The methyltransferase Setdb2 mediates virus-induced susceptibility to bacterial superinfection

Abstract

Immune responses are tightly regulated to ensure efficient pathogen clearance while avoiding tissue damage. Here we report that Setdb2 was the only protein lysine methyltransferase induced during infection with influenza virus. Setdb2 expression depended on signaling via type I interferons, and Setdb2 repressed expression of the gene encoding the neutrophil attractant CXCL1 and other genes that are targets of the transcription factor NF-κB. This coincided with occupancy by Setdb2 at the Cxcl1 promoter, which in the absence of Setdb2 displayed diminished trimethylation of histone H3 Lys9 (H3K9me3). Mice with a hypomorphic gene-trap construct of Setdb2 exhibited increased infiltration of neutrophils during sterile lung inflammation and were less sensitive to bacterial superinfection after infection with influenza virus. This suggested that a Setdb2-mediated regulatory crosstalk between the type I interferons and NF-κB pathways represents an important mechanism for virus-induced susceptibility to bacterial superinfection.

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Figure 1: Setdb2 expression is induced in an IFNAR1-dependent manner upon infection with influenza virus and stimulation of TLRs.
Figure 2: Setdb2GT/GT macrophages show increased expression of a subset of NF-κB target genes, including Cxcl1.
Figure 3: Setdb2 binds to the Cxcl1 promoter region and associates with H3K9me3.
Figure 4: Setdb2GT/GT mice show exacerbated lung inflammation in a model of LPS-induced neutrophilia.
Figure 5: Setdb2 mediates influenza virus–induced susceptibility to superinfection with S. pneumoniae.

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Acknowledgements

We thank the Superti-Furga laboratory (Research Center for Molecular Medicine of the Austrian Academy of Sciences) for antibody to Zbp1; S. Schüchner and E. Ogris (Max F. Perutz Laboratories Monoclonal antibody facility in Vienna) for help with generating the antibody to Setdb2; E. Southon and M.E. Palko for technical help in generating the Setdb2GT/GT mice; Y. Guo for technical help; B. Marzolf and P. Troisch (microarray core facility of the Institute for Systems Biology) for technical help in generating microarray data; M. Farlik, T. Penz, M. Schuster and C. Bock (Biosequencing Facility of the Research Center for Molecular Medicine of the Austrian Academy of Sciences) for technical help and advice for generating RNA-Seq data; M. Müller and B. Strobl (University of Veterinary Medicine Vienna) for Irf7−/− and Stat1−/− mice; M. Gorna, F. Grebien, L. Heinz, T. Karonitsch, M. Rebsamen, C. Rosenberger and S. Saluzzo for advice; and D. Barlow, R.A. Flavell, A.N. Hegazy, A. Jamieson, R. Medzhitov and G. Superti-Furga for discussions. Supported by the German Academic Exchange Service (C.S.), the Intramural Research Program of the National Cancer Institute, Center for Cancer Research, of the US National Institutes of Health (E.K.F., A.L.T., L.T. and D.E.S.), the European Molecular Biology Organization (ALTF 48-2008 to A.Be.; and ALTF 314-2012 to R.K.K.), the Austrian Academy of Sciences (A.Bh.), the Swiss National Science Foundation (PP00P3_152928 to D.M.), the Klaus-Tschira Foundation (D.M.), the Gebert-Rüf Foundation (D.M.), the Ohio State University Comprehensive Cancer Center (D.E.S.), the US National Institutes of Health (R01AI032972, R01AI025032 and U19AI100627 to A.A.), the Swiss Foundation for Medical-Biological Stipends (A.Be.), the Austrian Academy of Sciences (A.Be.) and the Austrian Science Fund (P-25360 to A.Be.).

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Contributions

C.S. designed experiments, performed in vitro and in vivo studies and wrote the manuscript; E.K.F., A.L.T., L.T. and D.E.S. generated the Setdb2GT/GT mouse and provided advice; B.V. performed ChIP, flow cytometry and in vivo experiments; U.R. performed in vitro experiments and generated the monoclonal antibody to Setdb2; S.S., L.B., I.M.G., L.K., A.L., A.Bh. and K.K. performed in vitro experiments; B.B. and M.M.E. performed in vivo experiments and provided advice; R.K.K. performed bioinformatics analyses; F.S., V.L., J.S. and S. Ku. provided reagents and/or advice; I.M., A.H., D.M. and S.Kn. did histological analyses and provided reagents and/or advice; D.E.S. and A.A. supervised the study and provided reagents and advice; and A.Be. supervised the study, designed experiments, performed in vitro and in vivo experiments and wrote the manuscript.

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Correspondence to Andreas Bergthaler.

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C.S. and A.Be. are the authors of a patent application related to this work.

Integrated supplementary information

Supplementary Figure 1 Induction of Setdb2 and Zbp1 by PAM3 is TLR2 dependent.

WT and Tlr2–/–Cd36–/– BMDMs were treated with PAM3 (two different batches from Invivogen), LPS or left unstimulated (US). (a) Setdb2 or (b) Zbp1 mRNA expression was quantified by real-time PCR and displayed as fold induction compared to untreated WT cells 8 h after stimulation. The experiment was performed in biological triplicates (mean ± s.e.m.). Statistical significance was calculated by unpaired t-test. Significant p-values were indicated as follows: * p≤0.001, ** p≤0.0001.

Supplementary Figure 2 Expression kinetics of PKMT-encoding genes in BMDMs after stimulation with poly(I:C).

WT BMDMs were stimulated with poly(I:C) and gene expression levels were determined at the indicated time points by microarray. Fold gene induction compared to unstimulated cells is shown. The top three up-regulated genes are highlighted. The data is derived from systemsimmunology.org (Supplementary Table 3).

Supplementary Figure 3 Generation of Setdb2GT/GT mice.

(a) Schematic of recombinant Setdb2 genetrap targeting strategy. (b) Southern blot of transfected ES cells. (c) Genotyping PCR of Setdb2 genetrap (Setdb2GT/GT) mice.

Supplementary Figure 4 No difference between poly(I:C)-stimulated wild-type and Setdb2GT/GT BMDMs in the degradation of IκBα.

WT and Setdb2GT/GT BMDMs were stimulated with poly(I:C) for indicated times points and IκBα expression/degradation was analyzed by immunoblot using the antibody sc-371. Immunoblot against Actin served as loading control. One out of two similar experiments is shown.

Supplementary Figure 5 Blockade of IFNAR1 leads to increased CXCL1 expression upon stimulation with poly(I:C).

WT and Setdb2GT/GT BMDMs were treated with 20μg/ml IFNAR1-specific antibody (clone MAR1-5A3, anti-IFNAR) or with isotype control followed by poly(I:C) stimulation. (a) Setdb2 and (b) Cxcl1 expression was quantified by real-time PCR after 2 h of stimulation. Relative expression compared to the housekeeping gene Ef1a is shown. The experiment was performed in biological triplicates (mean ± s.e.m.). One out of two similar experiments is shown. Statistical significance was calculated by unpaired t-test. Significant p-values were indicated as follows: * p≤0.01, ** p≤0.001.

Supplementary Figure 6 Time kinetics of the expression of Cxcl1 and Setdb2 in BMDMs after stimulation with poly(I:C).

WT BMDMs were stimulated with poly(I:C) and Cxcl1 and Setdb2 mRNA expression was determined by microarray at the indicated time points. The data was derived from systemsimmunology.org.

Supplementary Figure 7 Setdb2GT/GT alveolar macrophages express increased levels of Cxcl1 and CXCL1 upon infection with influenza virus.

Alveolar macrophages derived from the BAL fluid of naïve WT and Setdb2GT/GT mice were seeded on 96-well tissue culture plates and subsequently infected with influenza virus (PR8) (MOI 100) or left uninfected (UI). 12 h after stimulation, (a) Cxcl1 mRNA expression was quantified by real-time PCR and is displayed as fold induction compared to untreated WT cells. (b) CXCL1 protein was quantified by ELISA. Results from one out of two similar experiments are shown (mean ± s.e.m.). Statistical significance was calculated by unpaired t-test. Significant p-values were indicated as follows: * p≤0.05, ** p≤0.01, *** p≤0.001.

Supplementary Figure 8 Cytokine profiling of mice infected with influenza virus or S. pneumoniae or superinfected with both pathogens.

WT and Setdb2GT/GT mice were either left uninfected (UI), infected with Streptococcus pneumoniae (SP) (~2x104 CFU), infected with a sublethal dose of influenza virus (PR8), or superinfected with SP (~2x104 CFU) (PR8+SP) on day 5 after PR8 infection. BAL fluid was harvested 16 h after SP infection respectively 5 d and 16 h for the groups infected with PR8 or PR8+SP. Levels of (a) Cxcl2, (b) IL-6, (c) IL-10 and (d) CXCL1 were determined by ELISA. Scatter blots represent individual mice (mean ± s.e.m.). (a-c) Results from two pooled experiments are shown. No statistically significant differences between WT and Setdb2GT/GT groups of the respective infections were detected by unpaired t-test.

Supplementary Figure 9 Cellular lung profiling of wild-type and Setdb2GT/GT mice.

WT and Setdb2GT/GT mice were either (a-b) left uninfected (UI), (c-d) infected with influenza virus for 5 d and 16 h, (e-f) infected with SP for 16 h, or (g-h) superinfected with SP for 16 h on day 5 after influenza virus infection (compare Supplementary Fig. 8 legend for respective infectious doses). (a, c, e, g) Representative FACS plots with gating strategies for neutrophils (Neutr), monocytes/macrophages/dendritic cells (Mac/DC), alveolar macrophages (AM), NK cells (NK), T cells and B cells are shown. Scatter plots represent total cell numbers per lung from individual mice. Mean gate frequencies ± s.e.m. are displayed within the FACS plots. (b, d, f, h) Scatter plots represent relative percentages of live CD45+ cells in BAL fluid of individual mice. (i) Representative backgating plot of the population of AMs from CD45+ live lung cells. Scatter blots represent data derived from individual mice (mean ± s.e.m.). (a-d, f-h) Pooled data of two or more experiments are shown. Statistical significance was calculated by unpaired t-test. Significant p-values were indicated as follows: * p≤0.01, ** p≤0.01, *** p≤0.0001.

Supplementary Figure 10 No difference in pathogen loads of Setdb2GT/GT mice and those of wild-type mice after single infection with either influenza virus or S. pneumoniae.

(a) WT and Setdb2GT/GT mice were infected by a sublethal dose of influenza virus (PR8). 5 d later, mice were sacrificed and total RNA was prepared from the right lung lobes. Viral loads were quantified by real-time PCR for the M gene. (b) WT and Setdb2GT/GT mice were infected with ~2x103 respectively ~4x104 CFU of SP. 2 d later, mice were sacrificed and bacterial burden was analyzed by colony formation assay on blood agar plates. Lysates from total lung tissue were analyzed. Scatter blots represent data derived from individual mice pooled from 1-3 independent experiments (mean ±_s.e.m.). No statistically significant differences between WT and Setdb2GT/GT groups of the respective infections were detected by unpaired t-test.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–10 and Supplementary Tables 1–3 (PDF 1431 kb)

Supplementary Table 1

Microarray data of lung tissue from influenza virus infected compared to mock treated wild type mice. (XLSX 94 kb)

Supplementary Table 2

Predicted transcription factor binding sites of Setdb2. (XLSX 65 kb)

Supplementary Table 3

Microarray data of poly(I:C) stimulated wild type BMDMs (XLSX 48 kb)

Supplementary Table 4

RNAseq data of poly(I:C) stimulated WT and Setdb2GT/GT BMDMs. (XLSX 95 kb)

Supplementary Table 5

List of NF-κB target genes (XLSX 37 kb)

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Schliehe, C., Flynn, E., Vilagos, B. et al. The methyltransferase Setdb2 mediates virus-induced susceptibility to bacterial superinfection. Nat Immunol 16, 67–74 (2015). https://doi.org/10.1038/ni.3046

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