CARD15/NOD2 is required for Peyer's patches homeostasis in mice

Abstract

Background: CARD15/NOD2 mutations are associated with susceptibility to Crohn's Disease (CD) and Graft Versus Host Disease (GVHD). CD and GVHD are suspected to be related with the dysfunction of Peyer's patches (PP) and isolated lymphoid follicles (LFs). Using a new mouse model invalidated for Card15/Nod2 (KO), we thus analysed the impact of the gene in these lymphoid formations together with the development of experimental colitis.

Methodology/principal findings: At weeks 4, 12 and 52, the numbers of PPs and LFs were higher in KO mice while no difference was observed at birth. At weeks 4 and 12, the size and cellular composition of PPs were analysed by flow cytometry and immunohistochemistry. PPs of KO mice were larger with an increased proportion of M cells and CD4(+) T-cells. KO mice were also characterised by higher concentrations of TNFalpha, IFNgamma, IL12 and IL4 measured by ELISA. In contrast, little differences were found in the PP-free ileum and the spleen of KO mice. By using chamber experiments, we found that this PP phenotype is associated with an increased of both paracellular permeability and yeast/bacterial translocation. Finally, KO mice were more susceptible to the colitis induced by TNBS.

Conclusions: Card15/Nod2 deficiency induces an abnormal development and function of the PPs characterised by an exaggerated immune response and an increased permeability. These observations provide a comprehensive link between the molecular defect and the Human CARD15/NOD2 associated disorders: CD and GVHD.

Figure 1. Nod2 and postnatal development of gut associated lymphoid formations.

(A) PP count on the whole intestines of KO (▪) and WT (□) mice at birth and at weeks 4, 12 and 52. (B) Number of isolated LFs identified on microscopic examination of the small intestine at weeks 12 and 52. (C and D) Number of cells per PP and spleen at weeks 4, 12 and 52). (E) Number of apoptotic cells identified by caspase-3 immunostaining at week 12. Data represent the means±SEM of 8 mice per group. *P<0.05; **P<0.01, ***P<0.001.

Figure 2. Nod2 and Peyer's patch cellular composition.

(A and B) Relative proportions of T-, B- and dendritic cells from PPs and spleens of KO (▪) and WT (□) mice at week 12. T-, B- and dendritic cells were investigated by flow cytometry using antibodies to CD3, B220 and CD11c. (C) Relative proportion of polymorphonuclear neutrophils was analyzed using Ly-6G antibody. (D) M cells number inside the follicle associated with epithelium was investigated by immuno-histochemistry. Arrows indicated the presence of M-cell inside the follicle associated with epithelium. Data represent the means±SEM of 8 mice per group. **P<0.01.

Figure 3. Nod2 and T-cells subset in PP and spleen from mice at 12 weeks of age.

Relative proportion of CD4⁺, CD8⁺ and CD4⁻CD8⁻ T-cells from PPs (A) and spleen (B) of KO (▪) and WT (□) mice. At week 12, CD3⁺ T-cells were stained with antibodies to CD3, CD4, and CD8. Data were gated for CD3⁺ T-cells. Data represent the means±SEM of 8 mice per group. *P<0.05.

Figure 4. Nod2 invalidation increases cytokine expression in PP.

Expressions of Il-1β, IFNγ, TNFα, IL-12, IL-4 were determined by ELISA in PPs, Ileum and spleens from KO (▪) and WT (□) mice at weeks 4 (left panel) and 12 (right panel). Data represent the means±SEM of 8 mice per group. *P<0.05; **P<0.01.

Figure 5. Paracellular permeability and bacterial translocation are increased in Nod2 invalidated mice.

Ussing-chamber and Real-time PCR experiments were performed on PP and PP-free ileum from WT (□) and KO (▪) at week 12. (A) Paracellular permeability was analysed by FITC-dextran flux from PP and PP-free ileum under basal condition. (B) mRNA expression levels of TJ proteins (ZO-1, ZO-2 and Occ) from PP were analysed by real-Time-PCR under basal condition. (C) Bacterial translocation of chemically killed fluorescent Escherichia Coli K-12. (D) Translocation of the viable non enteropathogen Escherichia Coli strain J53. (E) Translocation of a chemically killed fluorescent Staphylococcus Aureus. (F) Translocation of chemically killed fluorescein-conjugated Saccharomyces cerevisiae. Data represent the means±SEM of 8 mice per group. *P<0.05 and **P<0.01, significantly different from WT.

Figure 6. Nod2 invalidation increased the suceptibility of TNBS induced colitis.

(A) Body weight, (B) Macroscopic damage score, (C) cytokines levels were assessed in 12 week old mice. These parameters were determined three days after intracolonic administration of TNBS. Values are mean (SEM) (n = 8 in each group). *P<0.05 and **P<0.01 between WT and KO mice.

Figure 7. Targeting disruption of the murine Nod2 gene by homologous recombination.

(A) Generation of the Card15/Nod2 KO allele: targeting strategy. The restriction maps of the Card15/Nod2+allele (5′ portion), the Card15/Nod2 KO targeting fragment, and the modified Card15/Nod2 allele after homologous recombination and Cre-mediated recombination of an Hygromycin selection cassette are shown. Exons 1, 2, and 3 (black boxes) and restriction sites used for cloning and screening (X) XbaI, (A) AgeI, (P) PmlI, (S) SalI, (N) NotI are indicated. The Card15/Nod2 KO targeting fragment comprises the EGFP gene (Clontech) in frame with Card15/Nod2 ATG and the floxed PGKHygromycin (Clontech) selection cassette, introduced between the AgeI site downstream of Card15/Nod2 exon 1 and the SalI site upstream of exon 1 in the Card15/Nod2 orientation. All loxP sites are represented by open triangles. Recombination of loxP1 and loxP2 results in the loxP1+2 site. In the first step of the strategy, the Card15/Nod2 locus was targeted with the Card15/Nod2 KO targeting fragment. In the second step, the PGKHygromycin selection cassette was removed by Cre recombinase. The double-headed arrows indicate the DNA fragments resulting from digestions with different enzymes expected to hybridize with probes A, B or Hyg. Also depicted are combinations of PCR primers P1-4 that detect the different Card15/Nod2 alleles. (B) Southern blot analysis of restricted DNA resulting from digestion with XbaI.Hybridation with Probe A and B show complete integration of the targetting fragment after homologous recombination. Probe Hyg show the differents integrations of targetting fragments after homologous recombination. (C) Genotyping of Nod2-deficient mice by PCR. Genomic DNA from mice was amplified by PCR to detect the disrupted sequence (PCR product of 459 bp). (D) Expression of Nod2 mRNA in spleen. RT-PCR was performed on purified mRNA from the spleen of WT and KO mice. As expected, no signal was observed in KO mice. GAPDH expression was used as positive control of expression.