Non-canonical NF-κB signaling promotes intestinal inflammation by restraining the tolerogenic β-catenin-Raldh2 axis in dendritic cells

  1. Alvina Deka
  2. Naveen Kumar
  3. Meenakshi Chawla
  4. Namrata Bhattacharya
  5. Sk Asif Ali
  6. Swapnava Basu
  7. Bhawna
  8. Upasna Madan
  9. Shakti Kumar
  10. Bhabatosh Das
  11. Debarka Sengupta
  12. Amit Awasthi
  13. Soumen Basak

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Abstract

Dendritic cell (DC) dysfunctions exacerbate intestinal pathologies. However, the mechanisms compromising DC-mediated immune controls remain unclear. We found that intestinal DCs from mice subjected to experimental colitis possessed heightened non-canonical NF-κB signaling, which activates the RelB:p52 heterodimer. Genetic inactivation of this pathway in DCs alleviated inflammation in colitogenic mice. Unexpectedly, RelB:p52 deficiency diminished the transcription of Axin1, a critical component of the β-catenin destruction complex. This reinforced β-catenin-driven expression of Raldh2, which imparts tolerogenic DC attributes by promoting retinoic acid (RA) synthesis. Indeed, DC-specific non-canonical NF-κB impairment improved the colonic frequency of Tregs and IgA + B cells, which fostered luminal IgA and eubiosis. Introducing β-catenin haploinsufficiency in non-canonical NF-κB-deficient DCs moderated Raldh2 activity, reinstating colitogenic sensitivity in mice. Finally, IBD patients displayed a deleterious non-canonical NF-κB signature in intestinal DCs. In sum, we establish a DC network that integrates non-canonical NF-κB signaling to subvert RA metabolic pathway in fueling intestinal inflammation.

Significance (100)

Distorted dendritic cell (DC) functions have been implicated in aberrant intestinal inflammation; however, the underlying mechanism remains obscure. We discovered that the non-canonical NF-κB pathway exacerbates inflammation in the colitogenic gut by downmodulating β-catenin-driven synthesis of Raldh2 in DCs. Raldh2 represents a key enzyme involved in the production of tolerogenic retinoic acid in intestinal DCs. Beyond regulating immune genes, therefore, non-canonical NF-κB signaling appears to instruct retinoic acid-mediated control of gut health. While we illustrate a DC network integrating immune signaling and micronutrient metabolic pathways in the intestine, our finding may have broad relevance for nutritional interventions in inflammatory ailments.

eToC

Deka and Kumar et al . illustrate a DC-circuitry that exacerbates intestinal inflammation in IBD patients and colitogenic mice. Non-canonical NF-κB signaling restrains β-catenin in DCs to downmodulate Raldh2, which promotes tolerogenic RA synthesis, leading to diminished Treg and IgA + cell frequencies in the gut.

Highlights

  • Aberrant intestinal inflammation is associated with and exacerbated by non-canonical NF-κB signaling in DCs.

  • Non-canonical signaling restrains the tolerogenic β-catenin-Raldh2 axis in DCs by upregulating Axin1.

  • DC-specific RelB:p52 impairment promotes β-catenin-dependent Treg accumulation in the gut.

  • A DC defect of non-canonical signaling causes β-catenin-dependent increase in luminal sIgA, fostering the gut microbiome.

One sentence

The non-canonical NF-κB pathway fuels intestinal inflammation by waning the tolerogenic β-catenin-Raldh2-retinoic acid axis in DCs.

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  1. Review Commons

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    Reply to the reviewers

    __Reviewer #1 (Evidence, reproducibility and clarity (Required)):____ __ Deka and colleagues report that a non-canonical NFKb signaling operates in DCs in the context of inflammation and inhibits a tolerogenic mechanism driven by b-catenin-Raldh2. The following comments are made to clarify the findings presented.

    The authors re-analyzed published scRNAseq data from DSS colitis to identify the expression of Relb and NFKB2 in myeloid cells. 1a- The authors are encouraged to expand this analysis to other published datasets.

    We sincerely appreciate the comment from the knowledgeable reviewer. Unfortunately, we did not find any other publicly available scRNAseq dataset from DSS-treated mice. To circumvent this problem, we instead examined a previously published microarray-based bulk transcriptomic dataset obtained using FACS-sorted DCs isolated from the mouse colon (GSE58446, Muzaki et al. 2016; doi:10.1038/mi.2015.64). We consistently found an increased expression of Relb, and to some extent Nfkb2, mRNAs in intestinal DCs upon DSS treatment (Fig R1). Because microarray analysis lacks the quantitative attributes that scRNAseq offers, we provided this newly analysed dataset for reviewer's eyes and refrained from including this data in the manuscript per se. Of note, we have also provided our own experimental data directly demonstrating p100 processing in DC isolated from colitogenic gut. (Fig 1F)

    Importantly, we could identify an additional scRNAseq dataset derived colitogenic human ulcerative colitis patients (GSE162335, Devlin et al. 2021, doi.org/10.1053/j.gastro. 2020.12.030). Interrogation of this dataset indeed confirmed that RELB and NFKB2 mRNAs were majorly expressed in intestinal DCs and not in intestinal macrophages and that IBD was associated with increased expression of multiple RelB-important genes in intestinal DCs. These analyses further supported the notion that heightened non-canonical NF-kB signalling in DCs could be fuelling aberrant gut inflammation. We have now incorporated this newly acquired data in the supplementary Figure S5A-S5C. (line# 456-460)

    1b. Additionally, the expression of Relb and NFKB2 in other cells - especially other myeloid cells -should be explored and included, even if then the authors later choose to test their function in DCs.

    Adhering to this brilliant suggestion, we have now further interrogated the mouse scRNAseq dataset (GSE148794 ; Ho et al., 2021) to compare macrophages and DCs for the expression of the non-canonical signal transducers. Indeed, we found a relatively insignificant level of Relb and Nfkb2 mRNAs in intestinal macrophages in comparison to intestinal DCs. Our data suggested that the non-canonical NF-kB pathway is likely to play a more prominent role in DCs than in macrophages in the gut. This new analysis has now been presented in the revised draft in Figure 1B. (revised text line#136-140) This comparison indeed proved useful in motivating subsequent in-depth analyses of the non-canonical NF-kB pathway in DCs in the context of experimental colitis.

    1c. Please note that there is a transition from Fig 1A to Fig 1B to focus on DCs, which is not apparent from the figure.

    Please find our response to #1b.

    Please include scale bars for all histological analyses.

    We thank the reviewer for alerting us. The scale bars were already included in the histological analyses; we have now appropriately highlighted them in this revised version for better visual clarity.

    In Fig S1I, the authors show that loss of body weight upon DSS treatment in Nfkb2DCD11c is indistinguishable from control. Why is the starting weight at 110%? Please clarify.

    We sincerely apologize for this inadvertent error. We have now rectified the axis label, representing the starting weight at day 0 as 100% (currently Figure S1J).

    In Figure 2, please indicate the database/s used for the identification of top biological pathways.

    We used "WikiPathways subset of cellular processes" available at www.gsea-msigdb.org/gsea/msigdb/mouse/collections.jsp?targetSpeciesDB=Mouse#M8 for the pathway enrichment analysis presented in Figure 2B. We also utilized a previously published RA-target gene set for the gene set enrichment analysis presented in Figure 2C (Balmer and Blomhoff, 2002; 10.1194/jlr.r100015-jlr200). While this information was included in the materials and methods section in the original draft, we have now included these descriptions in the figure legend for further clarity. (please see revised figure legends 2B and 2C)

    The authors show a more significant expansion in Tregs upon DSS treatment when non-canonical NFKb is ablated in DCs. Is this at the expense of a reduction of specific Th cells? Can the authors also report the number of cells beyond the % of cells?

    In response to the reviewer's comment, we have examined the abundance of Th17 cells in the colon of our knockout mice. As also observed earlier upon DC-specific ablation of NIK function (Jie et al., 2018), disruption of non-canonical NF-kB signaling in DCs in RelbDDC or Nfkb2DDC mice led to a reduced frequency of RoRgt+ Th17 cells in the LP (Figure S3F, new data). Our in vitro (Figure 2I) and in vivo (Figure 3F-G, 6B-6C) studies conclusively linked DC-intrinsic non-canonical NF-kB signaling to intestinal Treg via the RA pathway. Therefore, we conjectured that the observed decline in the Th17 compartment in our knockouts could be secondary to Treg expansion. We have now further discussed this point in the revised manuscript. (line#296-300)

    As the reviewer suggested, in addition to the Treg frequency, we have also presented the number of intestinal FoxP3+ CD4 T cells in the supplement (Figure S3E). Our data revealed a similar increase in the total Treg numbers in the mouse colon upon ablation of the non-canonical NF-kB pathway in DCs. (line#296-300)

    In figure 6A, it appears that not only the amount of beta-catenin expressed but also the percentage of beta-catenin positive MNL DCs is significantly expanded upon ablation of non-canonical NFkb. Please verify and if so, include.

    We thank the reviewer for this very insightful comment. We have now catalogued MLN DCs into b-cateninlow and b-cateninhigh compartments. Indeed, we found a substantial more than two-fold increase in the frequency of b-cateninhigh DCs in RelbDDC mice. Accordingly, we have revised Figure 6A and emphasised this point in the text.

    (line#428-430)

    In analogy to comment #1 above, please expand the analyses in human samples to include the expression of Relb and Nfkb2 to other myeloid cells.

    Adhering to the valuable suggestion by the reviewer, we have now analysed the scRNAseq dataset (SCP 259) comparing DCs, macrophages, and inflammatory and cycling monocytes present in the human gut for the expression of RELB and NFKB2 mRNA (Figure 7B). Consistent with our observation involving the mouse colon, we found that mRNAs encoding these non-canonical signal transducers were mostly expressed in DCs among various MNPs. This point has also been emphasized in the revised draft. (line#446-448)

    Reviewer #1 (Significance (Required)):

    Strengths of the manuscript include the conceptual novelty of the intersection between non-canonical NFkb and the tolerogenic b-catenin-Raldh2 axis. And additional strength is the methodical approach, which includes various immunological and biochemical assessments as well as genetic perturbations to dissect such relationships. While it remains unknown the relevant triggers for the non-canonical axis described, this study advances our mechanistic understanding on how activation of this axis overrides regulatory mechanisms in DCs. As such, this manuscript should be of broad interest to immunologists and in particular mucosal immunologists. We sincerely thank the reviewer for lauding our work as conceptually novel and methodical. The encouragement from the knowledgeable reviewer would certainly motivate us further to identify the relevant trigger of this pathway in the gut.

    Reviewer #2 (Evidence, reproducibility and clarity (Required)):

    The following issues are noted.

    - all animal strains and their provenance should be described and properly referenced (for example, there are at least two CD11c-Cre strains with different specificity). Along the same lines, the specificity of Cre recombination should be confirmed, at least in major cell types (DCs vs T effector or regulatory cells).

    We sincerely appreciate the reviewer's attention to these important details. We would like to point out that in our original draft, Table 1 in the Materials and Methods section provides information on the source and the identifier of all mouse strains used. In particular, we utilized CD11c-Cre mice with the identifier 008068 from the Jackson Laboratories. Alternately known as B6.Cg-Tg (Itgax-cre)1-1Reiz/J, this strain displays Cre-mediated recombination in more than 95% of conventional DCs while exhibiting only minor recombination in lymphocytes (low-activated T cells (www.jax.org/strain/008068). Importantly, our immunoblot analyses revealed efficient depletion of RelB in specifically splenic CD11c+ cells of RelbDDC mice with only a negligible reduction in CD11c- cells (Figure S1E). Our analyses involving Nfkb2DDC mice also assured of similar gene disruption specificity (Figure S1H). Notably, our results were consistent with those documented on RelbDDC and Nfkb2DDC strains earlier (Andreas et al., 2019). To further address the reviewer's concern pertaining to the T-cell compartment, we have now compared splenic CD4+ cells from Nfkb2fl/fl and Nfkb2DDC mice for the expression of p100 (Figure S1I, newly added in the revised draft). Our results confirmed that CD11c-Cre-driven ablation of the non-canonical NF-kB pathway did not perturb p100 expressions in T cells. Taken together, these allow us to emphasize that knockout phenotypes observed in our study were attributed to non-canonical NF-kB deficiency in DCs. We have accordingly modified the text to highlight gene deletion specificities in our knockouts. (line#166-167, 180-182)

    - the DSS model is prone to "batch effects" of individual cages, and proper comparison between genotypes is possible only if mice of different genotypes (eg littermates) are housed together in the same cages. The authors should clearly confirm whether this was the case, and if not, key experiments should be repeated in this setting.

    As mentioned in the materials and methods section of the original draft, littermate male mice of indicated genotypes were indeed cohoused for at least one week prior to experiments. We have now further emphasised this point in the legend of Figure 1.

    - BMDCs represent a heterogeneous mixture of DCs and macrophages (Helft et al., Immunity 2015). These populations should be clearly defined and compared between genotypes, to make sure that they do not underlie the observed gene expression differences.

    The knowledgeable reviewer has raised a very pertinent issue. We would like to emphasize that instead of generating BMDCs using GM-CSF alone following the protocol prescribed by Helft et al. (2015), we differentiated bone marrow cells to BMDCs using a cocktail of GM-CSF+IL4 adhering to the protocol published by Jin and Sprent (2018). Following the reviewer's suggestion, we have now compared BMDCs generated in these two protocols in our laboratory. As reported earlier (Jin and Sprent, 2018), unlike BMDCs generated using GM-CSF alone, BMDCs generated using the GM-CSF+IL4 cocktail did not contain CD115high macrophage-like cells (Figure S2C). (line#230-232) However, they displayed equivalent expressions of the DC marker CD135 on their surface. Moreover, when we compared BMDCs derived from Relbfl/fl and RelbDCD11c mice in flow cytometry analyses, we found comparable surface expression of CD135, assuring intact BMDC generation from bone marrow cells ex vivo in spite of the absence of RelB (Figure S2I). (line#266-268) These studies argue that macrophage-like cells did not contribute to the observed gene expression differences between WT and RelB-deficient BMDCs.

    • the analysis of DCs in mutant strains (e.g. in Fig. 3) would benefit from a better definition of populations, e.g. resident vs migratory DCs in the MLN, the Notch2-dependent CD103+ CD11b+ DCs in the LP and MLN, etc. Again, this would be important to justify differences in gene expression (e.g. Fig. 3D).

    We sincerely appreciate the comment from the knowledgeable reviewer. In a landmark paper from Prof. Fiona Powrie's group (Coombes et al., 2007), it was earlier demonstrated that CD103+ DCs present in the intestine migrate to local MLNs and play a key role in producing RA and supporting Tregs. While our BMDC data strongly supported a cell-intrinsic mechanism underlying Raldh2 upregulation upon non-canonical NF-kB deficiency (Figure 2F), our in vivo studies (Figure 3D-3E) did not entirely rule out also a possible expansion of RA-producing CD103+ DC compartment in our knockout mice. Although the proposition that non-canonical NF-kB signaling regulates the generation of specific intestinal DC subsets seems attractive, we must point out that previous studies showed a relatively unaltered frequency of CD103+ cells among steady-state migratory DCs in skin-draining lymph nodes (Döhler et al., 2017). Nevertheless, following the reviewer's suggestion, we now plan to perform advanced flow cytometry analyses to compare Relbfl/fl and RelbDCD11c mice for the frequency of CD103+CD11b-, CD103+CD11b+ and CD103-CD11b+ DCs in the intestine. To this end, we have already optimized our experimental protocol for staining intestinal DCs with anti-CD103 antibody (BD Bioscience). In the coming weeks, we are expecting to gather adequate numbers of littermate knockout mice to perform a side-by-side comparison. [NOTE: also the section - "Description of the planned revisions"]

    • the analysis of b-catenin protein expression and cellular localization at the single-cell level (e.g. by IF) would greatly strengthen the mechanistic connection between NF-kB and Wnt/b-catenin pathways.

    Adhering to the reviewer's suggestion, we have now performed immunofluorescence assay (IFA) to capture the impact of RelB deficiency on b-catenin expression and cellular localization. Because BMDCs pose challenges for IFA owing to their non-adherent nature, we instead examined mouse embryonic fibroblasts (MEFs), which provide for a genetically amenable model cell system. As presented below (Figure R2), our IFA data conclusively demonstrated an increased cellular abundance and nuclear localization of b-catenin in Relb-/- MEFs. While we are truly excited to find that our proposed mechanism is functional in another cell type, we feel that the inclusion of MEF data in the main manuscript, which describes DC-mediated immune controls, may cause significant distractions for the general audience. Accordingly, we have provided this data for the reviewer's eyes only.

    Minor:

    • The reanalysis of previous single-cell data is in Figs. 1 and 7 are much less convincing or exciting than the new experimental data relegated to the supplements. The distribution of the results between main and experimental figures may be reconsidered in this light.

    We concur with the knowledgeable reviewer that our scRNAseq analyses may have appeared less convincing in the original draft. In response to comments by reviewer-1 and reviewer-3, we have now added additional data panels (Figure 1B, Figure 7B and Figure 7G) and examined additional publicly available datasets (Figure S5). In the revised draft, these analyses helped us to more firmly establish a link between non-canonical NF-kB signaling in DCs to aberrant intestinal inflammation in mice and humans.

    However, we slightly diverge that many key experimental datasets were relegated to the supplement. Except for the FITC-dextran experiment, data from all other experimental analyses were presented in the main text (Figure 1). To suitably manage space in our figure panels, we opted to present quantified data averaged from experimental replicates in the main text while providing representative raw data in the supplement. Besides, immunoblot analyses confirming DC-specific ablation of target genes in our knockouts were placed in the supplement. Notably, these knockout strains were also examined earlier (Andreas et al., 2019). Those studies, along with our own analyses (Figure S1E, S1H and S1I - additional data), confirmed the most efficient gene deletion in CD11c+ cells. While maintaining these data in the supplement for want of space, we have now cited this reference in the main text to emphasize that knockout phenotypes observed in our study were attributed to non-canonical NF-kB dysfunctions in DCs.

    Reviewer #2 (Significance (Required)):

    The manuscript by Deka et al. explores the role of the non-canonical NF-kB pathway, specifically of its key mediators RelB and NF-kB2, in dendritic cells (DCs) during intestinal inflammation. The key strength of the paper is the demonstration that DC-specific deletion of RelB or NF-kB2 leads to improved acute or chronic DSS colitis. It is also shown that reducing the dose of b-catenin rescues the phenotype of RelB deletion, providing an important genetic connection between NF-kB and Wnt/b-catenin pathways. As such, the work is novel, important and of potential significance to the field.

    We express our deepest gratitude to the reviewer for his/her valuable time and insightful comments. We are indeed extremely excited that the knowledgeable reviewer finds our work novel, important and of potential significance to the field. These positive comments would inspire us to look further into potential interventions targeting the non-canonical NF-kB pathway in human ailments.

    __ Reviewer #3 (Evidence, reproducibility and clarity (Required)):__

    Summary:

    This manuscript from Deka et al. investigates the role of dendritic cell noncanonical NFκB signaling on intestinal inflammation. Based on prior data showing altered DC function in intestinal inflammation, they interrogated existing scRNAseq data and found that DSS treatment (which yields chemical colitis) increased the expression of non-canonical NFkB family members in dendritic cells. This led to the generation of a DC-specific RelB deficient mouse and use of a DC specific NFkB2 deficient mouse, each of which showed varying degrees of protection from chemical colitis.

    Overall, they do a very nice job identifying a mechanism by which noncanonical NFκB signaling in dendritic cells contributes to intestinal inflammation via transcriptional regulation of Axin1, downregulation of β-catenin, restraint of Raldh2 synthesis, impaired retinoic acid synthesis and subsequent decrease in protective Tregs, IgA+ B cells, and microbial dysbiosis. The importance of this pathway is well supported by their focused targeting of β-catenin. After pharmacologic inhibition of β-catenin showed restoration of Raldh2 abundance, they made a DC-specific β-catenin haploinsufficiency RelBDCD11c mouse which showed impaired Raldh2 activity with restoration of colonic Tregs and fecal sIgA. When challenged with DSS, the protective phenotype seen with the RelBDCD11c was lost and the colitis phenotype returned to that of the Relbfl/fl control, further solidifying the role of β-catenin, Raldh2 and RA on intestinal inflammation. Additionally, the discussion provides a robust mechanistic explanation for the phenotypic differences between the RelB and Nfkb2 genotypes, drawing on the authors' deep knowledge of the non-canonical NFκB pathway.

    Major comments:

    Although they propose a novel mechanism by which dendritic cells can contribute to intestinal inflammation, it is in a model of acute epithelial injury that accentuates the contribution of the innate immune system. Would recommend including a discussion of the limitations of this model.

    We most sincerely thank the knowledgeable reviewer for raising this important issue. We argue that while erosive epithelial injury initiates colitis in the DSS model, T cells were shown to aggravate intestinal pathologies, particularly at DSS doses used in our study (Kim et al., 2006; doi: 10.3748/wjg.v12.i2.302). Furthermore, our chemically-induced colitis model offered a convenient tool for genetically dissecting the DC-intrinsic role of the non-canonical NF-kB pathway in the intestine. However, we agree entirely that no single animal model fully captures the clinical complexities of human IBD and that other models of experimental colitis should also be employed in the future to assess the generalisability of the proposed DC mechanism in regulating intestinal inflammation. In particular, future studies ought to examine composite knockout strains in the T-cell transfer model of experimental colitis to establish further the role of non-canonical NF-kB signaling in DCs in alleviating intestinal inflammation. As suggested by the reviewer, we have now articulated this point in the discussion section. (line# 553-557)

    The human work (Figure 7) shows solid evidence of heightened non-canonical NFκB signaling in DCs via abundance of RELB and NFKB2 along with a few RelB important genes, however, the RA-specific pathway identified in the mouse work is not strongly corroborated by the human data. There is demonstration of one β-activated gene (CCND1) showing decreased expression in IBD patients, however no other gene along with RA pathway was clearly identified to be differentially expressed as one would predict from the mouse work.

    We sincerely thank the knowledgeable reviewer for articulating this deficiency in our analyses of single-cell RNA-seq data derived from IBD patients (SCP259, Smillie et al., 2019). We would like to clarify that many well-known b-catenin target genes, including MYC, were not detectable in this dataset. Nevertheless, to address the reviewer's concern, we subjected this dataset to GSEA using a previously published list of RA target genes (Balmer et al., 2002). Our analyses revealed a significant enrichment of RA targets among genes that were downmodulated in DCs derived from inflamed colonic tissues of IBD patients as compared to those from non-inflamed tissues (Figure 7G, newly added in the revised version). We have now discussed this data in the result section. (line#470-475) These studies further substantiated the inverse correlation between noncanonical NF-kB signalling and the RA pathway in DCs in the inflamed human gut.

    Minor comments: 1. Their NFκB2DCD11c mouse underwent a regimen of chronic DSS treatment after acute DSS treatment only displayed subtle phenotypic changes. Was the same chronic colitis regimen also tested in the RelBDCD11c ?

    Indeed, we also examined RelbDCD11c mice in the chronic DSS treatment regime. As compared to Relbfl/fl mice, these knockout mice displayed significantly less bodyweight changes upon chronic DSS challenge. Because* RelbDCD11c* mice readily showed acute DSS phenotype, we did not further pursue investigations involving this strain in the chronic DSS settings and rather focused on Nfkb2DCD11c mice to illustrate chronic DSS phenotypes.

    In the introduction, it was stated that patients with UC have a marked reduction in intestinal DCs. If DCs (particularly non-canonical NFκB signaling) promote inflammation, how do you explain a decrease in this cell type in patients with active disease?

    Depending on the expression of immunogenic or tolerogenic factors, DCs may both promote or subdue inflammation in the colon. We have now revisited the relevant reference published by Magnusson et al., (2016). Indeed, the authors noted a marked reduction in the intestine of the CD103+ DC subset, which has been majorly linked to tolerogenic RA synthesis. While it is generally thought that aberrant inflammation promotes the death of mononuclear phagocytes in the intestine, it seems that either a contraction of the tolerogenic DC compartment or downmodulation of tolerogenic pathways in DCs incites gut inflammation in IBD patients. We have now revised the text in the introduction section to clarify this point. (line#81-83)

    The focus on retinoic acid is interesting, however may be oversimplifying the role of non-canonical NFκB in DCs on the mucosal immune system. It must also be mentioned that there is crosstalk between the non-canonical and canonical NFκB signaling systems, for example Nfkb2 is capable of functioning as a IkB protein and inhibiting RelA-p50 (from the last author's prior work - Basak et al, Cell, 2007). Thus would include some mention of possible effects on the canonical system that contribute to intestinal inflammation.

    We thank the reviewer for raising this important point. As mentioned in the introduction section of our original draft, the canonical NF-kB pathway in DCs aggravates experimental colitis mice (Visekruna, A. et al. 2015). Indeed, Nfkb2-dependent crosstalk was shown to modulate inflammatory RelA activity in a variety of cell types (Basak et al., 2007; Shih et al., 2009; Chawla et al., 2021). Although such cross-regulatory RelA controls by non-canonical NF-kB signaling are yet to be established in DCs, our studies involving RelB-deficient cells confirmed an essential role of p100-mediated RelB regulations in DC functions. We admit that further studies are required to determine if, independent of RelB, p100 directs immunogenic DC attributes via also RelA or another factor. We have now elaborated on p100-mediated crosstalks in the discussion section. (line#561-562)

    In the single-cell DSS data they analyzed, there was a distinct DC population seen with DSS colitis treatment. Although they are categorized as cDC2s, what genes separate them from the other DC populations?

    We curated a list of genes from Brown et al., (2019) to categorize cDC1 and cDC2 subsets in our study. We would like to clarify that the list was provided in Supplementary Table 1 in our original draft. In view of the reviewer's comment, we have now referred to this Table in the legend of Supplementary Figure 1 and also in the main text. (line#153)

    Why was the RNAseq work on BMDCs (that identified RA metabolism as a top-ranking differentially expressed pathway) done only on Nfkb2-/- BMDCs and not RelB-/-? RelB-/- had a more pronounced protected phenotype in the cell type-specific knockout and is a cleaner target (does not have the IkB capability of Nfkb2).

    We broadly agree with the knowledgeable reviewer that comparing WT and Relb-/- BMDCs for global gene expressions could have been worthwhile. We would like to clarify that we initially utilized a dataset derived using Nfkb2-/- BMDCs already available in the laboratory. These analyses were instrumental in developing a notion that non-canonical NF-kB signalling could be modulating Radh2 expression in DCs. Because previous studies involving germline Relb-/- mice suggested a role of RelB in the nonhematopoietic niche in instructing myeloid development (Briseño et al., 2017), we focused our subsequent analyses on BMDCs generated using bone marrow cells from cell type-specific knockouts. Indeed, we could confirm elevated Raldh2 expressions in BMDCs generated from both RelbDDC and Nfkb2DDC mice. Taken together, our studies suggested that Nfkb2-encoded p100 controlled Raldh2 expressions in DCs by providing RelB:p52 and less so as a regulator of the RelA activity. Although we admit that further studies are required to determine if, independent of RelB, p100 directs immunogenic DC attributes via also RelA or another factor. We have now deliberated this point in the discussion section. (line#566-567)

    Reviewer #3 (Significance (Required): As a physician-scientist who clinically cares for patients with inflammatory bowel disease, and scientifically studies signaling within innate immune cells, this manuscript does a rigorous job of identifying a mechanism by which canonical NFκB signaling in dendritic cells contributes to intestinal inflammation. This study would be very informative for both basic and translational researchers as it identifies a clear pathway by which the innate immune system contributes to intestinal inflammation, and opens up room for inquiry into triggers of non-canonical NFκB in IBD and modulation of the RA pathway as a potential novel therapeutic target.

    We are humbled that the knowledgeable reviewer finds our work to be informative for basic and translational research. These encouragements would undoubtedly motivate us further to identify the relevant trigger of this pathway in the gut and explore potential interventions.

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    Referee #3

    Evidence, reproducibility and clarity

    Summary:

    This manuscript from Deka et al. investigates the role of dendritic cell noncanonical NFκB signaling on intestinal inflammation. Based on prior data showing altered DC function in intestinal inflammation, they interrogated existing scRNAseq data and found that DSS treatment (which yields a chemical colitis) increased expression of non-canonical NFkB family members in dendritic cells. This led to the generation of a DC specific RelB deficient mouse and use of a DC specific NFkB2 deficient mouse, each of which showed varying degrees of protection from chemical colitis. Overall, they do a very nice job identifying a mechanism by which noncanonical NFκB signaling in dendritic cells contributes to intestinal inflammation via transcriptional regulation of Axin1, downregulation of β-catenin, restraint of Raldh2 synthesis, impaired retinoic acid synthesis and subsequent decrease in protective Tregs, IgA+ B cells, and microbial dysbiosis. The importance of this pathway is well supported by their focused targeting of β-catenin. After pharmacologic inhibition of β-catenin showed restoration of Raldh2 abundance, they made a DC specific β-catenin haploinsufficiency RelBCD11c mouse which showed impaired Raldh2 activity with restoration of colonic Tregs and fecal sIgA. When challenged with DSS, the protective phenotype seen with the RelBCD11c was lost and the colitis phenotype returned to that of the Relbfl/fl control, further solidifying the role of β-catenin, Raldh2 and RA on intestinal inflammation. Additionally, the discussion provides a robust mechanistic explanation for the phenotypic differences between the RelB and Nfkb2 genotypes, drawing on the authors' deep knowledge of the non-canonical NFκB pathway.

    Major comments:

    1. Although they propose a novel mechanism by which dendritic cells can contribute to intestinal inflammation, it is in a model of acute epithelial injury that accentuates the contribution of the innate immune system. Would recommend including a discussion of the limitations of this model.
    2. The human work (Figure 7) shows solid evidence of heightened non-canonical NFκB signaling in DCs via abundance of RELB and NFKB2 along with a few RelB important genes, however the RA specific pathway identified in the mouse work is not strongly corroborated by the human data. There is demonstration of one β-activated gene (CCDN1) showing decreased expression in IBD patients, however no other gene along with RA pathway was clearly identified to be differentially expressed as one would predict from the mouse work.

    Minor comments:

    1. Their NFκB2CD11c mouse underwent a regimen of chronic DSS treatment after acute DSS treatment only displayed subtle phenotypic changes. Was the same chronic colitis regimen also tested in the RelBCD11c ?
    2. In the introduction, it was stated that patients with UC have a marked reduction in intestinal DCs. If DCs (particularly non-canonical NFκB signaling) promote inflammation, how do you explain a decrease in this cell type in patients with active disease?
    3. The focus on retinoic acid is interesting, however may be oversimplifying the role of non-canonical NFκB in DCs on the mucosal immune system. It must also be mentioned that there is crosstalk between the non-canonical and canonical NFκB signaling systems, for example Nfkb2 is capable of functioning as a IkB protein and inhibiting RelA-p50 (from the last author's prior work - Basak et al, Cell, 2007). Thus would include some mention of possible effects on the canonical system that contribute to intestinal inflammation.
    4. In the single cell DSS data they analyzed, there was a distinct DC population was seen with DSS colitis treatment. Although they are categorized as cDC2s, what genes separate them from the other DC populations?
    5. Why was the RNAseq work on BMDCs (that identified RA metabolism as a top ranking differentially expressed pathway) done only on Nfkb-/- BMDCs and not RelB-/-? The RelB-/- had a more pronounced protected phenotype in the cell type specific knockout, and is a cleaner target (does not have the IkB capability of Nfkb2).

    Significance

    As a physician scientist who clinically cares for patients with inflammatory bowel disease, and scientifically studies signaling within innate immune cells, this manuscript does a rigorous job of identifying a mechanism by which canonical NFκB signaling in dendritic cells contributes to intestinal inflammation. This study would be very informative for both basic and translational researchers as it identifies a clear pathway by which the innate immune system contributes to intestinal inflammation, and opens up room for inquiry into triggers of non-canonical NFκB in IBD and modulation of the RA pathway as a potential novel therapeutic target.

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    Referee #2

    Evidence, reproducibility and clarity

    The following issues are noted.

    • all animal strains and their provenance should be described and properly referenced (for example, there are at least two CD11c-Cre strains with different specificity). Along the same lines, the specificity of Cre recombination should be confirmed, at least in major cell types (DCs vs T effector or regulatory cells).
    • the DSS model is prone to "batch effects" of individual cages, and proper comparison between genotypes is possible only if mice of different genotypes (eg littermates) are housed together in the same cages. The authors should clearly confirm whether this was the case, and if not, key experiments should be repeated in this setting.
    • BMDCs represent a heterogeneous mixture of DCs and macrophages (Helft et al., Immunity 2015). These populations should be clearly defined and compared between genotypes, to make sure that they do not underlie the observed gene expression differences.
    • the analysis of DCs in mutant strains (e.g. in Fig. 3) would benefit from better definition of populations, e.g. resident vs migratory DCs in the MLN, the Notch2-dependent CD103+ CD11b+ DCs in the LP and MLN, etc. Again, this would be important to justify differences in gene expression (e.g. Fig. 3D).
    • the analysis of b-catenin protein expression and cellular localization at single-cell level (e.g. by IF) would greatly strengthen the mechanistic connection between NF-kB and Wnt/b-catening pathways.

    Minor:

    • the reanalyses of previous single-cell data in Figs. 1 and 7 are much less convincing or exciting than the new experimental data relegated to the supplements. The distribution of the results between main and experimental figures may be reconsidered in this light.

    Significance

    The manuscript by Deka et al. explores the role of the non-canonical NF-kB pathway, specifically of its key mediators RelB and NF-kB2, in dendritic cells (DCs) during intestinal inflammation. The key strength of the paper is the demonstration that DC-specific deletion of RelB or NF-kB2 lead to improved acute or chronic DSS colitis. It is also shown that reducing the dose of b-catenin rescues the phenotype of RelB deletion, providing an important genetic connection between NF-kB and Wnt/b-catenin pathways. As such, the work is novel, important and of potential significance to the field.

  4. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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    Referee #1

    Evidence, reproducibility and clarity

    Deka and colleagues report that a non-canonical NFKb signaling operates in DCs in the context of inflammation and inhibits a tolerogenic mechanism driven by b-catenin-Raldh2. The following comments are made to clarify the findings presented.

    1. The authors re-analyzed published scRNAseq data from DSS colitis to identify the expression of Relb and NFKB2 in myeloid cells.
      • a. The authors are encouraged to expand this analysis to other published datasets.
      • b. Additionally, the expression of Relb and NFKB2 in other cells - especially other myeloid cells -should be explored and included, even if then the authors later choose to test their function in DCs.
      • c. Please note the there is a transition from Fig 1A to Fig1B to focus on DCs, which is not apparent from the figure.
    2. Please include scale bars for all histological analyses.
    3. In Fig S1I, the authors show that loss of body weight upon DSS treatment in Nfkb2deltaCD11c is indistinguishable from control. Why is the starting weight at 110%? Please clarify.
    4. In Figure 2, please indicate the database/s used for identification of top biological pathways.
    5. The authors show a more significant expansion in Tregs upon DSS treatment when non-canonical NFKb is ablated in DCs. Is this at the expense of a reduction of specific Th cells? Can the authors also report the number of cells, beyond the % of cells?
    6. In figure 6A, it appears that not only the amount of beta-catenin expressed, but also the percentage of beta-catenin positive MNL DCs is significantly expanded upon ablation of non-canonical NFkb. Please verify and if so, include.
    7. In analogy to the comment #1 above, please expand the analyses in human samples to include the expression of Relb and Nfkb2 to other myeloid cells.

    Significance

    Strengths of the manuscript include the conceptual novelty of the intersection between non-canonical NFkb and the tolerogenic b-catenin-Raldh2 axis. And additional strength is the methodic approach, which includes various immunological and biochemical assessments as well as genetic perturbations to dissect such relationships. While it remains unknow the relevant triggers for the non-canonical axis described, this study advances our mechanistic understanding on how activation of this axis overrides regulatory mechanisms in DCs. As such, this manuscript should be of broad interest to immunologists and in particular mucosal immunologists.

  5. Version published to 10.1101/2023.12.03.569755v2 on bioRxiv
  6. Version published to 10.1101/2023.12.03.569755v1 on bioRxiv