A cross-institutional Journal Club Initiative
Human intestinal epithelial cells can internalize luminal fungi via LC3-associated phagocytosis
Cohen-Kedar, S. et al. (BioRxiv) DOI: 10.1101/2022.12.14.520372
Human intestinal epithelial cells
Here the authors demonstrate that primary human intestinal epithelial cells and intestinal epithelial cell lines express Dectin-1, facilitating SYK-independent and NAPDH oxidase-dependent phagocytosis the commensal fungi Candida albicans or the fungal cell wall product Zymosan by MUC2+ and MUC2-cells. The presented data shows that similar to macrophages and other antigen-presenting cells, human epithelial cells are capable of sampling fungi to direct them to lysosomal degradation via LC3-associated phagocytosis(LAP)in cells lines and primary human epithelial cells. Using pH sensitive probes, conjugated to zymosan, or fluorescently labelled C. albicans, the team demonstrates co-localization of Rubicon and LC3 with phagocytosed fungi in epithelial cells and cell lines supporting the execution of LAP. Using chemical inhibitors, the team proceeds to block Dectin-binding of beta-1,3-glucans, abrogate internalization of Dectin-bound fungi through blockade of actin polymerization and the formation of the LAPosome by inhibition of NADPH oxidases in epithelial cells. Collectively, the presented results are clear and logically structured providing a new mechanism on how commensal microbes are sampled by the host.
The study primarily focuses on using human intestinal epithelial cell lines and human primary ileal or colonic organoids from healthy controls. A validation of this mechanism in an experimental animal model or transgenic system would have been a nice addition to the data and strengthened the results, opening confirmation in knock out mice or human knock-down cell lines. To circumvent this limitation, the authors made use of a broad collection of chemical inhibitors to conduct experiments supporting the proposed mechanisms of phagocytosis.
This authors demonstrate that Dectin-1/2 participates in the phagocytosis of fungi, but do not address if TLR-2 as receptor for fungal patterns may play an additional role in phagocytosis in their system.
The presented data would benefit from a consistent quantification of phagocytosis across all models used in the manuscript.
Isotype controls or staining controls for antibody stains should have been added to the supplementary data.
While the authors discuss the implication of cytokines and antigen-presentation, data showing MHCII expression, or cytokines released by human organoids would have been a fantastic addition strengthening to the manuscript.
It remains open if the the mechanism described by the authors in human intestinal epithelial organoids is active exclusively on commensal fungi. Would a pathogenic fungal preparation drive the same form of phagocytosis? Would LAP also function if the commensal fungal preparations were viable?
It would be phenomenal to see if autologous T cells (primarily CD4+) can be activated by intestinal epithelial cells that phagocytosed the fungal commensal or by their culture supernatant following phagocytosis. Induction of anergy, expansion, cytokine production, or blockade of T cell proliferation should be considered as possible read out for such an experiment. These may be not possible, but would be a great addition to the described biology.
The characterization of the human primary epithelial organoids would benefit from a more detailed characterization. While demonstrating that MUC2+ cells are able to phagocytose fungi, other cells are capable of doing so too. What are the identities of these cells? Is LAP be a commonly shared mechanism of fungal sampling by all epithelial cells or is it restricted to a subset of lineages?
Do Dectin-1 and -2 expression change in the context of inflammation? Would exposure of organoids to fungal toxins or inflammatory cytokines promote/enhance LAP?
Lastly, a direct comparison of LAP by professional antigen-presenting cells and intestinal epithelial cells would be informative to elaborate on the possible relevance of this pathway in the intestinal tract
The article demonstrates that intestinal epithelial cell-expressed Dectin-1 (and possibly Dectin-2), mediate the uptake of fungal particles and whole dead commensal fungi. The proposed mechanism of antigen-sampling opens the opportunity for therapeutic interventions in diseases driven by fungi or by Dectin-1/2 activation. Besides these relevant findings, the manuscript inspires investigations into the contribution of intestinal epithelial cells as antigen-presenting cells and the contribution to humoral immunity. With limited understanding on how intestinal antigens are taken up by the host, these findings position epithelial cells at the interphase of microbes and the immune system with important implications to understand intestinal homeostasis, immune tolerance and inflammation.
Reviewed by Arthur Mortha (University of Toronto, Department of Immunology) as part of the cross-institutional journal club of the Immunology Institute of the Icahn School of Medicine, Mount Sinai and the Kennedy Institute of Rheumatology, University of Oxford.