Pooled CRISPR screening identifies m6A as a positive regulator of macrophage activation

doi:10.1126/sciadv.abd4742

. 2021 Apr 28;7(18):eabd4742.

doi: 10.1126/sciadv.abd4742. Print 2021 Apr.

Pooled CRISPR screening identifies m⁶A as a positive regulator of macrophage activation

Jiyu Tong ^{1

2

3}, Xuefei Wang ^{1

2}, Yongbo Liu ^{1

2}, Xingxing Ren ⁴, Anmin Wang ⁴, Zonggui Chen ⁵, Jiameng Yao ¹, Kaiqiong Mao ^{1

2}, Tingting Liu ^{6

7}, Fei-Long Meng ^{6

7}, Wen Pan ⁴, Qiang Zou ¹, Jun Liu ⁸, Yu Zhou ⁵, Qiang Xia ⁹, Richard A Flavell ^{10

11}, Shu Zhu ¹², Hua-Bing Li ^{13

2

14}

Affiliations

¹ Shanghai Institute of Immunology, State Key Laboratory of Oncogenes and Related Genes, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China.
² Shanghai Jiao Tong University School of Medicine-Yale Institute for Immune Metabolism, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China.
³ Department of Pediatrics and Key Laboratory of Birth Defects and Related Diseases of Women and Children of MOE, West China Second University Hospital, Sichuan University, China.
⁴ Hefei National Laboratory for Physical Sciences at Microscale, the Chinese Academy of Sciences Key Laboratory of Innate Immunity and Chronic Disease, School of Basic Medical Sciences, Division of Life Sciences and Medicine, University of Science and Technology of China, 230027 Hefei, China.
⁵ Institute for Advanced Studies, State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan 430072, China.
⁶ State Key Laboratory of Molecular Biology, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai 200031, China.
⁷ University of Chinese Academy of Sciences, Beijing 100049, China.
⁸ School of Life Sciences, Peking University, Beijing 100871, China.
⁹ Department of Liver Surgery and State Key Laboratory of Oncogenes and Related Genes, Ren Ji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200127, China. xiaqiang@medmail.com.cn zhushu@ustc.edu.cn richard.flavell@yale.edu huabing.li@shsmu.edu.cn.
¹⁰ Department of Immunobiology, Yale University School of Medicine, New Haven, CT 06520-8055, USA. xiaqiang@medmail.com.cn zhushu@ustc.edu.cn richard.flavell@yale.edu huabing.li@shsmu.edu.cn.
¹¹ Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT 06520-8055, USA.
¹² Hefei National Laboratory for Physical Sciences at Microscale, the Chinese Academy of Sciences Key Laboratory of Innate Immunity and Chronic Disease, School of Basic Medical Sciences, Division of Life Sciences and Medicine, University of Science and Technology of China, 230027 Hefei, China. xiaqiang@medmail.com.cn zhushu@ustc.edu.cn richard.flavell@yale.edu huabing.li@shsmu.edu.cn.
¹³ Shanghai Institute of Immunology, State Key Laboratory of Oncogenes and Related Genes, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China. xiaqiang@medmail.com.cn zhushu@ustc.edu.cn richard.flavell@yale.edu huabing.li@shsmu.edu.cn.
¹⁴ Department of Liver Surgery and State Key Laboratory of Oncogenes and Related Genes, Ren Ji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200127, China.

PMID: 33910903
PMCID: PMC8081357
DOI: 10.1126/sciadv.abd4742

Pooled CRISPR screening identifies m⁶A as a positive regulator of macrophage activation

Jiyu Tong et al. Sci Adv. 2021.

. 2021 Apr 28;7(18):eabd4742.

doi: 10.1126/sciadv.abd4742. Print 2021 Apr.

Authors

Jiyu Tong ^{1

2

3}, Xuefei Wang ^{1

2}, Yongbo Liu ^{1

2}, Xingxing Ren ⁴, Anmin Wang ⁴, Zonggui Chen ⁵, Jiameng Yao ¹, Kaiqiong Mao ^{1

2}, Tingting Liu ^{6

7}, Fei-Long Meng ^{6

7}, Wen Pan ⁴, Qiang Zou ¹, Jun Liu ⁸, Yu Zhou ⁵, Qiang Xia ⁹, Richard A Flavell ^{10

11}, Shu Zhu ¹², Hua-Bing Li ^{13

2

14}

Affiliations

¹ Shanghai Institute of Immunology, State Key Laboratory of Oncogenes and Related Genes, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China.
² Shanghai Jiao Tong University School of Medicine-Yale Institute for Immune Metabolism, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China.
³ Department of Pediatrics and Key Laboratory of Birth Defects and Related Diseases of Women and Children of MOE, West China Second University Hospital, Sichuan University, China.
⁴ Hefei National Laboratory for Physical Sciences at Microscale, the Chinese Academy of Sciences Key Laboratory of Innate Immunity and Chronic Disease, School of Basic Medical Sciences, Division of Life Sciences and Medicine, University of Science and Technology of China, 230027 Hefei, China.
⁵ Institute for Advanced Studies, State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan 430072, China.
⁶ State Key Laboratory of Molecular Biology, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai 200031, China.
⁷ University of Chinese Academy of Sciences, Beijing 100049, China.
⁸ School of Life Sciences, Peking University, Beijing 100871, China.
⁹ Department of Liver Surgery and State Key Laboratory of Oncogenes and Related Genes, Ren Ji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200127, China. xiaqiang@medmail.com.cn zhushu@ustc.edu.cn richard.flavell@yale.edu huabing.li@shsmu.edu.cn.
¹⁰ Department of Immunobiology, Yale University School of Medicine, New Haven, CT 06520-8055, USA. xiaqiang@medmail.com.cn zhushu@ustc.edu.cn richard.flavell@yale.edu huabing.li@shsmu.edu.cn.
¹¹ Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT 06520-8055, USA.
¹² Hefei National Laboratory for Physical Sciences at Microscale, the Chinese Academy of Sciences Key Laboratory of Innate Immunity and Chronic Disease, School of Basic Medical Sciences, Division of Life Sciences and Medicine, University of Science and Technology of China, 230027 Hefei, China. xiaqiang@medmail.com.cn zhushu@ustc.edu.cn richard.flavell@yale.edu huabing.li@shsmu.edu.cn.
¹³ Shanghai Institute of Immunology, State Key Laboratory of Oncogenes and Related Genes, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China. xiaqiang@medmail.com.cn zhushu@ustc.edu.cn richard.flavell@yale.edu huabing.li@shsmu.edu.cn.
¹⁴ Department of Liver Surgery and State Key Laboratory of Oncogenes and Related Genes, Ren Ji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200127, China.

PMID: 33910903
PMCID: PMC8081357
DOI: 10.1126/sciadv.abd4742

Abstract

m⁶A RNA modification is implicated in multiple cellular responses. However, its function in the innate immune cells is poorly understood. Here, we identified major m⁶A "writers" as the top candidate genes regulating macrophage activation by LPS in an RNA binding protein focused CRISPR screening. We have confirmed that Mettl3-deficient macrophages exhibited reduced TNF-α production upon LPS stimulation in vitro. Consistently, Mettl3 ^flox/flox;Lyzm-Cre mice displayed increased susceptibility to bacterial infection and showed faster tumor growth. Mechanistically, the transcripts of the Irakm gene encoding a negative regulator of TLR4 signaling were highly decorated by m⁶A modification. METTL3 deficiency led to the loss of m⁶A modification on Irakm mRNA and slowed down its degradation, resulting in a higher level of IRAKM, which ultimately suppressed TLR signaling-mediated macrophage activation. Our findings demonstrate a previously unknown role for METTL3-mediated m⁶A modification in innate immune responses and implicate the m⁶A machinery as a potential cancer immunotherapy target.

Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).

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Figures

Fig. 1

Fig. 1. CRISPR screening identifies METTL3 as a regulator of TNF-α production in macrophages.

(A) Scheme of pooled CRISPR-Cas9 screening of RBPs playing critical roles in macrophage activation. Briefly, Cas9-expressing Raw 264.7 cells were infected with lentivirus library containing sgRNAs targeting RBP genes in the mouse genome. After selection with puromycin for 7 days, the cells were stimulated with LPS and sorted by flow cytometry on the basis of the expression levels of TNF-α. (B) Venn diagrams showing the overlap between the top 100 ranked candidate genes enriched in TNF-α–Low and TNF-α–Hi populations in two replicate screens. (C) Volcano plot showing sgRNA-targeted genes enriched in the TNF-α–Hi (blue) and TNF-α–Low (red) populations. Known positive regulators (purple), negative regulators (green), and m⁶A modulators (black) of TNF-α production in macrophages are highlighted. (D) Protein level of METTL3 and the overall RNA m⁶A methylation levels in WT and Mettl3-KO Raw 264.7 cells were measured by Western blotting and m⁶A dot blot assay. (E) Expression of TNF-α in METTL3-depleted and control Raw 264.7 cells after LPS stimulation measured by flow cytometry. MFI, median fluorescence intensity; NT, not treated. (F) GO enrichment analysis of down-regulated transcripts in Mettl3-KO Raw 264.7 cells compared to WT control cells. (G) Heatmap illustrating the expression of transcripts downstream of the TLR4 signaling pathway in Mettl3-deficient and WT Raw 264.7 cells. (H) Expression of TNF-α in bone marrow–derived macrophages (BMDMs) from Mettl3^flox/flox;Lyzm-Cre and Mettl3^flox/flox control mice upon LPS stimulation measured by flow cytometry. Data are shown from two experiments (B and C), as a representative result of three independent experiments (D), or as means ± SEM (E and H).*P < 0.05 and ****P < 0.0001 (unpaired two-tailed Student’s t test).

Fig. 2

Fig. 2. Mettl3^flox/flox;Lyzm-Cre mice are more susceptible to S. typhimurium infection.

(A) Body weight of Mettl3^flox/flox;Lyzm-Cre (n = 14) and their Mettl3^flox/flox littermates (n = 10) measured 2 and 3 days after S. typhimurium infection. (B to F) Bacteria load of the feces (B and C), cecum (D), spleen (E), and liver (F) of infected Mettl3^flox/flox;Lyzm-Cre (n = 14) and Mettl3^flox/flox littermates (n = 10) measured by counting colony-forming units (CFU) in cultures of serially diluted homogenates of organs on MacConkey agar plates. Data are shown as representative results of three independent experiments (A to F) or as means ± SD of indicated determinants (B to F). *P < 0.05 and **P < 0.01 (unpaired two-tailed Student’s t test).

Fig. 3

Fig. 3. Mettl3^flox/flox;Lyzm-Cre mice exhibit faster tumor growth and a lower level of macrophage-derived TNF-α than WT littermates.

MC38 cells were subcutaneously injected into the flanks of Mettl3^flox/flox;Lyzm-Cre mice and their Mettl3^flox/flox littermates. The tumor was excised, and tumor-infiltrating cells were characterized by flow cytometry and real-time qPCR. (A) Representative images of tumors excised from Mettl3^flox/flox;Lyzm-Cre mice (n = 10) and Mettl3^flox/flox littermates (n = 10) 21 days after cell injection. Photo credit: Jiyu Tong, Shanghai Jiao Tong University School of Medicine. (B) Tumor growth in Mettl3^flox/flox;Lyzm-Cre mice (n = 10) and Mettl3^flox/flox littermates (n = 10). (C and D) Relative expression of Mettl3 (C) and Tnf-α (D) in TAMs from Mettl3^flox/flox;Lyzm-Cre mice (n = 4) and Mettl3^flox/flox littermates (n = 4) measured by real-time qPCR. (E and F) Flow cytometry profile and MFI of CD86-positive (E) and CD206-positive (F) TAMs from Mettl3^flox/flox;Lyzm-Cre mice (n = 4) and Mettl3^flox/flox littermates (n = 4). (G and H) Fractions of intratumoral PD-1–positive CD4⁺ T cells (G) and PD-1–positive CD8⁺ T cells (H) measured by flow cytometry (n = 3). (I and J) BMDMs from Mettl3^flox/flox;Lyzm-Cre mice and Mettl3^flox/flox littermates were stimulated with medium conditioned by MC38 tumor cells in vitro. The expression of TNF-α was measured by flow cytometry and shown as a histogram (I) and MFI (J). Arg-1 expression in TAMs from Mettl3^flox/flox;Lyzm-Cre mice and Mettl3^flox/flox littermates measured by real-time qPCR after TCM or IL-4 treatment. Data are shown as representative results of three independent experiments (A, E, F, and I), as means ± SD (B, G, and H), or as means ± SEM (C to F, J, and K). *P < 0.05 and ***P < 0.001 (unpaired two-tailed Student’s t test).

Fig. 4

Fig. 4. METTL3 deficiency impairs the TLR4 signaling pathway by modulating IRAKM expression.

(A) Activation of the TLR4 signaling pathway in Mettl3-KO and Mettl3-WT BMDMs upon LPS stimulation was analyzed by Western blotting of p65, JNK, p38, ERK, and IRAKM. (B) Relative expression of Tirap, Myd88, Traf6, Irak, Irak4, Trif, and Tram in Mettl3^flox/flox;Lyzm-Cre mice and Mettl3^flox/flox littermates measured by real-time qPCR. (C) Relative expression of Irakm in control (NT) and LPS-treated Mettl3^flox/flox;Lyzm-Cre and Mettl3^flox/flox littermates measured by real-time qPCR. (D) Knockdown efficiency of shRNA targeting Irakm in BMDMs measured by real-time qPCR (n = 3). (E) TNF-α synthesis in WT BMDMs transfected with control shRNA (sh-CTL) and Mettl3-deficient BMDMs transfected with sh-CTL or IRAKM shRNA (sh-IRAKM) measured by flow cytometry (n = 3). Data are shown as representative results of three independent experiments (A) or as means ± SEM (B to E). **P < 0.01 and ***P < 0.001 (unpaired two-tailed Student’s t test).

Fig. 5

Fig. 5. IRAKM expression is down-regulated by m⁶A modification.

(A) Specific m⁶A peaks enriched in the 3′UTR of Irakm mRNAs in Raw 264.7 macrophages profiled using MeRIP-seq. CDS, coding sequences. (B) m⁶A peaks enriched in the 3′UTR of Irakm mRNAs in WT cells were lost in Mettl3-KO Raw 264.7 cells. (C) WT or Mettl3-deficient BMDMs were treated with the transcription inhibitor actinomycin D, and the level of Irakm transcripts was measured over time. (D) Relative luciferase activity of pGL4-luc2 with WT-3′UTR (IRAKM-WT) or with IRAKM-3′UTR containing mutated m⁶A sites (IRAKM-MUT) transfected into HEK293T cells was measured. The firefly luciferase activity was normalized to Renilla luciferase activity. Data are shown as representative results of three independent experiments (A) or as means ± SEM (B to D). **P < 0.01 and ****P < 0.0001; NS, not significant (unpaired two-tailed Student’s t test).

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References

1. Kawai T., Akira S., Toll-like receptors and their crosstalk with other innate receptors in infection and immunity. Immunity 34, 637–650 (2011). - PubMed
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[1] Kawai T., Akira S., Toll-like receptors and their crosstalk with other innate receptors in infection and immunity. Immunity 34, 637–650 (2011). - PubMed

[2] Kawai T., Akira S., Toll-like receptors and their crosstalk with other innate receptors in infection and immunity. Immunity 34, 637–650 (2011). - PubMed

[3] Murray P. J., Macrophage polarization. Annu. Rev. Physiol. 79, 541–566 (2017). - PubMed

[4] Murray P. J., Macrophage polarization. Annu. Rev. Physiol. 79, 541–566 (2017). - PubMed

[5] Noy R., Pollard J. W., Tumor-associated macrophages: From mechanisms to therapy. Immunity 41, 49–61 (2014). - PMC - PubMed

[6] Noy R., Pollard J. W., Tumor-associated macrophages: From mechanisms to therapy. Immunity 41, 49–61 (2014). - PMC - PubMed

[7] Vitale I., Manic G., Coussens L. M., Kroemer G., Galluzzi L., Macrophages and metabolism in the tumor microenvironment. Cell Metab. 30, 36–50 (2019). - PubMed

[8] Vitale I., Manic G., Coussens L. M., Kroemer G., Galluzzi L., Macrophages and metabolism in the tumor microenvironment. Cell Metab. 30, 36–50 (2019). - PubMed

[9] DeNardo D. G., Ruffell B., Macrophages as regulators of tumour immunity and immunotherapy. Nat. Rev. Immunol. 19, 369–382 (2019). - PMC - PubMed

[10] DeNardo D. G., Ruffell B., Macrophages as regulators of tumour immunity and immunotherapy. Nat. Rev. Immunol. 19, 369–382 (2019). - PMC - PubMed

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Pooled CRISPR screening identifies m⁶A as a positive regulator of macrophage activation

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Pooled CRISPR screening identifies m⁶A as a positive regulator of macrophage activation

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