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

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.

In this manuscript, Rochereau and colleagues investigate the role of NOD2 in influencing the retrotranslocation of SIgA and SIgA-immune complexes into mouse (and a limited number of human) Peyer's patch tissues. While the study is intriguing, the investigators are quick to draw conclusions without sufficient experimental numbers and/or without important controls to justify claims about specificity of the SIgA transport in wild type and NOD2-/-mice. There long list of concerns compromises the ability of the authors to make conclusions possible connections to C rohn's disease.
The following constitutes a list of major/moderate concerns: 1. For studies related to Figure 1, the authors should stain for anti-SIgA (or anti-SC ), not anti-IgA alone, to make claims about retrotranslocation. From the present figure, one cannot make claims about whether the IgA was enriched from inside (interstitial fluids) or outside (retrotranslocation). It is not implausible that interstitial IgA accumulates in the PP of C D patients.
2. For studies related to Figure 2, the authors should specific the number of PP used per experiment. The legend suggests 6 mice per experiment. Does that translate to one PP per mouse? That is an extremely small sample size.
3. For studies related to Figure 2, only SIgA was used for transport/uptake experiments. The authors should use an inert antigen (e.g., BSA) and a control immunoglobulin (e.g., IgG) to test whether the effects observed are specific (or not) to SIgA. The same applies to the oral immunization studies, which could have been done with IgG-p24 complexes or deglycosylated SIgA (as done previously by the a 4. The results in Figure 3 A should be replotted and analyzed statistically to compare wild type mice to the NOD2 KO mice for common treatment condition. For example, wild type and NOD2 mice for the DSS + laminarin should be plotted side by side and then statistics applied between the two mouse strains, not among treatments for single mouse strain as is currently presented. Only then will they authors be able to make claims about NOD. Also, there is no indication of sample sizes for these experiments.
5. On line 157-158, the authors state that Salmonella challenge in the presence of Sal4SIgA worsens DAI. This is counter intuitive since that antibody and similar work by the authors has shown that anti-LPS SIgA are protective and promote agglutination in the lumen. Are the authors making the claim that the NOD2 mice take up SIgA-aggregates? This should be shown by microscopy.
6. The transcytosis studies with C aco-2 with M-like cells conversion are lacking important controls, including the appearance /demonstration of M-like cells and that transcytosis occurs exclusively via these cells following conversion of C aco-2 monolayers. These controls should be included as supplemental figures. 7. In Figure 4, the knockdown studies should be accompanied by an apical-to-basolateral transport control with a protein known to use those relevant pathways (e.g., a toxin). C o-localization studies by confocal microscopy are also required. Simply demonstrating the effect of a knock down on SIgA transport by ELISA (and with a few select pulldown assays) is not sufficient to make claims about cellular pathways of transport.

Manuscript number: NCOMMS-19-15971
Saint-Etienne, 20 th December 2019  Fig 3), then it would be difficult to determine the exact influence of NOD2 on this phenotype. This point is now detailed in the first major point of the reviewer 1. Figure 5 present convincing evidence of a role for Nod2 in IgA-complex transport. This data would add to the field and should be considered for publication.

The in vitro data showing increased SIgA transport in NOD2 knockdown cells and cells stimulated with MDP, provide strength to the initial findings of increased IgA+ cells in CD patients. Further, increased Dectin-1 and Siglec-5 expression in the knockdown cells and M-cells reinforce the involvement of NOD2 in IgA retrograde transport. Overall, the findings in CD patients with NOD2 mutations and data in
We greatly appreciate the positive appraisal of our work by Reviewer 1.

IgA retrograde transport is only mediated via M cells (Rochereau et al. Plos Biology 2013). We deleted NOD2 in a specific in vitro M-like cells model containing only enterocytes and M cells.
Using this specific model of FAE, we were able to monitor the role of NOD2 during IgA reverse transcytosis only in M cells.
Besides as the editor says "Exploration of cell type-specific roles of Nod2 is not a critical requirement from the editorial perspective.", we didn't went into cell-type specific roles.

Since the mice used were littermates, it would be important to confirm that the gut microbiota is not significantly different in the mice used in Fig 2 to show increased IgA transport into the PP. Alternatively, comparing the reactivity of the IgA between WT and Nod2KO mice to the gut microbiota (similar to what was done in McCarthy et al. 2011 JCI) may further indicate when the role of NOD2 is most critical.
We totally agree with this comment and have now performed these experiments as suggested by the reviewer. First, we verified the ability of IgA to bind to the same microbiota in littermate or NOD2 2KO mice, as previously described by McCarthy et al (Fig 2c). To quantify our observations, we also measured the MFI of IgA coated bacteria by flow cytometry (Fig 2b).
Major points: 1. The initial findings and data in Figure 5 present convincing evidence of a role for NOD2 in IgAcomplex retrograde transport, however, the in vivo data is underwhelming and does not provide direct evidence of a role for Nod2 in the sensing or immune response to these IgA-complexes. As this is the title and main conclusion of the paper, it is a concern. Could the data from Fig 2 (we think that you talked about the figure 3 as you mentioned previously) be represented to show WT and Nod2KO mice on the same plot to allow for statistical comparison? This is the only way to clearly show a role for Nod2 in the mice.
We agree with the reviewer 1 and we replotted the salmonella-IgA without laminarin conditions for littermate WT and NOD2 KO mice only (supplemental Fig. 1d). Statistical analysis of this new comparison revealed that there was a bias in our first analysis. All inflammatory parameters measured in the serum (IL-6, CRP and LPS) were taken at D5 post-infection but for neutrophil infiltrations, the colon histology was done at the time of mouse sacrifice (D9 for WT mice as they showed fewer clinical signs and D5 for NOD2 KO mice). Weight loss from D5 to D9 of WT mice indicates that they became increasingly sick. The Nancy score was not comparable between the WT and NOD2 KO mice. We reproduced the experiment on littermate WT and NOD2 KO mice focusing on the laminarin-free condition. In addition to colon histological analysis, we also measured IL-6, LPS and CRP at D5 in the blood. With these new data presented in supplemental figure 1d and implemented in figure 3a, 3b, and supplemental figure  1a and 1b, we now confirm the role of NOD2 in our observations.

Reviewer #2 (Remarks to the Author):
In this manuscript, Rochereau and colleagues investigate the role of NOD2 in influencing the retrotranslocation of SIgA and SIgA-immune complexes into mouse (and a limited number of human) Peyer's patch tissues. While the study is intriguing, the investigators are quick to draw conclusions without sufficient experimental numbers and/or without important controls to justify claims about specificity of the SIgA transport in wild type and NOD2-/-mice. There long list of concerns compromises the ability of the authors to make conclusions possible connections to Crohn's disease.
The following constitutes a list of major/moderate concerns: 1. For studies related to Figure 1, the authors should stain for anti-SIgA (or anti-SC), not anti-IgA alone, to make claims about retrotranslocation. From the present figure, one cannot make claims about whether the IgA was enriched from inside (interstitial fluids) or outside (retrotranslocation). It is not implausible that interstitial IgA accumulates in the PP of CD patients.
We thank the reviewer 2 for this point. Colocalization between SIgA and DC-SIGN (Fig. 1b) was already added and clearly show that counted SIgA-positive cells could come from a retrograde transport of IgA through M cells and a consecutive uptake by dendritic cells. A new colocalization (Fig. 1b) using anti-IgA and anti-secretory component (SC) staining has been now added and confirmed that IgA was not enriched from inside (interstitial fluids) but from the lumen (retrotranslocation). Figure 2, the authors should specific the number of PP used per experiment. The legend suggests 6 mice per experiment. Does that translate to one PP per mouse? That is an extremely small sample size.

For studies related to
This experiment was repeated on 6 mice per group but each point represents the average of 3 PP per mouse. This precision has been added in the corresponding figure legend. Figure 2, only SIgA was used for transport/uptake experiments. The authors should use an inert antigen (e.g., BSA) and a control immunoglobulin (e.g., IgG) to test whether the effects observed are specific (or not) to SIgA.

For studies related to
We have now performed these control experiments (Fig 2a).
The same applies to the oral immunization studies, which could have been done with IgG-p24 complexes or deglycosylated SIgA (as done previously by the authors).
The role of IgA as a vector carrying a protein such as p24 has already been published (Rochereau et al, EJI 2014 and JACI 2015). We also showed that IgG was not able to cross epithelium via M cells in vitro (Rochereau et al, Plos Biology 2013) and in vivo (in this article). In Figure 2d, we already showed control groups such as IgA alone and p24 alone. Moreover, we know that administration of IgG-p24 by oral or nasal route does not induce p24-specific antibodies in WT mice (data not shown).