Group 3 innate lymphoid cells produce the growth factor HB-EGF to protect the intestine from TNF-mediated inflammation

Abstract

Tumor necrosis factor (TNF) drives chronic inflammation and cell death in the intestine, and blocking TNF is a therapeutic approach in inflammatory bowel disease (IBD). Despite this knowledge, the pathways that protect the intestine from TNF are incompletely understood. Here we demonstrate that group 3 innate lymphoid cells (ILC3s) protect the intestinal epithelium from TNF-induced cell death. This occurs independent of interleukin-22 (IL-22), and we identify that ILC3s are a dominant source of heparin-binding epidermal growth factor-like growth factor (HB-EGF). ILC3s produce HB-EGF in response to prostaglandin E2 (PGE2) and engagement of the EP2 receptor. Mice lacking ILC3-derived HB-EGF exhibit increased susceptibility to TNF-mediated epithelial cell death and experimental intestinal inflammation. Finally, human ILC3s produce HB-EGF and are reduced from the inflamed intestine. These results define an essential role for ILC3-derived HB-EGF in protecting the intestine from TNF and indicate that disruption of this pathway contributes to IBD.

Extended Data Fig. 1. The role of ILC3s and IL-22 in TNF-induced intestinal epithelial cell death

a. Experimental design of the assay. b. Mice in (a) were examined for the depletion efficiency of ILC3s in the small intestine. (n=3). c. CD90.2 levels in ILC2s or ILC3s were determined in IgG control mice. (n=3 individual mice). d. Mice in (a) were examined for the depletion efficiency of ILC2s in the small intestine. (n=3). e. Representative immunofluorescence sections and quantifications of TUNEL staining of the small intestine from noted mice 4 hours post recombinant TNF injection (N=3 mice per group, n=24 fields quantified per group). f. Mice in (a) were examined for the neutralization efficiency of endogenous IL-22 (n=3 individual mice). Data are representative of two independent experiments, shown as the means ± S.E.M. Statistics are calculated by unpaired two-tailed Student’s t-test (b-d and f) or one-way ANOVA (e). Scale bars=50 μm.

Extended Data Fig. 2. Signature gene expression of ILC clusters in the scRNA-Seq data set

Small intestinal ILCs were sort-purified from PBS- or TNF-treated mice and determined by scRNA-Seq. UMAP shows the relative expression of signature genes in all clusters of each treatment condition.

Extended Data Fig. 3. ILC3s express HB-EGF in the large intestine

a. Flow cytometry plots show HB-EGF expression in large intestine lamina propria (LI-LP) of noted mice as measured by conversion of the fluorescent LacZ substrate fluorescein di-β-d-galactopyranoside (FDG). b. Graph of frequency of HB-EGF⁺ cells in SI-LP of noted mice as measured by LacZ activity (n=5 individual mice). c-e. Flow cytometry plots (c), graph of frequency (d) and quantification of cell number (e) of HB-EGF⁺ cells in LI-LP of noted mice as measured by LacZ activity (n=4 individual mice). In both small and large intestines, CD4⁺ T cells were gated on CD45⁺CD3ε⁺CD4⁺, macrophages were gated on CD45⁺CD11b⁺CD11c⁺MHC II⁺CD64⁺, dendritic cells were gated on CD45⁺CD11b⁺CD11c⁺MHCII⁺CD64⁻, ILC2s were gated on CD45⁺Lin⁻ CD127⁺CD90.2⁺KLRG1⁺, ILC3s were gated on CD45⁺Lin⁻CD127⁺CD90.2⁺KLRG1⁻CD45^dim CCR6⁺. Lineage 1: CD3ε, CD5, CD8α, NK1.1, TCRγδ. Lineage 2: CD11b, CD11c, B220. Data in a-e are representative of two or three independent experiments, shown as the means ± S.E.M. Statistics are calculated by one-way ANOVA.

Extended Data Fig. 4. HB-EGF protects from TNF-induced intestinal epithelial cell death

a-c. Cell death was induced in the murine rectum epithelial cell line CMT-93 by TNF and a cIAP inhibitor. Cell death was determined by representative flow cytometric analysis of cleaved-caspase 3 (a) or cleaved-caspase 8 (c) in the presence or absence of recombinant HB-EGF. Cell death was determined by representative flow cytometric analysis of cleaved-caspase 3 in the presence of various inhibitors (b). Data in a and b are representative of three independent experiments, data in c are pooled from three independent experiments, shown as the means ± S.E.M. Statistics are calculated by one-way ANOVA.

Extended Data Fig. 5. A PGE2-EP2 axis controls HB-EGF expression in ILC3s

a. qPCR analysis of Hbegf expression in sort-purified ILC3s cultured with or without TNF treatment, relative to Hprt (n=4 individual mice). b and c. UMAP show the expression of Ptgs2 (b) or Ptges (c) gene in all clusters of each treatment condition. The dotted line indicates CCR6⁺ ILC3 clusters. d. qPCR analysis of Hbegf expression in sort-purified ILC3s cultured with or without retinoic acid, relative to Hprt (n=6). e. Bar graph of the frequencies of IL-17A, IL-22, IFN-γ or GM-CSF in ILC3s in the presence or absence of PGE2 stimulation ex vivo (n=6). f. qPCR examination of Hbegf transcript in sort-purified ILC3s in the presence of various stimulus, relative to Hprt (n=4 individual mice). g. Flow cytometry plots with graph of the frequencies of HB-EGF⁺ immune cells in small intestine of Rorc^creHbegf^f/− mice with or without EP2 agonist in vivo treatment (n=3 individual mice of Mock and n=4 individual mice of EP2 agonist). h. Western blot analysis of pro-HB-EGF in sort-purified ILC2s or ILC3s from small intestine of Rag1^−/− mice with or without EP2 agonist in vivo treatment. i. Sex- and age- matched Ptger2^f/f and Rorc^crePtger2^f/f mice were examined for the deletion efficiency of EP2 in ILC3s in the small intestines by qPCR, relative to Hprt (n=6 individual mice). j. Sort-purified ILC3s were stimulated with IL-1β in the presence or absence of EP2 antagonist, Hbegf expression was then examined by qPCR (n=6 individual mice). Data in a and f-h are representative of two independent experiments, data in d, e, i and j are pooled from two independent experiments, shown as the means ± S.E.M. Statistics are calculated by unpaired (a, g and i) or paired (d and e) two-tailed Student’s t-test or one-way ANOVA (f and j). *, non-specific band.

Extended Data Fig. 6. ILC3-derived HB-EGF does not impact immune homeostasis in the healthy gut

a and b. Flow cytometry plots with graph of frequencies and quantification of cell numbers of ILC3s (a) or IL-22⁺ ILC3s (b) in the large intestine of Hbegf^f/f and Hbegf^△ILC mice at steady state (n=5 individual mice). c. Graph of frequencies and quantification of cell numbers of T cell subsets in the large intestine of Hbegf^f/f and Hbegf^△ILC mice at steady state (n=5 individual mice). Data in a-c are representative of two independent experiments, shown as the means ± S.E.M. Statistics are calculated by unpaired two-tailed Student’s t-test.

Extended Data Fig. 7. Loss of T cell-derived HB-EGF does not alter susceptibility to experimental intestinal inflammation

a-c. Hbegf^f/f and Hbegf^{△T cell} mice were given 3% DSS for 6 days, disease and recovery were monitored by weight loss (a), colon shortening (b) and H&E staining of the distal colon (c) at day 11 (n=5 individual mice). Data in a-c are representative of two independent experiments, shown as the means ± S.E.M. Statistics are calculated by unpaired two-tailed Student’s t-test. Scale bars=100 μm.

Extended Data Fig. 8. Intestinal epithelial cell responses in the absence of ILC3-specific HB-EGF

a-d. Hbegf^f/f and Hbegf^△ILC mice were challenged with 3% DSS for 6 days (n=6 individual mice). The functional ability of intestinal epithelium was analyzed, including anti-microbial peptides production (a), tight junction proteins expression (b), stem cell composition (c) and proliferative ability (d) (N=4 individual mice per group, n=27 crypts quantified per group). e. Schematic model of chronic colitis induction. Data in a-d are representative of two independent experiments, shown as the means ± S.E.M. Statistics are calculated by unpaired two-tailed Student’s t-test. Scale bars=50 μm.

Extended Data Fig. 9. ILC3s produce HB-EGF to protect the intestine from TNF

Here we define a novel pathway of tissue protection in the mammalian intestine. Briefly, ILC3s sense intestinal damage or inflammation through IL-1β release, which amplifies ILC3 responses through a PGE2-EP2 axis, subsequently promoting HB-EGF production and protection against TNF-induced cell death in the intestinal epithelium.

Figure 1.. ILC3s protect the intestinal epithelium from TNF-induced cell death.

a and b. Representative immunofluorescence sections and quantifications of cleaved-caspase 3 (CC3) (a) or TUNEL (b) staining of the small intestine from noted mice 4 hours post recombinant TNF injection (N=3 individual mice per group, n=18 fields per group). c and d. Representative immunofluorescence sections and quantifications of CC3 (c) or TUNEL (d) staining of small intestine from noted mice 4 hours post recombinant TNF injection (N=3 individual mice per group, n=18 fields per group). Data in a-d are representative of two independent experiments, shown as the means ± S.E.M. Statistics are calculated by one-way ANOVA. WT, wild type. Scale bars=50 μm.

Figure 2.. ILC3s are a major cellular source of HB-EGF in the intestine.

a-d. Total ILCs were sort-purified from the small intestine of PBS- or TNF-treated mice and analyzed by scRNA-Seq. Uniform manifold approximation and projection (UMAP) embedding of 11,556 cells (dots), colored by cluster (a). Heatmap showing expression Z-scores of the indicated genes in ILC2s and ILC3s from TNF-treated mice (b). Dot plot shows the mean expression (color) of Hbegf in clusters grouped by cell type as noted, dot size represents the proportion of cells in a cluster with the gene detected (c). UMAP shows the expression of Hbegf gene in all clusters of each treatment condition and the dotted line indicates CCR6⁺ ILC3 clusters (d). e. Expression of Hbegf in the indicated sort-purified immune cells as determined by qPCR (n=3 individual mice). Relative to Actb. f. Flow cytometry plots show HB-EGF expression in small intestine lamina propria of noted mice as measured by conversion of the fluorescent LacZ substrate fluorescein di-β-d-galactopyranoside (FDG). g and h. Flow cytometry plots (g) and quantification of cell number (h) of HB-EGF⁺ cells in the small intestine of noted mice as measured by LacZ activity (n=5 individual mice). Lineage 1: CD3ε, CD5, CD8α, NK1.1, TCRγδ. Lineage 2: CD11b, CD11c, B220. Data in e-h are representative of two independent experiments, shown as the means ± S.E.M. Statistics are calculated by one-way ANOVA. DN, double-negative of CCR6 and Ncr1.

Figure 3.. ILC3-derived HB-EGF protects the intestinal epithelium from TNF.

a and b. Cell death was induced in the murine rectum epithelial cell line CMT-93 by TNF and a cIAP inhibitor. Bar graph of frequencies of cleaved-caspase 3 (CC3) in the presence or absence of recombinant HB-EGF (a). Bar graph of frequencies of CC3 in the presence of various inhibitors (b). c-e. Representative immunofluorescence sections and quantifications of cleaved-caspase 3 (c) or TUNEL (d) or cleaved-caspase 8 (e) staining of small intestine 4 hours post recombinant TNF injection (N=3 individual mice per group. n=31 fields per group of c, n=20 fields per group of d, n=28 fields per group of e). Data in a and b are pooled from three independent experiments, shown as the means ± S.E.M, calculated by one-way ANOVA. Data in c-e are representative of two independent experiments, shown as the means ± S.E.M, calculated by unpaired two-tailed Student’s t-test. i, inhibition. Scale bars=50 μm.

Figure 4.. ILC3s produce PGE2 in response to IL-1β.

a. Volcano plot of differential expression between PBS and TNF in CCR6⁺ ILC3s of scRNA-Seq data set. b and c. Dot plot show the mean expression (color) of Ptgs2 (b) or Ptges (c) in clusters grouped by cell type as noted. d. Ptgs2 transcript in sort-purified ILC3s in the presence of various stimulus, relative to Hprt (n=3 individual mice). e. Flow cytometry plots with graph of frequency of COX-2⁺ ILC3s from the SI-LPs of wild type mice following ex vivo culture (n=4 individual mice). f. Histograms and bar graph examination of mPGES-1 expression in indicated cells from the small intestine of wild type mice (n=5 individual mice). g. Sort-purified ILC3s were stimulated with or without IL-1β and PGE2 in supernatants were determined by ELISA (n=10 individual mice). Data in d-f are representative of two or three independent experiments, data in g are pooled from three independent experiments, shown as the means ± S.E.M. Statistics are calculated by paired two-tailed Student’s t-test (e and g) or one-way ANOVA (d and f).

Figure 5.. PGE2 elicits ILC3-intrinsic HB-EGF production through the EP2 receptor.

a. qPCR analysis of Hbegf expression in sort-purified ILC3s with or without PGE2 treatment, relative to Hprt (n=4 individual mice). b. Dot plot shows the mean expression (color) of Ptger1-4 in clusters grouped by cell type as noted from the small intestine of TNF-treated mice. c. Sort-purified ILC3s were stimulated with PGE2 in the presence of EP2 or EP4 antagonist, Hbegf expression was then examined by qPCR, relative to Hprt (n=6 individual mice). d. Flow cytometry plots with graph of the frequencies of HB-EGF⁺ ILC3s in small intestine of Rorc^creHbegf^f/+ mice with or without EP2 agonist in vivo treatment (n=3 individual mice in Mock, n=4 individual mice in EP2 agonist). e. ILC3-intrinsic HB-EGF of indicated mice at steady state was determined by qPCR, relative to Hprt (n=6 individual mice). f and g. Representative immunofluorescence sections and quantifications of cleaved-caspase 3 (CC3) (f) (N=3 individual mice per group, n=21 fields per group) or TUNEL (g) (N=3 mice per group, n=21 fields per group) staining of the small intestine from noted mice 4 hours post recombinant TNF injection. Data in a, d, f and g are representative of two or three independent experiments, data in c and e are pooled from two independent experiments, shown as the means ± S.E.M. Statistics are calculated by paired (a) or unpaired (d and e) two-tailed Student’s t-test or one-way ANOVA (c, f and g). Scale bars=50 μm.

Fig 6.. ILC3-derived HB-EGF protects from acute intestinal damage and inflammation.

a-f. Mice were given 3% DSS for 6 days, followed by normal water for recovery. Disease and recovery were monitored by weight loss (a), colon shortening (b), hematoxylin and eosin (H&E) staining with histopathology scores of the distal colon (c), lamina propria neutrophil infiltration (d) and CD4⁺ T cell response (e) at day 10 (n=6 individual mice). Intestinal epithelial cell death was examined by TUNEL staining at day 6 (f) (N=4 individual mice per group, n=16 fields per group). g-i. Wild-type mice were exposed to 3% DSS in the drinking water for 7 days and treated with PBS or 1 mg/kg recombinant HB-EGF intraperitoneally every other day starting on day 2 of DSS exposure. Mice were examined for weight loss (g) over the course of treatment, and colon length (h) and H&E staining with histopathology scores of the distal colon (i) were assessed on day 10 (n=4 individual mice in PBS, n=5 individual mice in rHB-EGF). Data are representative of two or three independent experiments, shown as the means ± S.E.M. Statistics are calculated by unpaired two-tailed Student’s t-test. Scale bars=50 μm in f. Scale bars=100 μm in c and i.

Fig 7.. HB-EGF-producing ILC3s protect from chronic intestinal inflammation and are altered in IBD.

a. Expression of Hbegf in the indicated sort-purified immune cells from large intestine of IL-10-deficient mice as determined by qPCR (n=3 individual mice). Relative to Hprt. b-f. Mice were orally infected with H. hepaticus and blockade of the regulatory response with anti-IL10R antibody (n=5 individual mice). Colon shortening (b), hematoxylin and eosin (H&E) staining with histopathology scores of the distal colon (c) (n=4 individual mice), lamina propria neutrophil infiltration (d) and CD4⁺ T cell response (e), and intestinal epithelial cell death was examined by TUNEL staining (f) (N=4 mice per group, n=25 fields per group) at day 21. g. Sort-purified ILC3s from human tonsils were stimulated with or without PGE2 and HBEGF transcript was determined by qPCR (n=7 individual donors). h-j, Human lamina propria lymphocytes were sort-purified from adjacent non-inflamed versus matched inflamed surgical resection tissues from the small intestine of Crohn’s disease patients (n=11 individual donors). HBEGF in the indicated cells of non-inflamed tissues were determined by qPCR, relative to ACTB (h). HBEGF in the NKp44⁻ILC3s or NKp44⁺ILC3s of adjacent versus inflamed tissues were determined by qPCR (i). Quantification of NKp44⁻ILC3s or NKp44⁺ILC3s numbers per gram of tissue between adjacent and inflamed tissues (j). Data in a-f are representative of two independent experiments, data in g-j are pooled from two or three independent experiments, shown as the means ± S.E.M. Statistics are calculated by one-way ANOVA (a) or unpaired two-tailed Student’s t-test (b-f), statistic in g and j are performed using two-tailed Wilcoxon matched-pairs test (paired), statistics in h and i are performed using two-tailed Mann–Whitney U-test (unpaired), Scale bars=50 μm in f. Scale bars=100 μm in c.