Non-redundant functions of group 2 innate lymphoid cells

doi:10.1038/s41586-022-05395-5

. 2022 Nov;611(7937):794-800.

doi: 10.1038/s41586-022-05395-5. Epub 2022 Nov 2.

Non-redundant functions of group 2 innate lymphoid cells

Katja J Jarick¹, Patrycja M Topczewska¹, Manuel O Jakob¹, Hiroshi Yano^{2

3

4}, Mohammad Arifuzzaman^{2

3

4}, Xuemei Gao¹, Sotiria Boulekou¹, Vladislava Stokic-Trtica¹, Pierre S Leclère¹, Alexandra Preußer¹, Zoe A Rompe¹, Anton Stamm¹, Amy M Tsou^{2

5}, Coco Chu², Frederik R Heinrich⁶, Gabriela M Guerra⁶, Pawel Durek⁶, Andranik Ivanov⁷, Dieter Beule⁷, Sofia Helfrich¹, Claudia U Duerr¹, Anja A Kühl⁸, Christina Stehle^{6

9}, Chiara Romagnani^{6

9

10}, Mir-Farzin Mashreghi⁶, Andreas Diefenbach^{1

6}, David Artis^{2

3

4

11}, Christoph S N Klose¹²

Affiliations

¹ Department of Microbiology, Infectious Diseases and Immunology, Charité-Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Berlin, Germany.
² Jill Roberts Institute for Research in Inflammatory Bowel Disease, Weill Cornell Medicine, New York, NY, USA.
³ Joan and Sanford I. Weill Department of Medicine, Division of Gastroenterology and Hepatology, Weill Cornell Medicine, Cornell University, New York, NY, USA.
⁴ Department of Microbiology and Immunology, Weill Cornell Medicine, Cornell University, New York, NY, USA.
⁵ Division of Pediatric Gastroenterology, Hepatology and Nutrition, Weill Cornell Medical College, New York, NY, USA.
⁶ Deutsches Rheuma-Forschungszentrum (DRFZ), an Institute of the Leibniz Association, Berlin, Germany.
⁷ Core Unit Bioinformatics, Berlin Institute of Health at Charité-Universitätsmedizin Berlin, Berlin, Germany.
⁸ iPATH.Berlin - Core Unit of the Charité Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Berlin, Germany.
⁹ Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Department of Gastroenterology, Infectious Diseases and Rheumatology, Hindenburgdamm 30, 12203, Berlin, Germany.
¹⁰ Leibniz-Science Campus Chronic Inflammation, Berlin, Germany.
¹¹ Friedman Center for Nutrition and Inflammation, Joan and Sanford I. Weill Department of Medicine, Department of Microbiology and Immunology, Weill Cornell Medicine, Cornell University, New York, NY, USA.
¹² Department of Microbiology, Infectious Diseases and Immunology, Charité-Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Berlin, Germany. christoph.klose@charite.de.

PMID: 36323785
PMCID: PMC7614745
DOI: 10.1038/s41586-022-05395-5

Non-redundant functions of group 2 innate lymphoid cells

Katja J Jarick et al. Nature. 2022 Nov.

. 2022 Nov;611(7937):794-800.

doi: 10.1038/s41586-022-05395-5. Epub 2022 Nov 2.

Authors

Affiliations

¹ Department of Microbiology, Infectious Diseases and Immunology, Charité-Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Berlin, Germany.
² Jill Roberts Institute for Research in Inflammatory Bowel Disease, Weill Cornell Medicine, New York, NY, USA.
³ Joan and Sanford I. Weill Department of Medicine, Division of Gastroenterology and Hepatology, Weill Cornell Medicine, Cornell University, New York, NY, USA.
⁴ Department of Microbiology and Immunology, Weill Cornell Medicine, Cornell University, New York, NY, USA.
⁵ Division of Pediatric Gastroenterology, Hepatology and Nutrition, Weill Cornell Medical College, New York, NY, USA.
⁶ Deutsches Rheuma-Forschungszentrum (DRFZ), an Institute of the Leibniz Association, Berlin, Germany.
⁷ Core Unit Bioinformatics, Berlin Institute of Health at Charité-Universitätsmedizin Berlin, Berlin, Germany.
⁸ iPATH.Berlin - Core Unit of the Charité Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Berlin, Germany.
⁹ Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Department of Gastroenterology, Infectious Diseases and Rheumatology, Hindenburgdamm 30, 12203, Berlin, Germany.
¹⁰ Leibniz-Science Campus Chronic Inflammation, Berlin, Germany.
¹¹ Friedman Center for Nutrition and Inflammation, Joan and Sanford I. Weill Department of Medicine, Department of Microbiology and Immunology, Weill Cornell Medicine, Cornell University, New York, NY, USA.
¹² Department of Microbiology, Infectious Diseases and Immunology, Charité-Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Berlin, Germany. christoph.klose@charite.de.

PMID: 36323785
PMCID: PMC7614745
DOI: 10.1038/s41586-022-05395-5

Abstract

Protective immunity relies on the interplay of innate and adaptive immune cells with complementary and redundant functions. Innate lymphoid cells (ILCs) have recently emerged as tissue-resident, innate mirror images of the T cell system, with which they share lineage-specifying transcription factors and effector machinery¹. Located at barrier surfaces, ILCs are among the first responders against invading pathogens and thus could potentially determine the outcome of the immune response². However, so far it has not been possible to dissect the unique contributions of ILCs to protective immunity owing to limitations in specific targeting of ILC subsets. Thus, all of the available data have been generated either in mice lacking the adaptive immune system or with tools that also affect other immune cell subsets. In addition, it has been proposed that ILCs might be dispensable for a proper immune response because other immune cells could compensate for their absence^3-7. Here we report the generation of a mouse model based on the neuromedin U receptor 1 (Nmur1) promoter as a driver for simultaneous expression of Cre recombinase and green fluorescent protein, which enables gene targeting in group 2 ILCs (ILC2s) without affecting other innate and adaptive immune cells. Using Cre-mediated gene deletion of Id2 and Gata3 in Nmur1-expressing cells, we generated mice with a selective and specific deficiency in ILC2s. ILC2-deficient mice have decreased eosinophil counts at steady state and are unable to recruit eosinophils to the airways in models of allergic asthma. Further, ILC2-deficient mice do not mount an appropriate immune and epithelial type 2 response, resulting in a profound defect in worm expulsion and a non-protective type 3 immune response. In total, our data establish non-redundant functions for ILC2s in the presence of adaptive immune cells at steady state and during disease and argue for a multilayered organization of the immune system on the basis of a spatiotemporal division of labour.

PubMed Disclaimer

Conflict of interest statement

Declaration of interests

D.A. has contributed to scientific advisory boards at Pfizer, Takeda, FARE, and the KRF. The other authors declare no competing interests.

Figures

**Extended data 1 (related to Figure 1). Gating strategy for flow cytometry.**
a, Generation of genetic construct of the *Nmur1*^Cre-T2A-GFP BAC transgene. 70^th bp of exon 2 to 29^th bp downstream of the TGA stop codon of exon 3 was replaced by a cassette containing iCre and eGFP linked with P2A and T2A. (86,388,138-86,386,296) were replaced through homologous recombination. The FRT-flanked kanamycin selection cassette was deleted after FLP-mediated recombination. Removal of LoxP and LoxP511 sites on BAC vector backbone. b, Expression of the *Nmur1* gene in GFP⁺and GFP’ cells purified by FACsorting from the intestinal lamina propria. c, Gating strategy of bone marrow precursors. Lineage: CD3, CD5, CD19, Ly6G, FcεRIa. d, Gating strategy of intestinal lymphoid populations. e, Gating strategy of intestinal myeloid populations. Lineage: CD3, CD5, CD19, FcεRIa. f, Gating strategy of intestinal eosinophils, 2 alternative gates shown. Lineage: CD3, CD5, CD19, Ly6G. g, Gating strategy of γδ T cells, regulatory T cells (Treg) and TH2 cells from the mesenteric lymph node. h, Gating strategy of mast cells and basophils from the peritoneal lavage. Lineage: CD3, CD5, CD19, Ly6G

**Extended data 2 (related to Figure 1). Nmur1^Cre-T2A-GFP specifically labels ILC2s in steady state and during type 2 inflammation.**
a, Flow-cytometric plots of GFP and ST2 in lung ILC2s (left) and T_H2 cells (right) of *Nmur1*^Cre-T2A-GFP mice. Numbers denote percentage of cells inside the gate. b, GFP expression in T_H2 cells and ILC2s of *Nmur1*^Cre-T2A-GFP mice, quantification of (a). c, GFP expression in mLN CD45^- cells (black) and ILC2s (gray). d, Quantification of (c). e, Quantification of GFP expression across organs and cell types as displayed in the heatmap of Fig. 1d. f, RFP and ST2 in lung ILC2s (left) and T_H2 cells (right) of *Nmur1*^Cre-T2A-GFP x *Rosa26*^Rfp/Rfp F1 offspring. Numbers denote percentage of cells inside the gate. g, RFP expression in T_H2 cells and ILC2s, quantification of (f). h, RFP expression in mLN CD45^- cells (black) and ILC2s (gray) of *Nmur1*^Cre-T2A-GFP x *Rosa26*^Rfp/Rfp F1 offspring. i, Quantification of (h). j, RFP expression across organs and cell types as displayed in the heatmap of Fig. 1h. k, Heatmap of flow-cytometric quantification of GFP and RFP expression in TH2 and Treg cells. Values represent median percentage of positive cells of parental gate, n=3 mice per group. l-q, Immunofluorescence micrographs of RFP expression in *Nmur1* C^re-T2A^-GFP _xRosa26^Rlp/Rlp F1 offspring stained for alpha smooth muscle actin (α-SMA), CD45 and DAPI in the small intestine (l), mesenteric lymph nodes (m), lung (n), mesenteric fat (o), femoral muscle (p) and large intestine (q). r, GFP and Cre (RFP) expression seven days after *Nippostrongylus brasiliensis* (N.b.) helminth infection across immune cell populations in *Nmur1*^Cre-T2A-GFP x *Rosa26^Rfp/Rfp* F1 offspring. s,t, GFP (s) and RFP (t) expression in *Nmur1*^Cre-T2A-GFP *Rosa26*^Rfp/+ mice, seven days after infection with N.b.. Data in (r-t) are representative of two independent experiments with 3 mice per group.

**Extended Data 3, (related to Figure 2). Nmur1^Cre-T2A-GFPGata3^flox/flox and Nmur1^Cre-t2A-GFP id2^flox/flox mice lack ILC2s in all tissues investigated.**
a, Flow-cytometric plots of ILC2s gated from live CD45⁺ Lin^- (CD3, CD5, CD19) cells as CD127⁺ KLRG1⁺ or CD127⁺ ST2⁺ across organs of *Nmur1*^Cre-T2A-GFP *Gata3*^flox/flox mice and littermate controls. Numbers denote percentage of cells inside the gate. b, Quantification of (a). Mean +/- SD, Student’s t-test. **c-d**, Heatmaps displaying Log2-fold changes of the median cell frequencies among live CD45⁺ cells across different populations and organs of *Nmur1*^Cre-T2A-GFP *Gata3*^flox/flox (c) and *Nmur1*^Cre-T2A-GFP *Id2*^flox/flox (d) mice, compared to the respective littermate controls. Results are representative for 2 independent experiments with 3-4 mice per group. Littermate controls for (a-c) were *Gata3^flox/+* or *Nmur1*^Cre-T2A-GFP *Gata3*^flox/+, for (d) *Id2*^flox/+ or *Nmur1*^Cre-T2A-GFP *Id2*^flox/+. e, Quantification of relative T_Hh2 (top) and Treg (bottom) numbers in *Nmur1*^Cre-T2A-GFP *Id2*^flox/flox mice and *Id2*^flox/flox littermate controls. Results are representative for 3 independent experiments with 3-6 mice per group. Mean +/- SD, Student’s t-test. f, Cytokine secretion by cultured *Nmur1*^Cre-T2A-GFP *Id2*^flox/flox immune cells isolated from lung and intestine, stimulated with IL-2, IL-7, IL-25 and IL-33 or PMA/Ionomycin for 8 h, measured in the supernatant by cytometric bead assay. Mean +/-SD, two-way ANOVA with Šídák’s multiple comparison tests, data are representative of two independent experiments with 3-4 mice per group. g, Serum IL-5 in untreated *Id2*^flox/flox and *Nmur1*^Cre-T2A-GFP *Id2*^flox/flox animals as determined by the BD Cytometric Bead Array Mouse IL-5 Enhanced Sensitivity Flex Set, Student’s t-test. ns not significant, *p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. (a-g) One symbol represents data from one mouse.

**Extended Data 4 (related to Figure 3). ILC2s regulate eosinophils in tissue.**
Analysis of eosinophils across organs in the steady state. a, Eosinophils were gated from live CD45⁺ Lin^- (CD3, CD5, CD19, Ly6G) cells using Siglec F and CCR3, across organs of *Nmur1*^Cre-T2A-GFP *Id2*^flox/flox mice and littermate controls. b, Relative ILC2 and eosinophil numbers in bone marrow chimera of *Nmur1*^Cre-T2A-GFP *Id2*^flox/flox (KO) and *Id2*^flox/flox (WT) recipients transplanted with bone marrow of *Nmur1*^Cre-T2A-GFPId2^flox/flox (KO) or *Id2*^flox/flox (WT) donors. c, Quantification of eosinophils in *IL5^-/-* mice and respective *IL5^+/-* littermate controls. d, Flow-cytometric plots of granulocyte/macrophage progenitor (GMP) gated from live CD45⁺ Lin^- (CD3, CD5, CD19, Gr1, B220) Sca-1^- c-Kit^hi cells as CD34⁺CD16/CD32^hi and relative quantification in bone marrow of *Nmur1*^Cre-T2A-GFP *Id2*^flox/flox mice and *Id2*^flox/flox littermate controls. e, Quantification of relative Eosinophil precursors (EoP) and GMPs in the bone marrow of *IL-5-/-* compared to WT controls. f, Flow-cytometric quantification of relative numbers of bone marrow-derived eosinophils gated from live CD45⁺ Lin^- (CD3, CD5, CD19) CD11b⁺ cells as SSC^hi and Siglec F⁺. BM cells were cultured with either SCF and FLT3L, IL-5, ILC2P or supernatant from ILC2s for 10 days. g,h Mean fluorescence intensity of CCR3 on eosinophils in *Nmur1*^Cre-T2A-GFP *Id2*^flox/flox (g) and *Il5-/-* (h) mice and respective littermate controls. b-e, g-h One symbol represents data from one mouse (b-e, g-h) or one well (f), mean +/- SD, c-**e, g-h** Student s t-Test. b, f Oneway ANOVA with Tukey’s multiple comparisons, ns not significant, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

**Extended Data 5 (related to Figure 3). ILC2 deletion regulates myeloid and epithelial populations in the lung.**
a, Bubble plot of marker genes of global cell populations in scRNA sequencing of myeloid and epithelial cells in the steady state lung of *Nmur1*^Cre-T2A-GFP *Id2*^flox/flox mice and littermates. b, Marker genes of subclusters of interstitial macrophages. c, Marker genes of DC subclusters. d, Marker genes in subclusters of alveolar type 2 epithelial cells. e-g, Subclusters in e macrophages, f dendritic cells and g alveolar type 2 epithelial cells. h, Macrophage UMAPs color-coded gray-to-red with genes downregulated in Macrophages of *Nmur1*^Cre-T2A-GFP *Id2*^foxl/flox mice. i, Macrophage UMAPs color-coded with genes upregulated in macrophages of *Nmur1*^Cre-T2A-GFP *Id2*^flox/flox mice. j, DC UMAP color-coded by Ccl24 expression. k, UMAPs of alveolar type 2 epithelial cells color-coded with genes downregulated in alveolar type 2 epithelial cells in *Nmur1*^Cre-T2A-GFP *Id2*^flox/flox mice. l, Statistical evaluation of genes displayed in f-h. Cells from four mice per group were pooled. Littermate controls for (a-l) were *Id2*^flox/flox or *Id2*^flox/+.

**Extended Data 6 (related to Figure 3). ILC2s regulate myeloid cells in type-2 inflammatory allergy models of the lung.**
**a-d**, Flow-cytometric quantification of cell population in *Nmur1*^Cre-T2A-GFP *Id2*^flox/flox mice and littermate controls sensitized with *A. alternata* extract for three consecutive days, analyzed 7 days after the first dose. a, absolute numbers of lung ILC2s, lung an BAL eosinophils and total CD45+ cells in the BAL. b, Relative and absolute numbers of T_H2 cells in the lung. c,d, relative (c) and absolute (d) numbers of myeloid populations from the lung interstitium, and alveolar macrophages from the BAL. Data in a-d are representative of two independent experiments with 4-6 animals per group. **e-i**, Analysis of *Nmur1*^Cre-T2A-GFPId2^flox/flox mice and littermate controls sensitized with papain for three consecutive days, analyzed 7 days after the first dose. e, Absolute numbers of ILC2s in the lung interstitium, and eosinophils in the lung interstitium and BAL. f, Eosinophils are visualized by modified Sirius red stain in the lung. g, Relative and absolute numbers of T_H2 cells in the lung. h,i, relative (h) and absolute (i) numbers of myeloid populations from the lung interstitium, and alveolar macrophages from the BAL. Data in e and g-i are pooled from two independent experiments with 4-5 mice per group. Untreated controls for (e-i) were *Id2*^flox/flox, papain-treated littermate controls were *Id2*^flox/flox, *Id2*^flox/+ or *Nmur1*^Cre-T2A-GFP *Id2*^flox/+. One symbol represents data from one mouse, mean+/- SD, Oneway ANOVA with Tukey’s multiple comparison tests, ns: not significant, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. BAL, bronchoalveolar lavage.

**Extended Data 7 (related to Figure 3). ILC2s regulate myeloid cells in type-2 inflammatory allergy models of the lung.**
**a-e,** Analysis of *Nmur1*^Cre-T2A-GFP *Id2*^flox/flox mice (cKO) and littermate controls (Ctrl) sensitized and challenged with house dust mite. a, Lung ILC2s (top), BAL eosinophils (middle) and Lung T_H2 cells (bottom) were gated from live CD45⁺ Lin^- (CD11b-CD11c-FcεR1a-B220-CD3-5-) cells. The second gate in the eosinophil plots marks alveolar macrophages. b, Quantification of (a). c, Periodic-acid-Schiff staining of lungs. d, Gating of cytokine-expressing T helper cells isolated from the lung. e, Quantification of (d). f-j, Resident and inflammatory eosinophils from the lungs of papain- and *A. alternata*-treated mice. f, Flow-cytometric plots showing resident and inflammatory eosinophils from lung after Papain treatment of *Nmur1*^Cre-T2A-GFP *Id2^flox/flox* mice (cKO) and littermate controls (Ctrl). g, Relative and absolute numbers of resident and inflammatory eosinophils. h, Flow-cytometric plots showing resident and inflammatory eosinophils from lung after *Altenaria alternata* treatment of *Nmur1*^Cre-T2A-GFP *Id2*^flox/flox mice (cKO) and littermate controls (Ctrl). i, Relative and absolute numbers of resident and inflammatory eosinophils. Data are representative for two independent experiments with 3-5 mice per group (f-i). Symbols represent data from one mouse, mean +/- SD, One-way ANOVA with Tukey’s multiple comparisons (b-e) or Student’s t-test (g,i), ns not significant, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

**Extended Data 8 (related to Figure 3). Eosinophils are altered in ILC2-deficient mice.**
**a-f**, Flow-cytometric analysis of eosinophils and ILC2s in the BAL and lung interstitium of *Id2*^flox/flox, *Nmur1*^Cre-T2A-GFP *Id2*^flox/flox mice treated with either PBS or recombinant IL-5, seven days after allergy induction by intranasal application of *Alternaria alternata* extract. a,b Eosinophils in the lung interstitium, c,d Eosinophils in the BAL, e,f ILC2s in the lung interstitium. Symbols represent data from one mouse, data are pooled from two independent experiments. Mean +/- SD, Student’s t-test, ns not significant, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. g Flow-cytometric plots showing the gating strategy for the analysis and sorting of eosinophils from lung after Papain treatment of *Nmur1*^Cre-T2A-GFP *Id2*^flox/flox mice (cKO) and littermate controls (Ctrl). Data are representative of one experiment with 5 mice per group. h, Pathways from tmod gene set enrichment analysis (GSEA) of bulk RNA sequencing from eosinophils of 4 *Nmur1*^Cre-T2A-GFP *Id2*^flox/flox mice vs 4 WT control animals. Bars are color-coded according to Databases: Gene Ontology (GO, red), Reactome (gray), Hallmark (white), tmod (purple), KEGG (pink). Genes differentially expressed in eosinophils after papain treatment of *Nmur1*^Cre-T2A-GFP *Id2*^flox/flox mice (cKO) and littermate controls (Ctrl) were used for the analysis. i, Volcano plot showing differentially expressed genes of interest in eosinophils after papain treatment of *Nmur1*^Cre-T2A-GFP *Id2*^flox/flox mice (cKO) and littermate controls (Ctrl) j, Heatmaps of genes differentially expressed in eosinophils after papain treatment of *Nmur1*^Cre-T2A-GFP *Id2*^flox/flox mice (cKO) and littermate controls (Ctrl). Each heatmap corresponds to one pathway shown in (h).

**Extended Data 9 (related to Figure 4). ILC2-deficient mice are highly susceptible to *N. brasiliensis* infection**
*Nmur1*^Cre-T2A-GFP *Id2*^flox/flox mice (cKO) and littermate controls (Ctrl) were analyzed 7 (a-c) or 11 days (i-m) after infection with *N.b.*, alongside untreated WT controls. a, T_H2 cells. b,c, Neutrophils in the lung. Littermate controls for (a-c) were *Id2*^flox/+ or *Nmur1*^Cre-T2A-GFP *Id2*^flox/+. **d-h**, *Nmur1*^iCre-GFP *Gata3*^flox/flox mice (cKO) and littermate controls (Ctrl) 7 days after *N. b.* infection alongside untreated WT controls. **d-e**, ILC2s (top) and eosinophils (bottom) in the mLN (d) or mLN and lung (e). f, Worm burden g, Goblet- and tuft cells per villus, mucus area quantified from UEA-I stain, all as shown in (h). h, left: Periodic acid-Schiff staining; middle: intestinal tuft cell staining with Dclk1 (red), DAPI (blue); right: intestinal mucus stained with UEA-I (green), DAPI (blue). Littermate controls for (d-h) were *Gata3*^flox/+. i,j, ILC2s and eosinophils in the mLN (i) or mLN and lung (j) 11 days after infection. k, T_H2 cells. l, Goblet- and tuft cells per villus, mucus area quantified from UEA-I stain as shown in (m). m, intestine as shown in (h), 11 days after infection. **n-r** Infection of BM chimeras of C57BL/6 recipients transplanted with bone marrow of *Id2*^flox/flox(Ctrl), *Nmur1*^Cre-T2A-GFP *Id2*^flox/flox (cKO) donors, or bone marrow of cKO donors supplemented with sorted ILC2 precursors (cKO + ILC2p) from C57BL/6 donors, with *N.b..*n, Periodic acid-Schiff staining of small intestine seven days after *N. b.* infection. o, Goblet cells per villus. p,q, Neutrophils in the lung. r, T_H2 cells. s, ILC3s, LTi-like ILC3s and NCR⁺ ILC3s 7 days after *N.b.* infection of *Nmur1^Cre-T2A-GFP Id2^flox/flox* mice (cKO) and littermate controls (Ctrl). (a-s) One symbol represents data from one mouse, one-way ANOVA with Tukey’s multiple comparisons, ns not significant, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

**Extended Data 10 (related to Figure 4). Gene expression in epithelial cells of ILC2-deficient mice during *N. brasiliensis* infection**
a, Heatmap showing scaled expression of marker genes for colonic cell populations in scRNA Seq Data shown in Fig 4m. b, Violin plots showing normalized count distributions of differentially expressed genes *Reg3b, Reg3g, Muc-2, Clca1* and *Ang4* in colonic epithelial cells between infected *Nmurl*^Cre-T2A-GFPId2 ^flox/flox mice (cKO) and littermate controls (Ctrl) in all clusters.

**Figure 1. *Nmurl*^Cre-T2A-GFP faithfully labels ILC2s.**
a, Flow cytometric histograms of GFP expression in ILC2s of *Nmur1*^Cre-T2A-GFP (green) and C57BL/6 WT mice (gray) across organs. ILC2s were gated on live CD45⁺ Lin^- (CD3, CD5, CD19) CD127⁺ and KLRG1⁺ or ST2⁺. b, Quantification of ILC2s expressing GFP as in (a). c, Flow cytometric histograms of GFP expression in *Nmur1*^Cre-T2A-GFP mice across immune cell populations in the steady state. d, Heatmap of flow-cytometric quantification of GFP-expressing cells, across immune cell populations and organs. Values represent percentage of positive cells of parental gate, median of three mice. X, not determined. e, Flow cytometric histograms of Cre expression, measured by RFP fluorescence in *Nmur1*^Cre-T2A-GFP x *Rosa26*^Rfp/RfP F1 offspring, in ILC2s across organs in the steady state. f, Quantification of ILC2s expressing RFP as in (e). g, Flow cytometric histograms of Cre (RFP) expression across immune cell populations in the steady state. h, Heatmap of flow-cytometric quantification of Cre-expressing cells, across immune cell populations and organs. SI small intestine, mLN mesenteric lymph nodes, BM bone marrow, PL peritoneal lavage. Each symbol represents data from one mouse, data are representative of two experiments with three mice per group. Mean +/- SD, Student’s t-test, ** p<0.01, *** p<0.001, **** p<0.0001.

**Figure 2. *Nmurl*^Cre-T2A-GFP *Id2*^flox/flox mice lack ILC2s in all tissues investigated.**
a, Flow-cytometric plots of ILC2s gated from live CD45⁺ Lin^- (CD3, CD5, CD19) cells as CD127⁺ KLRG1⁺ or CD127⁺ ST2⁺ across organs of *Nmur1*^Cre-T2A-GFP *Id2*^flox/floxmice (cKO) and littermate controls (Ctrl). Numbers denote percentage of cells inside the gate. b,c, Quantification of (a), Student’s t-test. d, Heatmap displaying Log2-fold changes of the median cell frequencies among live CD45⁺ cells across different populations and organs of *Nmur1*^Cre-T2A-GFP *Id2*^flox/flox mice, compared to littermate controls. X, not determined. e, IL-5 secretion measured in the supernatant of cultured immune cells isolated from lung or intestine of *Nmur1*^Cre-T2A-GFP *Id2*^flox/flox mice and littermate controls, stimulated with IL-2, IL-7, IL-25 and IL-33 or PMA/Ionomycin for 8 h. Each symbol represents data from one mouse, mean +/- SD, two-way ANOVA with Šídák’s multiple comparison tests, data are representative of two independent experiments with 3-4 mice per group. ns not significant, *p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. Littermate controls for a-d were *Id2*^flox/+ and *Nmur1*^Cre-T2A-GFP *Id2*^flox/+combined.

**Figure 3. ILC2-deficient mice have reduced eosinophil counts and fail to develop eosinophilia in allergic asthma models**
a, Lung eosinophils in untreated *Nmur1*^Cre-T2A-GFP *Id2*^flox/flox and littermate controls gated from live CD45⁺Lin^- (CD3, CD5, CD19, Ly6G) cells using Siglec F and CCR3. b, Percentage and absolute numbers of eosinophils across organs. c, Eosinophil numbers in bone marrow chimeras of *Nmur1*^Cre-T2A-GFP *Id2*^flox/flox (KO) and *Id2*^flox/flox (WT) recipients transplanted with bone marrow of KO or WT donors. d, Pearsons correlation and linear regression of ILC2s and eosinophils frequencies. R², coefficient of determination; p, P value. e, Eosinophil-lineage-committed progenitor (EoP) gated from live CD45⁺ Lin^- (CD3, CD5, CD19, Gr1, B220) Sca1^- CD34⁺ and relative quantification in bone marrow of *Nmur1*^Cre-T2A-GFP *Id2*^flox/flox mice compared to littermate controls. f, UMAP displaying cell population clusters of scRNA-sequenced lung myeloid (blue shades) and epithelial (red shades) cells of untreated *Nmur1*^Cre-T2A-GFP *Id2*^flox/flox mice and littermate controls (*Id2*^flox/+ or *Id2*^flox/flox). g, Violin plots of differentially expressed genes in populations marked by triangles in Extended Data figure 5 e-g. **h-j**, Lung eosinophils of *Nmur1*^Cre-T2A-GFP *Id2*^flox/flox mice and littermate controls, 7 (**h,i**) or 17 (j) days after allergy induction by intranasal application of *Alternaria alternata* (h), papain (i), or house dust mite (j) and percentage of the indicated cell subsets. Untreated littermate controls in h and j were *Id2*^flox/flox, papain-treated littermate controls were *Id2*^flox/+ or *Id2*^flox/flox or *Nmur1*^Cre-T2A-GFP *Id2*^flox/+). BAL, bronchoalveolar lavage. Data are representative of the following number of independent experiments and mice per group: four experiments with 3-5 mice (a,b), two experiments with 4-5 mice(c,) 3-5 (e), 3-5 mice (h,j) or pooled from two experiments with 3-4 mice (i). Mean +/- SD, Student’s t-test (b,e) or one-way ANOVA with Tukey’s multiple comparisons (c, h-j), ns not significant, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

**Figure 4. ILC2-deficient mice are susceptible to *N. brasiliensis* infection**
**a,b** Natural and inflammatory ILC2s and eosinophils in the mLN of untreated WT animals and infected *Nmur1*^Cre-T2A-GFP *Id2*^flox/flox ILC2-deficient mice (cKO) and littermate controls (Ctrl). b, Relative ILC2- and eosinophil numbers in mLN and lung. c, Worm burden in untreated WT animals and infected *Nmur1*^Cre-T2A-GFP *Id2*^flox/flox ILC2-deficient mice or littermate controls. d, Goblet- and tuft cell numbers per villus and quantification of UEA-I⁺ pixels. e, Periodic acid-Schiff staining of murine small intestine seven days after infection (left). Immunofluorescence micrographs of murine small intestine, tuft cells (Dclk1, red, middle) mucus (UEA-I, green, right) and DAPI (blue). Data in a-e are representative of two independent experiments with 3-4 mice per group. f, Worm burden as in (c), 11 days after infection. **g-i**, Infection of BM chimeras with *Nippostrongylus brasiliensis.* C57BL/6 recipients were transplanted with bone marrow of *Id2*^flox/flox (Ctrl), *Nmur1*^Cre-T2A-GFP *Id2*^flox/flox (cKO) donors, or bone marrow of *Nmur1*^Cre-T2A-GFP *Id2*^flox/flox(cKO) donors supplemented with sorted ILC2 precursors from C57BL/6 donors (cKO + ILC2p). g, ILC2s and eosinophils in the mLN. h, ILC2s and eosinophils in mLN and lung. i, Worm burden 7 days after infection. Data are representative of two independent experiments with 3-6 mice per group. j, Serum cytokines 7 days after infection as in a-e. Dotted lines indicate detection limits. a-j One-way ANOVA with Tukey’s multiple comparisons. ns not significant, * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. k, UMAP displaying cell population clusters of scRNA-sequencing of colon epithelium from *N.b.* infected *Nmur1*^Cre-T2A-GFP *Id2*^flox/flox mice and littermate controls. l, Differentially expressed genes in colonic epithelial cells of infected *Nmur1* C^re-T2A-GFP *Id2*^flox/flox mice (cKO) compared to littermate controls (Ctrl) in clusters expressing these genes (see extended data 10). Untreated littermate controls in (a-l) were C57BL/6, *N. b.*-infected littermate controls were *Id2*^flox/+ or *Nmur1*^Cre-T2A-GFP *Id2*^flox/+).

See this image and copyright information in PMC

Comment in

Pinning down unique ILC2 functions.
Flemming A. Flemming A. Nat Rev Immunol. 2022 Dec;22(12):717. doi: 10.1038/s41577-022-00807-z. Nat Rev Immunol. 2022. PMID: 36357706 No abstract available.

Cited by

Transcriptional profiling identifies IL-33-expressing intestinal stromal cells as a signaling hub poised to interact with enteric neurons.
Topczewska PM, Savvopoulou A, Cosovanu C, Klose CSN. Topczewska PM, et al. Front Cell Dev Biol. 2024 Aug 1;12:1420313. doi: 10.3389/fcell.2024.1420313. eCollection 2024. Front Cell Dev Biol. 2024. PMID: 39149516 Free PMC article.
Moniezia benedeni drives the SNAP-25 expression of the enteric nerves in sheep's small intestine.
Huang Z, Yao W, He W, Pan J, Chai W, Wang B, Jia Z, Fan X, Wang W, Zhang W. Huang Z, et al. BMC Vet Res. 2024 Jul 1;20(1):283. doi: 10.1186/s12917-024-04140-6. BMC Vet Res. 2024. PMID: 38956647 Free PMC article.
Unveiling the Neuron-mediated Group 2 Innate Lymphoid Cell Activation in Human Asthma.
Kabata H, Ueki S. Kabata H, et al. Am J Respir Crit Care Med. 2024 Sep 15;210(6):701-703. doi: 10.1164/rccm.202404-0844ED. Am J Respir Crit Care Med. 2024. PMID: 38820208 Free PMC article. No abstract available.
IL-33 controls IL-22-dependent antibacterial defense by modulating the microbiota.
Röwekamp I, Maschirow L, Rabes A, Fiocca Vernengo F, Hamann L, Heinz GA, Mashreghi MF, Caesar S, Milek M, Fagundes Fonseca AC, Wienhold SM, Nouailles G, Yao L, Mousavi S, Bruder D, Boehme JD, Puzianowska-Kuznicka M, Beule D, Witzenrath M; CAPNETZ Study Group; Löhning M, Klose CSN, Heimesaat MM, Diefenbach A, Opitz B. Röwekamp I, et al. Proc Natl Acad Sci U S A. 2024 May 28;121(22):e2310864121. doi: 10.1073/pnas.2310864121. Epub 2024 May 23. Proc Natl Acad Sci U S A. 2024. PMID: 38781213
Serum monocyte chemotactic protein 1 and soluble mannose receptor aid predictive diagnosis of pediatric sepsis.
Song D, Zheng X. Song D, et al. Am J Transl Res. 2024 Mar 15;16(3):964-972. doi: 10.62347/FZMM3162. eCollection 2024. Am J Transl Res. 2024. PMID: 38586091 Free PMC article.

See all "Cited by" articles

References

1. Vivier E, et al. Innate Lymphoid Cells: 10 Years On. Cell. 2018;174:1054–1066. doi: 10.1016/j.cell.2018.07.017. - DOI - PubMed
1. Klose CS, Artis D. Innate lymphoid cells as regulators of immunity, inflammation and tissue homeostasis. Nature immunology. 2016;17:765–774. doi: 10.1038/ni.3489. - DOI - PubMed
1. Vely F, et al. Evidence of innate lymphoid cell redundancy in humans. Nature immunology. 2016;17:1291–1299. doi: 10.1038/ni.3553. - DOI - PMC - PubMed
1. Rankin LC, et al. Complementarity and redundancy of IL-22-producing innate lymphoid cells. Nature immunology. 2016;17:179–186. doi: 10.1038/ni.3332. - DOI - PMC - PubMed
1. Colonna M. Innate Lymphoid Cells: Diversity, Plasticity, and Unique Functions in Immunity. Immunity. 2018;48:1104–1117. doi: 10.1016/j.immuni.2018.05.013. - DOI - PMC - PubMed

Publication types

Actions
Actions

MeSH terms

Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions
Actions

Substances

Actions
Actions
Actions
Actions
Actions

Grants and funding

LinkOut - more resources

Full Text Sources
Other Literature Sources
- H1 Connect
Medical
- MedlinePlus Health Information
Molecular Biology Databases
- Mouse Genome Informatics (MGI)

[1] Vivier E, et al. Innate Lymphoid Cells: 10 Years On. Cell. 2018;174:1054–1066. doi: 10.1016/j.cell.2018.07.017. - DOI - PubMed

[2] Vivier E, et al. Innate Lymphoid Cells: 10 Years On. Cell. 2018;174:1054–1066. doi: 10.1016/j.cell.2018.07.017. - DOI - PubMed

[3] Klose CS, Artis D. Innate lymphoid cells as regulators of immunity, inflammation and tissue homeostasis. Nature immunology. 2016;17:765–774. doi: 10.1038/ni.3489. - DOI - PubMed

[4] Klose CS, Artis D. Innate lymphoid cells as regulators of immunity, inflammation and tissue homeostasis. Nature immunology. 2016;17:765–774. doi: 10.1038/ni.3489. - DOI - PubMed

[5] Vely F, et al. Evidence of innate lymphoid cell redundancy in humans. Nature immunology. 2016;17:1291–1299. doi: 10.1038/ni.3553. - DOI - PMC - PubMed

[6] Vely F, et al. Evidence of innate lymphoid cell redundancy in humans. Nature immunology. 2016;17:1291–1299. doi: 10.1038/ni.3553. - DOI - PMC - PubMed

[7] Rankin LC, et al. Complementarity and redundancy of IL-22-producing innate lymphoid cells. Nature immunology. 2016;17:179–186. doi: 10.1038/ni.3332. - DOI - PMC - PubMed

[8] Rankin LC, et al. Complementarity and redundancy of IL-22-producing innate lymphoid cells. Nature immunology. 2016;17:179–186. doi: 10.1038/ni.3332. - DOI - PMC - PubMed

[9] Colonna M. Innate Lymphoid Cells: Diversity, Plasticity, and Unique Functions in Immunity. Immunity. 2018;48:1104–1117. doi: 10.1016/j.immuni.2018.05.013. - DOI - PMC - PubMed

[10] Colonna M. Innate Lymphoid Cells: Diversity, Plasticity, and Unique Functions in Immunity. Immunity. 2018;48:1104–1117. doi: 10.1016/j.immuni.2018.05.013. - DOI - PMC - PubMed

Save citation to file

Email citation

Add to Collections

Add to My Bibliography

Your saved search

Create a file for external citation management software

Your RSS Feed

Non-redundant functions of group 2 innate lymphoid cells

Affiliations

Non-redundant functions of group 2 innate lymphoid cells

Authors

Affiliations

Abstract

Conflict of interest statement

Figures

Comment in

Similar articles

Cited by

References

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources

Full Text Sources

Other Literature Sources

Medical

Molecular Biology Databases

Abstract

Conflict of interest statement

Figures

Comment in

Similar articles

Cited by

References

Publication types

MeSH terms

Substances

Related information

Grants and funding

LinkOut - more resources

Full Text Sources

Other Literature Sources

Medical

Molecular Biology Databases