Genetic diversity of Collaborative Cross mice implicates FFAR3 as a target for ILC2 anti-inflammatory reprogramming

Domain: biology
Published: 2026-01-03
Editorial outcome: minor_revision

Abstract
Pulmonary group 2 innate lymphoid cells (ILC2s) are key drivers of Type 2 inflammation in diseases like asthma, yet the molecular mechanisms regulating their function are incompletely understood. Using the genetically diverse Collaborative Cross (CC) mouse panel, we mapped a quantitative trait locus (QTL) that governs ILC2 prevalence in the lung after aeroallergen exposure. This QTL induces a large population of ILC2s in the lung that are resistant to activation and have diminished Type 2 effector function. We identified free fatty acid receptor 3 (Ffar3) as a gene responsible for this effect and demonstrated that FFAR3 signaling reprograms ILC2s to an anti-inflammatory state by promoting their survival, reducing Type 2 cytokine production, and enhancing IL-10 expression. This anti-inflammatory state is dependent on IL-2 signaling, is characterized by decreased ST2 expression, and is distinct from previously described IL-10-producing ILC2 phenotypes. FFAR3-dependent reprogramming is mediated by epidermal growth factor receptor (EGFR) upregulation, and FFAR3’s anti-inflammatory effect is partially conserved in human ILC2s.

Introduction
Asthma is one of the most prevalent chronic diseases in the United States. A myriad of factors including the rising rates of allergic diseases, modern Western diets and lifestyles, urbanization, and antibiotic usage all contribute to its development1. In addition to environmental contributors, asthma has numerous genetic determinants of susceptibility, highlighting the complexity of the immunologic mechanisms that govern disease pathogenesis2. Investigating the specific molecular regulators of asthma has allowed for the development of targeted biological therapeutics, which have proven effective in treating poorly-controlled and severe asthma3–5. The heterogeneity of the disease and the recent success of these targeted therapies demonstrates the vital importance of uncovering new molecular regulators of asthma to expand our therapeutic target repertoire.

Mouse research is commonly used to identify disease targets, but most of this research relies on a small number of traditional inbred strains. While restricting work to a few strains has practical benefits, research conducted in this manner ignores the phenotypic heterogeneity that exists within the mouse species and potentially limits the breadth of discovery. For a heterogeneous disease like asthma in the diverse human population, it is possible that the contributions of important genetic regulators are not appropriately represented in just a few mouse genotypes. We endeavored to implicate disease-relevant targets that may have been missed due to this limited sampling by harnessing the genetic diversity in the mouse species with the Collaborative Cross (CC) recombinant inbred (RI) mouse panel, one of the most ambitious mouse genetics projects undertaken in recent history.

The CC is an RI panel created from 8 founders, 5 classical laboratory strains (A/J, C57BL/6J, 129S1/SvImJ, NOD/LtJ, NZO/HiLtJ) and 3 wild derived strains (CAST/EiJ, PWK/PhJ, and WSB/EiJ). Owing to these diverse founder strains, the panel’s breeding architecture encompasses roughly 90% of known genetic variation in laboratory mice6. These founders were crossed to yield the CC RI strains. The genotype of each CC RI strain is unique and contains genetic character from each of the 8 founders. The genomes of these recombinant strains have been reconstructed as probabilistic mosaics of founder haplotypes, meaning that one can know from which of the 8 founders a specific position on the genome was inherited7,8. The vast genetic diversity in the population and high-marker density makes the CC an excellent tool for the high resolution of mapping complex traits9–13.

In this study, we performed a quantitative trait locus (QTL) mapping experiment to leverage the genetic diversity of the CC to identify disease-relevant targets not previously described. QTL mapping studies involve quantifying phenotypic differences among a large set of progeny and use the segregation of genetic markers in those progeny to identify genomic loci that associate with the measured phenotype14. In principle, this approach can experimentally associate genomic regions as regulators of any complex trait, provided that the phenotypic trait being observed is quantifiable and continuous (i.e. not qualitative or discrete). In addition, the success of a QTL mapping study is determined by the density of genomic markers used, the number of genetically distinct progeny, the phenotypic differences across these progeny, and the effect sizes of the genes regulating the trait15,16. To uncover genetic regulators of asthma pathogenesis with the QTL method in the CC, we selected a quantitative phenotype relating to group 2 innate lymphoid cells (ILC2s).

The adaptive immune system had traditionally been considered primarily responsible for the pathologic Type 2 inflammation that characterizes many variants of asthma. However, this adaptive immunity-centric view was challenged with the first descriptions of group 2 innate lymphoid cells (ILC2s) in 201017–19. ILC2s are tissue resident immune cells derived from the common lymphoid progenitor that are capable of secreting large quantities of Type 2 cytokines, namely IL-5 and IL-13, which in turn act to propagate eosinophil survival and goblet cell mucin production, respectively. ILC2s do not participate in antigen recognition for activation; instead, they are activated primarily by alarmin cytokines like IL-33, thymic stromal lymphopoietin (TSLP), and IL-25 released by the airway epithelial cells in response to aeroallergens20. ILC2s play a pivotal role in the rapid induction of inflammation seen in allergen-exacerbated asthma21,22. Many key regulators of ILC2 biology, including secreted factors, cell surface receptors, and transcription factors have been extensively described and associated with asthma pathogenesis in humans23–26. For this reason, ILC2s represent a high yield cell type where the discovery of novel regulators has a high likelihood of identifying a target with real therapeutic potential for asthma.

In this study, we used the diversity of the CC to identify genes that are regulators of ILC2 function. To do this, we mapped a locus that associated with the prevalence of ILC2s in the lung after aeroallergen challenge, a quantitative and continuous phenotype that is a function of ILC2 activation. We demonstrate a successful attempt at mapping a QTL relating to ILC2 prevalence and effector function using the CC, allowing us to establish the role of a gene in ILC2 biology and asthma pathogenesis.

Results
Different strains of the CC have different ILC2 responses to aeroallergen challenge
In this study, we use the Alternaria alternata fungal extract (AltEx) airway challenge model to induce the Type 2 inflammation that mirrors what would occur during allergen-exacerbated asthma27–29. This AltEx contains the Alta 1 fungal protein antigen, which has been associated with asthma exacerbations30,31. Administration of AltEx into the lungs rapidly induces ILC2 activation32, and the short 4-day course precludes the involvement of the adaptive immune system. The precise quantitative phenotype that we planned to measure in the CC strains was ILC2 prevalence in the lung after 4 days of AltEx challenge. ILC2 prevalence was measured as the percentage of all live cells in the lung that were ILC2s, allowing for comparisons between strains that have differences in total lung cells (Fig. 1A).

We obtained 48 unique CC founder and recombinant strains and subjected each to our AltEx model. Whole lungs were harvested from the mice on day four and quantified by flow cytometry (Fig. 1B). ILC2s were identified according to the gating strategy defined in Supplementary Fig. 1. There existed a wide range of ILC2 prevalence among the strains of the CC, suggesting that QTL mapping would likely be successful. Strain differences persisted when quantifying ILC2s after challenge with total cell numbers in the lung (Supplementary Fig. 2A), and we observed comparable variability in baseline ILC2 prevalence and number in the CC mice without challenge (Supplementary Fig. 2B & C). Expansion ratios for each strain with naïve and challenged ILC2 quantification data are presented in Supplementary Table 1. The vast phenotypic differences among the tested strains confirm the broad genetic diversity within the CC panel and broadly highlight the importance of mouse strain background when considering any experimental model.

QTL mapping with CC strains identifies a 3.43 Mb locus on mouse chromosome 7 that associates with ILC2 prevalence after aeroallergen challenge
We used the phenotypic data gathered in Fig. 1B, in conjunction with CC haplotype data from the Most Recent Common Ancestor 8-State haplotype files33,34, to perform the QTL mapping analysis according to previously described methods8. The basics of the analysis involve grouping each of the recombinant strains based on founder diplotypes at a specific marker. Whether or not a difference in the quantitative phenotype exists between the groups is then determined by an unconstrained regression. Phenotypic differences based on founder diplotype at a markers suggest that the marker influences the quantitative phenotype of interest. This process is repeated across all markers and the resultant QTL map defines loci that regulate the phenotype, with statistical significance thresholds determined by permutation test.

QTL mapping returned a significant locus on mouse chromosome 7 that regulated ILC2 prevalence after aeroallergen challenge (Fig. 2A). This QTL spanned 3.43 megabases starting from position 30.538129 and contained 72 protein coding genes, fully detailed in Table 1. It was the only peak in our analysis to reach significance, and it exceeded the 95% permutation threshold. Henceforth, this locus will be referred to in this manuscript as the Ch.7 ILC2 Locus. The Ch.7 ILC2 Locus did not appear to contain any previously described regulators of ILC2 function, suggesting that the QTL had identified an uncharacterized target.

Founder haplotype effect analysis reveals that the presence of the QTL is driven almost exclusively by the CAST/EiJ founder haplotype, meaning that recombinant strains inheriting this locus from the CAST/EiJ founder have elevated ILC2 numbers after aeroallergen challenge compared to recombinant strains that inherited the locus from all other founders (Fig. 2B). In our initial phenotyping study presented in Fig. 1B, all CAST/EiJ mice died during the AltEx challenge, preventing the quantification of ILC2s in this founder strain.

We sought to identify the specific gene or set of genes within the Ch.7 ILC2 Locus that is responsible for the QTL effect, and therefore, a regulator of ILC2 biology. Our following experimental approach had two aims: 1.) fully characterize the nature of the ILC2-related phenotype that the Ch.7 ILC2 Locus regulates, and 2.) narrow the list of candidate genes in the locus to identify and validate a specific ILC2 target. The best approach to tackle both aims was a direct comparison of two CC strains with opposite phenotypic extremes from Fig. 1B. We identified a strain with high ILC2 prevalence after challenge that inherited its locus from CAST/EiJ and a strain with low ILC2 prevalence after challenge that inherited its locus from a negative contributing founder strain. We hypothesized that: 1.) the differences between the ILC2s in these high and low strains more completely describe the phenotype governed by the QTL, as it is likely that the QTL’s physiologic effects extend beyond simply the quantity of ILC2s after challenge, and 2.) a gene in the Ch.7 ILC2 Locus that influences ILC2 prevalence after challenge exhibits expression differences between a strain with very high ILC2 prevalence and a CAST/EiJ haplotype at the Ch.7 ILC2 Locus and a strain with low ILC2 prevalence and a negatively contributing founder haplotype at the Ch.7 ILC2 Locus.

CC030/GeniUnc mice have more numerous and transcriptionally distinct ILC2s at baseline compared to C57BL/6J
The two strains that we utilized for the high and low phenotypic comparison were the CC030/GeniUnc (henceforth CC030) recombinant strain and the C57BL/6J founder strain. We selected CC030 because it presented with the second highest ILC2 prevalence after challenge of the tested strains, and it possessed 100% CAST/EiJ lineage for the entirety of the QTL. The only strain with a higher prevalence was CC001/Unc, but this strain did not have complete haplotype homogeneity for the Ch.7 ILC2 Locus34. We selected C57BL/6J as our low phenotype reference strain because of its low ILC2 prevalence phenotype, locus haplotype homogeneity (inherent to the strain being a founder), and negative haplotype contribution (Fig. 2B).

We first sought to more completely characterize the phenotype that is regulated by the Ch.7 ILC2 Locus. Unchallenged CC030 mice had significantly higher ILC2 prevalence and total ILC2 numbers in the lung compared to unchallenged C57BL/6J mice (Fig. 3A−C), suggesting that the ILC2 prevalence phenotype regulated by the Ch.7 ILC2 Locus is present at baseline and is not a function of increased proliferation in response to aeroallergen challenge. To assess whether this elevation in ILC2 percentage in CC030 vs. C57BL/6J was: 1.) restricted to the lung and 2.) dependent on female sex, we quantified ILC2s in the lung, bone marrow, colon, and gonadal adipose tissue from female and male CC030 and C57BL/6J mice (Supplementary Fig. 3A). ILC2 prevalence and total numbers of ILC2s were elevated in the lungs of both male and female CC030 mice compared to their respective sexes of C57BL/6J mice. This pattern of CC030 mice exhibiting elevated ILC2s compared to C57BL/6J in a sex-independent manner was not observed in the other tissues, suggesting that the quantitative phenotype mapped with the Ch.7 ILC2 Locus is unique to the lung. Despite being more numerous, CC030 ILC2s exhibit less than half the expression of ST2, the receptor for ILC2’s primary activating cytokine IL-33, compared to C57BL/6J ILC2s (Fig. 3D and E). The decrease of ST2 expression in the CC030 ILC2s suggests that they may be less capable of responding to IL-33 alarmin signals, implying decreased IL-33-mediated inflammatory potential.

Inspired by the differences in ST2 expression, we endeavored to more broadly characterize the differences between the ILC2s of these strains. We performed bulk RNA-sequencing on naïve CC030 and C57BL/6J ILC2s sorted from the lungs via flow cytometry (Fig. 3F). Although these cells were isolated using identical ILC2 markers, they were drastically different by their gene expression profiles. This difference was appreciated in the quantity of differentially expressed genes (Fig. 3G), sample distance matrix (Fig. 3H), and principal component analysis (PCA) (Fig. 3I). The full list of differentially expressed genes provided in Supplementary Data 1. We then performed gene set enrichment analysis (GSEA) with WikiPathways annotated gene sets to better understand the specific cellular pathways and processes that were different between the ILC2s of these strains. A host of signaling pathways relevant to ILC2 activation and Type 2 inflammatory function were significantly enriched in the C57BL/6J ILC2s. Some of the most striking differences were in TSLP signaling, IL-7 signaling, IL-2 signaling, and the ST2 Signaling/IL-1 structural pathway (Fig. 3J). Although C57BL/6J had only one fourth of the number of upregulated genes compared to CC030, seemingly most of the traditional ILC2 inflammatory pathways were represented. To capture other differences between these cells in an unbiased manner, we performed hierarchical clustering of the top 30 Gene Ontology: Biological Processes pathways most enriched in C57BL/6J and CC030 ILC2s, ranked by normalized enrichment score (Fig. 3K). The most enriched gene sets in C57BL/6J ILC2s cluster together as pathways related to protein translation, mitochondrial complex assembly, RNA processing, ribosome biogenesis, and oxidative phosphorylation. This GSEA result emphasizes that C57BL/6J ILC2s are more transcriptionally, translationally, and metabolically active compared to CC030 ILC2s, suggesting their increased general readiness for activation and the propagation of Type 2 inflammation. In contrast, CC030 ILC2s are most transcriptionally enriched for pathways related to cell-cell interactions, cell membrane ion transport, and detection of chemical stimuli. Taken together, these data highlight the myriad of differences between ILC2s of these two strains (full GSEA lists in Supplementary Data 2-4). CC030 ILC2s have a larger population size at baseline, but their gene expression profiles suggest that they have a significant downregulation of canonical ILC2 inflammatory processes. Despite these differences, we assert that this population in CC030 should still be classified as ILC2s, as they are defined by GATA3+ and show a high percentage of ST2+, despite a lower ST2 mean fluorescence intensity (MFI) compared to C57BL/6J ILC2s (Fig. 3A, D).

CC030 mice have a greater number of ILC2s that are less proliferative and less inflammatory than C57BL/6J in vitro and in vivo
We established from our initial phenotyping experiment that CC030 have a very high prevalence of lung ILC2s after AltEx challenge compared to C57BL/6J. This large ILC2 population is present at baseline in CC030 mice, and our data suggest that CC030 ILC2s have reduced Type 2 effector function compared to C57BL/6J. To test this hypothesis, we isolated ILC2s from naïve lungs of C57BL/6J and CC030 female mice by flow cytometry according to the gating strategy defined in Supplementary Fig. 4A, and we cultured them at equal numbers from both strains with IL-33, TSLP, and IL-2. After 4 days, the number of C57BL/6J ILC2s was significantly increased compared to CC030 ILC2s (Fig. 4A). We incubated ILC2s from both strains with CFSE in an identical culture system and harvested cells after 48h to assess proliferative differences. C57BL/6J ILC2s showed a greater degree of CFSE dilution (Fig. 4B), indicating greater proliferation (Fig. 4C) and division (Fig. 4D) compared to CC030 ILC2s. These results confirmed that C57BL/6J ILC2s have an increased ability to proliferate in response to identical cytokine stimulation compared to CC030 ILC2s, as was suggested by greater ST2 expression on ILC2s in C57BL/6J. C57BL/6J ILC2s produced more Type 2 cytokines in culture than CC030 ILC2s. We detected significantly greater IL-13 in the supernatant (Fig. 4E) and observed greater IL-13 production on a per-cell basis by intracellular cytokine staining (Fig. 4F) in C57BL/6J ILC2s compared to CC030 ILC2s. These findings are in accordance with our proliferation data, showing that C57BL/6J ILC2s have greater intrinsic inflammatory capability than CC030 ILC2s.

The reduced proliferative capacity of the CC030 ILC2s compared to C57BL/6J prompted us to contemplate the mechanism by which the large ILC2 population exists in CC030 mice. A group previously described that NK cells deficient in Lysine-specific demethylase 6A (UTX) had an elevated population size in the spleen but exhibited decreased cell fitness. UTX deficient NK cells had decreased apoptotic function, contributing to elevated NK cell numbers35. We hypothesized that the large ILC2 population size in CC030 could be attributed to decreased apoptotic activity, and we quantified the percentage of live C57BL/6J and CC030 ILC2s that were positive for activated caspases after 2 days of culture with IL-33, TSLP, and IL-2. CC030 ILC2s had a decreased percentage of active caspase positive cells compared to C57BL/6J ILC2s (Fig. 4G, H). To further compare apoptotic activity between the two strains, we performed surface staining with Annexin V and viability staining with propidium iodide (PI) to identify early apoptotic (Annexin V+/PI-) and late apoptotic/dead (Annexin V+/PI+) ILC2s (Fig. 4I). Interestingly, CC030 ILC2s had significantly more early apoptotic cells than C57BL/6J, but they had significantly fewer late apoptotic cells and showed an overall greater viability (Fig. 4J, K). This result may suggest that CC030 mice initiate apoptosis of ILC2s, but they become arrested in the early stages of this process and do not progress through programmed cell death as completely as C57BL/6J. Corroborating these results, we identified multiple pro-apoptotic pathways enriched in C57BL/6J ILC2s compared to CC030 from our RNA-sequencing analysis between naïve CC030 and C57BL/6J ILC2s. (Fig. 4L). These results offer an explanation as to how CC030 ILC2s are less proliferative and less responsive to inflammatory cytokines compared to C57BL/6J, yet CC030 have a significantly larger population of ILC2s at baseline. A decreased ability to complete the process of apoptosis may compromise the overall inflammatory fitness of the ILC2 population, leading to an increase in cell number with diminished Type 2 effector function.

We then assessed if this same phenotypic distinction exists between the strains in vivo. We challenged CC030 and C57BL/6J mice in our 4-day AltEx challenge model (Fig. 1A), however, we increased the AltEx dosage to 8 μg from 5 μg to elicit a more robust Type 2 response, as our initial challenge did not produce detectable IL-5 and IL-13 in the lung homogenates of C57BL/6J mice. CC030 mice had significantly increased ILC2 prevalence compared to C57BL/6J after AltEx challenge (Fig. 4M), reconfirming our initial findings. We hypothesized that the higher number of ILC2s in CC030 was specific to ILC2s and not to innate lymphoid cells in general, so we compared ILC2s (Lin-, CD90+, CD127+, GATA3+) as a percentage of Lin-, CD90+ cells, which we term ILCs (Fig. 4N). Approximately 50% of the ILCs in the lung of CC030 mice were ILC2s, while ILC2s made up less than 10% of ILCs in C57BL/6J, suggesting that the higher percentage of ILC2s in CC030 mice compared to C57BL/6J mice is not due to a broad expansion of the innate lymphoid compartment. Despite having more than 15 times as many ILC2s, CC030 mice produced 5 times less IL-5 and 30 times less IL-13 compared to C57BL/6J mice by ELISA in the lung homogenate after Alt Ex challenge (Fig. 4O, P). CC030 mice had significantly decreased total eosinophils in the BAL after Alt Ex challenge compared to C57BL/6J mice, demonstrating a functional consequence of reduced IL-5 production (Fig. 4Q).

Intrigued by the diminished ILC2-driven Type 2 response in CC030 mice, we aimed to more broadly characterize the inflammatory profile of CC030 lungs at baseline and after challenge with different allergens. We quantified neutrophils, lymphocytes, eosinophils, and macrophages in the BAL and further quantified lymphocyte subtype by flow cytometry in the lung according to the gating strategy in Supplementary Fig. 5A without challenge, with papain challenge, and with Alternaria challenge. At baseline, CC030 mice have significantly more total ILC2s, CD4+ T cells, Tregs, BAL macrophages, and BAL neutrophils compared to C57BL/6J, but they have decreased CD8+ T cells and show no difference in total B cells, BAL eosinophils, or BAL lymphocytes (Supplementary Fig. 5B).

We then challenged C57BL/6J and CC030 mice with AltEx (4-days, 8 μg) and papain (3-days, 10 μg) to profile each strains immune response to different protease-containing allergens (Supplementary Fig. 5C). CC030 mice had significantly more ILC2s than C57BL/6J mice after papain challenge, mirroring our AltEx results. While AltEx challenge resulted in nearly significantly lower ILC2-expansion ratio and total IL-5, and significantly lower total IL-13 for CC030 compared to C57BL/6J, papain challenge did not reveal any differences in these outcomes. The paucity of IL-5 and IL-13 with papain challenge suggests an inability of papain to elicit a robust Type 2 innate response at this dose, which was chosen as the highest dose that did not cause hemorrhage in our model. CC030 mice also had a greater number of B cells, CD8+ T cells, and CD4+ T cells with AltEx challenge but only a greater number of CD4+ T cells with papain. There was no significant difference in total Tregs with either allergen. In the BAL fluid, CC030 mice had significantly decreased eosinophilia but increased neutrophilia compared to C57BL/6J with AltEx challenge, with the neutrophilia alone being conserved in papain challenge. These results suggest that CC030 mice have a diminished innate Type 2 response to Alternaria that compensates with neutrophilia over eosinophilia, and some elements of this response are conserved in exposure to other protease containing antigens like papain.

Ffar3 is a gene candidate responsible for the QTL effect, thereby regulating ILC2 number and effector function
We leveraged the differences between CC030 and C57BL/6J mice to gain a better understanding of the phenotype that is regulated by the Ch.7 ILC2 Locus. Our next objective was to identify a specific targetable gene or subset of genes within the Ch.7 ILC2 Locus that could be responsible for the QTL effect. We developed a strategy to refine our list of 72 QTL genes from two hypotheses: 1.) a gene in the QTL that regulates ILC2 prevalence is differentially expressed between a strain with high ILC2 prevalence (CC030) and a strain with low ILC2 prevalence (C57BL/6J), and 2.) the gene in question is expressed in ILC2 and has an ILC2 intrinsic effect. Of the 72 protein coding genes, 50 were differentially expressed between ILC2s from CC030 and C57BL/6J mice. We were most interested in implicating genes that are relevant to ILC2 biology in humans, so we gated our gene list to only include those genes expressed in human ILC2s36. This yielded 12 human orthologs. The ultimate goal of this study was to uncover regulators of ILC2-mediated allergic inflammation that could be targeted for therapeutic benefit, and we therefore narrowed our selection to only those genes that are druggable targets37. We evaluated the 4 remaining genes to identify those with previously documented function on immune cells, which left only 2 genes: Cd22 and Ffar3. A diagram of this selection process is provided in Fig. 4R, and a detailed list of genes after each selection step is provided in Supplementary Fig. 6A. We then investigated the effects of both genes in regulating ILC2 prevalence and effector function. We used knockout mice and blocking antibodies to interrogate the role of CD22 on ILC2s, and while CD22 is expressed on ILC2s, we concluded from our experiments that it does not have an appreciable impact on ILC2 numbers or effector function. These experiments are provided in Supplementary Fig. 7.

With Cd22 eliminated from our list, we turned our attention to free fatty acid receptor 3 (Ffar3). FFAR3 is a 7-transmembrane domain containing, lipid-binding Class A G-protein coupled receptor38. The receptor is expressed on pancreatic beta cells, epithelial cells of the gut, neuroendocrine tissues, and a host of other tissues throughout the body39–41. Microbiome-derived short-chain fatty acids (SCFAs) (acetate (C2), propionate (C3), butyrate (C4)) act as ligands for the receptor, with propionate and butyrate having increased binding affinity compared to acetate. These same ligands activate the closely related free fatty acid receptor 2 (FFAR2), but this receptor shows increased affinity for acetate over propionate and butyrate42,43. While the FFAR3 receptor has been extensively studied in the context of diabetes and metabolism, more recent attention has been paid to the contribution of Ffar3 to the regulation of inflammation44–47. SCFAs have a significant role in immune modification, through effects on epithelial barrier function48,49, macrophages50, B cells and plasma cells51,52, helper T cell effector function53, and regulatory T cell differentiation54,55. Most of the reported associations between SCFA signaling and immune regulation have implicated FFAR2 as the responsible receptor56. However, important studies have specifically linked the SCFA-FFAR3 axis to the regulation of Type 2 inflammation in asthma and allergic disease. Gut microbiome-derived SCFAs downregulated Thelper type 2 (T 2) cell effector function in an FFAR3, not FFAR2, dependent manner57. Two groups reported that FFAR3 was highly expressed in T 2 cells in eosinophilic esophagitis patients compared to healthy controls and described the receptor’s critical role in regulating IL-5 and IL-13 responses58,59. Others have reported the effect of short chain fatty acids on influencing ILC2 biology, but they did not implicate FFAR3 as a regulating receptor of any SCFA-dependent phenomena in the lung60–62. One study in particular established the expression of Ffar3 in pulmonary ILC2s in mice, but their data suggested that SCFAs modulated ILC2s primarily in a receptor-independent manner, likely through histone de-acetylase inhibition63. With known interaction between SCFAs and the immune system, and evidence supporting the role of FFAR3 in regulating adaptive Type 2 inflammation, we hypothesized that FFAR3 signaling influenced ILC2 function and aimed to clarify the role of this receptor on ILC2s.

We first sought to confirm the expression of Ffar3 in naïve CC030 ILC2s, which is demonstrated by the normalized read counts from our RNA-sequencing data (Fig. 4S). We isolated ILC2s from the naïve lungs of five CC030 female mice by flow cytometry sorting and extracted RNA. We confirmed the presence of Ffar3 transcript by q-PCR in line with previous reports (Fig. 4T), and we report its expression on ILC2s from a variety of tissue sources.

FFAR3 signaling expands ILC2 numbers and reprograms ILC2s to an anti-inflammatory effector state in vitro
C57BL/6J mice did not express FFAR3 on their ILC2s in our RNA-sequencing experiment (Fig. 4S), and we were therefore concerned that available Ffar3 KO strains on this background would have been suboptimal in investigating the effect of this receptor on ILC2s. As such, we were restricted to working with wild-type ILC2s and modulating FFAR3 directly in CC030 mice. To understand the effects of FFAR3 agonism on ILC2 activation and effector function, we cultured flow-sorted ILC2s from CC030 mice with the activating cytokines IL-33, TSLP, and IL-2. We added to the culture either AR420626, a highly specific FFAR3 agonist64, or DMSO vehicle (Fig. 5A).

After 6 days of culture, AR42062 increased the number of ILC2s nearly 3-fold compared to vehicle controls (Fig. 5B). Despite this large increase in ILC2 numbers, AR420626 significantly decreased the ability of these ILC2 to produce IL-5 and IL-13, measured by the concentration of cytokines in the supernatant (Fig. 5C) and expression on a per-cell basis via intracellular cytokine staining (Fig. 5D, E). In addition to decreasing Type 2 cytokine production, FFAR3 agonism significantly decreased ST2 expression on ILC2s (Fig. 5F). AR420626-treated ILC2s had a greater than 3-fold decrease in the percentage of cells that were ST2+ (Fig. 5G), and the MFI of ST2 on positive cells was decreased by more than 60% (Fig. 5H). This decrease in ST2 MFI with FFAR3 agonism mirrors the observed decrease in ST2 expression in CC030 ILC2s compared to C57BL/6J. In contrast to decreased production of inflammatory cytokines, AR42062-stimulated ILC2s experienced a ~12-fold increase in the secretion of the anti-inflammatory cytokine IL-10 compared to vehicle controls (Fig. 5I).

We hypothesized that the FFAR3 signaling-dependent increase in the cell number would not be a function of proliferation, but of resistance to apoptosis, reflecting our observations in CC030 ILC2s (FFAR3 expression) compared to C57BL/6J (no FFAR3 expression). AR420626 treated ILC2s had a similar fraction of Ki-67+ cells compared to vehicle controls (Fig. 5J). While the difference was statistically significant, the small magnitude of change (~2%) suggests that it is unlikely to be biologically meaningful (Fig. 5K). Further supporting this observation was the lack of a significant difference in proliferation or division after 48h of culture by CFSE dilution (Fig. 5L, M).

FFAR3 agonism increased the number of ILC2s but did not meaningfully increase proliferation. From this result, we hypothesized that the difference in ILC2 number was instead due to an FFAR3-signaling dependent decrease in apoptosis progression. The persistence of less fit cells that would be otherwise eliminated through programmed cell death could explain both the elevated ILC2 population size without proliferation difference and the decreased Type 2 effector function observed in AR420626-treated ILC2s. Similar to what was observed in CC030 vs. C57BL/6J, AR420626-treated ILC2s had a modestly higher percentage of cells in early apoptosis but a roughly 3-fold decrease in the percentage of cells advancing to late apoptosis and cell death (Fig. 5. N, P). This considerable increase in cell viability supports the idea that FFAR3 signaling is not expanding cell number through stimulating cell proliferation, but through the maintenance of a large cell population by the inhibition of programmed cell death.

These in vitro data show that the effect of FFAR3 agonism mirrors what was observed in the differences between CC030 and C57BL/6J, highlighting an increase in ILC2 cell number with a concomitant decrease in Type 2 effector function, and even revealing an increase in the ability of FFAR3 agonist-treated ILC2s to produce the anti-inflammatory cytokine IL-10. This effect is particularly interesting, as ILC2 cell number after activation and Type 2 cytokine secretion traditionally correlate65–67. ILC2s increasing cell number, decreasing Type 2 cytokine secretion, and increasing secretion of IL-10 is a response that, to the best of our knowledge, has not yet been categorized and possibly represents a unique ILC2 effector state.

We observed a lack of Ffar3 expression in C57BL/6J ILC2s in our initial sequencing experiment, and we therefore expected C57BL/6J ILC2s to have diminished or no response to FFAR3 agonism. We cultured C57BL/6J ILC2s according to our model in Fig. 5A and did not observe a decrease in IL-5 or IL-13, a difference in ILC2 number, or an effect on apoptosis. (Supplementary Fig. 8A-G). Similarly, AR420626 treatment had no effect on C57BL/6J ILC2 proliferation, measured by CFSE dilution (Supplementary Fig. 8H-J). Thio et al. demonstrated expression of Ffar3 on ILC2s from IL-33 pre-treated Rag2−/− mice, but they surprisingly did not observe any effect of AR420626 treatment on cytokine production from ILC2s stimulated by IL-33 in vitro63.

Our results suggested that differences in baseline FFAR3 signaling on ILC2s between C57BL/6J and CC030 mice were attributable to differences in receptor expression, but we aimed to investigate possible in vivo differences in SCFAs, the endogenous FFAR3 ligands, as well. We quantified acetate, propionate, and butyrate in the serum of C57BL/6J and CC030 mice at baseline via mass spectrometry, and CC030 mice had significantly lower serum concentrations of acetate compared to C57BL/6J, while there were no differences in propionate or butyrate (Supplementary Fig. 9). The lower concentration of FFAR3 ligands in the serum of CC030 mice suggested that the increased FFAR3 signaling on CC030 ILC2s is dependent on increased receptor expression, not SCFA concentration.

FFAR3 signaling elicits a unique IL-10-producing effector state in ILC2s
We were intrigued by the anti-inflammatory cytokine secretion profile that FFAR3 agonism imparted on ILC2s, and we sought to further characterize this effector state. We investigated whether this IL-10 producing phenotype was dependent the on the specific combination of stimulatory cytokines used to activate ILC2s in our culture system. A previous report from Seehus et al. demonstrated that IL-2 was necessary for IL-10 induction in ILC2s68. We cultured CC030 ILC2s with AR420626 or vehicle in the presence or absence of IL-2, and we observed that IL-2 was necessary for AR420626 to increase cell number (Fig. 6A), decrease IL-5 production (Fig. 6B), and induce IL-10 production (Fig. 6C). Given that FFAR3’s anti-inflammatory effect is IL-2 dependent, we hypothesized that FFAR3 signaling may increase IL-2 signaling through upregulation of the high-affinity IL-2 receptor alpha (CD25). We confirmed our hypothesis by detecting greater CD25 expression by flow cytometry on ILC2s treated with AR420626 compared to vehicle controls (Fig. 6D, E). These data suggest that, like previously described IL-10-producing ILC2s, FFAR3 acts in an IL-2 dependent manner and may induce this state through an upregulation of CD25.

Given the concomitant production of IL-10 and decrease in Type 2 cytokines, it seemed possible that IL-10 was acting in an autocrine and or paracrine manner to inhibit IL-5 and IL-13 production in ILC2s. We used an IL-10 blocking antibody to determine if the reciprocal changes in anti-inflammatory and inflammatory cytokines were dependent or independent processes. IL-10 blockade had no effect on ILC2 number, IL-5 production, or IL-13 production (Fig. 6F–I). Our results corroborate a previous report that showed IL-10 signaling having no effect on IL-5 production by ILC2s69.

To understand whether this anti-inflammatory state elicited by FFAR3 agonism was unique or similar to previously described ILC2 effector states, we compared AR420626 head-to-head with known inducers of IL-10 expression like retinoic acid (RA)68,70–72. AR420626 induced similar IL-10 expression to RA, but interestingly, RA significantly increased IL-5 secretion compared to vehicle, while AR420626 significantly decreased IL-5 secretion compared to vehicle (Fig. 6J, K). In fact, RA-stimulated ILC2s produced roughly 3-fold more IL-5 than those treated with the FFAR3 agonist. While a benchmark IL-10 inducer like RA increases IL-10 expression with concurrent increase in IL-5, AR420626 induces IL-10 while decreasing IL-5. This opposite regulation of Type 2 cytokines, which is independent of autocrine and or paracrine IL-10 signaling, suggests that the ILC2 effector state induced by FFAR3 signaling is unique in its anti-inflammatory nature; it increases anti-inflammatory cytokine production while decreasing pro-inflammatory cytokine production.

We compared AR420626-treated ILC2s with other IL-10-producing ILC2s in an unbiased manner to further assess the apparent distinctiveness of this effector state. We performed bulk-RNA-sequencing on AR420626 and vehicle treated ILC2s after 6 days of culture. We compared the transcriptional signatures of our FFAR3 agonist-treated ILC2s against those of other IL-10+ ILC2s from previously published sequencing experiments68,73. There are apparent transcriptional differences between AR4206262-treated ILC2s, IL-10+ ILC2s from Seehus et al., and IL-10+ ILC2s from Howard et al. To assess global similarity between the 3 sets of ILC2s, we performed PCA analysis on normalized read counts from each experiment using a batch correction method that retained variation associated with IL-10+ status (Fig. 6L). It is apparent from the PCA plot that the other two IL-10+ ILC2 effector states are much more similar to each other than they are to our FFAR3-dependent state, suggesting a uniqueness to our examined phenotype.

FFAR3 reprograms ILC2s to an anti-inflammatory effector state by increasing EGFR expression
From our bulk RNA-sequencing data, we endeavored to gain further insight into the mechanism by which FFAR3 signaling reprogrammed ILC2s (Fig. 7A). Amongst all differentially expressed genes (Supplementary Data 5), Egfr had the greatest positive fold-change, and the second lowest p value when comparing AR420626-treated to vehicle-treated ILC2s (Fig. 7B). Egfr codes for the epidermal growth factor receptor (EGFR), a known regulator of cell growth that has been implicated in a host of neoplastic disorders74. A prior report established the expression of EGFR on regulatory T cells (Tregs), and it demonstrated that amphiregulin (an EGFR ligand) signaling through Treg-intrinsic EGFR was critical for enhancement of Treg suppressor function, including IL-10 production75. ILC2 have been reported to secrete amphiregulin76,77, and thus, an autocrine/paracrine amphiregulin-EGFR signaling axis on our cultured ILC2s could explain the reprogramming of FFAR3-agonist treated ILC2s to an anti-inflammatory, IL-10 producing phenotype.

AR42062-treated cells had significantly greater Egfr expression compared to vehicle controls by RNA-sequencing (Fig. 7C), and we confirmed this finding by flow cytometry (Fig. 7D, E). We established causality between EGFR signaling and FFAR3 signaling through a repeat of our culture experiment with the EGFR inhibitor gefitinib78. We observed that AR4206 expanded ILC2 numbers compared to vehicle controls, but AR4206 with the addition of gefitinib reduced ILC2 numbers back to the level of vehicle controls (Fig. 7F). Similarly, while AR4206 treatment of ILC2s increased IL-10 secretion compared to vehicle controls, AR4206 and gefitinib reduced IL-10 secretion compared to AR4206 alone (Fig. 7G). EGFR blockade negated the AR4206-dependent decrease in IL-5 production, demonstrated by the increase in IL-5 expression by intracellular cytokine staining in gefitinib and AR4206-treated ILC2s compared to only AR4206-treated ILC2s (Fig. 7H, I). We confirmed that ILC2s secrete amphiregulin in our culture system (Fig. 7J). Blockade of amphiregulin in AR420626-treated ILC2s limited IL-10 induction to a modest but statistically significant degree (Fig. 7K), suggesting that amphiregulin, and possibly other EGFR ligands, are signaling through EGFR in an autocrine and paracrine manner. Given that FFAR3 signaling upregulates EGFR on ILC2s, and that inhibition of EGFR signaling reversed many of the observed effects of FFAR3 signaling, we propose that FFAR3 is partially regulating ILC2s through an EGFR-dependent mechanism, seemingly mirroring the effect of EGFR promoting increased IL-10 secretion in Tregs.

FFAR3 signaling partially reprograms human ILC2s towards an anti-inflammatory effector state in vitro
Our gene selection process considered expression on human ILC2s to maintain the translational potential of any discovery, and so we aimed to validate the effect of FFAR3 signaling on human ILC2s directly. Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors, and ILC2s were flow sorted as Lineage-, CD45+, CD127+, CRTH2+ cells (Fig. 8A). We cultured these human ILC2s with the activating cytokines IL-33, TSLP, and IL-2 with the FFAR3-specific agonist AR420626 at concentrations of 1, 5, and 10 μM or DMSO vehicle control. AR420626 had no effect on cell number at 1 μM, but 5 μM and 10 μM significantly decreased ILC2s after 6 days of culture compared to vehicle controls (Fig. 8B). Neither of the 3 concentrations of AR420626 decreased ILC2 viability, suggesting that the drug’s effects were not a function of toxicity (Fig. 8C). AR420626 treatment demonstrated a decrease in IL-5 and IL-13 production, with 10 μM decreasing both cytokines approximately 5-fold (Fig. 8D, E). Despite this marked decrease in Type 2 inflammatory cytokines, AR402626 did not induce IL-10 production at any concentration in these human ILC2s (Fig. 8F). At 5 μM, AR420626 had a more modest effect on decreasing cell number compared to decreasing IL-5 and IL-13 secretion, suggesting a potentially greater effect on cytokine secretion than cell expansion.

We confirmed EGFR expression on human ILC2s, but we observed a significant degree of heterogeneity in the response of EGFR expression to FFAR3 signaling (Fig. 8G). Of 8 examined samples, 3 experienced increases in EGFR MFI of at least 10%, while all other remained identical between AR420626 and vehicle treated ILC2s. Interestingly, there was a trend towards an increase in EGFR expression on cells at Day 6 of culture compared to Day 4 (Fig. 8H), suggesting that EGFR expression is induced through the process of ILC2 activation. Despite the variability in FFAR3-dependent changes in EGFR expression, the EGFR inhibitor gefitinib trended towards reversing the effects of AR420626 in limiting Type 2 cytokine production. While adding gefitinib at multiple concentrations to AR420626-treated ILC2s had no appreciable effect on cell number or viability, there was a near significant reversal of IL-5 production with gefitinib treatment (p=0.063) (Fig. 8I–L), suggesting that EGFR blockade may undo the anti-inflammatory effects imparted on ILC2s by FFAR3 signaling.

Taken together, these results demonstrate that the anti-inflammatory effect of FFAR3 signaling is partially conserved between mice and humans. AR420626 treatment significantly decreased Type 2 effector function for ILC2s of both species, but the ability of AR420626 to increase IL-10 production was not conserved in the human ILC2s that we tested. We confirm that human ILC2s express EGFR as in mouse, and we suggest that FFAR3 signaling may act in a partially-EGFR dependent manner.

Methods
All research presented in this manuscript complies with all relevant ethical regulations, approved by the Vanderbilt University Medical Center Institutional Animal Care and Use Committee (protocol number: M1800150-01) and the Vanderbilt University Medical Center Institutional Review Board (202162).

Mice
All Collaborative Cross (CC) recombinant strains used in this manuscript (CC001/Unc, 002/Unc, 003/Unc, 004/TauUnc, 005/TauUnc, 006/TauUnc, 007/Unc, 010/GeniUnc, 011/Unc, 012/GeniUnc, 013/GeniUnc, 016/GeniUnc, 019/TauUnc, 021/Unc, 023/GeniUnc, 024/GeniUnc, 025/GeniUnc, 027/GeniUnc, 030/GeniUnc, 031/GeniUnc, 035/Unc, 036/Unc, 037/TauUnc, 038/GeniUnc, 039/Unc, 040/TauUnc, 041/TauUnc, 043/GeniUnc, 044/Unc, 051/TauUnc, 053/Unc, 057/Unc, 059/TauUnc, 060/Unc, 061/GeniUnc, 071/TauUnc, 072/TauUnc, 074/Unc, 078/TauUnc, 080/TauUnc, 081/Unc) were obtained from the UNC Systems Genetics Core Facility. For clarity, strain suffixes were omitted throughout the manuscript. CC founder strains 129S1/SvlmJ, A/J, C57BL/6J, CAST/EiJ, NOD/ShiLtJ, and WSB/EiJ mice were obtained from Jackson Laboratory. C57BL/6-Cd22tm1Lam/J (CD22KO) mice were obtained from Jackson Laboratory. CC030 mice, C57BL/6J, and CD22KO mice were bred and maintained in our mouse colony. All in vivo and in vitro mouse experiments utilized age-matched mice 8-16 weeks old. Mice used for experiments are female unless otherwise indicated. Animal sex was considered in the study design. Mice were housed in a temperature-controlled room at 22.2 °C on a half daylight-dark cycle. Mice were fed a regular diet (PicoLab Laboratory Rodent Diet 5LOD) and tap water was available ad libitum. Mouse experiments were approved by the Institutional Animal Care and Use Committee (IACUC) at Vanderbilt University Medical Center and were conducted according to the guidelines for the Care and Use of Laboratory Animals prepared by the Institute of Laboratory Animal Resources, National Research Council.

Aeroallergen challenge
Mice were anesthetized with intraperitoneal injection of a combination ketamine and xylazine solution. 5 min after anesthesia, mice were challenged with intratracheal administration of Alternaria extract (Greer) or papain (Millipore) dissolved in 80 μL of phosphate buffered saline (PBS). 5 μg of Alternaria extract was used for the initial phenotyping of CC strains presented in Fig. 1, but subsequent challenges employed a dosage of 8 μg to allow for the detection of inflammatory cytokines. 10 μg of papain were used, as this was the highest dose that did not elicit alveolar hemorrhage in a titration experiment. Intratracheal administration was performed with an IV catheter tip fixed on a 1 mL TB syringe (BD). Mice were allowed to recuperate from anesthesia on a heating pad before being returned to cages. AltEx challenge was carried out for 4 consecutive days, while papain challenge was carried out for 3 consecutive days.

Isolation of cells from mouse tissues
All mice were euthanized with intraperitoneal injection of a lethal dosage of pentobarbital solution and allowed to expire. Mouse lungs were placed in a digestion solution (RPMI 1640, 5% FBS, 1 mg/mL Type IV Collagenase from Clostridium histolyticum (Sigma), and 20 μg/mL of DNase I) and minced with scissors. Lungs were digested for 35 min at 37 °C with rotation. Digestion was neutralized with EDTA, and lungs were subsequently ground through cell strainers to obtain a single cell suspension. Mouse bone marrow was obtained from the femurs and tibias of mice after euthanasia. Bones were cleared of muscle and connective tissue, and the ends of the long bones were cut with a scalpel. Bones were placed into 0.5 mL tubes with puncture holes in the bottom. These punctured tubes were fit into 1.5 mL tubes and centrifuged, allowing for the collection of the bone marrow effluent. Bone marrow cells were then passed through cell strainers. Gonadal adipose tissue was excised bilaterally from the mice after euthanasia. Adipose tissue was minced and suspended in digestion buffer (2 mg/mL Collagenase (Sigma C-6885) with 1% FBS in PBS). Digestion was carried out at 37 °C with rotation for 45 min. Digested adipose tissue was passed through a cell strainer and diluted with neutralization buffer (2 mM EDTA with 1% FBS in PBS). Samples were centrifuged, and the supernatant with adipocytes was removed. Colons were harvested from mice and washed with cold PBS. Colons were then opened longitudinally and cut into 0.5 cm pieces, which were subsequently incubated with RPMI containing Penicillin/Streptomycin (Pen/Strep), 5 mM EDTA, 20 mM HEPES, 5% FBS and 1 mM DTT for 40 min at 37 °C with shaking. Contents were then poured through cells strainers, with the flow through discarded. Remaining tissue pieces were shaken vigorously in cold RPMI with Pen/Strep, 5 mM EDTA, and 20 mM HEPES and passed through a cell strainer, again discarding flow through. Tissue pieces were minced in a beaker and incubated in RPMI Pen/Strep, 5 mM EDTA, and 20 mM HEPES, 0.1 mg/mL Liberase TL (Roche), 0.05% DNase I (Sigma D5025) with stirring for 30 min at 37 °C. Contents were pulled through a 10 mL syringe and poured through a cell strainer. Cells were subsequently washed with RPMI with Pen/Strep, 5 mM EDTA, and 20 mM HEPES, and 0.05% DNase. Cells were then isolated with a 40%/90% Percoll gradient. Erythrocyte lysis was carried out for all samples with ammonium chloride-based RBC lysis solution (Tonbo) for 5 min before neutralization with cold PBS.

Analytical flow cytometry for ILC2s and lymphocytes from mouse tissues
Viability staining was carried out in PBS with Live/Dead Aqua Fixable Viability dye for 25 min. Surface staining for single cell suspensions was carried out in FACS staining buffer (3% FBS in PBS). Prior to staining, cells were incubated with Fc block (BD) for 10 min. Surface staining for ILC2 was carried out with Lineage staining cocktail (α-CD3, α-CD4, α-FCεR1, Hematopoietic Lineage Labeling Cocktail, anti-mouse biotin (Miltenyi)), α-CD19, α-CD45, α-CD90, α-CD127, α-ST2, α-ICOS, α-CD25, α-CD22 for 20 min. Streptavidin conjugation to biotinylated antibodies was carried out for 15 min. Surface staining for lymphocytes was carried out with α-CD19, α-TCRβ, α-CD4, α-CD8, and α-CD25. Fixation and permeabilization was carried out overnight with the FoxP3 Transcription Factor Staining Buffer Kit (eBioscience, cat: 00-5523-00). Intracellular staining for GATA3 and FoxP3 was carried out for 1 h in Perm Buffer (eBioscience). Analysis was conducted in FACS staining buffer on a BD 5-Laser Fortessa or 4-laser Cytek Aurora. A full list of antibodies used in the study is provided in Supplementary Data 7.

QTL mapping
We utilized 235 mice from 47 Collaborative Cross (CC) strains for quantitative trait locus (QTL) mapping. Genotype data, consisting of a probability matrix with 36 genotype calls (8 homozygous and 28 heterozygous) across 76,689 SNPs, were obtained from https://csbio.unc.edu/CCstatus/index.py?run=FounderProbs, which were then converted into an 8-state allele probability matrix. QTL mapping was performed at each SNP using the qtl2 package in R (version 4.2.1)94. A single-QTL genome scan was conducted by regressing the phenotypic trait of interest on the genotype probabilities at each SNP, and LOD (logarithm of the odds) scores were calculated. Genome-wide significance was evaluated empirically through 1000 permutations, and phenotypes with top candidate loci that exceeded a genome-wide significance threshold of 0.05 were identified as significant.

Flow cytometry sorting of mouse ILC2
Single cell suspensions were obtained from the naïve lungs of mice according to the harvest procedure outlined in this methods section. For the flow sorting experiment described in Fig. 3, ILC2 were sorted separately as biological replicates from the whole lungs of 3 C57BL/6J female mice and 3 CC030 female mice. Single cell suspensions from lungs were enriched for ILC2 with the Lineage Cell Depletion kit, mouse (Miltenyi, cat: 130-110-470) and the CD25 MicroBead Kit, mouse (Miltenyi, cat: 130-091-072), utilizing LS columns (Miltenyi) for both separations. ILC2 for this experiment were identified by surface staining as Lymphocytes (FSC-A vs. SSC-A), singlets by pulse gating, Live/Dead Aqua-, Lineage- (α-CD3, α-CD4, α-FCεR1, Hematopoietic Lineage Labeling Cocktail, anti-mouse biotin (Miltenyi)), CD90+, CD127+, CD25+, ICOS+. Sorting was performed on a BD FACS Aria III run on the slowest setting with a 100 μM nozzle. Due to equipment availability changes, ILC2 sorted for assessment of Ffar3 expression (Fig. 4) and culture (Figs. 4–7) were sorted on a BD FACS Symphony S6 and defined as Lymphocytes (FSC-A vs. SSC-A), singlets by pulse gating, Live/Dead Aqua-, Lineage- (α-CD3, α-CD4, α-FCεR1, Hematopoietic Lineage Labeling Cocktail, anti-mouse biotin (Miltenyi)), CD90+, CD127+, ST2+. Cells were again sorted on the lowest setting with a 100 μM nozzle.

Low-input library preparation and next generation sequencing
ILC2 were sorted into Single Cell Lysis Buffer (Takara), and subsequent library preparation and sequencing was performed at the Vanderbilt Technologies for Advanced Genomics (VANTAGE) core lab. The SMART-Seq v4 Ultra Low Input RNA Kit (Takara, cat: 634888) was used to isolate RNA. cDNA was generated from extracted RNA through reverse transcription. cDNA was fragmented, blunt-ended, and adenylated for adaptor ligation and subsequently PCR amplified. Amplified cDNA libraries were quality controlled by bioanalyzer. The libraries were sequenced using the NovaSeq 6000 with 150 bp paired end reads targeting 50 M reads per sample. RTA (version 2.4.11; Illumina) was used for base calling and analysis was completed using MultiQC v1.7.

RNA-sequencing data analysis
We conducted RNA-Seq analysis on both C57BL/6J and CC030 mice. Sequencing data quality was initially assessed using FastQC95, followed by read trimming with fastp96. For the C57BL/6J samples, reads were aligned to the mm10 reference genome using STAR97 with default parameters. For the CC030 samples, reads were aligned to the mm9 reference pseudogenome using STAR with default parameters, after which lapels98 was used to convert the alignments to mm10 reference coordinates. Gene expression counts were subsequently quantified across all samples using HTSeq99. Differential gene expression analysis was performed on the resultant read count matrices using the DESeq2 R package100. For the bulk-RNA sequencing experiment presented in Figs. 6, 7, paired differential gene expression analysis was conducted, as biological replicates were cultured separately for both conditions (vehicle vs. agonist). Normalized counts of genes, sample distance matrices, and principal component analyses were conducted with DESeq2, and results were visualized with ggplot2101 and pheatmap102. Differentially expressed genes were visualized in volcano plots with EnhancedVolcano103. Mouse genes were converted to human orthologs with biomaRt104,105. ClusterProfiler was used to perform gene-set enrichment analysis using Gene Ontology: Biological Processes and WikiPathways curated gene sets and to generate the resultant tree plot106. A significant enrichment for the IL-1R signaling pathway is represented as ST2 signaling, as no specific ST2 Wikipathways gene set exists, but the IL-1R signaling gene set describes identical signaling to ST2 downstream of the cell surface receptor. Transcriptomic comparison in Fig. 6 compared the normalized count matrices for ILC2 samples from 3 experiments: IL-10+ ILC2s from Seehus et al. (2017, GSE81882), IL-10+ ILC2s from Howard et al. (2020, GSE158983), and AR420626-treated ILC2s from this manuscript (GSE288176). For the PCA plot in Fig. 6, values in the normalized count matrices were log2 transformed to stabilize variance. We corrected for inter-experiment batch effects with ComBat from the sva R package107. A design matrix was specified to retain variation associated with IL-10+ vs. IL-10- status (condition) while adjusting for experiment (batch). The ComBat corrected expression matrix was then subset to IL-10+ samples. PCA analysis included group-wise convex hulls and centroids to highlight sample clustering by experiment.

CFSE labelling for proliferation quantification
ILC2 were resuspended in 0.1% FBS in PBS immediately after flow cytometry sorting. CFSE was diluted to the cell suspension for a final concentration of 1.5 μM and allowed to incubate at room temperature for 8 min. An equal volume of pre-warmed FBS was added, and the cells were incubated at 37 °C for 10 min to allow for the efflux of unbound CFSE. Cells were washed with 2% FBS in PBS prior to culturing. A small sample of CFSE-labelled cells was saved and immediately fixed with the FoxP3 Transcription Factor Staining Buffer Kit (eBioscience, cat: 00-5523-00) for the Day 0 control.

ILC2 culture
All ILC2 culture experiments were carried out in RMPI 1640 with 10% FBS, Penicillin/Streptomycin, HEPES, Sodium Pyruvate, and β-mercaptoethanol. Cells were plated in 96-well non-tissue culture treated U-bottom plates (Corning) in a total volume of 250 μL per plate. For mouse experiments, ILC2 were cultured at a density of 3000 cells per well. Due to low numbers, human PBMC-derived ILC2 were cultured at varying cell densities, with cells equally split from each donor between conditions. ILC2 in all experiments were stimulated immediately upon plating with IL-33 (10 ng/mL, mouse (PeproTech), human (PeproTech)), TSLP (10 ng/mL, mouse (PeproTech), human (PeproTech)), and IL-2 (100 U/mL, human (NIH), used for both mouse and human cells). Experiments involving AR420626 (Sigma) and Gefitinib (Sigma) and retinoic acid (Sigma) utilized DMSO vehicle controls at appropriate concentrations. The concentration of AR420626 in culture was 10 μM unless otherwise specified. Gefitinib was used at concentrations of 5 μM and 10 μM for mouse ILC2 culture and 0.1 μM and 0.5 μM for human ILC2 culture. Retinoic acid was used at a concentration of 1 μM. α-IL-10 antibody (R&D) or isotype control were added at 10 μM, and α-amphiregulin or isotype control was added at 20 μM. Cells were harvested after 48h for proliferation assessment via CFSE, after 4 days for strain comparison between C57BL/6J and CC030, and 6 days for CC030 drug-treatment and human ILC2 experiments.

Active caspase staining
ILC2 were harvested from culture and immediately placed in 1 mL of pre-warmed culture media without cytokines. BD Pharmigen Yellow-Green Caspase Probe was added for a final dilution of 1:100,000. Cells were incubated at 37 °C for 45 min protected from light. Cells were washed with media and centrifuged, then resuspended in pre-warmed media and allowed to incubate for an additional 15 min at 37 °C to allow for the outward diffusion of unbound probe.

Apoptosis detection by annexin V and propidium iodide (PI) staining
Apoptosis detection by Annexin V and PI was carried out with the BD Pharmigen FITC Annexin V Apoptosis Detection Kit I (cat: 556547). ILC2s were harvested from culture at Day 6 and washed in room temperature PBS. ILC2s were resuspended in 100 μL 1x Binding Buffer with Annexin V-FITC (1:500) and PI (1:400) and incubated for 15 min. After incubation, 400 μL of 1x Binding Buffer was added and cells were analyzed by flow cytometry.

Flow cytometry analysis of cultured ILC2
All ILC2 harvested from culture were stained for viability, and human ILC2s underwent surface staining for EGFR (antibody) for 15 min prior to fixation. Mouse ILC2 were surface stained with ST2 and CD25. Cells were fixed with the FoxP3 Transcription Factor Staining Buffer Kit (eBioscience, cat: 00-5523-00). Intracellular cytokine staining and Ki-67 staining was carried out for 45 min, and cells were not restimulated prior to intracellular cytokine staining. Mouse EGFR staining was performed by incubating cells with concentration of the unconjugated α-EGFR antibody (Invitrogen) for 30 min. Cells were washed and subsequently incubated with a 1:1,000,000 dilution of the secondary antibody for an additional 30 min. 123count eBeads (Invitrogen) were utilized to enumerate ILC2. Cultured cells were analyzed on a 4-laser Cytek Aurora spectral flow cytometer.

Cytokine detection by ELISA
Cell supernatants were harvested, and IL-5, IL-13, IL-10, and amphiregulin were detected by DuoSet ELISA development systems kits (R&D, cat: IL-5: DY405(Mouse) DY205(Human), DY413(Mouse) DY213(Human), DY417(Mouse) DY217B(Human), DY989(Mouse)) for ILC2 culture. Cytokine detection in mouse lungs was carried out on lung homogenate. Briefly, the left lobe of the lung was isolated from euthanized mice and homogenized in bead homogenizer tubes with silicone beads and Dulbecco’s Minimal Essential Media. Homogenization was carried out in a Bead Beater machine, and supernatants of lung homogenate were collected. IL-5 and IL-13 were detected in mouse lung homogenates by Quantikine (R&D) ELISA kits (cat: M5000, M1300CB). For detected values under the lowest point on the standard curve, one half of the lowest point on the curve was assigned for that sample.

Cell counts and differential with bronchoalveolar lavage (BAL)
Mice were euthanized and tracheostomy was performed with scissors. A blunt needle in a flexible rubber adaptor was inserted into the trachea and 800 μL of sterile PBS was injected into the lungs with a 1 mL TB syringe. Fluid was withdrawn and cell counts were performed on the sample. 100 μL of the sample were spun onto a glass slide with 20 μL FBS in a CytoSpin machine. Slides were stained with hematoxylin and eosin, and cell differentials were performed by lab members blinded to the experimental groups.

Ffar3 detection by qRT-PCR
The single cells from the whole lungs of naïve female mice were isolated as biological replicates, and lineage negative cells were enriched with the Lineage Cell Depletion kit, mouse (Miltenyi, cat: 130-110-470). ILC2 were isolated with the flow cytometry sorting strategy outlined in this methods section and were sorted directly into lysis buffer. The CellAmp Whole Transcriptome Amplification kit (Takara, cat: 3734) and TaKaRa Ex Taq Hot Start Version (Takara, cat: RR006A) were used for RNA isolation and cDNA preparation. Quantitative real-time PCR was carried out with TaqMan Gene Expression assays: Gapdh (Mm99999915) and Ffar3 (Mm02621638_s1) on a QuantStudio 5 Real-Time PCR System (Applied Biosystems) instrument for 45 cycles. Relative gene expression was quantified by calculating 2-ΔΔCT. ΔCT was calculated from the Gapdh housekeeping gene run with each sample.

α-CD22 antibody treatment
C57BL/6J and CC030 mice were injected intraperitoneally with 250 μg of the α-CD22 blocking and depleting antibody Cy34.1 (BioXCell) or IgG1 Isotype control (BioXCell) in 100 μL of PBS. Mouse lungs were harvested according to the protocol outlined in this methods section 8 days after antibody or isotype control injection.

Mass spectrometry-based quantification of short-chain fatty acids
Short-chain fatty acids (SCFAs) in serum were quantified by LC-HRMS following derivatization with dansylhydrazine and EDC. Samples were spiked with deuterated internal standards, extracted, derivatized, and analyzed on a Thermo Q Exactive HF Orbitrap mass spectrometer with Vanquish Horizon HPLC. Quantification was performed using targeted selected ion monitoring (t-SIM) with a mass tolerance of ±5 ppm and calibration against external standards. Chromatographic separation was achieved using a BEH C18 column with a 15-minute gradient and ammonium acetate/acetic acid buffer system.

Human ILC2 isolation
Phlebotomy was performed on healthy, non-asthmatic participants to recover 50 mL of blood from each donor. Human primary cells used in this study were obtained as de-identified samples under an IRB-approved protocol managed by collaborating investigators. Sex and/or gender information was not provided to the experimenters and therefore could not be incorporated into study design or analysis. As a result, sex-disaggregated analyses were not performed. Informed consent was obtained from all participants. Additional details about participant recruitment and exclusion are available in the reporting summary. PBMCs were isolated using SepMate tubes with Lymphoprep (StemCell). Red blood cells lysis was carried out with ammonium chloride-based RBC Lysis Buffer (Tonbo). Lineage negative cells were enriched with the Lineage Cell Depletion Kit, human (Miltenyi, 130-092-211) and stained for viability with the LIVE/DEAD Fixable Aqua Dead Cell Stain Kit (Invitrogen, cat: L34957) in PBS. Surface staining for ILC2 was carried out for 20 min in 3% FBS with anti-human lineage cocktail (Biolegend), α-CD45, α-CD127, and α-CRTH2. ILC2 were sorted on a BD FACS Symphony S6 cell sorter as Lineage-, CD45+, CD127+, CRTH2+.

Statistical analysis
All statistical analysis outside of QTL mapping and transcriptomic analysis was carried out in GraphPad Prism 10 software. Comparisons of two groups with assumed normal distributions were evaluated with a Student’s t-test. Comparisons of 3 or more groups with assumed normal distributions and two independent variables and were assessed with a two-way analysis of variance (ANOVA) with Tukey or Sidak’s multiple comparisons testing. Comparisons of 3 or more groups with assumed normal distributions and one independent variable and were assessed with a one-way analysis of variance (ANOVA) with Tukey or Sidak’s multiple comparisons testing. All measurements represent independent biological replicates unless otherwise indicated in figure legends; paired donor experiments are described as such. Exact sample sizes (n) for each experimental condition are provided in the corresponding figure legends. Non-transcriptomic data did not include covariates in statistical analyses. Data are presented as mean ± standard deviation (SD) unless otherwise specified. P values are reported in the figure legends where applicable. Effect sizes were not calculated. For human ILC2 experiments (Fig. 8B–F) with large variability between donors, the distribution of paired differences was assessed for normality using the Shapiro–Wilk test. When the differences were approximately normally distributed, significance was evaluated with a two-tailed paired Student’s t-test. When the Shapiro–Wilk test indicated non-normality, significance was instead evaluated with the Wilcoxon signed-rank test, a nonparametric test that evaluates whether the median difference between paired samples differs from zero while accounting for both the magnitude and direction of changes across donors. For Wilcoxon tests with small sample sizes (n≤7), p-values reflect the exact distribution of rank sums. All tests were two-sided, and p<0.05 was considered statistically significant. To evaluate consistent directional effects across donors in small-sample experiments (Fig. 8I–L), a one-sided binomial sign test was used to assess whether the number of donors showing an increase in IL-5 or IL-13 following co-treatment with AR420626 and gefitinib (compared to AR420626 alone) exceeded what would be expected by chance. For each donor, the maximum value observed across the two gefitinib concentrations was compared to the AR420626-alone condition. The test was performed with a null probability of 0.5 (i.e., 50% chance of increase), and significance was interpreted at α=0.05.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Rusznak et al. demonstrate in this manuscript that FFAR3 is a factor to expand lung ILC2 by aeroallergens by comparison CC030 and C57BL/6J strain mice showing high and low expansions and that FFAR3 does this by upregulating EGFR. Many interpretations in this article are unreasonable and the present data are not enough to draw these interpretations.\nMajor\n1. In Figure 1 and Figure 2, the authors have drawn interpretations about the mouse strains and the chromosome loci.\nHowever, they did it only based on the ILC2 numbers expanded by Alternaria extract. If they want to compare the ability to\nexpand ILC2, they should count the basal numbers of ILC2 and calculate the ratios of ILC2 expansion.\n2. We cannot exclude the possibility that the acquired immunity interferes ILC2 expansion in Alternaria extract-treated mice.\nIf the authors want to identify the ability of ILC2 to expand by itself, they should use Rag1/2-deficient mice to exclude\ninvolvement of the acquired immunity.\n3. There is no data that FFAR3 is responsible for the different numbers of ILC2 among mouse strains. To draw such\nconclusion, the authors have to stimulate or block FFAR3 in vivo using genetic or pharmacological methods leading to\nupregulation or downregulation of the ILC2 numbers, respectively. Moreover, the authors should examine expression of\nFFAR3 in lung ILC2 of each mouse strain and show that it is parallel with expanded numbers of ILC2 shown in Figure 1.\n4. Higher number of ILC2 in CC030 compared to C57BL/6J is detected only in lungs. However, there is no explanation\nabout it. Expression or behaviors of FFAR3 in other organs are different from those of lungs in CC030 mice?\n5. The authors have drawn an interpretation that CC030 mice have decreased apoptotic activity based on the result that\nactive caspase positive cells were decreased in CC030 mice compared to C57BL/6J mice. It is unreasonable and not\nenough. The authors have to estimate and compare apoptotic activities in both lung ILC2 from CC030 mice and C57BL/6J\nmice.\n6. The authors have drawn conclusion that CC030 mice have an ability to expand ILC2 by decreasing apoptotic activity. In\ncontrast, they show that FFAR3 expand ILC2 by upregulating EGFR. The action of EGF is cell proliferation, not inhibition of\napoptosis. These two interpretations are contradictory.\n7. The human data in Figure are just negative. These do not support the mouse data.\nMinor\n8. If the used dose of Alternaria extract for CAST/EiJ mice in Figure 1 is lethal, the authors should use a lower, not lethal,\ndose.\n9. In Figure 3K, there is no comparison with CC030.\n10. If C57BL/6J mice do not express FFAR3, the authors should use another mouse strain showing poorly expanded ILC2\nlike C57BL/6J mice.\n11. In Figure 6, the authors should show cell fraction data of alive and apoptosis cells.", "reviewer_2": "In the manuscript by M. Rusznak et al., the authors have undertaken an elegant approach to identify the free-fatty acid\nreceptor 3 (Ffar3/GPR41) as an important regulator of ILC2 cell behaviour.\nVia quantitative trait locus mapping to the collaborative Cross (CC) mouse panel, the authors identified a locus on Ch7,\nwhich was associated with ILC2 abundance following Alternaria extract challenge. Subsequent experiments pinpointed the\nfree-fatty acid receptor 3 (FFAR3) as the key mediator in reprogramming ILC2s. Finally, the authors propose that the FFAR3\nmediated effects occurred via an EGFR-dependent mechanism, and that FFAR3 may represent a potential therapeutic target\nfor Type 2 inflammation in humans.\nThe manuscript is well written and the figures clearly presented. Although, the data is generally supportive of the authors’\nconclusions, a few open points remain.\nFig1a, shows ILC2s from different collaborative Cross strains quantified as a percentage of live cells in the lung post\nAlternaria challenge. Considering that this model involves both eosinophil (EOS) and lymphocyte recruitment, which could\nsignificantly affect the relative percentages, please provide the ILC2 data as the total number of ILC2s in the lung.\nFurthermore, what were the baseline ILC2 levels in naïve mice across the different strains? Finally, if available, please\ninclude data on EOS, neutrophil and lymphocyte numbers post challenge. This would be important to distinguish ILC2-\nspecific changes from general immune cell recruitment.\nDo CC030 mice have any other response to Alternaria challenge e.g. neutrophil counts, or is the entire immune response to\nAlternaria repressed?\nPlease speculate on the reasons behind the lower expression of ST2 in CC030 ILC2, is there any relationship to the Ch.7\nILC2 Locus?\nBased on the authors hypothesis concerning the importance of FFAR3, and consequently EGFR signalling in ILC2 cells, it\nwould be expected that C57BL/6J ILC2 would not respond to AR420626, as they do not express Ffar3. To support this\nhypothesis, please provide data demonstrating the response (or lack thereof) of C57BL/6J ILC2s to AR420626 for key\nreadouts, such as type 2 cytokine levels (e.g., IL-5 and IL-13), cell numbers, proliferation, and apoptosis. This data will be\ncrucial in confirming the role of FFAR3 in mediating ILC2 responses through EGFR signaling.\nAdditionally, please confirm the absence of Ffar3 expression in C57BL/6J ILC2 by qPCR and ideally flow cytometry in Fig\n5DE.\nIt is unclear why the authors presented the data in in Fig 8 “as normalised to vehicle”, when all other figures give absolute\nvalues for all graphs. Please present this figure as absolute values.\nThe connection and data presentation between Fig 8F and Extended Data Figure 6 is currently confusing. It appears that\nDonor 4 From (Extended Data Figure 6) is also presented in Fig 8F the main manuscript, and was chosen for the main figure\nas it was the one donor from four that exhibited a similar increase in EGFR receptor expression in response to AR420626 as\nobserved in mouse ILC2. For transparency, please include in the main figure a summary graph showing the difference\nbetween EGFR levels between Vehicle and AR420626 after 6 days of culture.\nPlease show whether the AR420626 effect in human ILC2 is dependent on EGFR signalling.\nIn contrast to mouse ILC2, which increased total numbers in response to AR420626, human ILC2 responded with a\ndecrease. Please discuss the reasons behind this difference.\nIn the 2018 paper by Thio et al. (DOI: 10.1016/j.jaci.2018.02.032), the authors detail the role of short-chain fatty acids\n(SCFAs) in allergic airway inflammation and the induction of GPR41/FFAR3 in ILC2s by IL-33. This conflicts with the\nstatement in the current study regarding the lack of knowledge on FFAR3's role in regulating ILC2 biology in the lung.\nPlease revise the associated statements to accurately reflect the prior knowledge presented by Thio et al.\nFurthermore, the implications of Thio et al.'s findings should be thoroughly discussed in the context of the current study.\nNotably, the previous study did not observe a reduction in IL-13 and IL-5 levels when ILC2s were treated with the FFAR3\nagonist AR420626. This raises important questions about the regulatory mechanisms of FFAR3 in ILC2 function and its\npotential differential effects on cytokine production.\nMinor\nFrom the results, it is slightly unclear whether the Ch.7 ILC2 Locus was the only locus that exceeded the significance\nthreshold. Please clarify in the text.\nPlease clarify whether strains CC026 and F-A/J also possessed the Ch.7 ILC2 Locus.\nFig 7B please label the top10 up and down genes and not just Egfr\nPlease change “dose” to “concentration”, when talking about in vitro experiments.\nThe citation for Ref 35 is incomplete.\nIn Extended Data Figure 3, there is no concentration-dependent manner observed, please correct the phrasing or include\nadditional concentrations.", "reviewer_3": "In the manuscript “Genetic diversity of Collaborative Cross mice reveals FFAR3 as a target for ILC2 anti-inflammatory\nreprogramming,” Rusznak et al. propose a novel role for FFAR3 in regulating ILC2 responses. By examining genetically\ndiverse mouse strains, they identify a locus that influences ILC2 abundance in the lung following allergen exposure. Despite\nincreased ILC2 numbers, these cells show lower activation and diminished type 2 cytokine production. The study further\nhighlights FFAR3 as crucial for inducing a less inflammatory ILC2 state with enhanced IL-10 production. While these\nfindings are promising and the manuscript is broadly well-constructed, there are several critical issues and several points\nthat require further investigation to increase robustness of study:\n1. All experiments rely on one allergen (Alternaria alternata). Testing additional allergens (e.g., papain or HDM) would\nstrengthen manuscript and clarify whether this phenotype is general or unique to A. alternata.\n2. Because ILC2s in CC030 mice appear less responsive to IL-33, experiments should be conducted under diverse cytokine\nconditions, possibly excluding IL-33 or adding other stimuli to confirm whether the observed response (lower proliferation\nand cytokine production) depends on specific culture conditions.\n3. The study often reports small sample sizes (e.g., n=3) from what appear to be single experiments (not independent\nexperiments), raising concerns about statistical power. More independent experiments with larger n are needed, particularly\nfor critical conclusions drawn from subtle cell-number differences.\n4. Different mouse strains can have distinct microbiomes that influence ILC2 phenotypes, especially since FFAR3 responds\nto microbiome-derived fatty acids (FFAs such as acetate, butyrate, propionate act as ligands for this receptor). The authors\nshould detail backcrossing or husbandry methods used to control for these variables.\n5. Additional data on how FFAR3 agonism alters ILC2 phenotype—particularly regarding pathways related to IL-10—would\nstrengthen the study. Discrepancies between Figures 6 and 7 in IL-10 expression levels were also unclear.\n6. The claim that FFAR3 signaling maintains cell populations by inhibiting programmed cell death requires more\nmechanistic support, as the reported changes in caspase expression and viability appear modest.\n7. For the human ILC2 experiments, raw cell numbers and viability assessments at the endpoint should accompany fold-\nchange data. Moreover, it remains unclear whether IL-10 induction, observed in mice, extends to human ILC2s. This is\ncentral/key for the overall stated significance of FFAR3/possible therapeutic interventions proposed in discussion etc.\n8. To strengthen novelty, the authors could compare the transcriptional signature of CC030 ILC2s to known ILC210 profiles,\nincluding those in studies that link IL-10 production with altered metabolism. For example :\n• Seehus CR, Kadavallore A, Torre B, Yeckes AR, Wang Y, Tang J, Kaye J. Alternative activation generates IL-10 producing\ntype 2 innate lymphoid cells. Nat Commun. 2017 Dec 1;8(1):1900. doi: 10.1038/s41467-017-02023-z. PMID: 29196657;\nPMCID: PMC5711851.\n• Howard E, Lewis G, Galle-Treger L, Hurrell BP, Helou DG, Shafiei-Jahani P, Painter JD, Muench GA, Soroosh P, Akbari\nO. IL-10 production by ILC2s requires Blimp-1 and cMaf, modulates cellular metabolism, and ameliorates airway\nhyperreactivity. J Allergy Clin Immunol. 2021 Apr;147(4):1281-1295.e5. doi: 10.1016/j.jaci.2020.08.024. Epub 2020 Sep 6.\nPMID: 32905799.\n9. In vivo validation of targeting FFAR3 therapeutically would provide proof-of-principle of the authors’ suggestion that\ntargeting FFAR3 may mitigate allergic inflammation. Otherwise discussion needs to be tempered.\n10. With IL-10 production highlighted, testing other known IL-10–inducing factors would confirm FFAR3’s role in driving this\nphenotype. Do they also upregulate FFAR3? How do they compare in inducing IL-10 head-to-head with FFAR3 agonist?\n11. The potential co-expression of amphiregulin also remains an intriguing avenue for CRISPR or siRNA-based mechanistic\nstudies. Is amphiregulin and IL-10 co-expressed? Does knocking down amphiregulin decrease IL-10 production?\nCollectively, these points suggest that deeper mechanistic and translational data will strengthen the manuscript. Addressing\nthese concerns will provide a more robust foundation for the authors’ conclusion that FFAR3 is a key regulator of anti-\ninflammatory reprogramming in ILC2s."}, "author_response": "Rusznak et al.\nWe greatly appreciate the opportunity to have had our manuscript reviewed at Nature\nCommunications and want to thank the reviewers and editor for their very insightful and helpful\ncomments. We have performed the many additional experiments requested and believe that the\nmanuscript is much stronger as a result of the reviewers’ suggestions. We are grateful for the\nopportunity to submit this revised manuscript which we hope will now be of sufficient impact to\nwarrant publication in Nature Communications. Please let us know if there are further questions\nthat we can answer.\nReviewer 1\nRusznak et al. demonstrate in this manuscript that FFAR3 is a factor to expand lung ILC2 by\naeroallergens by comparison CC030 and C57BL/6J strain mice showing high and low\nexpansions and that FFAR3 does this by upregulating EGFR. Many interpretations in this article\nare unreasonable and the present data are not enough to draw these interpretations.\nWe appreciate the reviewer’s concern for some of the interpretations that we have drawn from\nthe data, and we are grateful for the opportunity to revise the manuscript to address these\nconcerns. We have significantly expanded on our original data, and we have made corrections\nto many of our interpretations highlighted below. We hope that our responses may offer some\nclarification on the claims and rationale presented in the manuscript.\nMajor Comments\n1.) In Figure 1 and Figure 2, the authors have drawn interpretations about the mouse strains\nand the chromosome loci. However, they did it only based on the ILC2 numbers expanded\nby Alternaria extract. If they want to compare the ability to expand ILC2, they should count the\nbasal numbers of ILC2 and calculate the ratios of ILC2 expansion.\nR1C1: The reviewer raises an important point regarding the quantitative phenotype that we are\nmapping in the experiment. The reviewer is correct that mapping ILC2 expansion would require\na ratio of ILC2 prevalence after aeroallergen challenge over ILC2 prevalence at baseline for\nevery strain. This phenotype would be very useful in describing the ability of ILC2s to expand\ntheir numbers in response to aeroallergen challenge; however, the phenotype that we are\ndescribing in this manuscript is the prevalence of ILC2s in the setting of challenge, not their\ndegree of expansion. For this reason, we believe that our quantitative phenotype of choice is\nvalid for the conclusions that we have drawn. ILC2 prevalence after challenge is a quantitative\nreadout that is a function of both ILC2s at baseline and expansion in response to challenge. Our\nrationale for using this phenotype was that we could perform our mapping on a single value\nfrom a single batch of experiments that reflects the total degree of an immune cell’s prevalence\nin the setting of challenge. We recognize that the high ILC2 prevalence phenotype that we\ndescribe in CC030s is one that is also present at baseline, and we further explore the steady-\nstate differences in ILC2s between CC030 and C57BL/6J in Figure 3.\nWe agree that the expansion ratio is a critically important quantitative phenotype, and while it is\nnot the one that we mapped, it would be very helpful to present. We now provide the ILC2\nexpansion ratios for the 42 strains for which we were able to obtain baseline and post-challenge\nILC2 phenotype data. Due to strain availability for the second batch, we could not calculate\nexpansion ratios for all 48 strains. We have added these new data in Supplementary Table 1,\nand we have added discussion of these data to the results section with the following text:\n“Strain differences persisted when quantifying ILC2s after challenge with total cell numbers in\nthe lung (Supplementary Figure 2.A), and we confirmed strain variability in ILC2 prevalence and\nnumber in the CC mice at baseline (Supplementary Figure 2.B&C). Expansion ratios for each\nstrain with naïve and challenged ILC2 quantification data are presented in Supplementary\nTable 1.”\n2.) We cannot exclude the possibility that the acquired immunity interferes ILC2 expansion in\nAlternaria extract-treated mice. If the authors want to identify the ability of ILC2 to expand by\nitself, they should use Rag1/2-deficient mice to exclude involvement of the acquired immunity.\nR1C2: The reviewer highlights an important limitation of using Collaborative Cross strains to\nassess innate immune phenotypes. The gold standard mouse model for attributing a phenotype\nto the innate immune response is the Rag1/Rag2-/- mouse, where contribution from the adaptive\nimmune system can be confidently excluded due to the lack of T and B cells. Given that the\nexperiments presented in this manuscript relied on using 48 strains of Collaborative Cross mice\nto perform QTL mapping, it would have been unfeasible to generate Rag1/Rag2-/- on every one\nof the 48 different strain backgrounds. Further, introducing BALB/c or C57BL/6 genes from the\nRag1/Rag2-/- mice that were crossed with the Collaborative Cross strains would have introduced\na great deal of genetic heterogeneity into the experiment which would have made the\nexperiment impossible as it would have required many generations of breeding to replicate the\nCollaborative Cross genotypes that we did study.\n3.) There is no data that FFAR3 is responsible for the different numbers of ILC2 among mouse\nstrains. To draw such conclusion, the authors have to stimulate or block FFAR3 in vivo using\ngenetic or pharmacological methods leading to upregulation or downregulation of the ILC2\nnumbers, respectively. Moreover, the authors should examine expression of FFAR3 in lung ILC2\nof each mouse strain and show that it is parallel with expanded numbers of ILC2 shown in\nFigure 1.\nR1C3: The most direct way to demonstrate Ffar3’s causal role in the high-abundance ILC2\nphenotype in CC030 mice would have been to knockout the gene on the CC030 background.\nWe attempted to create a global Ffar3-/- mouse with a CRISPR/Cas9 approach. Despite\neffective embryo recovery, editing, and transfer into 5 pseudo-pregnant females, only 3\ndeceased pups were recovered, all of which were positive for the targeted Ffar3 deletion. These\nresults suggest that global deletion of this gene might be embryonically lethal on the CC030\nbackground. A full report of this gene deletion attempt is provided in Supplementary Data 5.\nWe thank the reviewer for your suggestion that Ffar3 expression should be evaluated in all\nstrains to correlate expression with ILC2 abundance. There is a wide range of ILC2 abundance\nbetween the CC strains that we phenotyped for our study, however, our results do not suggest\nthat the entirety of this variation can be attributed to the Ch.7 ILC2 Locus or Ffar3. While the\nCh.7 ILC2 Locus was the only QTL that reached significance, the presence of other peaks\nsuggests that its effect size is not enough to explain the entirety of the variation observed\nbetween the strains. A hypothetical QTL is present at a locus when the phenotype of interest is\naffected by different genotypes at the locus (i.e. founder haplotype). At a genomic locus that\ndoes not regulate the phenotype, that locus may be inherited from any of the founders and it will\nnot affect said phenotype. At a locus that does regulate the phenotype (QTL), one or more of\nthe founder genotypes will have either a statistically significant positive or negative contribution\nto that trait.\nOur founder haplotype effect analysis from Figure 2.B describes how the different founder\nhaplotypes contribute to the presence of a QTL for ILC2 abundance at the Ch.7 ILC2 Locus.\nThe CAST/EiJ founder haplotype has a strongly positive QTL effect, while all other founder\nhaplotypes have very weak positive or negative contributions. This QTL is driven by the\nCAST/EiJ founder haplotype versus other founder haplotypes that behave similarly, meaning\nthat the CAST/EiJ haplotype is responsible for the majority of the variation in ILC2 abundance\nthat can be attributed to this QTL. This result can be interpreted as: inheriting the Ch.7 ILC2\nLocus from CAST/EiJ will increase a founder strain’s ILC2 abundance compared to inheriting\nthat locus from any other founder strain. As most of the 48 strains that we phenotype do not\nhave the CAST/EiJ haplotype at the QTL, the locus is not contributing meaningfully to their ILC2\nabundance. Yet, there is a large phenotypic range even amongst the strains with other\nhaplotypes, suggesting that there are other sources of phenotypic variability outside of locus,\nwhich is to be expected given the large number of genes that are known to regulate ILC2\nbehavior.\nIn summary, it would be accurate to say that the CAST/EiJ founder haplotype at the Ch.7 ILC2\nLocus positively contributes to ILC2 abundance, and we suggest that the most relevant\nconsequence of inheriting the CAST/EiJ haplotype of the Ch.7 ILC2 Locus is higher Ffar3\nexpression. This would mean that higher Ffar3 expression positively contributes to ILC2\nabundance, but a majority of the variation between the strains in ILC2 abundance is not\nattributable to our QTL. This is demonstrated by the fact that the 7 other founder haplotypes had\nminimal positive or negative contribution to the QTL effect in Figure 2.B, meaning that for the\nCC strains with the other 7 haplotypes at the Ch.7 ILC2 Locus, the specific haplotype that they\ninherit (WSB/EiJ vs. NOD/ShiLtJ etc.), does not make much of a difference with respect to the\nQTL. However, there is still substantial variation between these other strains with different\nhaplotypes at the locus. Therefore, while we assert that Ffar3 regulates ILC2 abundance, the\nother CC strains with different founder haplotypes at the Ch.7 ILC2 Locus would not necessarily\nhave their ILC2 abundance correlating with their Ffar3 expression, as the variation in these\nstrains is explained by other genetic factors.\n4.) Higher number of ILC2 in CC030 compared to C57BL/6J is detected only in lungs. However,\nthere is no explanation about it. Expression or behaviors of FFAR3 in other organs are different\nfrom those of lungs in CC030 mice?\nR1C4: We appreciate the reviewer inquiring about what may be driving the elevated numbers of\nILC2s in the lung compared to other tissues. To assess whether Ffar3 expression differences\nmay be responsible for the observed difference in ILC2 abundance, we isolated ILC2s from\nlung, bone marrow, adipose tissue, and colon by flow cytometry and isolated RNA. We then\ncreated cDNA libraries from these samples for analysis via qPCR with Gapdh as a\nhousekeeping gene.\nWe have added these data in Figure 4.T and discuss them in the results section. There were no\nsignificant differences in Ffar3 expression between ILC2s from any of the 4 tissues, although\nthere may be a trend towards lower expression in colonic ILC2s compared to lung ILC2s. We\nmay conclude from these data that Ffar3 expression alone is not able to account for the\ndifferences in these tissues. Despite comparable levels of gene expression, local differences in\nligand concentration (SCFAs) may significantly influence the degree of FFAR3 signaling that is\noccurring at baseline, which may still contribute to the observed differences in ILC2 abundance.\n5.) The authors have drawn an interpretation that CC030 mice have decreased apoptotic activity\nbased on the result that active caspase positive cells were decreased in CC030 mice compared\nto C57BL/6J mice. It is unreasonable and not enough. The authors have to estimate and\ncompare apoptotic activities in both lung ILC2 from CC030 mice and C57BL/6J mice.\nR1C5: We appreciate the reviewer’s concern that the current data regarding strain differences in\napoptosis lack mechanistic depth. To address this concern, we have employed an Annexin V\nand propidium iodide-based apoptosis detection assay to more accurately detect and quantify\napoptosis in our ILC2 culture. Using this method, we can identify 3 distinct ILC2 populations. We\nterm Annexin V- and PI- cells as healthy ILC2s, Annexin V+ and PI- cells as early apoptotic\nILC2s, and Annexin V+ and PI+ cells as late apoptotic/dead ILC2s. With this method, we have\nanother way of quantifying apoptosis and differentiating between early and late phases of\nprogrammed cell death. We have added these experiments in Figure 4 I-K and have discussed\nthem in the results section.\nWe conclude from our new data in Figure 4 that CC030 ILC2s indeed have greater viability\nduring cell culture and resist progressing through apoptosis to cell death. CC030 mice have\nsignificantly higher overall viability with a decreased fraction of late apoptotic/dead cells\ncompared to C57BL/6J, but they also have an increased percentage of early apoptotic cells\ncompared to C57BL/6J. A possible explanation for this pattern of more early apoptotic cells and\nfewer late apoptotic cells is that apoptosis may become arrested in its early stages in CC030\nILC2s. While these programmed cell death processes are initiated to enough of a degree that\nphosphatidyl serine begins to lose its asymmetry and it can be detected with Annexin V staining,\nit is possible that this apoptotic process does not progress further towards complete cell death,\nor apoptotic change in these early stages may be reversed. In either situation, you would have a\ncomparatively large population of less fit or hypofunctional ILC2s that are less capable of\ncarrying out Type 2 effector function and may assume an alternative, less inflammatory effector\nstate.\n6.) The authors have drawn conclusion that CC030 mice have an ability to expand ILC2 by\ndecreasing apoptotic activity. In contrast, they show that FFAR3 expand ILC2 by upregulating\nEGFR. The action of EGF is cell proliferation, not inhibition of apoptosis. These two\ninterpretations are contradictory.\nR1C6: The reviewer raises an important concern regarding the somewhat unorthodox manner in\nwhich EGFR is behaving in our system. We agree with the reviewer that EGFR signaling on\nmany cell types, especially with EGF as the ligand, contributes to cell proliferation. There is\nevidence, however, that amphiregulin signaling through EGFR on lymphocytes does not have a\nproliferative effect and instead affects other cellular functions like cytokine secretion. Zais et al.\nshow in their 2018 manuscript that treatment of EGFR+ Tregs with 100ng/mL of amphiregulin\n(an EGFR ligand) significantly enhanced their suppressive function, but it had no effect on the\ntotal number of ILC2s.1 This published data is provided below:\nWe suggest in our manuscript that amphiregulin acting through EGFR on ILC2s is contributing\nto primarily non-proliferative cell changes. Florentin et al. published data which demonstrate that\namphiregulin signaling through EGFR in endothelial cells decreases apoptosis and cell death.2\nSurprisingly, endothelial cells with cell-specific deletion of Egfr experienced a greater degree of\nproliferation compared to WT controls, suggesting that the amphiregulin-EGFR signaling axis\nmay exert a broader range of downstream effects than previously appreciated. Similarly, the\nauthors demonstrate that amphiregulin decreases expression of the pro-apoptotic factor BCL2-\nassociated agonist of cell death (BAD) in a concentration dependent manner, offering some\nevidence as to how amphiregulin may be suppressing apoptosis.\nWhile there is published evidence to support the plausibility of our proposed function of\namphiregulin and EGFR in our system, we concur with the reviewer that we do not directly\nimplicate EGFR signaling in apoptosis resistance with our current data. We have adjusted the\nlanguage in the manuscript to acknowledge this limitation:\n“Finally, we suggest that FFAR3 signaling acts through an EGFR dependent mechanism, and\nwe show that FFAR3 signaling increases apoptosis resistance, but our data do not directly\nimplicate EGFR signaling as a mediator of apoptosis resistance in ILC2s.”\n7.) The human data in Figure are just negative. These do not support the mouse data.\nR1C7: We thank the reviewer for the concern about the human data not fully reflecting the\nmouse data. There are important elements of the murine ILC2 response to FFAR3 signaling that\nare conserved in humans, but there are some notable differences as well. An important\nconsideration is that the ILC2s used for the mouse studies were isolated from the lung, while the\nhuman ILC2s were isolated from PBMCs. We have added data in Figure 8.B-L along with\ntextual descriptions to the results section to address these concerns.\nMost importantly, we see that AR420626 treatment decreases Type 2 cytokine production in\nhuman ILC2s like in mouse. There is a large degree of heterogeneity, but we also observe an\nincrease in EGFR expression in ILC2s from a subset of donors. Additionally, we see that\ninhibition of EGFR with gefitinib trended towards a reversal of the AR420626-mediated\ndecrease in IL-5 and IL-13. Collectively, these suggest that FFAR3 signaling is acting to\ndecrease the Type 2 cytokine producing capability of these ILC2s, and there is data to suggest\nthat this might be occurring through an EGFR-mediated mechanism.\nDifferences between the murine and human phenotypes are that FFAR3 agonism did not\nexpand the number of human ILC2s, it did not affect viability, and it did not induce IL-10\nproduction. AR420626 did not significantly affect ILC2 number at 1μM or 5μM, but it reduced\nILC2 numbers at 10μM. Notably, AR420626 at 5μM had no significant effect on cell number, but\nit significantly reduced Type 2 cytokines, suggesting that there is a negative effect on IL-5 and\nIL-13 production out of proportion to the effect on cell number. Our mouse findings showed that\nFFAR3 agonism increases ILC2 viability, but our human cultures reveal consistently high\ndegrees of viability in both treatment groups. It is reassuring that while a decrease in ILC2\nnumber is observed with AR420626 treatment, there is no decrease in viability even at the\nhighest doses, suggesting against non-specific drug toxicity. Finally, we were unable to\nreproducibly induce IL-10 production in human ILC2s with AR420626 treatment. From the new\nmechanistic experiments that we present in Figure 6, we assert that the effect of FFAR3\nagonism on Type 2 cytokines and IL-10 are uncoupled from one another, meaning that they are\nseparate processes and the decrease in IL-5 and IL-13 is not dependent on IL-10. It is possible\nthat FFAR3 signaling reduces Type 2 cytokines in both species, but its effect on IL-10 is not\nconserved. It should be emphasized that the human ILC2s were derived from PBMCs, meaning\nthat there are many differences between the human and mouse ILC2s in our study that are\nattributable to their different tissues of origin. It is possible that the induction of IL-10 in ILC2s\nwith FFAR3 signaling is a phenotype that is specific to ILC2s imprinted by the pulmonary\nenvironment, and we were unable to capture this conservation because we were using ILC2s\nfrom a different human tissue.\nWe have addressed this limitation of our study with the following added text in the discussion:\n“Another limitation is that there are notable differences in the response to FFAR3 signaling\nbetween ILC2s from our mouse and human experiments, especially relating to responses in cell\nnumber and IL-10 production. It is possible that this incomplete degree of phenotypic\nconservation can be explained by the different tissue sources of ILC2s for the experiments; our\nmouse experiments used lung ILC2s while our human experiments used ILC2s from the\nperipheral blood. Additionally, some of our mechanistic data supports the notion that the\ndownstream responses of FFAR3 signaling in ILC2s (decreased Type 2 cytokines, increased\ncell number through apoptosis resistance, and increased IL-10) are uncoupled from one\nanother, and different arms of this response may be variably conserved across species and\nILC2 tissue types.”\nMinor Comments\n8.) If the used dose of Alternaria extract for CAST/EiJ mice in Figure 1 is lethal, the authors\nshould use a lower, not lethal, dose.\nR1C8: We agree with the reviewer that having data for the CAST/EiJ founder strain in the initial\nphenotyping experiment would have been helpful for drawing conclusions, and it was\nunfortunate that they did not survive our 4-day challenge protocol. We obtained further batches\nof CAST/EiJ mice to characterize their ILC2 responses in a modified Alt Ex challenge model that\nwould not cause excessive mortality. We modified the quantity of Alt Ex per intratracheal\nchallenge through multiple trial experiments, but CAST/EiJ mice continued to die in our 4-day\nmodel. Given that mice tended to die immediately after challenge, we concluded that the\nmortality was likely due to an acute complication of the challenge, and decreasing challenge\nfrequency may improve survival. We restricted our challenges to 8μg of Alt Ex on Day 0 and\nDay 2, harvesting the mice on Day 4, and we found that this dose was able to induce detectable\ncytokine production without significant mortality.\nBecause our initial Alt Ex challenge experiment for Figure 1.A was completed with the 4-day\nmodel, these results could not be incorporated into the analysis. However, it is interesting to\ninformally compare the ILC2 abundance in CAST/EiJ mice after a reduced-intensity challenge\nalongside C57BL/6J and CC030 ILC2 abundances from Figure 1.A. The CAST/EiJ founder\nstrain has similar ILC2 abundance to CC030 mice and significantly increased ILC2 abundance\ncompared to C57BL/6J. These data are presented below in Response to Reviewers Figure\n1.A While statistical significance is evaluated between groups, we caution direct comparison\ndue to the different challenge models used.\nResponse to Reviewers Figure 1. (A) Comparison of ILC2 abundance between\nC57BL/6J, CC030, and CAST/EiJ females after Alt Ex challenge. C57BL/6J and CC030\ngroups were challenged with 5μg of Alt Ex daily for 4 days, while CAST/EiJ mice were\nchallenged for every other day for a total of 2 challenges. Significance was assessed\nwith a one-way ANOVA with Tukey’s multiple comparison test. n=5 for C57BL/6J and\nCC030, n=4 for CAST/EiJ. Result is representative of one experiment. ns = not\nsignificant. ** = p<0.005.\n9.) In Figure 3K, there is no comparison with CC030.\nR1C9: We thank the reviewer for highlighting an aspect of our gene-set enrichment analysis that\ncould have been clearer in the text. The gene sets in the tree plot in Figure 3.K represent cell\nsignaling pathways that were positively enriched in C57BL/6J ILC2s compared to CC030,\nmeaning that the comparison to CC030 is inherent to the analysis.\nWe have included another tree plot in Figure 3.K that shows gene sets which were positively\nenriched in CC030 compared to C57BL/6J. It is interesting to note that the pathways most\nupregulated in CC030 ILC2s center around cell-to-cell interactions and membrane-based ion\ntransport. This could suggest that CC030 ILC2s are engaging in some sort of homotypic\ninteractions that may be affecting their collective phenotype.\nWe now have the following text to clarify the two analyses:\n“To capture other differences between these cells in an unbiased manner, we performed\nhierarchical clustering of the top 30 Gene Ontology: Biological Processes pathways most\nenriched in C57BL/6J and CC030 ILC2s, ranked by normalized enrichment score (Figure 3.K).\nThe most enriched gene sets in C57BL/6J ILC2s cluster together as pathways related to protein\ntranslation, mitochondrial complex assembly, RNA processing, ribosome biogenesis, and\noxidative phosphorylation. This GSEA result emphasizes that C57BL/6J ILC2s are more\ntranscriptionally, translationally, and metabolically active compared to CC030 ILC2s, suggesting\ntheir increased general readiness for activation and the propagation of Type 2 inflammation. In\ncontrast, CC030 ILC2s are most transcriptionally enriched for pathways related to cell-cell\ninteractions, cell membrane ion transport, and detection of chemical stimuli.”\n10.) If C57BL/6J mice do not express FFAR3, the authors should use another mouse strain\nshowing poorly expanded ILC2 like C57BL/6J mice.\nR1C10: C57BL/6J mice were chosen as a reference strain due to the C57BL/6J founder\nhaplotype’s negative QTL effect (Figure 2.B) at the Ch.7 ILC2 Locus. By picking the founder\nstrain rather than a recombinant strain, we can be certain that the entirety of the genome is\nderived from the C57BL/6J founder. We found in our initial experiments that C57BL/6J mice did\nnot express Ffar3 in their ILC2s, but it would have been difficult to know which of the low ILC2\nabundance strains express Ffar3 on their ILC2s and which ones don’t. As discussed in the\nresponse to comment #3, it is not guaranteed that Ffar3 expression would correlate with ILC2\nabundance for strains at the low end of the spectrum.\n11.) In Figure 6, the authors should show cell fraction data of alive and apoptosis cells.\nR1C11: We have included apoptosis data for Figure 5.N-P with the Annexin V/PI staining\nprotocol that allows us to differentiate between early apoptotic (live) and late apoptotic (dead)\ncells.\nReviewer 2\nIn the manuscript by M. Rusznak et al., the authors have undertaken an elegant approach to\nidentify the free-fatty acid receptor 3 (Ffar3/GPR41) as an important regulator of ILC2 cell\nbehaviour.\nVia quantitative trait locus mapping to the collaborative Cross (CC) mouse panel, the authors\nidentified a locus on Ch7, which was associated with ILC2 abundance following Alternaria\nextract challenge. Subsequent experiments pinpointed the free-fatty acid receptor 3 (FFAR3) as\nthe key mediator in reprogramming ILC2s. Finally, the authors propose that the FFAR3\nmediated effects occurred via an EGFR-dependent mechanism, and that FFAR3 may represent\na potential therapeutic target for Type 2 inflammation in humans.\nThe manuscript is well written and the figures clearly presented. Although, the data is generally\nsupportive of the authors’ conclusions, a few open points remain.\nWe appreciate the reviewer’s enthusiasm for the work presented in the manuscript, and we are\nthankful for the opportunity to address the reviewer’s concerns with additional experiments and\ndiscussion. The reviewer’s recommendations have substantially increased the robustness and\nimpact of our study.\nMajor Comments\n1.) Fig1a, shows ILC2s from different Collaborative Cross strains quantified as a percentage of\nlive cells in the lung post Alternaria challenge. Considering that this model involves both\neosinophil (EOS) and lymphocyte recruitment, which could significantly affect the relative\npercentages, please provide the ILC2 data as the total number of ILC2s in the lung.\nFurthermore, what were the baseline ILC2 levels in naïve mice across the di:erent strains?\nR2C1: We appreciate the reviewer’s insightful comments regarding our use of ILC2 abundance\nas our quantitative phenotype and some of the limitations of this choice. It is an important point\nthat these ILC2 percentages may be increased or decreased based on changes in the relative\nabundance of other immune cells after Alternaria challenge. To address this concern, we have\nprovided the total numbers of ILC2s for each of the strains presented in Figure 1.A. These total\nILC2 counts are derived from the same experiment, and they are presented in the new\nSupplementary Figure 2.A. While CC030 mice remain the second highest strain when ranked\nby total ILC2s, there are noticeable differences in the rank of other strains throughout the range.\nBecause of these differences, it is important to emphasize that the quantitative phenotype to\nwhich we associated the Ch.7 ILC2 Locus was ILC2 abundance (percentage) after Alt Ex\nchallenge. While we go on to demonstrate a broader effect of the QTL and Ffar3, the mapping\nportion of this study is specifically relevant to abundance calculated by percentage.\nWe further thank the reviewer for inquiring about the ILC2s present at baseline throughout all of\nour strains. A limitation of our mapping study is that it only takes into account ILC2 abundance\nafter challenge. This single phenotype is useful for comparing the post-activation numbers of\nILC2s, but quantifying ILC2s in naïve mice adds important insight into differences between their\nbaseline states. This is especially important as our data in Figure 3 suggest that many of the\nphenotypic differences between C57BL/6J and CC030 ILC2s are present at baseline.\nWe have provided the baseline ILC2 percentages and total numbers for 52 strains of the\nCollaborative Cross in Supplementary Figure 1.B&C. Due to the challenged and naïve\nexperiments being performed at separate times, the availability of strains for acquisition from the\nUNC Systems Genetics Core was different, and so we could not obtain naïve ILC2 data for the\nexact same selection of strains presented in Figure 1.A. Nonetheless, there is a robust range of\ndifferences between strains in both ILC2 abundance and total number at baseline. It is\ninteresting to note that CC030 mice again have the second highest ILC2 number, but they are\nnearly identical to the highest strain. These results support our conclusions from Figure 3 that\nthe high ILC2 abundance present in CC030 is largely driven by a phenotype that is present at\nbaseline.\n2.) Finally, if available, please include data on EOS, neutrophil and lymphocyte numbers post\nchallenge. This would be important to distinguish ILC2-specific changes from general immune\ncell recruitment.\nR2C2: We agree with the reviewer that it would be valuable to include data on the different\ntypes of immune cells that are induced by Alt Ex challenge, as these likely have significant\nvariation between the tested CC strains. Regrettably, we did not quantify these other cell types\nin our challenge experiment from Figure 1. We have repeated our Alt Ex challenge experiment\nto more comprehensively profile the differences in immune response between CC030 mice and\nC57BL/6J to short-course allergen challenge. These data address the reviewer’s third comment\nand are discussed in detail below in section R2C3.\n3.) Do CC030 mice have any other response to Alternaria challenge e.g. neutrophil counts, or is\nthe entire immune response to Alternaria repressed?\nR2C3: This is an important question posed by the reviewer regarding the global differences in\nthe immune response of CC030 to allergen challenge. We demonstrate that CC030 mice have\nbroadly diminished innate Type 2 inflammatory response, evidenced by their decreased levels\nof Type 2 cytokines in vivo, decreased eosinophilia, and diminished Type 2 effector function in\ncultured ILC2s compared to C57BL/6J. While the Type 2 response is suppressed, it is possible\nthat these mice compensate by driving other types of inflammation. We investigated the immune\nprofiles of CC030 and C57BL/6J mice at baseline and in response to both Alternaria extract\nchallenge as well as papain. These new data are provided in Supplementary Figure 5.\nIn the naïve state, we confirmed the increased number of ILC2s in CC030 compared to\nC57BL/6J, and we further examined differences in lymphocyte subsets. There was no significant\ndifference in total B cells between the two strains, but CC030 mice did have more total CD4+\nT cells and specifically Tregs, while C57BL/6J had more CD8+ T cells. In the BAL fluid, CC030\nmice had more total macrophages at baseline compared to C57BL/6J. While C57BL/6J had\nvirtually no cells other than macrophages present on the differential, CC030 mice had\ndetectable neutrophils in the BAL without any allergen challenge (Supplementary Figure 5.B).\nAllergen challenge revealed a broadly reduced Type 2 inflammatory response with Alternaria for\nCC030, but this difference was less appreciable with papain (Supplementary Figure 5.C).\nInterestingly, there were no differences between strains in papain-induced inflammation except\nin that CC030 mice had a greater number of total CD4+ cells and neutrophils compared to\nC57BL/6J. With Alternaria, however, CC030 mice exhibited a greater number of total B cells,\nCD8+ T cells, CD4+ T cells, and neutrophils. CC030 mice had significantly fewer eosinophils,\nwhich aligns with the strain’s lower ILC2 expansion ratio, IL-5, and IL-13.\nIt is clear from this experiment that the innate Type 2 response in CC030 mice is suppressed,\nbut it does not appear that the entire immune response to Alternaria is inhibited. Allergen-\nchallenged CC030 mice favor a profile of increased lymphocytes and neutrophils, suggesting\nthat this may be a compensatory response in a strain-specific phenotype where Type 2\nimmunity is blunted. Just as the increased ILC2 phenotype is present in the CC030 mice at\nbaseline, the neutrophilic predominance is similarly present in the absence of allergen\nchallenge.\n4.) Please speculate on the reasons behind the lower expression of ST2 in CC030 ILC2, is\nthere any relationship to the Ch.7 ILC2 Locus?\nR2C4: We are grateful to the reviewer for bringing attention to the relationship between ST2 and\nthe Ch.7 ILC2 Locus. As the reviewer mentioned, CC030 mice have significantly lower\nexpression of ST2 at baseline compared to C57BL/6J. Our assertion throughout the manuscript\nis that much of the CC030 phenotype, especially related to diminished Type 2 inflammatory\npotential, can be attributed to the Ch.7 ILC2 Locus, and specifically, Ffar3. On the reviewer’s\nrecommendation, we assessed if ST2 expression was regulated by the locus through agonism\nof FFAR3. This new data is provided in Figure 5.F-H.\nWe were encouraged to find that FFAR3 agonism with AR420626 in cultured CC030 ILC2s\ndrastically decreased ST2 expression. The total percentage of ILC2s that were ST2+ decreased\nmore than 3-fold, and the MFI of ST2 on ST2+ positive ILC2s also decreased more than 2.5-\nfold. It was intriguing that ST2 expression, another feature of ILC2 inflammatory reactivity, was\ninhibited with FFAR3 modulation. This may explain the decrease in IL-5 and IL-13 observed with\nAR420626 treated ILC2s, and it is another way in which FFAR3 agonism mimics the observed\nphenotype in CC030 mice. The precise mechanism of how FFAR3 signaling regulates ST2\nexpression would be an interesting avenue for further investigation.\n5.) Based on the authors hypothesis concerning the importance of FFAR3, and consequently\nEGFR signalling in ILC2 cells, it would be expected that C57BL/6J ILC2 would not respond to\nAR420626, as they do not express Ffar3. To support this hypothesis, please provide data\ndemonstrating the response (or lack thereof) of C57BL/6J ILC2s to AR420626 for key readouts,\nsuch as type 2 cytokine levels (e.g., IL-5 and IL-13), cell numbers, proliferation, and apoptosis.\nThis data will be crucial in confirming the role of FFAR3 in mediating ILC2 responses through\nEGFR signaling. Additionally, please confirm the absence of Ffar3 expression in C57BL/6J ILC2\nby qPCR and ideally flow cytometry in Fig 5DE.\nR2C5: The reviewer highlights an important aspect of the manuscript’s claim regarding the\nstrain differences in ILC2s that contributed to our selection of Ffar3 as our primary gene\ncandidate. In our initial RNA-sequencing experiment, we observed that C57BL/6J ILC2s, which\nhad very low numbers in the lung at baseline in the 1-3x103 range, did not express Ffar3. Given\nthat C57BL/6J ILC2s did not express Ffar3 in the naïve state, we hypothesized that they would\nnot respond to AR420626 treatment, and we performed new experiments in Supplementary\nFigure 8 to test this hypothesis. We isolated ILC2s from pooled C57BL/6J mice (lungs of four\nmice were combined for one biological replicate), and we cultured them with IL-33, TSLP, and\nIL-2 +/- 10μM AR420626 in paired biological replicates.\nThere was no significant difference between vehicle and AR420626 treated C57BL/6J ILC2s in\nIL-5 or IL-13 in the supernatant, total ILC2s, total live ILC2s, early apoptotic percentage, or late\napoptotic/dead percentage (Figure 8.A-G). In a separate experiment, there was no difference in\nproliferation between vehicle and AR420626 treated ILC2s 48 hours after culture in CFSE\ndilution (Figure 8.H-J). The cytokine and cell number results support the conclusion that\nC57BL/6J ILC2s do not express Ffar3 at baseline, and they are therefore unresponsive to\nFFAR3 signaling. We would not expect the ILC2s to respond with changes in proliferation even\nif FFAR3 signaling was occurring, given that our CC030 culture data showed no difference in\nCFSE dilution with FFAR3 agonism.\nWe have added the following text to the manuscript to describe these findings: “We observed a\nlack of Ffar3 expression in C57BL/6J ILC2s in our initial sequencing experiment, and we\ntherefore expected C57BL/6J ILC2s to have diminished or no response to FFAR3 agonism. We\ncultured C57BL/6J ILC2s according to our model in Figure 5.A and did not observe a decrease\nin IL-5 or IL-13, a difference in ILC2 number, or an effect on apoptosis. (Supplementary Figure\n8.A-G). Similarly, AR420626 treatment had no effect on C57BL/6J ILC2 proliferation, measured\nby CFSE dilution (Supplementary Figure 8.H-J).”\nWe were unable to perform qPCR on these ILC2s, as the low cell yields allowed only for cell\nculture, and we felt that the functional assays were more important to address the reviewer’s\nquestion. To support our claim that C57BL/6J ILC2s do not express Ffar3 in the naïve state, we\nhave compiled data from 4 other published RNA-sequencing data sets that isolated RNA from\nC57BL/6J ILC2s. We were careful to ensure that these data were collected on ILC2s that were\nnot pre-expanded in vivo, cultured, or activated in any way. We have presented the normalized\nread counts for Ffar3 from Yang et al. (GSE125584), Langlais et al. (GSE180075), Ricardo-\nGonzales et al. (GSE117568), and Huang et al. (GSE237815) alongside our data in Response\nto Reviewers Figure 2. Ffar2 expression, which we found to be present in our experiment, has\nbeen included as a reference for each of the other datasets. The other experiments found zero\n(Yang et al. & Langlais et al.) or very minimal (Ricardo-Gonzales et al. (0, 0, 0, 3.3, 10.9) &\nHuang (0, 0.07) Ffar3 expression on C57BL/6J ILC2s.\nResponse to Reviewers Figure 2. Normalized read counts of Ffar2 and Ffar3 in RNA\nfrom pre-published data of non-stimulated, non-cultured C57BL/6J lung ILC2s isolated\nby flow cytometry. Read counts were accessed and downloaded from the NCBI Gene\nExpression Omnibus. Studies were included where bulk RNA-sequencing was\nperformed on ILC2s isolated from the lungs of C57BL/6J mice without any in vivo\nexpansion (e.g. IL-33 administration, allergen challenge) or culture (e.g. in vitro\nstimulation with cytokines). Ffar2 normalized read counts are provided as a reference.\nFinally, we were unable to reliably detect FFAR3 protein with flow cytometry. Unfortunately,\nthere are no monoclonal antibodies raised against murine FFAR3 that have been appropriately\nvalidated in flow cytometry to our knowledge. We attempted staining FFAR3 with a polyclonal\nantibody (ThermoFisher Cat: FFAR3-FITC) conjugated to FITC previously optimized for\nimmunofluorescence, and a primary human antibody (ThermoFisher Clone: 1D10B7, Cat:\n66811-1-IG). We were unable to detect any positive signal in a heterogeneous lung tissue\nsample with the polyclonal antibody, and we could not optimize our human antibody, as our\navailable secondary antibodies bound non-specifically to the other antibodies that we used for\nsurface staining to identify ILC2s.\n6.) It is unclear why the authors presented the data in in Fig 8 “as normalised to vehicle”, when\nall other figures give absolute values for all graphs. Please present this figure as absolute\nvalues.\nR2C6: We thank the reviewer for pointing out that our initial mode of presentation for these data\nwas unclear. We had initially chosen this format given the large degree of variability in\nexpansion and cytokine production of human ILC2s between subjects. We noted that the\nmagnitude of the effect of AR420626 was quite consistent between individual donors, but the\nindividual variability made the data difficult to interpret.\nWe have added to the number of replicates (donors) for our AR420626 treatment experiments in\nFigure 8.B-F, and we now present all of these data with absolute values on log scales to more\nclearly show the data while preserving the natural variability that exists between human\nsamples.\n7.) The connection and data presentation between Fig 8F and Extended Data Figure 6 is\ncurrently confusing. It appears that Donor 4 From (Extended Data Figure 6) is also presented in\nFig 8F the main manuscript, and was chosen for the main figure as it was the one donor from\nfour that exhibited a similar increase in EGFR receptor expression in response to AR420626 as\nobserved in mouse ILC2. For transparency, please include in the main figure a summary graph\nshowing the difference between EGFR levels between Vehicle and AR420626 after 6 days of\nculture.\nR2C7: We appreciate the reviewer’s careful reading, and we recognize that our previous\npresentation was not clear. The donor highlighted by the reviewer was in fact selected to be\npresented in the main Figure 8 as the example of a donor who responded to AR420626 by\nincreasing EGFR expression. We agree that it would be clearer and more transparent to present\nEGFR expression for all donors in the main figure, highlighting that it was a heterogeneous\nresponse.\nWe have updated Figure 8.G to show EGFR expression for every donor for which it was\nmeasured. We defined a meaningful upregulation of EGFR as an increase in MFI of at least\n10%. Of the 8 donors examined in 2 experimental batches, 3 donors’ ILC2s responded to\nAR420626 by upregulating EGFR to varying degrees. These data highlight the heterogeneity of\nthe response of human ILC2s and suggest that FFAR3-dependent upregulation of EGFR is\nconserved in humans, although with significant subject-to-subject variation.\n8.) Please show whether the AR420626 effect in human ILC2 is dependent on EGFR signalling.\nR2C8: We thank the reviewer for their helpful suggestion to clarify whether FFAR3 signaling\nacts through EGFR on human ILC2s like in mouse. While there is donor-specific variability in\nwhether or not FFAR3 signaling modifies EGFR expression, the ILC2s of all tested donors\nexpressed EGFR (Figure 8.G). We also show that there is a trend towards ILC2s increasing\nEGFR expression with time spent in culture with activating cytokines like IL-33, TSLP and IL-2\n(Figure 8.H).\nWe performed a new experiment in Figure 8.I-L where we culture human ILC2s with IL-33,\nTSLP, and IL-2 with DMSO vehicle, AR420626 alone, or AR420626 + varying concentrations of\nthe EGFR inhibitor gefitinib (0.1μM or 0.5μM). EGFR inhibition via gefitinib did not reverse the\nAR420626-mediated decrease in ILC2 number, and it did not have an effect on ILC2 viability\n(Figure 8.I&J). Gefitinib treatment did, however, trend towards reversing the FFAR3-dependent\ndecreases in IL-5 and IL-13. In each of the donors (n=4 for IL-5, n=3 for IL-13 because one\ndonor had all conditions under limit of detection for IL-13 ELISA), the AR420626 + gefitinib\ncondition had higher IL-5 and IL-13 than the AR420626 alone condition. The gefitinib\nconcentration at which the maximal increase in IL-5 and IL-13 occurred varied between the\ndonors, but gefitinib increased IL-5 an average of 2-fold and IL-13 an average of 1.88-fold.\nTo evaluate whether the observed increases in IL-5 and IL-13 with gefitinib were consistent\nacross donors, we performed a binomial sign test comparing each donor’s IL-5 or IL-13 level in\nthe AR420626-alone condition to the highest value observed with either concentration of\ngefitinib. This approach asks whether the number of donors showing an increase with gefitinib\nexceeds what would be expected by chance (i.e., 50% probability). All tested donors exhibited\nhigher IL-5 (4/4) and IL-13 (3/3) levels with at least one dose of gefitinib compared to AR420626\nalone. Binomial testing yielded p = 0.0625 for IL-5 and p = 0.125 for IL-13, indicating a\nreproducible trend toward reversal of AR420626-mediated suppression despite limited sample\nsize.\n9.) In contrast to mouse ILC2, which increased total numbers in response to AR420626,\nhuman ILC2 responded with a decrease. Please discuss the reasons behind this difference.\nR2C9: The thank the reviewer for this important observation, and it prompts discussion about\nthe degree of conservation of our phenotype between mouse and human ILC2s. The most\nstraightforward explanation for the opposite direction of the response in ILC2 number between\nour mouse and human experiments is the fact that they use ILC2s taken from different tissues\nwithin their respective organisms. Our mouse experiments were all performed using ILC2s\nisolated from the lung, while our human experiments used ILC2s isolated from the peripheral\nblood. It is a well-documented phenomenon that ILC2s exhibit a significant degree of tissue\nheterogeneity and their phenotypes are substantially molded by the environments in which they\nreside.3 While human lung ILC2s perhaps from deceased tissue donors would have allowed for\nthe most appropriate inter-species comparison, this was not possible for our study due to\npractical and technical considerations.\nIt would appear that FFAR3’s negative effect on Type 2 cytokine production is the most robustly\npreserved element of this phenotype, as it remains present in ILC2s across species and tissues\nof origin. It is likely that the three major effects of FFAR3 signaling that we observe in CC030\nILC2s (decreased Type 2 cytokines, increased cell number through apoptosis resistance, and\nincreased IL-10) are independent downstream consequences of FFAR3 signaling. Our new\nFigure 6.F-I shows that the downregulation of IL-5 and IL-13 is not a result of increased\nautocrine/paracrine IL-10 signaling, and our human data shows Type 2 cytokine effects and\nvariable EGFR responses without increasing cell number. Furthermore, our data from the\nC57BL/6J mice in Experiment 2 (Supplementary Figure 8), discussed in R2C5, shows an\nincrease in cell number via apoptosis suppression but no Type 2 cytokine response. We suggest\nthat the different aspects of the FFAR3-mediated response in ILC2s may be uncoupled from\none another, and the different responses are variably conserved between ILC2s of different\ngenotypes, species, and tissues. It is reassuring, however, that the most therapeutically\nbeneficial aspect of FFAR3-signaling, decreasing IL-5 and IL-13 production, is present in mouse\nand humans across multiple ILC2 tissue types.\nWe have added discussion around this topic in the manuscript in the discussion which reads:\n“Another limitation is that there are notable differences in the response to FFAR3 signaling\nbetween ILC2s from our mouse and human experiments, especially relating to responses in cell\nnumber and IL-10 production. It is possible that this incomplete degree of phenotypic\nconservation can be explained by the different tissue sources of ILC2s for the experiments; our\nmouse experiments used lung ILC2s while our human experiments used ILC2s from the\nperipheral blood. Additionally, some of our mechanistic data supports the notion that the\ndownstream responses of FFAR3 signaling in ILC2s (decreased Type 2 cytokines, increased\ncell number through apoptosis resistance, and increased IL-10) are uncoupled from one\nanother, and different arms of this response may be variably conserved across species and\nILC2 tissue types.”\n10.) In the 2018 paper by Thio et al. (DOI: 10.1016/j.jaci.2018.02.032), the authors detail the\nrole of short-chain fatty acids (SCFAs) in allergic airway inflammation and the induction of\nGPR41/FFAR3 in ILC2s by IL-33. This conflicts with the statement in the current study regarding\nthe lack of knowledge on FFAR3's role in regulating ILC2 biology in the lung. Please revise the\nassociated statements to accurately reflect the prior knowledge presented by Thio et al.\nR2C10: We sincerely apologize for the oversight regarding the previously published data\ndemonstrating Ffar3 expression in ILC2s by Thio et al. We have amended the manuscript to\nreflect this prior knowledge with the following text:\n“One study in particular established the expression of FFAR3 on pulmonary ILC2s in mice, but\ntheir data suggested that SCFAs modulated ILC2s primary in a receptor-independent manner,\nlikely through histone de-acetylase inhibition.4”\n11.) Furthermore, the implications of Thio et al.'s findings should be thoroughly discussed in the\ncontext of the current study. Notably, the previous study did not observe a reduction in IL-13 and\nIL-5 levels when ILC2s were treated with the FFAR3 agonist AR420626. This raises important\nquestions about the regulatory mechanisms of FFAR3 in ILC2 function and its potential\ndifferential effects on cytokine production.\nR2C11: We agree with the reviewer regarding the importance of discussing our findings in the\ncontext of this previous report that examined the response of FFAR3 expressing ILC2s to\nAR420626 at the same concentration that we used in our paper. Thio et al. demonstrate in their\nImportantly, these mice were pre-expanded with in vivo-administered IL-33 prior to isolation, and\nupon stimulation with IL-33 in culture, they upregulated Ffar3 more than 4-fold after 6 hours.\nThese data suggest that ILC2 activation may upregulate Ffar3 expression. These data from\nFigure 6 in their manuscript are presented below:\nIt is very intriguing, albeit somewhat perplexing, that they report an ILC2 experiment where they\ndetect Ffar3 transcript in their ILC2s but do not observe a response to AR420626 in IL-5 or IL-13\nin culture. They did investigate differences in proliferation by Ki-67 positivity which showed no\neffect of AR420626, and this aligns with our data in both CC030 and C57BL/6J ILC2s. Notably,\nthey did not report cell number or apoptosis data, and it would be interesting to know if these\nreadouts were affected in their culture model. All of this is contrasted with CC030 ILC2s that\nreproducibly show cell number increases, apoptotic resistance, decreased IL-5 and IL-13, and\nIL-10 induction in response to treatment with the FFAR3 agonist AR420626.\nIt is interesting to consider what might be contributing to the observed differences in response to\nAR420626 between ILC2s that express Ffar3. It is important to recognize that Thio et al.’s and\nour experimental setups use different mouse strain backgrounds with ILC2s in different baseline\nstates pre-culture: Thio et al. use ILC2s from BALB/c that have been pre-expanded in vivo with\nIL-33 and we use ILC2s from CC030 mice in a naïve, unstimulated state (Figures 4-7). It is\npossible that the Ffar3 expression in the ILC2s from Thio et al. is transient and low (only relative\nexpression to ILC2s cultured without IL-33 was reported), resulting in only minimal signaling\ntaking place. Another notable difference is that their culture system only uses IL-33 to stimulate\nILC2s. From our data in Figure 6.B, it seems that IL-2 signaling is necessary for AR420626 to\ndecrease IL-5 secretion. We have added the following text to the manuscript to present some of\nthis discussion:\n“…and the report by Thio et al. demonstrated expression of Ffar3 in ILC2s from IL-33 pre-\ntreated Rag2−/− mice.4 While they established the expression of the gene in ILC2s, they\nsurprisingly did not observe any effect of AR420626 treatment (10μM) on Type 2 cytokine\nproduction from ILC2s stimulated by IL-33 in vitro. There are some notable differences in the\nexperimental approaches that may explain this discrepancy. Thio et al. use ILC2s that have\nbeen pre-expanded in vivo with IL-33 prior to isolation, altering their baseline phenotype.\nAdditionally, they use only IL-33 to activate their ILC2s in culture, while our culture system used\nIL-33, TSLP, and IL-2. Our data suggest that the anti-inflammatory action of FFAR3 signaling is\ndependent on IL-2, and therefore, this effect was possibly not observed in previously published\nexperiments.”\nMinor Comments\n12.) From the results, it is slightly unclear whether the Ch.7 ILC2 Locus was the only locus that\nexceeded the significance threshold. Please clarify in the text.\nR2C12: We appreciate the reviewer’s concern relating to the lack of clarity about which loci\nachieved significance in the QTL mapping experiment. The Ch.7 ILC2 Locus was the only locus\nto reach any of the permutation thresholds (80%, 90%, or 95%), and it exceeded the 95%\npermutation threshold.\nWe have clarified this point in the manuscript with the following text:\n“It was the only peak in our analysis to reach significance, and it exceeded the 95% permutation\nthreshold.”\n13.) Please clarify whether strains CC026 and F-A/J also possessed the Ch.7 ILC2 Locus.\nR2C13: The reviewer requests an important clarification. The Ch.7 ILC2 Locus is technically a\ngenomic interval on mouse chromosome 7 from position 30.538129 to 33.94594, so every\nmouse strain possesses this locus. The difference between the strains is the founder haplotype\nthat the strain has at the Ch.7 ILC2 Locus. Put simply, while every strain has the locus, the\nfounder haplotype at the locus is what makes the difference. Our QTL mapping and subsequent\nfounder haplotype analysis (Figure 2) revealed that it was the CAST/EiJ founder haplotype that\nwas primarily responsible for the significant QTL at the Ch.7 ILC2 Locus, i.e. strains that\ninherited the Ch.7 ILC2 Locus chromosomal interval from the CAST/EiJ founder had\nsignificantly higher ILC2 abundance.\nThe implication of the reviewer’s request is very important. CC026 and F-A/J have the locus,\nlike all strains, but they have different founder haplotypes at the locus. CC026 has inherited the\nentirety of the Ch.7 ILC2 Locus from the A/J founder strain. This information can be obtained\nfrom the Locus Probabilities tool available on the UNC Systems Genetics Core website, at\nhttps://csbio.unc.edu/CCstatus/index.py?run=locus. This tool will detail the founder haplotype\nprobabilities for all CC recombinant strains for any given gene or chromosomal interval. An\nexample of CC026 is provided below, showing that there is a 100% chance that the entirety of\nthe Ch.7 ILC2 Locus (based on chromosomal interval) was inherited from A/J:\nEach of the 8 columns represent the different founder haplotypes, with the first column (yellow)\nrepresenting F-A/J. The 100.00 denotes that the entirety of the locus was certainly inherited\nfrom A/J, while other strains may have variable percentages if different portions of the locus\nwere inherited from more than one founder.\nThe A/J strain, which had the 4th highest ILC2 abundance, just after CC026, must definitionally\nhave 100% A/J founder haplotype at the Ch.7 ILC2 Locus because it is a founder strain, so the\nentire genome is A/J founder haplotype. One might be tempted to conclude that two strains with\nthe A/J founder haplotype at the Ch.7 ILC2 Locus having high ILC2s might suggest that the A/J\nhaplotype is driving the QTL. However, by the analysis in Figure 2.B, the A/J haplotype has a\nremarkably neutral contribution to the QTL at the Ch.7 ILC2 Locus, visualized by the yellow line\nbeing very close to 0 over the range of the locus:\nThe likely explanation is that the F-A/J haplotype does contribute to high ILC2 abundance, but\nnot at the Ch.7 ILC2 Locus. It is very possible that other intervals of the CC026 genome that\nwere inherited from A/J could be contributing to other peaks on the QTL map from Figure 2.A\nthat did not quite reach significance. This highlights an important point regarding this analysis.\nNot all of the variation that exists in ILC2 abundance between CC strains can be explained by\nthe Ch.7 ILC2 Locus. ILC2 abundance is a complex trait that is regulated by many different\ngenes that are outside of the Ch.7 ILC2 Locus, and some of the other CC strains with high ILC2\nabundance might have this phenotype because of other loci positively contributing to this trait.\n14.) Fig 7B please label the top10 up and down genes and not just Egfr.\nR2C14: We have changed the new Figure 8.A to show the 10 most upregulated and\ndownregulated genes in AR420626 treated ILC2s compared to vehicle controls.\n15.) Please change “dose” to “concentration”, when talking about in vitro experiments.\nR2C15: We apologize for the oversight regarding terminology. We now use “concentration”\nthroughout the manuscript.\n16.) The citation for Ref 35 is incomplete.\nR2C16: We appreciate the reviewer’s attention to detail regarding the incomplete citation. That\ncitation was a truncated version of the DOQTL R package citation that was described in Gatti et\nal.’s 2014 paper, which is citation #8. We have consulted standard referencing guidelines, which\ndescribe that the most appropriate method for citation of the package is to cite the original paper\nthat published the package. Therefore, we now only provide the reference for the 2014 paper.\n17.) In Extended Data Figure 3, there is no concentration-dependent manner observed, please\ncorrect the phrasing or include additional concentrations.\nR2C17: We concur with the reviewer that the provided data does not demonstrate concentration\ndependent response. We have removed the data.\nReviewer 3\nIn the manuscript “Genetic diversity of Collaborative Cross mice reveals FFAR3 as a target for\nILC2 anti-inflammatory reprogramming,” Rusznak et al. propose a novel role for FFAR3 in\nregulating ILC2 responses. By examining genetically diverse mouse strains, they identify a\nlocus that influences ILC2 abundance in the lung following allergen exposure. Despite increased\nILC2 numbers, these cells show lower activation and diminished type 2 cytokine production. The\nstudy further highlights FFAR3 as crucial for inducing a less inflammatory ILC2 state with\nenhanced IL-10 production. While these findings are promising and the manuscript is broadly\nwell-constructed, there are several critical issues and several points that require further\ninvestigation to increase robustness of study:\nWe are encouraged by the reviewer’s optimism for the potential impact of our findings. We\nthank the reviewer for identifying important issues and opportunities to strengthen the\nmanuscript. We have performed the experiments requested by the reviewer, and we are\nconfident that this additional work has strengthened the rigor and breadth of our findings.\n1.) All experiments rely on one allergen (Alternaria alternata). Testing additional allergens (e.g.,\npapain or HDM) would strengthen manuscript and clarify whether this phenotype is general or\nunique to A. alternata.\nR3C1: We agree with the reviewer’s suggestion for an opportunity to strengthen the manuscript\nand broaden the applicability of our in vivo findings. To assess whether the CC030 phenotype of\ndiminished Type 2 innate inflammation was broadly present or restricted to Alternaria extract\nchallenge, we performed parallel experiments with Alt Ex challenge and papain challenge.\nWe selected 10μg administered over 3 consecutive days, as this was the highest dosage of\npapain that did not induce alveolar hemorrhage in our C57BL/6J mice. We performed a new\nchallenge experiment with both antigens, as well as unchallenged mice to establish baseline\nimmune profiles, in Supplementary Figure 5. Our Alt Ex groups for this new experiment largely\nvalidated our previous results that CC030 mice have significantly diminished ILC2-mediated\nType 2 responses to short-course allergen challenge. A more comprehensive evaluation of the\nimmune response to Alternaria revealed that while CC030 respond with diminished eosinophilia,\nthey have an enhanced neutrophilic response compared to C57BL/6J.\nOverall, the immune response to papain challenge was diminished compared to Alternaria in\nboth C57BL/6J and CC030, especially with regards to Type 2 cytokine production. However,\npapain challenge still revealed a greater number of total ILC2s and CD4+ T cells in CC030\ncompared to C57BL/6J. Notably, the increased neutrophilic response of CC030 mice to\nAlternaria was also seen with papain, evidenced by the increased number of total neutrophils in\nthe BAL after papain challenge.\nTaken together, some elements of the CC030 response (increased ILC2s, increased CD4+\ncells, and increased neutrophilia) are also present with papain challenge. The response to\npapain was significantly milder in both strains, and so differences in minimal cytokine\nexpression could not be appreciated. While it is possible that the CC030 phenotype is\ngeneralizable across other allergens that can elicit innate allergic inflammation, it is most robust\nwith Alternaria challenge.\nWe have added the following text to the manuscript regarding these results:\n“We then challenged C57BL/6J and CC030 mice with Alt Ex (4-days, 8μg) and papain (3-days,\n10μg) to profile each strain’s immune response to different protease-containing allergens.\nC57BL/6J mice had significantly more ILC2s than CC030 mice after papain challenge, mirroring\nour Alt Ex results. While Alt Ex challenge resulted in nearly significant decreases in ILC2-\nexpansion ratio and total IL-5, and significant decreases in total IL-13 for CC030 compared to\nC57BL/6J, papain challenge did not reveal any differences in these outcomes. The paucity of IL-\n5 and IL-13 with papain challenge would suggest an inability of papain to elicit a robust Type 2\ninnate response at this dose, which was the highest dose that did not cause alveolar\nhemorrhage in our model. CC030 mice also had a greater number of B cells, CD8+ T cells, and\nCD4+ T cells with Alt Ex challenge and a greater number of CD4+ T cells only with papain.\nThere was no significant difference in total Tregs with either allergen. In the BAL fluid, CC030\nmice had significantly decreased eosinophilia but increased neutrophilia compared to C57BL/6J\nwith Alt Ex challenge, with the neutrophilia alone being conserved in papain challenge. These\nresults suggest that CC030 mice have a diminished innate Type 2 response to Alternaria that\ncompensates with neutrophilia over eosinophilia, and some elements of this response are\nconserved in exposure to other protease containing antigens like papain (Supplementary\nFigure 5.C).”\n2.) Because ILC2s in CC030 mice appear less responsive to IL-33, experiments should be\nconducted under diverse cytokine conditions, possibly excluding IL-33 or adding other stimuli to\nconfirm whether the observed response (lower proliferation and cytokine production) depends\non specific culture conditions.\nR3C2: We are grateful to the reviewer for suggesting this line of inquiry, as it led us to uncover\nmore about the possible mechanisms that explain the anti-inflammatory effect of FFAR3\nsignaling in ILC2s. We were similarly curious as to whether this anti-inflammatory, IL-10\nproducing phenotype was dependent on the presence of specific cytokines. Previous reports\nhave suggested that IL-10 production in ILC2s was dependent on, or at least greatly enhanced\nby, IL-2 signaling.5,6\nTo assess whether our anti-inflammatory ILC2 phenotype was dependent on IL-2, we performed\nan in vitro culture experiment with different cytokine conditions examining AR420626\nresponsiveness in the presence or absence of IL-2. These new experiments are presented in\nFigure 6.A-E. We were intrigued to find that the anti-inflammatory effect of FFAR3 signaling on\nILC2s was dependent on IL-2. AR420626-mediated increases in ILC2 number, decreases in IL-\n5, and production of IL-10 only occurred in cytokine conditions containing IL-2 (Figure 6.A-C).\nWithout IL-2 (only IL-33 + TSLP), vehicle and AR420626 had no difference in total IL-5\nproduction, and the AR420626 group even trended towards a marginal increase in production of\nthe inflammatory cytokine. The IL-10 induction was seemingly the most dependent on IL-2, as\nthere was virtually no IL-10 production outside of the IL-2 + AR420626 group in this experiment.\nIntrigued by the apparent dependence on IL-2 of our FFAR3-mediated phenotype, we evaluated\nwhether FFAR3 signaling may somehow affect IL-2 responsiveness in ILC2s. We hypothesized\nthat AR420626 signaling would upregulate CD25 expression on ILC2s, thereby increasing IL-2\nsignaling. We confirmed our hypothesis by quantifying CD25 expression on ILC2s cultured with\nIL-33, TSLP, and IL-2 and we found that AR420626 treatment significantly increased CD25\nexpression roughly 2-fold (Figure 6.D-E).\nWe have added the following text to the manuscript to describe these experiments:\n“We were intrigued by the anti-inflammatory cytokine secretion profile that FFAR3 agonism\nimparted on ILC2s, and we sought to further characterize this effector state. We investigated\nwhether this IL-10 producing state was dependent the stimulatory cytokines used to activate\nILC2s in our culture system. A previous report from Seehus et al. demonstrated that IL-2 was\nnecessary for IL-10 induction in ILC2s.7 We cultured CC030 ILC2s with AR420626 or vehicle in\nthe presence or absence of IL-2, and IL-2 was necessary for AR420626 to increase cell number\n(Figure 6.A), decrease IL-5 production (Figure 6.B), and induce IL-10 production (Figure 6.C).\nGiven that FFAR3’s anti-inflammatory effect is IL-2 dependent, we hypothesized that FFAR3\nsignaling may increase IL-2 signaling through upregulation of the high-affinity IL-2 receptor\nalpha (CD25). We confirmed our hypothesis by observing greater CD25 expression by flow\ncytometry on ILC2 in the presence of IL-33, TSLP, and IL-2 treated with AR420626 compared to\nvehicle controls (Figure 6.D-E). These data suggest that, like previously described IL-10-\nproducing ILC2s, FFAR3 acts in an IL-2 dependent manner and may induce this state through\nan upregulation of CD25.”\n3.) The study often reports small sample sizes (e.g., n=3) from what appear to be single\nexperiments (not independent experiments), raising concerns about statistical power. More\nindependent experiments with larger n are needed, particularly for critical conclusions drawn\nfrom subtle cell-number differences.\nR3C3: We appreciate the reviewer’s concern regarding some experiments with small samples\nsizes and lack of clarity on the number of independent experiments that were performed to\nrepresent each. We have updated the manuscript with repeated independent experiments for\nthose that had small samples sizes, and we have increased the number of biological replicates\nto improve power and confidence in our results. We have also made sure to clearly and\ndefinitively state in the figure legends the number of biological replicates for each experiment\nwith the number of independent experiments that were performed, including whether the data\nare combined from independent experiments or one experiment representing the results of\nmultiple independent experiments.\nUpon improving the power and rigor, the original Figure 4.O had heterogeneous results that\ndecreased our confidence in meaningful differences. This experiment was not essential to the\nstory, and in the interest of rigor and reproducibility, we removed it from our manuscript.\n“Differences in ILC2 responses in the Alt Ex model might have been influenced by strain\ndifferences in alarmin cytokine release and airway anatomy, so we employed an intra-peritoneal\nIL-33 challenge model to identically stimulate CC030 and C57BL/6J ILC2s in vivo. Direct IL-33\nchallenge grew the number of ILC2s in C57BL/6J but did not in CC030 (Figure 4.O).”\n4.) Different mouse strains can have distinct microbiomes that influence ILC2 phenotypes,\nespecially since FFAR3 responds to microbiome-derived fatty acids (FFAs such as acetate,\nbutyrate, propionate act as ligands for this receptor). The authors should detail backcrossing or\nhusbandry methods used to control for these variables.\nR3C4: The reviewer highlights an important point regarding differences in microbiomes between\nmice. Much of our study centers around strain differences FFAR3, but the reviewer is correct\nthat the endogenous ligands of FFAR3, short chain fatty acids (SCFAs), are an integral part of\nthe signaling axis in vivo. A unique element of the multiple SCFA-FFAR signaling axes is that the\nligands are not produced by the host, but rather by the microbiomes which colonize the host.\nTherefore, the composition of the microbiome is a critical determining factor for FFAR signaling\nwithin an animal.\nIt is important to consider that ultimately the QTL mapping study that we performed revealed the\nCh.7 ILC2 Locus, and thereby Ffar3, due to genetic differences in the receptor between the\nstrains. So, while levels of SCFAs may vary between the strains and influence FFAR signaling,\nthese differences would not contribute to the formation of a QTL at a locus containing the genes\nfor FFAR receptors. The fact that a significant QTL was present in the locus containing Ffar3\nsuggests that it is genetic differences in the receptor (either expression or other genetic\nmechanisms) that are principally responsible for the phenotype.\nNonetheless, in comparing the ILC2 phenotypes of two strains, it is important to consider the\ncontribution of possible SCFA differences secondary to variable microbiomes. We did not\nperform any backcrossing in our facility, as fully inbred strains were obtained directly from UNC.\nWe ensured that mice used in the study had at least two weeks to acclimate before the initiation\nof any experiment. All mice were kept on the same rack within the same room of our housing\nfacility, fed the same diet from the same communal source and had access to the same water\ndelivery system on the rack.\nDespite these measures to ensure a consistent environment and limit possible microbiome\nvariation, there are genetic factors inherent to the strains that may contribute to microbiome\ndifferences. For this reason, we elected to quantify SCFAs in the serum to test for differences\nbetween C57BL/6J and CC030 mice. We quantified acetate, propionate, and butyrate via mass\nspectrometry.\nBriefly, short-chain fatty acids (SCFAs) in serum were quantified by LC-HRMS following\nderivatization with dansyl hydrazine and EDC. Samples were spiked with deuterated internal\nstandards, extracted, derivatized, and analyzed on a Thermo Q Exactive HF Orbitrap mass\nspectrometer with Vanquish Horizon HPLC. Quantification was performed using targeted\nselected ion monitoring (t-SIM) with a mass tolerance of ±5 ppm and calibration against external\nstandards. Chromatographic separation was achieved using a BEH C18 column with a 15-\nminute gradient and ammonium acetate/acetic acid buffer system.\nResponse to Reviewers Figure 4. (A) Quantification of acetate, propionate, and\nbutyrate in the serum of naïve C57BL/6J and CC030 female mice. Statistical significance\nwas assessed with an un-paired Student’s t-test. ns = not significant, ****=p<0.00005.\nIt was clear from our experiment that there exist substantial differences in the SCFA profiles of\nC57BL/6J and CC030 mice. In both strains, acetate makes up the overwhelming majority of the\nSCFAs, and C57BL/6J have a more than 4-fold higher concentration of acetate in their blood\ncompared to CC030 mice. This is particularly interesting, as our study suggests that elevated\nFFAR3 signaling in CC030 ILC2s at baseline contributes to their unique anti-inflammatory\nphenotype, which is re-created with FFAR3 agonists. Clearly, the increase in FFAR3 signaling in\nCC030 mice cannot be explained by an overabundance of the ligand, as SCFA levels are\nsubstantially lower compared to C57BL/6J. Seemingly, the CAST/EiJ haplotype at the Ch.7\nILC2 Locus in CC030 mice confers such an increase in FFAR3 signaling that CC030 ILC2s\nexperience substantially more of it despite significantly lower exposure to the ligands.\n5.) Additional data on how FFAR3 agonism alters ILC2 phenotype—particularly regarding\npathways related to IL-10—would strengthen the study. Discrepancies between Figures 6 and 7\nin IL-10 expression levels were also unclear.\nR3C5: We thank the reviewer for this suggestion in further investigating the FFAR3-induced\nphenotype in relation to IL-10. The added studies performed at the reviewer’s request to\naddress this point have strengthened the manuscript.\nGiven that FFAR3 signaling both induces IL-10 production and decreases IL-5 and IL-13,\nwe hypothesized that the anti-inflammatory IL-10 may be acting in an autocrine and or paracrine\nmanner to reduce IL-5 and IL-13 expression. We tested this hypothesis by blocking IL-10\nsignaling with the use of an α-IL-10 antibody, and this new data is presented in Figure 6.F-I.\nThe α-IL-10 antibody had no effect on the total number of ILC2s, IL-5 production, or IL-13\nproduction. We gathered from these data that FFAR3’s effect on decreasing Type 2 cytokine\nproduction and increasing IL-10 production were two independent processes, and\nautocrine/paracrine IL-10 signaling could not explain the altered phenotype.\nWe were encouraged to find that our results had been independently validated by Miyamoto et\nal. in their 2019 paper. The authors similarly observe a decrease in IL-5 expression with their IL-\n10-producing ILC2 phenotype. However, when they block IL-10 with an antibody or add IL-10 to\nthe culture, there is no effect on IL-5, suggesting that an ILC2’s production of IL-5 is\nindependent of IL-10 signaling. Their data is presented below:\nWe further investigated the IL-10-producing phenotype elicited by AR420626 treatment through\ncomparison with other IL-10-inducing agents (retinoic acid) and through analysis of\ntranscriptomic similarity to other, pre-published IL-10 signatures. These points will be\ncomprehensively addressed in R3C8 and R3C10.\nThe reviewer raises an important point regarding the differences between figures in the relative\ndifference in IL-10 production between vehicle and AR420626 treated ILC2s and total IL-10\nproduced. There are a few factors that may contribute to difference in IL-10 results between\nFigure 5.I (previously Figure 6) and Figure 7.F. Firstly, we have observed a degree of variation\nin the quantity of IL-10 produced in our cultures between experimental batches. While the\nultimate result is always the same (AR420626-treated ILC2s produce more IL-10 than vehicle),\nthe magnitude of this difference has been variable. Additionally, we believe that the magnitude\nof the difference is affected by the concentration of DMSO that is in the culture system. We have\nnoticed with both of our intendent experiments for Figure 7 with gefitinib, and our experiment in\nFigure 6 with retinoic acid (to be discussed in detail in R3C10), higher quantities of DMSO\nvehicle in the culture due to the presence of other compounds seems to diminish this difference.\nWe again reiterate that the difference is reproducibly present, but the magnitude is subject to\ninter-experiment variation.\n6.) The claim that FFAR3 signaling maintains cell populations by inhibiting programmed cell\ndeath requires more mechanistic support, as the reported changes in caspase expression and\nviability appear modest.\nR3C6: We acknowledge the reviewer’s concern about the need for more mechanistic support\nregarding our claim of increased ILC2 number via apoptosis resistance rather than increased\ncell proliferation. We have provided a new experiment in Figure 5.N-P using an Annexin V /\npropidium iodide (PI) based method to more effectively detect and quantify apoptosis in its\nvarious stages. Using this method, we can identify 3 distinct ILC2 populations. We term Annexin\nV- and PI- cells as healthy ILC2s, Annexin V+ and PI- cells as early apoptotic ILC2s, and\nAnnexin V+ and PI+ cells as late apoptotic/dead ILC2s.\nThe data in Figure 5.N-P show that AR420626 treatment increases the percentage of ILC2s\nthat are undergoing early apoptosis, but it drastically decreases the proportion of cells that\nprogress through the early stage of apoptosis into late apoptosis and cell death. The increase in\nviability imparted by FFAR3 signaling as measured with this method is striking. The reproducible\nlack of meaningful difference in proliferation by CFSE dilution and Ki-67+ positivity, paired with\nthe significant decrease in late apoptotic cells, suggests that the difference in cell number\ninduced by FFAR3 signaling can be attributed to survival differences rather than differences in\ncell division.\nThis interpretation is further supported by the specific patterns of staining within the Annexin\nV+/PI+ quadrant. Annexin V binds phosphatidyl serine that loses membrane asymmetry during\napoptosis and begins to appear on the cell membrane surface. PI positivity indicates membrane\npermeability that suggests more advanced apoptosis and cellular decay. It is particularly\nintriguing that the Annexin V+/PI+ late apoptotic/dead cell quadrant is heterogeneous and has 3\ndistinct populations within it. These populations can be appreciated in the top right (double\npositive) quadrant in the flow cytometry plot from Figure 5.N reproduced below:\nThe difference in Annexin V+/PI+ cells between vehicle and AR420626 ILC2s occurs mostly in\nthe weakly PI+ population in the top right quadrant (indicated with the black rectangle). Given\nthat PI positivity indicates membrane compromise, these Annexin V+, weakly PI+ positive cells\nlikely represent cells that are experiencing an intermediate degree of cell membrane\ncompromise compared to the distinctly more PI+, likely necrotic, cells above them. AR420626\nreduces cells in this initial stage of membrane compromise, supporting the notion that\nAR420626 treatment is reducing progression to late apoptosis, not just generally preventing cell\ndeath.\n7.) For the human ILC2 experiments, raw cell numbers and viability assessments at the\nendpoint should accompany fold-change data. Moreover, it remains unclear whether IL-10\ninduction, observed in mice, extends to human ILC2s. This is central/key for the overall stated\nsignificance of FFAR3/possible therapeutic interventions proposed in discussion etc.\nR3C7: We agree with the reviewer about the importance of showing viability data for each of the\nhuman ILC2 experiments to demonstrate that effects on cell number and cytokines are not\nmerely due to non-specific drug toxicity. We now provide the total numbers of ILC2s in each\nexperiment as well as viability data in Figure 8.B&C. AR420626 did not affect viability in any of\nour human ILC2 experiments.\nWe measured IL-10 production in our human ILC2 cultures and unfortunately found that\nAR420626 had no effect at the 3 concentrations that we tested (Figure 8.F). While the ability of\nFFAR3 signaling to decrease Type 2 cytokines is present in both mouse and human ILC2s,\nour\ndata would suggest that the receptor’s effect on IL-10 production is not conserved. A possible\nexplanation for the differences in the AR420626-induced phenotype is that our mouse and\nhuman experiments use ILC2s taken from different tissues within their respective organisms.\nOur mouse experiments were all performed using ILC2s isolated from the lung, while our human\nexperiments used ILC2s isolated from the peripheral blood. It is a well-documented\nphenomenon that ILC2s exhibit a significant degree of tissue heterogeneity and their\nphenotypes are substantially molded by the environments in which they reside.3 While human\nlung ILC2s from deceased tissue donors perhaps could have allowed for the most appropriate\ninter-species comparison, this was not possible for our study due to practical and technical\nconsiderations. It is entirely possible that the ability of ILC2s to produce IL-10 in response to\nFFAR3 signaling is a reaction that is specific to the environment of the lung, and therefore\nhuman ILC2s taken from other tissues would not capture this biological feature. Limitations of\nour human ILC2 model have been added to the discussion with the following text:\n“Another limitation is that there are notable differences in the response to FFAR3 signaling\nbetween ILC2s from our mouse and human experiments, especially relating to responses in cell\nnumber and IL-10 production. It is possible that this incomplete degree of phenotypic\nconservation can be explained by the different tissue sources of ILC2s for the experiments; our\nmouse experiments used lung ILC2s while our human experiments used ILC2s from the\nperipheral blood. Additionally, some of our mechanistic data supports the notion that the\ndownstream responses of FFAR3 signaling in ILC2s (decreased Type 2 cytokines, increased\ncell number through apoptosis resistance, and increased IL-10) are uncoupled from one\nanother, and different arms of this response may be variably conserved across species and\nILC2 tissue types.”\n8.) To strengthen novelty, the authors could compare the transcriptional signature of CC030\nILC2s to known ILC210 profiles, including those in studies that link IL-10 production with altered\nmetabolism. For example :\n• Seehus CR, Kadavallore A, Torre B, Yeckes AR, Wang Y, Tang J, Kaye J. Alternative\nactivation generates IL-10 producing type 2 innate lymphoid cells. Nat Commun. 2017\nDec 1;8(1):1900. doi: 10.1038/s41467-017-02023-z. PMID: 29196657; PMCID:\nPMC5711851.\n• Howard E, Lewis G, Galle-Treger L, Hurrell BP, Helou DG, Shafiei-Jahani P, Painter JD,\nMuench GA, Soroosh P, Akbari O. IL-10 production by ILC2s requires Blimp-1 and cMaf,\nmodulates cellular metabolism, and ameliorates airway hyperreactivity. J Allergy Clin\nImmunol. 2021 Apr;147(4):1281-1295.e5. doi: 10.1016/j.jaci.2020.08.024. Epub 2020\nSep 6. PMID: 32905799.\nR3C8: We thank the reviewer for their excellent recommendation on how we may further\ndemonstrate the novelty of the ILC2 phenotype that we describe in this manuscript. We have\nfollowed the reviewer’s recommendation and obtained the read counts for the RNA-sequencing\nexperiments presented in the manuscripts by Seehus et al. and Howard et al.7,8 We used the\nnormalized read counts of samples derived from IL-10+ ILC2s to compare with our AR420626-\ntreated ILC2s. These new analyses are presented in Figure 6.L.\nWe investigated transcriptomic similarities and differences between the samples with a PCA\nanalysis. Our method for this analysis is highlighted with the following text which has been\nadded to our methods section:\n“The PCA plot in Figure 6 was generated through comparison of the normalized count matrices\nfor ILC2 samples from 3 experiments (IL-10+ ILC2s from Seehus et al. (2017, GSE81882), IL-\n10+ ILC2s from Howard et al. (2020, GSE158983), AR420626-treated ILC2s from this\nmanuscript (GSE288176). Values in the normalized count matrices were log -transformed to\n2\nstabilize variance. We corrected for inter-experiment batch effects with ComBat from the sva R\npackage. A design matrix was specified to retain variation associated with IL-10+ vs. IL-10-\nstatus (condition) while adjusting for experiment (batch). The ComBat corrected expression\nmatrix was then subset to IL-10+ samples. PCA analysis included group-wise convex hulls and\ncentroids to highlight sample clustering by experiment.”\nIt is clear from our analysis that there are notable transcriptional differences between the anti-\ninflammatory, IL-10-producing state that we present and the other IL-10-producing states that\nhave been reported in the literature. Our PCA analysis is particularly helpful in evaluating these\ndifferences, as we use a method to correct for intra-experiment batch effects by retaining\nvariation associated with IL-10 status (made possible with the inclusion of IL-10- (Seehus and\nHoward) and vehicle-treated (our study) ILC2 samples). This reduces variation that exists due to\ndifferences in experimental approach or mouse strain and retains the unique features of the IL-\n10-producing state from each experiment in relation to the control samples. When grouping\nsamples by this method, it is clear that the IL-10+ ILC2s from Seehus et al. and Howard et al.\nare transcriptionally very similar to one another, as seen by the close proximity of centroids of\nthe two groups. In contrast, AR420626 ILC2s from our experiment demonstrate greater distance\nfrom the other two experiments on the PCA plot, suggesting a greater degree of transcriptional\ndistinction from previously described IL-10+ ILC2 states. We appreciate the reviewer’s\nsuggestion, as these experiments have helped to further solidify the novelty of the anti-\ninflammatory ILC2 state that we describe in this manuscript.\n9.) In vivo validation of targeting FFAR3 therapeutically would provide proof-of-principle of the\nauthors’ suggestion that targeting FFAR3 may mitigate allergic inflammation. Otherwise\ndiscussion needs to be tempered.\nR3C9: We concur with the reviewer that targeting FFAR3 in vivo would provide important proof-\nof-principle for FFAR3 as a therapeutic option for allergic inflammation. We sought to directly\ntarget FFAR3 through our attempt at creating a global Ffar3-/- mouse with a CRISPR/Cas9\nediting method on the CC030 background. Successful generation of Ffar3-/- on the C57BL/6J\nbackgrounds has been reported in the literature, and so we were encouraged that global\ndeletion on the CC030 line would be possible.9,10 Despite effective embryo recovery,\nediting, and transfer into 5 pseudo-pregnant females, only 3 deceased pups were recovered,\nall of which were positive for the targeted Ffar3 deletion. These results suggest that global\ndeletion of this gene might be embryonically lethal on the CC030 background. A full report of this\ngene deletion attempt is provided in Supplementary Data 5.\nGiven that we were not able to directly target FFAR3 in vivo, we have tempered discussion in\nthe manuscript around FFAR3’s therapeutic potential in allergic inflammation. We have removed\nthe following text from the manuscript:\n“ILC2s are critical mediators of Type 2 inflammation in allergen-exacerbated asthma, and a new\nsignaling mechanism that modulates this inflammation may have therapeutic implications.”\n“These data suggest that ILC2s could be targeted with FFAR3-specific agonists, or even with\nSCFA supplementation, to reduce Type 2 inflammation in an appropriate clinical setting, such as\nasthma.”\n“In addition to the potential clinical utility,”\n10.) With IL-10 production highlighted, testing other known IL-10–inducing factors would confirm\nFFAR3’s role in driving this phenotype. Do they also upregulate FFAR3? How do they compare\nin inducing IL-10 head-to-head with FFAR3 agonist?\nR3C10: This is an excellent suggestion by the reviewer that builds off previous questions\nregarding the uniqueness of the IL-10 producing state that we elicit with FFAR3 signaling. We\nshowed in Figure 6 that AR420626-induced IL-10 production is dependent on IL-2 signaling,\nwhich was similar to IL-10 producing ILC2s described in other reports. However, through our\ntranscriptomic analyses, we demonstrated that our ILC2s possess a unique transcriptomic\nsignature that distinguishes them from previously described IL-10+ ILC2s. Per the reviewer’s\nsuggestion, we investigated AR420626 head-to-head with retinoic acid, which is a well-known\ninducer of IL-10.7\nWe repeated our ILC2 culture system with IL-33, TSLP, IL-2 and DMSO vehicle, AR420626\n(10μM) and retinoic acid (1μM) to assess their cytokine responses head-to-head with new data\npresented in Figure 6.J&K. We were intrigued to find that AR420626 and retinoic acid secreted\nsimilar quantities of IL-10, but we were most encouraged by the IL-5 data. While AR420626\ndecreased IL-5 production compared to vehicle as we have repeatedly seen, retinoic acid\nsignificantly increased IL-5 expression compared to vehicle. This suggests that while AR420626\nand retinoic acid are both inducers of IL-10, they have opposite effects when it comes to Type 2\ninflammatory cytokines like IL-5. As we previously investigated, these two processes are\nindependent, and our data suggest that FFAR3 signaling causes a more comprehensive anti-\ninflammatory reprogramming than other known IL-10 inducers in ILC2s. We feel that this finding\neffectively supports the novelty of our finding, as we are unaware of other modulators that can\nsimultaneously increase IL-10 expression while decreasing Type 2 cytokines.\nWe investigated the reviewer’s question regarding whether retinoic acid upregulated FFAR3 on\nILC2s.Given these differences in response, we hypothesized that retinoic acid acted in an\nFFAR3-independent manner and did not influence its expression. We extracted RNA from ILC2s\ntreated with DMSO vehicle, AR420626, or retinoic acid, and we found that neither factor\nupregulated Ffar3 expression in the ILC2s. Interestingly, it seems that ILC2 activation and\nculture for 6 days markedly reduces Ffar3 expression in all conditions. This may suggest that\nthe re-programming experienced by ILC2s from AR420626 may occur in the earlier phases of\nculture. The qPCR data for this experiment is presented below:\nResponse to Reviewers Figure 5. (A) Quantification of Ffar3 by qPCR in ILC2s\nisolated by flow cytometry sorting from CC030 mice (naïve) and cultured with IL-33,\nTSLP, and IL-2 with DMSO vehicle, AR420626, or retinoic acid. ΔCT values for Ffar3 in\neach sample were calculated to the housekeeping gene Gapdh in the naïve condition,\nbut all cultured cells had no amplification for Ffar3. Relative expression was calculated\nwith 2-ΔΔCT. Significance was assessed with Student’s t-test. ns=not significant.\n****=p<0.00005.\n11.) The potential co-expression of amphiregulin also remains an intriguing avenue for CRISPR\nor siRNA-based mechanistic studies. Is amphiregulin and IL-10 co-expressed? Does knocking\ndown amphiregulin decrease IL-10 production?\nR3C11: The reviewer raises important questions regarding the proposed mechanism of\namphiregulin and EGFR signaling on ILC2s. We demonstrate that AR420626 increases the\nexpression of EGFR, and we hypothesized that ILC2s are secreting EGFR ligands, like\namphiregulin, to activate EGFR in an autocrine and/or paracrine manner. We measured\namphiregulin in our ILC2 culture system, and while it is expressed in both conditions, we found\nthat AR420626 decreases amphiregulin secretion. While AR420626 treatment does not increase\namphiregulin, it is possible that the large increase in receptor expression outweighs the\ndecrease in ligand, thereby still causing greater overall EGFR signaling.\nWe hypothesized that amphiregulin was signaling through EGFR to induce IL-10 expression, so\nwe blocked amphiregulin signaling in our culture with an α-amphiregulin antibody, expecting that\nthis would decrease IL-10 expression compared to isotype control. This hypothesis was\nsupported by our data, which showed that amphiregulin blockade significantly decreased IL-10\nexpression in AR420626 treated groups. This decrease is modest, and the individual paired\nsamples are shown in Figure 7.K. It is possible that this effect is so modest due to the fact that\nEGFR signaling may continue through other EGFR ligands that are secreted by ILC2s, so while\namphiregulin may be neutralized, other secreted proteins are able to continue signaling. This\nwould be an intriguing line of future investigation to evaluate which EGFR ligands these ILC2s\nare secreting in our culture system.\nCollectively, these points suggest that deeper mechanistic and translational data will strengthen\nthe manuscript. Addressing these concerns will provide a more robust foundation for the\nauthors’ conclusion that FFAR3 is a key regulator of anti-inflammatory reprogramming in ILC2s.\nWe concur with the reviewer that addressing these points has significantly deepened the\nmechanistic insight and overall robustness of the paper. We are grateful for the careful review\nand constructive suggestions.\nReferences\n1. Zaiss, D. M. W. et al. Amphiregulin enhances regulatory T cell-suppressive function via the\nepidermal growth factor receptor. Immunity 38, 275–284 (2013).\n2. Florentin, J. et al. Loss of Amphiregulin drives inflammation and endothelial apoptosis in\npulmonary hypertension. Life Sci Alliance 5, e202101264 (2022).\n3. Spits, H. & Mjösberg, J. Heterogeneity of type 2 innate lymphoid cells. Nat Rev Immunol 22,\n701–712 (2022).\n4. Thio, C. L.-P., Chi, P.-Y., Lai, A. C.-Y. & Chang, Y.-J. Regulation of type 2 innate lymphoid\ncell-dependent airway hyperreactivity by butyrate. J Allergy Clin Immunol 142, 1867-\n1883.e12 (2018).\n5. Seehus, C. R. et al. Alternative activation generates IL-10 producing type 2 innate lymphoid\ncells. Nat Commun 8, 1900 (2017).\n6. Reid, K. T. et al. Cell therapy with human IL-10-producing ILC2s limits xenogeneic graft-\nversus-host disease by inhibiting pathogenic T cell responses. Cell Reports 44, (2025).\n7. Seehus, C. R. et al. Alternative activation generates IL-10 producing type 2 innate lymphoid\ncells. Nat Commun 8, 1900 (2017).\n8. Howard, E. et al. IL-10 production by ILC2s requires Blimp-1 and cMaf, modulates cellular\nmetabolism, and ameliorates airway hyperreactivity. J Allergy Clin Immunol 147, 1281-\n1295.e5 (2021).\n9. Ren, Z. et al. G protein coupled receptor 41 regulates fibroblast activation in pulmonary\nfibrosis via Gαi/o and downstream Smad2/3 and ERK1/2 phosphorylation. Pharmacol Res\n191, 106754 (2023).\n10. Li, J. et al. GPR41 deficiency aggravates type 1 diabetes in streptozotocin-treated mice by\npromoting dendritic cell maturation. Acta Pharmacol Sin 45, 1466–1476 (2024).\nResponse to Reviewers – Round 2\nRusznak et al.\nWe greatly appreciate the opportunity to submit these final revisions. We thank the reviewers for\ntheir thoughtful recommendations and careful attention to our manuscript.\nReviewer #1 (Remarks to the Author):\nThe content has been significantly improved in the revised version. It can be easily read and the\nimportance of FFAR3 in the anti-inflammatory reprogramming of ILC2 has been clearly\ndescribed.\nWe greatly appreciate the reviewer’s assessment that the revisions have significantly improved\nthe manuscript.\nI want the authors to correct one point. The potential physiological role of functional regulation of\nILC2 by FFAR3 has not been mentioned in the present version. I assume that anti-inflammatory\nregulation by the short-chain fatty acid/FFAR3 system may be equipped to dampen\ninflammation and/or that microbes may secrete FFAR3 ligands to escape from the host defense\nsystem via FFAR3. The authors should mention the possibility quoting appropriate reference(s).\nWe agree with the reviewer’s assessment that that manuscript would benefit from exploring the\npossible physiologic role of an SCFA-FFAR3 axis relating to ILC2s. We have added the\nfollowing text to the discussion:\n“This raises the question of what may be the physiological role of a signaling axis that makes\nILC2 effector states responsive to microbe-derived SCFAs. We speculate that this may be a way\nin which ILC2s, an integral part of the innate Type 2 inflammatory response that evolved to\ncombat helminthic infections, may tune their inflammatory readiness based on the state of the\nmicrobiome. Many helminths, such as Ascaris lumbricoides have life cycles that involve the gut\nand the lung, and infections are known to perturb the microbiome and modulate SCFA\nlevels.83,84 One could imagine the SCFA-FFAR3 axis acting as an early warning system: in\nsettings of microbiome eubiosis, high SCFAs levels shift ILC2s to an anti-inflammatory or\nmaintenance effector state, but helminthic infection and subsequent microbiome dysbiosis may\ndrop SCFA levels and shift pulmonary ILC2s to a pro-inflammatory state of high readiness\nwhere they are primed to respond in the event of systemic infection.”\nReviewer #2 (Remarks to the Author):\nThe authors have responded positively to the prior reviews and have addressed my concerns\nsatisfactorily.\nWe thank the reviewer for the positive comments on our revision.\nMinor comments\nPlease add the %variance to the x and y axis labels in Fig 6L\nWe have added % variance to the x and y axis labels in Figure 6L.\nReviewer #3 (Remarks to the Author):\nI greatly appreciate the effort the research team put forward to address prior concerns.\nThe revisions have been thoughtful and fully address prior comments. I believe the manuscript\nis now suitable for publication.\nWe are grateful that the reviewer found our revisions to be thoughtful and comprehensive.\nI have only two minor requests:\n1. Please include Response to Reviewer Figure 4, either within the Methods section or as a\nSupplemental Figure, and add one or two sentences in the main text describing these data, as\nthey are important to document.\nWe have now included Response to Reviewers Figure 4 as a supplemental figure. We agree\nthat highlighting the differences short-chain fatty acid levels between strains is important to the\nmanuscript. It has been added as Supplemental Figure 9, and it has been referenced in the\nmain text as below:\n“Our results suggested that differences in baseline FFAR3 signaling on ILC2s between\nC57BL/6J and CC030 mice were attributable to differences in receptor expression, but we\naimed to investigate possible in vivo differences in SCFAs, the endogenous FFAR3 ligands, as\nwell. We quantified acetate, propionate, and butyrate in the serum of C57BL/6J and CC030\nmice at baseline via mass spectrometry, and CC030 mice had significantly lower serum\nconcentrations of acetate compared to C57BL/6J, while there were no differences in propionate\nor butyrate (Supplementary Figure 9). The lower concentration of FFAR3 ligands in the serum of\nCC030 mice suggested that the increased FFAR3 signaling on CC030 ILC2s is dependent on\nincreased receptor expression, not SCFA concentration.”\n2. I would suggest tempering the heading “FFAR3 signaling reprograms human ILC2s towards\nan anti-inflammatory effector state in vitro”, to “FFAR3 signaling partially reprograms human\nILC2s towards an anti-inflammatory effector state in vitro”, to not overstate the conclusion.\nWe agree with the reviewer that the language in the headings should be tempered to reflect the\npartial nature of the conservation between mouse and human ILC2s with respect to FFAR3’s\neffects. We have amended the text to read “partially reprograms” in the results heading and\nFigure 8 legend."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The content has been significantly improved in the revised version. It can be easily read and the importance of FFAR3 in the anti-inflammatory reprogramming of ILC2 has been clearly described.\nI want the authors to correct one point.\nThe potential physiological role of functional regulation of ILC2 by FFAR3 has not been mentioned in the present version. I assume that anti-inflammatory regulation by the short-chain fatty acid/FFAR3 system may be equipped to dampen inflammation and/or that microbes may secrete FFAR3 ligands to escape from the host defense system via FFAR3. The authors should mention the possibility quoting appropriate reference(s).", "reviewer_2": "The authors have responded positively to the prior reviews and have addressed my concerns satisfactorily.\nMinor comments\nPlease add the %variance to the x and y axis labels in Fig 6L", "reviewer_3": "I greatly appreciate the effort the research team put forward to address prior concerns. The revisions have been thoughtful and fully address prior comments. I believe the manuscript is now suitable for publication.\nI have only two minor requests:\n1. Please include Response to Reviewer Figure 4, either within the Methods section or as a Supplemental Figure, and add one or two sentences in the main text describing these data, as they are important to document.\n2. I would suggest tempering the heading “FFAR3 signaling reprograms human ILC2s towards an anti-inflammatory effector state in vitro”, to “FFAR3 signaling partially reprograms human ILC2s towards an anti-inflammatory effector state in vitro”, to not overstate the conclusion."}, "author_response": "Rusznak et al.\nWe greatly appreciate the opportunity to submit these final revisions. We thank the reviewers for their thoughtful recommendations and careful attention to our manuscript.\nReviewer #1 (Remarks to the Author):\nThe content has been significantly improved in the revised version. It can be easily read and the importance of FFAR3 in the anti-inflammatory reprogramming of ILC2 has been clearly described.\nWe greatly appreciate the reviewer’s assessment that the revisions have significantly improved the manuscript.\nI want the authors to correct one point. The potential physiological role of functional regulation of ILC2 by FFAR3 has not been mentioned in the present version. I assume that anti-inflammatory regulation by the short-chain fatty acid/FFAR3 system may be equipped to dampen inflammation and/or that microbes may secrete FFAR3 ligands to escape from the host defense system via FFAR3. The authors should mention the possibility quoting appropriate reference(s).\nWe agree with the reviewer’s assessment that that manuscript would benefit from exploring the possible physiologic role of an SCFA-FFAR3 axis relating to ILC2s. We have added the following text to the discussion:\n“This raises the question of what may be the physiological role of a signaling axis that makes ILC2 effector states responsive to microbe-derived SCFAs. We speculate that this may be a way in which ILC2s, an integral part of the innate Type 2 inflammatory response that evolved to combat helminthic infections, may tune their inflammatory readiness based on the state of the microbiome. Many helminths, such as Ascaris lumbricoides have life cycles that involve the gut and the lung, and infections are known to perturb the microbiome and modulate SCFA levels.83,84 One could imagine the SCFA-FFAR3 axis acting as an early warning system: in settings of microbiome eubiosis, high SCFAs levels shift ILC2s to an anti-inflammatory or maintenance effector state, but helminthic infection and subsequent microbiome dysbiosis may drop SCFA levels and shift pulmonary ILC2s to a pro-inflammatory state of high readiness where they are primed to respond in the event of systemic infection.”\nReviewer #2 (Remarks to the Author):\nThe authors have responded positively to the prior reviews and have addressed my concerns satisfactorily.\nWe thank the reviewer for the positive comments on our revision.\nMinor comments\nPlease add the %variance to the x and y axis labels in Fig 6L\nWe have added % variance to the x and y axis labels in Figure 6L.\nReviewer #3 (Remarks to the Author):\nI greatly appreciate the effort the research team put forward to address prior concerns.\nThe revisions have been thoughtful and fully address prior comments. I believe the manuscript is now suitable for publication.\nWe are grateful that the reviewer found our revisions to be thoughtful and comprehensive.\nI have only two minor requests:\n1. Please include Response to Reviewer Figure 4, either within the Methods section or as a Supplemental Figure, and add one or two sentences in the main text describing these data, as they are important to document.\nWe have now included Response to Reviewers Figure 4 as a supplemental figure. We agree that highlighting the differences short-chain fatty acid levels between strains is important to the manuscript. It has been added as Supplemental Figure 9, and it has been referenced in the main text as below:\n“Our results suggested that differences in baseline FFAR3 signaling on ILC2s between C57BL/6J and CC030 mice were attributable to differences in receptor expression, but we aimed to investigate possible in vivo differences in SCFAs, the endogenous FFAR3 ligands, as well. We quantified acetate, propionate, and butyrate in the serum of C57BL/6J and CC030 mice at baseline via mass spectrometry, and CC030 mice had significantly lower serum concentrations of acetate compared to C57BL/6J, while there were no differences in propionate or butyrate (Supplementary Figure 9). The lower concentration of FFAR3 ligands in the serum of CC030 mice suggested that the increased FFAR3 signaling on CC030 ILC2s is dependent on increased receptor expression, not SCFA concentration.”\n2. I would suggest tempering the heading “FFAR3 signaling reprograms human ILC2s towards an anti-inflammatory effector state in vitro”, to “FFAR3 signaling partially reprograms human ILC2s towards an anti-inflammatory effector state in vitro”, to not overstate the conclusion.\nWe agree with the reviewer that the language in the headings should be tempered to reflect the partial nature of the conservation between mouse and human ILC2s with respect to FFAR3’s effects. We have amended the text to read “partially reprograms” in the results heading and Figure 8 legend."}]

Decision letter