NOD2 deficiency increases retrograde transport of secretory IgA complexes in Crohn's disease

Abstract

Intestinal microfold cells are the primary pathway for translocation of secretory IgA (SIgA)-pathogen complexes to gut-associated lymphoid tissue. Uptake of SIgA/commensals complexes is important for priming adaptive immunity in the mucosa. This study aims to explore the effect of SIgA retrograde transport of immune complexes in Crohn's disease (CD). Here we report a significant increase of SIgA transport in CD patients with NOD2-mutation compared to CD patients without NOD2 mutation and/or healthy individuals. NOD2 has an effect in the IgA transport through human and mouse M cells by downregulating Dectin-1 and Siglec-5 expression, two receptors involved in retrograde transport. These findings define a mechanism of NOD2-mediated regulation of mucosal responses to intestinal microbiota, which is involved in CD intestinal inflammation and dysbiosis.

Fig. 1. NOD2 mutation increases SIgA reverse transcytosis in CD patients.

a The two first graph compares the frequency of IgA-positive cells between healthy donors (n = 8), CD WT NOD2 patients (n = 10) and CD patients expressing NOD2 polymorphisms (n = 10) in PP and in villi. The third graph shows the number of IgA positive PP cells in patients expressing different NOD2 polymorphisms. NOD2 polymorphisms (R702W, G908R, FS1007insC) were determined by qPCR and sequencing. The last graph shows the ratio PP/villi. Vertical bars show the mean value ± SEM. A nonparametric Mann–Whitney U-test was used (p values: **p < 0.01, ***p < 0.005). b Images obtained from patient biopsy samples taken from the terminal ileum. This experiment was repeated in all patients with similar results: healthy donors (n = 8), CD WT NOD2 patients (n = 10) and CD patients expressing NOD2 polymorphisms (n = 10). Biopsies were labeled with anti-human GFP-IgA2 and PE-GP2 to label M cells or PE-DC-SIGN or PE anti-human SC at room temperature for 2 h. The left panels show representative images from CD patients expressing NOD2 polymorphism, the middle panels, CD patients with WT NOD2, and the right panels depict images from healthy donors. Bars: 200 µm. On all pictures, dotted lines delineate the follicle–associated epithelium (FAE) separating the intestinal lumen and the lymphoid tissue (side view). Source data are provided as a Source Data file.

Fig. 2. NOD2 deficiency increases IgA retrograde transport in mice.

a Tissue section showing a PP obtained from a ligated intestinal loop in WT, NOD2 KO or WT mice after MDP-PAM stimulation following exposure to SIgA-Cy3 for 60 min. Mouse IgG and the BSA were used as negative controls. The graph shows the number of SIgA-positive cells per PP. This experiment was repeated on 6 mice per group and an average of 3 PP per mouse was done. Vertical bars show the mean value±SEM. One-way ANOVA followed by Bonferroni post hoc test was used (p values: *p < 0.05; ***p < 0.005). b Fecal bacteria from WT or Nod2KO mice were stained with anti-IgA-FITC and the MFI were calculated on IgA-bacteria. This experiment was repeated on 4 mice per group. Vertical bars show the mean value ± SEM. A nonparametric Mann–Whitney U-test was used (ns: not significant). c Lysed bacteria were used as the target antigens in a western blot, using serum (S) or fecal (F) supernatant from WT or NOD2 KO mice as primary antibody and anti–IgA-HRP as the secondary antibody. This experiment was repeated on eight cohoused mice (WT (n = 4), NOD2 KO (n = 4)) with similar results. NOD2 KO and littermate WT mice were cohoused and immunized orally with p24-SIgA. d Levels of Ag-specific mucosal IgA and e levels of serum IgG in littermate WT or NOD2 KO mice were shown. Vertical bars show the mean value ± SEM. n = 5 biologically independent mice per group. One-way ANOVA followed by Bonferroni post hoc test was used (p values: *p < 0.05; **p < 0.01). f Cytokine concentrations in serum and faeces were determined in triplicate by Luminex-multiplex cytokine assay in littermate WT (n = 3) and NOD 2 KO mice (n = 3). Cytokine profiles are shown as radar charts; each axis displays the mean quantity (pg/ml) of each cytokine after immunization with p24-SIgA, p24 and PBS. A nonparametric Mann–Whitney U-test was used. P values have been calculated by comparing the p24-SIgA group with the p24 group; *p < 0.05. Source data are provided as a Source Data file.

Fig. 3. SIgA-Salmonella amplify Salmonella-induced colitis in mice.

Cohoused mice were challenged orally either with DSS, PBS, Salmonella Typhimurium or Salmonella Typhimurium bound with murine IgA. They were left untreated or treated with 5% laminarin in drinking water for 3 days prior to colitis challenge. a Disease activity index (DAI) score was undertaken daily to evaluate the clinical progression of colitis. The DAI was the combined score of weight loss compared to initial weight, stool consistency, and bleeding. Vertical bars show the mean value±SEM. n = 5 biologically independent mice per group. One-way ANOVA followed by Bonferroni post hoc test was used (p values: *p < 0.05; ***p < 0.005). b Sections from the colon of mice showing neutrophil infiltrates in the lamina propria. This experiment was repeated on all mice showing similar results. c Nancy histological score applied in each colon section for littermate and NOD2 KO mice. n = 5 biologically independent mice per group. One-way ANOVA followed by Bonferroni post hoc test was used (p values: **p < 0.01). Source data are provided as a Source Data file.

Fig. 4. IgA reverse transcytosis after protein inhibition or stimulation.

After siRNA knockdown or muramyl dipeptide—Pam3Cys (MDP-PAM) stimulation, IgA2 conjugated with luciferase a transport were quantified in the inverted in vitro model of FAE. Vertical bars show the mean value ± SEM. n = 3 independent experiments. One-way ANOVA followed by Bonferroni post hoc test was used (p values: **p < 0.01; ***p < 0.005). b Immunoprecipitation made 30 or 60 min after IgA incubation. This experiment was repeated twice with similar results. c Immunofluorescence staining after IgA incubation (60 min), with FITC anti-human IgA and anti-Rabs mAbs or anti-EEA-1 mAbs followed by corresponding PE secondary Abs. Colocalization between IgA and endosomal proteins resulted in yellow dots present in EEA-1, Rab-5, and Rab-17 images. This experiment was repeated twice with similar results. Source data are provided as a Source Data file.

Fig. 5. NOD2 modulates the expression of Dectin-1 and Siglec-5 receptors.

Western blot showing the expression of proteins in an in vitro model containing (co-culture) or not (mono-culture) M-like cells before “−” or after “+” NOD2 blocking of transcription with siRNA knockdown (a) or NOD2 stimulation with MDP-PAM treatment (b). These experiments were repeated twice with similar results. Flow cytometry was used to examine the role of NOD2 on Dectin-1 and Siglec-5 expression in M like cells in vitro (c) (n = 3 independent experiments; Vertical bars show the mean value ± SEM; One-way ANOVA followed by Bonferroni post hoc test was used, p values: ***p < 0.005) and in vivo (d) (n = 4 independent experiments; Vertical bars show the mean value ± SEM; A nonparametric Mann–Whitney U-test was used, p values: *p < 0.05). e Total IgA concentration was determined by ELISA in serum and faeces of NOD2KO mice and littermate WT mice. n = 4 biologically independent mice. One-way ANOVA followed by Bonferroni post hoc test was used. Vertical bars show the mean value ± SEM. kd knockdown, stim stimulation. Source data are provided as a Source Data file.

Fig. 6. The retrograde transport of IgA-pathogen complexes across M cells is increased in CD patients as compared to healthy individuals.

This translocation of IgA is regulated by WT NOD2 upon decrease of Dectin-1 and Siglec-5 expression in M cells, ensuring proper homeostasis. This pathway is controlled by endosomal proteins such as EEA-1, Rab-5, and Rab-17. We would like to emphasize that this identified mechanism is likely one among others that is involved in the initiation and/or perpetuation of mucosal inflammation observed in CD patients.