
0.0
0.5
1.0
1.5
Mlck
2.0x106
1.5x106
Number of Caco-2/TC7 cells
1.0x106
0.5x106 1 2 3 4 5 6 7
Time (days) I J K
Empty vector
NOD2wt
NOD2100fs
Fold increase (2-ΔΔct)
0.0
0.5
1.0
1.5
Zo-1
Empty vector
NOD2wt
NOD2100fs
Fold increase (2-ΔΔct)
0.0
0.5
1.0
1.5
Occludin
Empty vector
NOD2wt
NOD2100fs
Akaline Phosphatase Activity (U/ml/min)
0.00
0.10
0.15
0.20
0.05

3. Supplementary Figure 3:B C D E WT KO 80 70 60 50 40 30 20 10
% of positive B220+ cells 40 30 20 10
% of positive CD3+ cells 8 6 4 2
% of positive
CD11c+ cells 60 40 20
% of positive CD4+ cells
% of positive CD8+ cells 40 30 20 10 F 8 6 4 2
% of positive CD3+ cells for Annexin V G 6 4 2
% of positive CD3+ cells for Caspase 3 H I
2.0
1.5
1.0
0.5
% of positive CD4+ cells for Caspase 3 8 7 6 5 4 3 2 1
% of positive CD8+ cells for Caspase 3 J 5 4 3 2 1
% of positive B220+ cells for Annexin V WT KO

4. Supplementary Figure 4:WT KO

5. Supplementary Figure 5:
0.8
0.6
0.4
0.2
Percentage of CD8+
positive for IFN-γ
Basal
Stimulated WT KO
Percentage of CD8+
positive for TNF-α
1.0
0.8
0.6
0.4
0.2
Basal
Stimulated WT KO

6. - + + + Supplementary Figure 6: Graphical abstract TNF-α IFN-γ
CD4+ T cells
MLCK
Nod23020insC
MDP +
TNF-R2
MDP
Nod2
RICK
TAK1
MLCK - +
Nod2 +
TNF-R2
TNF-R1
TNF-α
CD4+
T cells
IFN-γ

7. Supplementary Table 1: Peyer’s patches homeostasis.
Nod2+/+
Nod2-/-
Nod2+/+ CD4-
Nod2-/- CD4-
Nod2+/+ IFNγ-
Nod2-/- IFNγ-
Mlck
1.0±0.1
2.7±0.2**
1.1±0.2
0.9±0.3φφφ
0.9±0.2
Claudin 2
1.0±0.2
2.6±0.3***
1.1±0.1
0.9±0.2φφφ
1.3±0.2φφφ
Claudin 5
1.1±0.3
Claudin 8
0.9±0.3
Occludin
1.3±0.4
Zo-1
0.7±0.1**
1.0±0.1φφ
1.2±0.2φφ
Zo-2
0.6±0.1**
1.2±0.1φφ
1.2±0.4
0.9±0.3φφ
Tnf-r1
1.2±0.2
1.3±0.2
Tnf-r2
2.6±0.2**
0.9±0.1
0.9±0.2φφ
1.2±0.3
1.1±0.2φφ

8. Supplementary Table 2: Ileal mucosa homeostasis.
Nod2+/+
Nod2-/-
Nod2+/+ CD4-
Nod2-/- CD4-
Nod2+/+ IFNγ-
Nod2-/- 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
1.2±0.1
1.3±0.3
Claudin 5
0.9±0.1
0.9±0.2
1.1±0.3
1.2±0.4
Claudin 8
1.0±0.1
0.8±0.3
Occludin
0.6±0.2*
1.2±0.3
1.1±0.2φ
0.9±0.2φφ
Zo-1
1.1±0.1
0.8±0.2
0.8±0.1
Zo-2
0.5± 0.2*
1.2±0.2φφ
0.9±0.1φ
Tnf-r1
Tnf-r2
2.8±0.3*
0.9±0.3φφ

9. Supplementary Table 3: Summary of the in vivo experiments.
Permeability
Cytokine expression
Nod2 expression in intestinal epithelium
Impact of ML-7 treatment
Impact of MDP treatment
Nod2 is expressed in both compartments (WT→WT)
Normal
No change
Nod2 is absent in both compartments (KO→KO)
Increased
IFNγ, TNFα & IL-12 are increased
Not expressed
Normalized permeability and cytokine expression (versus KO→KO)
Nod2 is only expressed in the non-hematopoietic compartment
(KO→WT)
Normalized permeability and cytokine expression are (versus KO→KO)
Nod2 is only expressed in the hematopoietic compartment
(WT→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