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m6A RNA modification regulates innate lymphoid cell responses in a lineage-specific manner

Nature Immunology volume 24, pages 1256–1264 (2023)Cite this article

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Abstract

Innate lymphoid cells (ILCs) can quickly switch from a quiescent state to an active state and rapidly produce effector molecules that provide critical early immune protection. How the post-transcriptional machinery processes different stimuli and initiates robust gene expression in ILCs is poorly understood. Here, we show that deletion of the N6-methyladenosine (m6A) writer protein METTL3 has little impact on ILC homeostasis or cytokine-induced ILC1 or ILC3 responses but significantly diminishes ILC2 proliferation, migration and effector cytokine production and results in impaired antihelminth immunity. m6A RNA modification supports an increase in cell size and transcriptional activity in activated ILC2s but not in ILC1s or ILC3s. Among other transcripts, the gene encoding the transcription factor GATA3 is highly m6A methylated in ILC2s. Targeted m6A demethylation destabilizes nascent Gata3 mRNA and abolishes the upregulation of GATA3 and ILC2 activation. Our study suggests a lineage-specific requirement of m6A for ILC2 responses.

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Fig. 1: m6A is dispensable for mature ILC maintenance at steady state.
Fig. 2: m6A is required for cytokine-induced ILC2 responses.
Fig. 3: m6A is critical for ILC2-mediated antihelminth immunity.
Fig. 4: m6A targets Gata3 in activated ILC2s.
Fig. 5: m6A modulates Gata3 mRNA stability to facilitate ILC2 activation.

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Data availability

MeRIP–seq data are available in the Gene Expression Omnibus database (accession number GSE223084). Source data are provided with this paper.

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Acknowledgements

We thank all members of the Huang lab for discussions and assistance on experiments. We thank R. Flavell and I. Ivanov for providing critical mouse lines, J. Zhu for providing Gata3-NGFR-RV and NGFR-RV plasmids, Q. Yang for providing the ILC2/b6 cell line, Columbia Department of Microbiology and Immunology and the Columbia Stem Cell Initiative core facilities staff for assistance with cell sorting and S. Reiner for critical reading of the manuscript. This work was supported by NIGMS 1R35GM138805 (Y.H.), Columbia HICCC TBM Pilot Award Program (Y.H.) and NIAID 5RO1AI43897 (U.B.). Y.Z. was also supported by an AAI Career Reentry Fellowship.

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Author notes

  1. These authors contributed equally: Yingyu Zhang, Wanwei Zhang.

Authors and Affiliations

  1. Department of Microbiology & Immunology, Columbia University Medical Center, New York, NY, USA

    Yingyu Zhang, Wanwei Zhang, Jingyao Zhao, Takamasa Ito, Jiacheng Jin, Vincent Guichard, Sankar Ghosh, Lei Ding, Uttiya Basu & Yuefeng Huang

  2. Department of Medicine, Department of Systems Biology, Columbia University Medical Center, New York, NY, USA

    Alexis O. Aparicio & Xuebing Wu

  3. Department of Rehabilitation and Regenerative Medicine, Columbia Stem Cell Initiative, Columbia University Medical Center, New York, NY, USA

    Junsong Zhou & Lei Ding

  4. Department of Medicine, Division of Digestive and Liver Diseases, Columbia Center for Human Development, Columbia University Medical Center, New York, NY, USA

    Yinshan Fang & Jianwen Que

  5. Agricultural Research Service, Beltsville Human Nutrition Research Center, Diet, Genomics, and Immunology Laboratory, and Beltsville Agricultural Research Center, Animal Parasitic Diseases Laboratory, U.S. Department of Agriculture, Beltsville, MD, USA

    Joseph F. Urban Jr

  6. Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel

    Jacob H. Hanna

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Contributions

Y.Z. designed, performed and interpreted the experiments and drafted the manuscript. W.Z. analyzed and interpreted meRIP–seq data and drafted the initial manuscript. J. Zhao assisted with many experiments. T.I. and J.J. assisted with experiments. A.O.A. and X.W. designed and constructed gRNAs for targeted demethylation experiments. J. Zhou performed retrovirus packaging. V.G. performed western blotting. Y.F. and J.Q. assisted with METTL3 immunofluorescence staining. J.F.U. provided N. brasiliensis and editing input. J.H.H. provided Mettl3fl/fl mice. S.G. assisted with the experimental design. L.D. assisted with meRIP–seq and helped to design the experiments. U.B. assisted with targeted demethylation and helped to design the experiments. Y.H. designed the experiments, interpreted the data and finalized the manuscript. All authors contributed to the preparation of the manuscript.

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Correspondence to Yuefeng Huang.

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Nature Immunology thanks the anonymous reviewers for their contribution to the peer review of this work. Ioana Visan was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 Gene expression of m6A writers and associated proteins in ILC subsets.

a, Heat map of gene expression of m6A writers and associated factors in spleen NK, liver ILC1s, intestinal ILC2s and intestinal ILC3s re-analyzed from published RNA-seq data (access # GSE77695). b, c, The mRNA level of three key components of m6A writer complex in liver ILC1s, intestinal ILC2s and intestinal ILC3s sorted from naive mice (b) or cytokine-treated mice (c) (IL-12 + IL-18 treatment for ILC1 activation, IL-25 for ILC2 activation, IL-1β + IL-23 treatment for ILC3 activation) by RT-qPCR. ILC1s and ILC2s were from B6 mice and ILC3s were from Rorc-egfp mice. Left in b ****p < 0.0001 for ILC1s vs ILC2s, *p = 0.0317 for ILC3s vs ILC2s; middle in b *p = 0.0105 for ILC1s vs ILC2s, *p = 0.0206 for ILC3s vs ILC2s; right in b ***p = 0.0007 for ILC1s vs ILC2s, *p = 0.0156 for ILC3s vs ILC2s. Left in c ***p = 0.0004 for ILC1s vs ILC2s, **p = 0.0016 for ILC3s vs ILC2s; middle in c *p = 0.0104 for ILC1s vs ILC2s, ***p = 0.0002 for ILC3s vs ILC2s; right in c NS p = 0.1885 for ILC1s vs ILC2s, **p = 0.0011 for ILC3s vs ILC2s. Unpaired two-tailed t test. n = 3 in each group. Data are shown as mean ± s.d. Results are representative of two independent experiments in b and c.

Source data

Extended Data Fig. 2 Inducible deletion of Mettl3 leads to m6A deletion on mRNAs in ILCs.

a, Experimental strategy. b, Mettl3 mRNA level in intestinal ILCs pre-gated as live CD45+ Lin− Thy1+ and further gated as NK1.1+ ILC1s, KLRG1+ ILC2s and NK1.1− KLRG1− ILC3s of R26CreERMettl3fl/fl (Mettl3Δ/Δ) mice or Mettl3fl/fl littermate controls by RT-qPCR. ***p = 0.0001 for ILC1s, ***p = 0.0003 for ILC2s, ***p = 0.0002 for ILC3s. c, Immunoblot of METTL3 protein in ILC1s, ILC2s and ILC3s sorted from Mettl3Δ/Δ mice or littermates. GAPDH, internal control. d, Quantification of METTL3 protein levels in immunoblot. ***p = 0.0009 for ILC1s, ****p < 0.0001 for ILC2s, ****p < 0.0001 for ILC3s. e, METTL3 immunofluorescence staining of ILC1s, ILC2s and ILC3s sorted from Mettl3Δ/Δ mice or littermates; scale bar, 20 μm. f, Quantification of METTL3 fluorescence intensity in e. ****p < 0.0001 for ILC1s, ****p < 0.0001 for ILC2s, ****p < 0.0001 for ILC3s. g, Bioanalyzer results of the reverse transcripts of the m6A-methylated RNAs in ILC1s, ILC2s and ILC3s sorted from Mettl3Δ/Δ mice or littermates. h, Relative concentration of the reverse transcript products of m6A pull-downed mRNAs. ***p = 0.0005 for ILC1s, ***p = 0.001 for ILC2s, ***p = 0.0008 for ILC3s. Unpaired two-tailed t test. n = 4 in b, 3 in d and h, 135 ~ 204 in f in each group. Data are shown as mean ± s.d. Results are representative of two or three independent experiments in b, c, e, f and g.

Source data

Extended Data Fig. 3 Inducible deletion of Mettl3 leads to the depletion of ILC progenitors in bone marrow.

Representative flow cytometry (a) and quantification (b) of Common lymphoid progenitors (CLPs) gated as CD45+ Lin– c-Kit+ Sca1– IL-7R+ α4β7– Flt3+, ILC common progenitors (ILCPs) gated as CD45+ Lin– c-Kit + Sca1– IL-7R+ α4β7+ Flt3–, and ILC2 progenitors (ILC2Ps) gated as CD45+ Lin– CD25+ ST2+ IL-7R+ from Mettl3Δ/Δ mice or littermates. **p = 0.0026 for CLPs, **p = 0.0019 for ILCPs and *p = 0.0388 for ILC2Ps. Unpaired two-tailed t test. Mettl3fl/fl n = 4; Mettl3Δ/Δ n = 3 in b. Data are shown as mean ± s.d. Results are representative of three independent experiments.

Source data

Extended Data Fig. 4 Catalytic inhibition of METTL3 impairs ILC2 activation.

a, b, ELISA analysis of IL-13 and IL-5 in cell culture supernatant of ILC2s treated with DMSO, 30μM STM2457 or 100μM STM2457 without or with IL-25 plus IL-33. In a, ***p = 0.0003 for DMSO vs DMSO IL-25 + IL-33; **p = 0.0022 for DMSO IL-25 + IL-33 vs 30μM STM2457 IL-25 + IL-33; ***p = 0.0003 for DMSO IL-25 + IL-33 vs 100μM STM2457 IL-25 + IL-33. In b, **p = 0.0010 for DMSO vs DMSO IL-25 + IL-33; **p = 0.0087 for DMSO IL-25 + IL-33 vs 30μM STM2457 IL-25 + IL-33; **p = 0.0010 for DMSO IL-25 + IL-33 vs 100μM STM2457 IL-25 + IL-33. c, FACS analysis of Ki-67+ frequency of ILC2s. *p = 0.0467 for DMSO vs DMSO IL-25 + IL-33; **p = 0.0055 for DMSO IL-25 + IL-33 vs 30μM STM2457 IL-25 + IL-33; **p = 0.0058 for DMSO IL-25 + IL-33 vs 100μM STM2457 IL-25 + IL-33. d, Bioanalyzer results of the reverse transcripts of the m6A-methylated RNAs in ILC2s treated with DMSO or 100μM STM2457. e, Immunoblot of METTL3 protein in ILC2s treated with DMSO, 30μM STM2457 or 100μM STM2457. Unpaired two-tailed t test. n = 3 in each group. Data are shown as mean ± s.d. Results are representative of three independent experiments.

Source data

Extended Data Fig. 5 m6A supports an increase of cell size and transcription activity in activated ILC2s.

a, Representative image of Giemsa staining of liver ILC1s, small intestine ILC2s and ILC3s that were sorted from untreated (steady state) and IL-12 + IL-18-treated B6, IL-25-treated B6, or IL-1β + IL-23-treated Rorc-egfp reporter mice, respectively; scale bar, 100 μm. b, Quantification of cell diameter of the cells as in a. NS p = 0.0717 for ILC1s, ****p < 0.0001 for ILC2s and NS p = 0.2401 for ILC3s. c, Representative flow cytometry histograms of forward scatter of the cells as in a. d, Relative total RNA amount per cell of steady-state and activated ILC subsets as in a. **p = 0.0044 for ILC1s, ***p = 0.0001 for ILC2s and NS p = 0.0585 for ILC3s. e, Fold increase of activated ILC numbers relative to steady-state ILCs as in a. Activated ILC2s comprised ILC2s in the small intestine and iILC2s in lungs and MLNs of IL-25-treated mice. ****p < 0.0001. f, Representative flow cytometry histograms of forward scatter of intestinal ILC2s from IL-25-treated Mettl3Δ/Δ and Mettl3fl/fl mice that received three doses Tmx before IL-25 treatment. g, Fold increase of activated ILC2s relative to steady-state ILC2s as in f. ****p < 0.0001. Unpaired two-tailed t test. n = 30 in b, n = 3 ~ 5 in d, e and g. Data are shown as mean ± s.d. Results are representative of three independent experiments. .

Source data

Extended Data Fig. 6 KLRG1 is a marker for intestinal ILC2s.

a, Flow cytometry gating strategies of small intestine epithelial cells, B cells, CD4+ T cells, CD8+ T cells, NK/ILC1s, CD4+ ILC3s, CD4– ILC3s and ILC2s in B6 mice. b, KLRG1 expression on each indicated cell populations. Results are representative of two independent experiments.

Source data

Extended Data Fig. 7 Mettl3 deletion impairs ILC2 migration and cytokine production during helminth infection.

a, RT-qPCR of Mettl3 mRNA in Lin– Thy1+ KLRG1+ ILC2s and CD4+ T cells from the small intestine of Mettl3fl/fl and Mettl3ΔILC2 mice. b, Immunoblot of METTL3 protein in intestinal ILC2s as in a. c, Il13 and Il5 mRNA levels in the small intestine of steady-state Mettl3fl/fl and Mettl3ΔILC2 mice by RT-qPCR. Left NS p = 0.2577 and right NS p = 0.1643. d, Experimental strategy. Mettl3fl/fl and Mettl3Δ/Δ mice were pre-treated with Tmx as indicated, and then subcutaneously infected with 300 N. brasiliensis (N.b) larvae L3. e, Representative flow cytometry of CD45+ Lin– Thy1+/low ST2– KLRG1hi iILC2s in the lung on day 5 after infection. f, Quantification of iILC2s in the lung and MLNs of mice as in e. Left ***p = 0.0003 and right **p = 0.0051. g, Il5 and Il13 mRNA levels in the small intestine of mice as in e. Left **p = 0.0037 and right ***p = 0.0009. Unpaired two-tailed t test. n=3~5 in a, c, f and g. Data are shown as mean ± d.d. Results are representative of three independent experiments.

Source data

Extended Data Fig. 8 Mettl3 deletion doesn’t affect iILC2-to-ILC3 transition, or IL-7-triggered STAT5 phosphorylation.

a, Flow cytometry analysis of RORγt protein level in ST2+ nILC2s, RORγt+ ILC3s and IL-25-induced ST2– KLRG1hi iILC2s in the lungs of Mettl3ΔILC2 mice or littermates. b, Flow cytometry analysis of STAT5 phosphorylation level of intestinal ILC2s treated with or without 10 ng/mL IL-7 from Mettl3ΔILC2 mice or littermates. NS p = 0.8928. Unpaired two-tailed t test. n = 3 in each group in right b. Data are shown as mean ± d.d. Results are representative of three independent experiments.

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Extended Data Fig. 9 The transcripts encoding master transcription factors for ILC1s or ILC3s are not highly methylated.

a, Volcano plot for the comparation of genes enriched in m6A-bound fraction of liver ILC1s sorted from IL-12 + IL-18 treated B6 mice. b, Functional enrichment analysis on m6A-bound fraction in a by using gProfiler. Upper x-axis is -log10 adjusted p-value and lower x-axis is the number of methylated genes. c, Metascape analysis of m6A-bound fraction in a. Each node represents a functional term. The size of the node is proportional to the number of m6A-bound genes that fall into the corresponding term, and the color reflects its cluster identity. The edge represents the similarity between two terms. d, Heat map of selected genes of ILC1 meRIP-seq in a. e, Volcano plot for the comparation of genes enriched in m6A-bound fraction of intestinal ILC3s sorted from IL-1β + IL-23 treatedRorc-egfp mice. f, Functional enrichment analysis on m6A-bound fraction in e by using gProfiler. Upper x-axis is -log10 adjusted p-value and lower x-axis is the number of methylated genes. g, Metascape analysis of m6A-bound fraction in e. Each node represents a functional term. The size of the node is proportional to the number of m6A-bound genes that fall into the corresponding term, and the color reflects its cluster identity. The edge represents the similarity between two terms. h, Heat map of selected genes of ILC3 meRIP-seq in e.

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Zhang, Y., Zhang, W., Zhao, J. et al. m6A RNA modification regulates innate lymphoid cell responses in a lineage-specific manner. Nat Immunol 24, 1256–1264 (2023). https://doi.org/10.1038/s41590-023-01548-4

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