Abstract
Macrophages exhibit remarkable functional plasticity, a requirement for their central role in tissue homeostasis. During chronic inflammation, macrophages acquire sustained inflammatory ‘states’ that contribute to disease, but there is limited understanding of the regulatory mechanisms that drive their generation. Here we describe a systematic functional genomics approach that combines genome-wide phenotypic screening in primary murine macrophages with transcriptional and cytokine profiling of genetic perturbations in primary human macrophages to uncover regulatory circuits of inflammatory states. This process identifies regulators of five distinct states associated with key features of macrophage function. Among these regulators, loss of the N6-methyladenosine (m6A) writer components abolishes m6A modification of TNF transcripts, thereby enhancing mRNA stability and TNF production associated with multiple inflammatory pathologies. Thus, phenotypic characterization of primary murine and human macrophages describes the regulatory circuits underlying distinct inflammatory states, revealing post-transcriptional control of TNF mRNA stability as an immunosuppressive mechanism in innate immunity.
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Data availability
Datasets generated in this study have been deposited to the Gene Expression Omnibus (GSE210338, GSE210619, GSE210950, GSE268351, GSE268352, GSE268353, GSE268354 and GSE270634) and EGAS00001006485. Source data are provided with this paper.
Code availability
Code relevant to this project has been deposited in GitHub (https://github.com/Genentech/Haag_ng_2024) and Zenodo (https://zenodo.org/doi/10.5281/zenodo.13836037)93.
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Acknowledgements
We are grateful for the cooperation of Donor Network West and all of the organ and tissue donors and their families for giving the gift of life and the gift of knowledge by their generous donation. We thank W. A. Faubion at the Mayo Clinic for his long-standing collaboration and patient samples. We thank members of the Next Generation Sequencing Facility, I. Lehoux and Z. R. Li, for their support in construct design; Eclipse BioInnovations for performing m6A-seq and analysis; and L. Taraborrelli, R. M. Leitão, J. Lim and members of the Cancer Immunology and Immunology Discovery departments at Genentech for technical and intellectual support. The PheWAS analysis was conducted using the UK Biobank Resource under application number 44257.
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S.M.H., S.X., C.E., J.A.K., J.L., M. Callow, L.H., M.T., C.J.B. and C.C. designed and performed experiments, and analyzed and interpreted data. S.M.C., R.N.P. and S.Z.W. analyzed and interpreted data. A.L., J.-P.F. and M. Costa analyzed genome-wide CRISPR screen datasets. B.L.Y. and H.A.H. performed PheWAS analysis. E.F. and A.N. provided reagents. S.M., M.K., S.J.T. and K.G.-S. provided essential conceptual input. S.M.H. and A.M. conceptualized the study. S.M.H. wrote the paper with input from all authors. S.J.T. and A.M. oversaw the project.
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S.M.H., S.M.C., S.X., C.E., M. Callow, M. Costa, C.C., A.L., J.-P.F., M.K., L.H., E.F., A.N., S.Z.W., B.L.Y., H.A.H., R.N.P., K.G.-S., C.J.B., M.T., S.M. and S.J.T. are employees of Genentech. A.M. is an employee of Gilead Sciences. J.A.K. is an employee of Alector Therapeutics. J.L. is an employee of Sana Biotechnology.
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Extended data
Extended Data Fig. 1 Screening design in BMDMs.
a, Flow cytometry analysis of mBMDMs primed with IFN-γ in indicated concentrations. b, Flow cytometry analysis of intracellular TNFα and iNOS expression in mBMDMs stimulated as indicated. c, Heatmap of differentially expressed genes between each of the four comparisons in mBMDMs across 3 mice. d, Gating strategy to sort for ‘iNOS_high’ cells corresponding to Fig. 1b.
Extended Data Fig. 2 Quality controls for genome-wide CRISPR screen in mBMDMs.
a, sgRNA drop out of essential genes across 3 individual screens (replicates 1 - 3). Whiskers represent the minimum and maximum (unless points extend 1.5 * IQR from the hinge, then shown as individual points), the box represents the interquartile range, and the center line represents the median. b, gRNA-level log2-fold changes of representative genes. Vertical dotted gray line represents the median of control sgRNAs. Red and blue lines indicate enriched or depleted sgRNA rank for each indicated gene respectively. c, Flow cytometry of intracellular iNOS expression levels in perturbed mBMDMs as indicated (n = 2).
Extended Data Fig. 3 PCA and permuted energy distance test.
a, Individual gene ranking of PC1. b, variance ratio of PCA. c, Bona-fide monocyte and macrophage signature score in UMAP projection. d, scatter plot of the energy distance against the number of differentially expressed genes (DEG) calculated as pairwise comparison to NTC. Each dot represents a perturbation. Perturbations with significant effects (energy distance; FDR < 0.01, DEGs; FDR < 0.05) indicated in blue, non-significant in red. e, cluster map of cosine similarity between perturbations. Rows and columns are hierarchically clustered. Perturbations with significant effects as determined in (d) indicated in blue, non-significant in red.
Extended Data Fig. 4 LDA-derived programs and effect sizes across all perturbations.
a, LDA-derived programs. Feature plot of program expression scores and the top correlated genes within each program. b, Program enrichment upon IFN-γ priming. c, Genetic perturbation effects across programs compared to NTC control in IFN-γ primed conditions. b, c, Dot color represents effect size, and dot size corresponds to negative base 10 log (P value). P values determined by two-sided t-test.
Extended Data Fig. 5 Regulon analysis on perturbed hMDMs.
a, TF activity scores (color bar) of TFs (rows) in perturbed hMDMs (column) upon IFN-γ priming. P values determined by two-sided t-test. b, Motif enrichments were computed using the hypergeometric test as implemented in HOMER (log2FC > 1 and P value < 0.05) in ZC3H13- and WTAP-perturbed hMDMs against NTC-perturbed control cells in IFN-γ stimulated conditions (individual measurement of n = 3 donors). c, hMDMs cultivated in normoxic or hypoxic conditions for 24 h, followed by SPP1 measurements in supernatants using Luminex. Each symbol represents an individual measurement of n = 2 donors.
Extended Data Fig. 6 Additional cytokine secretion analysis.
a, Heatmap as shown in Fig. 3a with additional cytokine and chemokine values upon IFN-γ stimulation. Z-score scaling for each cytokine or chemokine shown for two individual donors. b, TNF expression in UMAP projection. c, Gene expression of CCL3, CCL4, and CXCL8 assessed by qPCR of perturbed hMDMs 18 h after IFN-γ priming with and without addition of TNFRII-Fc in culture medium; each symbol represents an individual donor (n = 5). Summary data of 3 individual experiments are shown as mean, with P values determined by one-way ANOVA with Sidak’s multiple comparisons test. d, Heatmap showing P03 genes in non-stimulated, IFN-γ primed and IFN-γ and TNFα co-stimulated cells in hMDMs, each column represents an individual donor (n = 3).
Extended Data Fig. 7 Perturbation of METTL3 and METTL14 in hMDMs elevate TNFα secretion.
a, Western blot of METTL3 perturbed with sgRNAs utilized in scCRISPR screen (sgMETTL3) and quantification of full length METTL3 (FL) (right). b, Western blot of METTL3 perturbed with sgRNAs targeting exon 5 (sgMETTL3_ex5.1 and sgMETTL3_ex5.2) and exon 6 (sgMETTL3_ex6). c, TNFα cytokine release and d, mean P03 of METTL3 perturbed cells using sgMETTL3_ex6. e, Western blot of METTL14 perturbed with sgRNAs utilized in scCRISPR screen (sgMETTL14) and (sgMETTL14_F). f, TNFα cytokine release in METTL14 perturbed hMDMs using sgMETTL14_F. a, c, d, f, Each symbol represents an individual donor (a, n = 3) (c, d and f n = 5). Summary data are shown as mean, with P values determined by Student two-sided t-test (c, d, f). a, representative Western blot image of 3 donors, b and e of 2 donors.
Extended Data Fig. 8 Genomic visualization of m6A-eCLIP.
a, Expression of P03 in NTC-, WTAP- and ZC3H13-perturbed hMDMs across indicated treatment conditions. Box plots represent the median and interquartile range, with whiskers indicating min and max values. b, c, Relative IL6 and IFNB1 mRNA levels upon ActinomycinD treatment in ZC3H13-depleted and NTC control cells. d, Normalized distribution of m6A peaks across 5’ UTR, CDS, and 3’UTR of perturbed hMDMs as indicated for two individual donors (1 and 2). Top motif enriched in m6A-eCLIP peaks derived from NTC control cells. e, Genomic visualization of normalized m6A-eCLIP signal along TNF, IL6 (f) and IFNB1 (g) in perturbed hMDMs as indicated for two individual donors (1 and 2); vertical red lines depict m6A residues h, TNFα cytokine levels determined by Luminex analysis of cell culture media of perturbed hMDMs as indicated 18 h after LPS or IFN-γ stimulation. a, b, c and h Each symbol represents an individual donor (a and h, n = 4) (b and c n = 3). h, P values determined by Students two-sided t-test.
Supplementary information
Supplementary Information
Supplementary Notes and Methods.
Supplementary Data 1
sgRNA sequences of genome-wide screen.
Supplementary Data 2
sgRNA sequences.
Supplementary Table 1
PheWas.
Source data
Source Data
Unprocessed western blots.
Source Data
Source data.
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Haag, S.M., Xie, S., Eidenschenk, C. et al. Systematic perturbation screens identify regulators of inflammatory macrophage states and a role for TNF mRNA m6A modification. Nat Genet 56, 2493–2505 (2024). https://doi.org/10.1038/s41588-024-01962-w
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DOI: https://doi.org/10.1038/s41588-024-01962-w
