Preprint Club
A cross-institutional Journal Club Initiative

Keywords

• Crohn’s disease

• Epithelial Cells

• IBD susceptibility genes

• Lipocalin-2

• Nitric Oxide Synthase 2

• Dual Oxidase 2

Main Findings

Crohn’s disease (CD) is a complex chronic inflammatory disorder that may affect any part of the gastrointestinal tract with extra-intestinal manifestations and associated immune dysregulation. While most single cell transcriptomic data regarding CD has largely focused on immune cell signatures, Li et al. combined bulk and single-cell RNA profiling and spatial transcriptomics to specifically study the epithelial cell compartment, which has been largely understudied in this disease. Using 170 patient samples, representing one of the largest cohorts of CD profiled, the authors identified a novel epithelial cell type, featuring high expression of LCN2, NOS2, and DUOX2, named ‘LND’. Compared to other enterocytes/colonocytes, LND cells had high expression of antimicrobial proteins, inflammatory cytokines, and antigen-presentation genes, suggesting a specialized immunoregulatory role. Furthermore, the authors also identified that LND cells only expand during chronic inflammation and used six publicly available datasets to verify LND emergence and expansion, corroborating the presence of LND cells and their induction in active CD. As CD is a complex disease that requires evolving clinical needs, this study suggests that LND cell populations could have clinical significance as biomarkers of disease severity and progression in CD.

Limitations

• Figure 2 demonstrates the proportional changes of each immune and stromal cell type in the ileum and colon. Given the role that T cell may play in Crohn’s Disease in both the terminal Ileum and the ascending colon, it would be very interesting to see if the proportion of proliferating T and T helper cells (TH1, TH2, and TH17) is different in patients who have inactive and active CD. Moreover, the authors should consider comparing across CD patients with different localisations of inflammation (i.e., ileal vs colonic CD). This would also be helpful for Figure 3.

• It is notable that the LND cells appear to be a distinct population from the mature epithelial cells. However, it is unclear whether these LND cells are activated epithelial cells or a new epithelial cell population. Perhaps the authors could cell sort for these LND cells to show the role of LND cells in mucosal immunity and how they are similar/different than mature epithelial cells.

• The authors may want to consider culturing LND cells ex vivo, perhaps by using organoids to demonstrate how LND cells can be developed from healthy organoids during stress/inflammation.

• The authors report the specimens taken from patients with CD and non-IBD, which are listed in supplement Table 1, but this was not included in the manuscript.

• It would be helpful if the authors determine or speculate what may be the LND cell equivalent in mice as many groups studying CD use murine models for their studies

• The microscopy images were very informative to demonstrate the presence of LND cells in the ileum and colon. The authors would benefit in making the images larger to easier see the images. While not necessarily needed, it would be nice if the authors could perform confocal to visibly see where the LND cells colocalize within the epithelia.

• While the authors describe the LND cells having antimicrobial properties, this population of epithelial cells only expands during active CD and appears to interact with immune cell populations. The authors may want to culture LND cells ex vivo and perform migration assays and ELISAs to corroborate their CellChat data to demonstrate that LND do indeed have similar macrophage-neutrophil interactions.

• Lipocalin is an important clinical parameter of disease activity. How does the production of lipocalin from LND compare to other immune cells? What triggers the emergence of this cell type? Are there mucosal specific triggers or rather classical inflammatory triggers?

• How can be distinguished between LNDs as a disease driver vs. as a bystander to the inflammatory hotspot?

Significance/Novelty

This manuscript is one of the most comprehensive single-cell atlas of 170 specimens from 83 individuals consisting of cells from the terminal ileum and ascending colon. In addition to confirming region-specific transcriptomics, the authors uncovered a new epithelial cell type named LND, due their high expression of LCN2, NOS2, and DUOX2, which expands during active Crohn’s disease. As the intestinal epithelium is known to play an important role in mucosal immunity, the presence of these LND cells in the ileum and colon raises the possibility that these epithelial cells are present in other tissue during inflammation in a different disease context, highlighting the novelty of this paper.

Credit

Reviewed by Nikolina Bradaric and Azuah Gonzalez as part of a cross-institutional journal club between the Vanderbilt University Medical Center (VUMC), Charité – Universitätsmedizin Berlin, the Medical University of Vienna and other life science institutes in Vienna.

The authors declare no conflict of interests in relation to their involvement in the review.