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Research Article
Systems Immunology

Comprehensive RNAi-based screening of human and mouse TLR pathways identifies species-specific preferences in signaling protein use

Jing Sun, Ning Li, Kyu-Seon Oh, Bhaskar Dutta, Sharat J. Vayttaden, Bin Lin, Thomas S. Ebert, Dominic De Nardo, Joie Davis, Rustam Bagirzadeh, Nicolas W. Lounsbury, Chandrashekhar Pasare, Eicke Latz, Veit Hornung, and Iain D. C. Fraser [email protected]
Science Signaling • 5 Jan 2016 • Vol 9, Issue 409 • p. ra3 • DOI: 10.1126/scisignal.aab2191
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Species-specific TLR signaling

Whether mice are a good model for the study of innate immune responses in humans is a topic of some debate. Sun et al. used an RNA interference (RNAi)–based screen to compare the responses of mouse and human macrophage reporter cell lines to various ligands of Toll-like receptors (TLRs), which are pattern recognition receptors that stimulate innate immune responses. In addition to confirming the conservation of many TLR signaling components between the two species, the study revealed important differences in how mouse and human macrophages use members of the IRAK family of kinases, findings that have implications for the study and treatment of human autoimmune diseases.

Abstract

Toll-like receptors (TLRs) are a major class of pattern recognition receptors, which mediate the responses of innate immune cells to microbial stimuli. To systematically determine the roles of proteins in canonical TLR signaling pathways, we conducted an RNA interference (RNAi)–based screen in human and mouse macrophages. We observed a pattern of conserved signaling module dependencies across species, but found notable species-specific requirements at the level of individual proteins. Among these, we identified unexpected differences in the involvement of members of the interleukin-1 receptor–associated kinase (IRAK) family between the human and mouse TLR pathways. Whereas TLR signaling in mouse macrophages depended primarily on IRAK4 and IRAK2, with little or no role for IRAK1, TLR signaling and proinflammatory cytokine production in human macrophages depended on IRAK1, with knockdown of IRAK4 or IRAK2 having less of an effect. Consistent with species-specific roles for these kinases, IRAK4 orthologs failed to rescue signaling in IRAK4-deficient macrophages from the other species, and only mouse macrophages required the kinase activity of IRAK4 to mediate TLR responses. The identification of a critical role for IRAK1 in TLR signaling in humans could potentially explain the association of IRAK1 with several autoimmune diseases. Furthermore, this study demonstrated how systematic screening can be used to identify important characteristics of innate immune responses across species, which could optimize therapeutic targeting to manipulate human TLR-dependent outputs.
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Supplementary Material

Summary

Fig. S1. Design and TLR ligand response of mouse and human macrophage reporter cell lines for siRNA screening applications.
Fig. S2. Effects of siRNA-mediated gene perturbations across the human and mouse TLR pathways.
Fig. S3. Human and mouse macrophages show both shared and distinct gene dependencies in TLR signaling.
Fig. S4. IRAK4 is required for the responses of human PBMCs to TLR ligands.
Fig. S5. Generation of IRAK4 rescue cell lines from IRAK4 KO IMMs and THP1 cells.
Fig. S6. Expression of kinase-deficient mouse and human IRAK4 in IRAK4 KO mouse IMMs and THP1 cells.
Fig. S7. Analysis of the IRAK1 dependency of human macrophages for TLR responses.
Fig. S8. CRISPR/Cas9-mediated targeting of the human IRAK1 and IRAK2 loci in THP1 cells.
Fig. S9. Expression distributions of IRAK1/Irak1, IRAK2/Irak2, and IRAK4/Irak4 mRNAs in human and mouse GEO data sets.
Table S1. Details of the siRNAs used to target the 126 human and mouse TLR pathway genes.
Table S2. siRNA z scores from screens of the 126 human and mouse TLR pathway genes.
Table S3. Ranking scores of human and mouse TLR pathway genes from the siRNA screens.
Table S4. Human versus mouse z score differences across matched TLR ligands and assay readouts.

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Science Signaling
Volume 9 | Issue 409
January 2016

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Received: 27 March 2015
Accepted: 11 December 2015

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Acknowledgments

We thank R. Germain, R. Gottschalk, and colleagues in the Laboratory of Systems Biology for helpful discussions and critical reading of the manuscript; S. Holland for assistance in obtaining blood from an IRAK4-deficient patient; X. Li for provision of the human IRAK4 kinase–deficient mutant; A. Miller for assistance with the bacterial infection assay; and R. Stahl for assistance in generating the mouse IRAK4-mCitrine retroviral plasmids. Funding: This work was supported by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases (NIAID) (to J.S., N.L., K.-S.O., B.D., S.J.V., B.L., J.D., N.W.L., and I.D.C.F.) and the BONFOR research commission at the University of Bonn (to D.D.N.). V.H. and T.S.E. are supported by grants from the German Research Foundation (SFB704 and SFB670) and the European Research Council (ERC-2009-StG 243046). E.L. is supported by grants from the NIH (R01HL112661), the Deutsche Forschungsgemeinschaft (SFB670), and the European Research Council (ERC InflammAct). E.L. and V.H. are members of the ImmunoSensation cluster of excellence. C.P. is supported by grants from the NIAID (AI082265) and The Welch Foundation (I-1820). R.B. is supported by an NIH Integrative Immunology Training program grant (5T32-AI005284-35). Author contributions: J.S., N.L., and I.D.C.F. designed the study; J.S. and N.L. performed the siRNA screen; B.D., J.S., N.L., and I.D.C.F. analyzed the siRNA screen data; J.S. performed all aspects of the IRAK studies; K.-S.O. performed immunoprecipitation of TLR signaling components; S.J.V. performed high-content imaging analysis of TLR pathway components; B.L. performed qRT-PCR analysis of siRNA-mediated knockdowns; T.S.E. and V.H. generated the THP1 IRAK KO cell lines; D.D.N. and E.L. generated the mouse IRAK4 rescue cell lines; J.D. acquired and processed the IRAK4-deficient patient samples; R.B. and C.P. acquired and processed the IRAK1 and IRAK2 KO BMDMs; N.W.L. and B.D. performed the analysis of IRAK expression in the GEO data; J.S. and I.D.C.F. wrote the paper; and all authors commented on the manuscript. Competing interests: The authors declare that they have no competing interests. Data and materials availability: Data from the RNAi-based screens have been deposited in the PubChem BioAssay database (http://pubchem.ncbi.nlm.nih.gov; AID 1159581 and AID 1159582).

Authors

Affiliations

Jing Sun
Signaling Systems Unit, Laboratory of Systems Biology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
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Ning Li
Signaling Systems Unit, Laboratory of Systems Biology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
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Kyu-Seon Oh
Signaling Systems Unit, Laboratory of Systems Biology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
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Bhaskar Dutta
Bioinformatics Team, Laboratory of Systems Biology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
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Sharat J. Vayttaden
Signaling Systems Unit, Laboratory of Systems Biology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
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Bin Lin
Signaling Systems Unit, Laboratory of Systems Biology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
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Thomas S. Ebert
Institute of Molecular Medicine, University Hospital, University of Bonn, 53127 Bonn, Germany.
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Dominic De Nardo
Institute of Innate Immunity, University Hospital, Biomedical Centre, University of Bonn, 53127 Bonn, Germany.
Inflammation Division, Walter and Eliza Hall Institute, Parkville,Victoria 3052, Australia.
Department of Medical Biology, The University of Melbourne, Parkville, Victoria 3010, Australia.
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Joie Davis
Laboratory of Clinical Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
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Rustam Bagirzadeh
Department of Immunology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
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Nicolas W. Lounsbury
Signaling Systems Unit, Laboratory of Systems Biology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
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Chandrashekhar Pasare
Department of Immunology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
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Eicke Latz
Institute of Innate Immunity, University Hospital, Biomedical Centre, University of Bonn, 53127 Bonn, Germany.
German Center for Neurodegenerative Diseases (DZNE), 53175 Bonn, Germany.
Department of Infectious Diseases and Immunology, University of Massachusetts Medical School, Worcester, MA 01605, USA.
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Veit Hornung
Institute of Molecular Medicine, University Hospital, University of Bonn, 53127 Bonn, Germany.
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Iain D. C. Fraser* [email protected]
Signaling Systems Unit, Laboratory of Systems Biology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
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Notes

*Corresponding author. E-mail: [email protected]

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