GM-CSF regulates ILC states and myeloid cell signaling during ulceration in Crohn’s disease

Reviewer #1 (Public review):

This study integrates Xenium spatial transcriptomics of paired inflamed and uninvolved Crohn's disease tissues with functional analyses in a csf2rb-/- larval zebrafish DSS intestinal injury model to investigate the spatial and cell-type-specific roles of GM-CSF. The work is limited mechanistically and adds little to an already disputed field: GM-CSF's role in intestinal inflammation is context-dependent and extensively studied in mice and humans, and this study does not resolve these controversies. The zebrafish appears to be a poor model for these questions: it lacks mammalian intestinal architecture, complex microbiota, and clearly validated functional ILC populations. Putative ILC1s are poorly defined based on stress-response gene modules, while ILC3s are somewhat better characterized, but overall, the system does not allow mechanistic insights into GM-CSF regulation of ILCs. The DSS experiments largely recapitulate the known protective effects of GM-CSF in epithelial injury without clarifying underlying mechanisms.

Figure 1

GM-CSF expression is extremely sparse, rarely exceeding 0.005 frequency even in inflamed regions. The authors should acknowledge this and discuss why. Xenium could be used to characterize the niche around GM-CSF-producing cells, but no new cellular circuit is revealed beyond known myeloid-lymphoid interactions.

Figure 2

Colon length in DSS colitis is not decreased in Csf2rb⁻/⁻ versus wild-type zebrafish under untreated conditions, suggesting endogenous GM-CSF has minimal impact. In Figure 2E, Tg(mpeg1:mCherry) larvae show staining in vessel- or epithelial-like structures expressing Csf2rb, which does not resemble macrophages and requires clarification. pSTAT5 is upregulated with GM-CSF treatment, but the responding cell types are unclear.

Figure 3

Putative ILC1s are defined by stress-response gene modules rather than canonical markers. Overlapping genes with human (HSP90AA1, UBB, MCL1, DOK2) do not indicate ILC1 identity, which is described by IL7R, KLRB1, or TBX21 expression in the human Xenium dataset. ILC2s were not detected, and Ifng expression is broadly distributed, making attribution to ILC1s uncertain. ILC3s are somewhat better defined, but overall, the data do not support mechanistic conclusions about GM-CSF regulation of ILC populations.

Reviewer #2 (Public review):

The authors show that GM-CSF prevents the loss of ILC3 populations and inhibits pro-inflammatory cytokine production during gut inflammation. They combine a preclinical model of gut inflammation in zebrafish with spatial transcriptomic analysis of samples from Crohn's disease patients. The data show that GM-CSF ameliorates gut inflammation by (1) curtailing the differentiation of disease-associated ILC1 and (2) by "boosting" the tissue repair function of ILC3.

The topic of the manuscript is interesting. However, there are various limitations that are summarized below.

(1) The main finding of the manuscript, that GM-CSF maintains ILC3 populations, is not analyzed in depth. Since the authors' own data and other publications show that the receptors for GM-CSF are expressed in myeloid cells, a better analysis of the transcriptional changes of these populations upon GM-CSF administration is needed.

(2) The authors could compare the transcriptome of macrophages and monocytes from inflamed and uninvolved sections in their Xenium dataset. In addition, investigating how zebrafish macrophages change due to the lack of GM-CSF and comparing them with the human findings would add to the data.

(3) Since the authors developed a novel mutation in zebrafish that is predicted to affect myeloid populations, a detailed characterization of the myeloid immune compartment in these organisms is missing.

(4) Niche analysis in the Xenium slides could provide direct evidence on how macrophages close to ILC3 are different from those closer to other cell types, like ILC1.

Author response:

We thank the editors and reviewers for their careful evaluation of our manuscript, “GM-CSF regulates ILC states and myeloid cell signaling during ulceration in Crohn’s disease.” We appreciate the constructive feedback and agree that strengthening the mechanistic understanding of GM-CSF signaling in the regulation of ILC populations will significantly improve the study.

The reviewers identified a key gap regarding the downstream mechanisms by which GM-CSF maintains ILC3 populations and limits ILC1 expansion. In response, we will focus our revision on defining the myeloid-mediated pathways downstream of GM-CSF that regulate ILC states.

Specifically, we plan to: 

(1) Characterize myeloid cell responses to GM-CSF signaling

We will perform additional analyses of both our Xenium spatial transcriptomics and zebrafish single-cell RNA-seq datasets to identify transcriptional changes in macrophages and monocytes associated with GM-CSF signaling. This will include differential gene expression and pathway enrichment analyses to uncover candidate signaling pathways (e.g., cytokine and STAT5-associated programs) that may mediate ILC regulation.

(2) Strengthen spatial niche analysis in human tissue

We will refine our Xenium-based analyses to better define the cellular microenvironments surrounding GM-CSF-producing cells, including higher-resolution visualization and quantification of receptor-expressing target cells and signaling niches within ulcerated regions.

(3) Further define immune cell populations in the zebrafish model

We will enhance the definition of ILC subsets by incorporating additional marker-based analyses and clarifying their relationship to human ILC populations. In parallel, we will more thoroughly characterize the myeloid compartment in csf2rb-deficient zebrafish to determine how GM-CSF signaling impacts these populations.

(4) Clarify analysis methods and presentation

We will address all points related to statistical testing, data visualization, and figure clarity raised by the reviewers, including the use of appropriate statistical comparisons for multi-group analyses and improved annotation of gene modules and data sources.

Together, these revisions will provide a clearer mechanistic framework linking GM-CSF signaling in myeloid cells to the maintenance of ILC3 populations and suppression of inflammatory ILC1 responses.

We believe these additions will substantially strengthen the manuscript and address the reviewers’ concerns. We appreciate the opportunity to revise our work and look forward to submitting a revised version.