Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2016 May 27;352(6289):aad7993.
doi: 10.1126/science.aad7993. Epub 2016 Apr 28.

Topoisomerase 1 inhibition suppresses inflammatory genes and protects from death by inflammation

Alex Rialdi #  1   2 , Laura Campisi #  1   2 , Nan Zhao  1   2 , Arvin Cesar Lagda  1   2 , Colette Pietzsch  3 , Jessica Sook Yuin Ho  4 , Luis Martinez-Gil  1   5 , Romain Fenouil  6 , Xiaoting Chen  7 , Megan Edwards  1 , Giorgi Metreveli  1   2 , Stefan Jordan  8 , Zuleyma Peralta  6 , Cesar Munoz-Fontela  9 , Nicole Bouvier  1 , Miriam Merad  8 , Jian Jin  10 , Matthew Weirauch  7 , Sven Heinz  11   12 , Chris Benner  12 , Harm van Bakel  6 , Christopher Basler  1 , Adolfo García-Sastre  1   2 , Alexander Bukreyev  3 , Ivan Marazzi  1   2
Affiliations

Topoisomerase 1 inhibition suppresses inflammatory genes and protects from death by inflammation

Alex Rialdi et al. Science. .

Abstract

The host innate immune response is the first line of defense against pathogens and is orchestrated by the concerted expression of genes induced by microbial stimuli. Deregulated expression of these genes is linked to the initiation and progression of diseases associated with exacerbated inflammation. We identified topoisomerase 1 (Top1) as a positive regulator of RNA polymerase II transcriptional activity at pathogen-induced genes. Depletion or chemical inhibition of Top1 suppresses the host response against influenza and Ebola viruses as well as bacterial products. Therapeutic pharmacological inhibition of Top1 protected mice from death in experimental models of lethal inflammation. Our results indicate that Top1 inhibition could be used as therapy against life-threatening infections characterized by an acutely exacerbated immune response.

PubMed Disclaimer

Figures

Fig. 1
Fig. 1. Top1 inhibition suppresses PAMP-induced gene expression
(A) Schematic representation of factors controlling different phases of RNAPII-mediated transcription. Chemical inhibitors FVD (red), (+)-JQ1 (green), and CPT (blue) are color-coded according to their protein targets. (B) Quantitative PCR (qPCR) results showing the expression levels of representative viral PAMP-induced genes IFNB and IFIT1, in response to the influenza PR8ΔNS1 virus infection in A549 cells, either untreated (−) or treated with DMSO or 5 μM inhibitors. (C) Heat map showing relative change in gene expression levels in A549 cells not transfected (UT) or transfected with a Top1-specific siRNA (siTop1), as compared to non-targeting control siRNA-treated (siCtrl) cells during infection with influenza PR8ΔNS1 for genes differentially expressed between siTop1 and siCtrl at 4 hours after infection (P < 0.01; ANOVA with post hocTukey HSD test). Known interferon-stimulated genes (ISGs) and cytokine-coding genes are indicated in the adjacent heat map. A table summarizing the top five pathways affected by Top1 depletion during infection is also shown (top right). (D) Expression levels of IFIT1 and IFIT2 genes in response to influenza PR8ΔNS1 infection in A549 cells treated with 0.5 μM CPT, 100 nM TPT, or DMSO at 4 hours after infection (left bars) or 16 hours after washout (white, right bars). (E) Mass spectrometry data showing representative virus-induced and housekeeping protein levels in response to influenza PR8ΔNS1 infection in A549 cells treated with 0.5 μM CPTor DMSO at 6 hours after infection. *P < 0.05, **P < 0.005 (Student's t test with Holm-Bonferroni sequential correction). Data are means ± SD from three [(B) to (D)] and two (E) independent experiments.
Fig. 2
Fig. 2. Topotecan (TPT) and camptothecin (CPT) suppress RNAPII at PAMP-induced genes
(A) Gene expression in human A549 cells, left untreated (−) or treated with 0.5 μM CPT, 100 nM TPT, or DMSO, at 4 hours after mock treatment or PR8ΔNS1 virus infection. (B) ChIP-qPCR analysis of endogenous RNAPII and Top1 at the promoters of IFIT1, IFIT2, and ACTB in A549 cells treated with 0.5 μM CPT or DMSO, at 4 hours after mock treatment or infection with influenza PR8ΔNS1. (C) ChIP-seq metaplot of endogenous RNAPII in A549 cells treated with 0.5 μM CPT or DMSO 6 hours after mock treatment or PR8ΔNS1 virus infection. Plots represent RNAPII occupancy at genes showing a factor of 2 up-regulation in their expression after infection. (D) ChIP-seq tracks of representative antiviral genes IFIT1 and IFIT2, and housekeeping genes ACTB and HPRT1. (E) Schematic representation of the chemical synthesis of TPT-A from TPT. (F) Chem-ChIP qPCR analysis of TPT-A occupancy across IFIT1, IFIT2, ACTB, and HPRT1 genes in A549 cells treated with DMSO or 100 nM TPT-A, at 6 hours after mock treatment or PR8ΔNS1 infection. *P < 0.05, **P < 0.005, ***P < 0.0005 (Student's t test with Holm-Bonferroni sequential correction). Data are means ± SD from three (A) and two [(B) and (F)] independent experiments.
Fig. 3
Fig. 3. Classification of SMARCA2/4-dependent PAMP-induced genes
(A) Expression of genes up-regulated by a factor of >2 after infection and suppressed by Top1 inhibition in A549 cells at 4 hours after mock treatment, infection with PR8ΔNS1 virus (infected), or stimulation with exogenous IFN-β (IFNB). Cells were dual-transfected with siRNAs targeting SMARCA2 and SMARCA4 (orange bars). SWI/SNF dependency was evaluated via transient knockdown of SMARCA2/4, and IRF3-dependent genes were compiled from the literature and cross-compared with a list of genes induced by IRF35D in STAT1−/− cells. (B) Table summarizing the results for genes in (A). Columns 2 and 3 show the effect of SMARCA2/4 knockdown on PR8ΔNS1 and exogenous interferon (IFN-β)–induced mRNA levels, respectively. Levels of mRNA are shown as a percentage of the mRNA level determined by qPCR in siCtrl-treated A549 cells (set at 100% for each gene). Column 4 classifies the genes: A and B (21/28 genes) are SWI/SNF-dependent (inducibility levels <50%), C and D (7/28) are SWI/SNF-independent (inducibility >50%). Color-coded legends for columns 2 and 3 are shown at the top right. *P < 0.05 (Student's t test with Holm-Bonferroni sequential correction). Data are means ± SD from three independent experiments.
Fig. 4
Fig. 4. Top1 inhibition suppresses PAMP-induced genes that require nucleosome remodeling for activation
(A) Left: ChIP-qPCR analysis of endogenous TBP, histone H3, and RNAPII at the promoters of classes A to D, housekeeping, and PAMP-induced Top1-independent genes (IRF1, KLF4) in A549 cells treated with 0.5 μM CPT or DMSO, at 6 hours after mock treatment or infection with influenza PR8ΔNS1. Right: Summation plots of each individual protein's occupancy (percent input). Inset: Correlation plot of gene expression (infected) versus RNAPII occupancy (infected) for genes shown in Fig. 3A. Data are means ± SD from two independent experiments. (B) Results of testing of 1630 ChIP-seq data sets for transcription factor (TF) enrichment at the promoters of Top1-affected genes during infection (see materials and methods). Negative log of the P value of each of these data sets (blue) and results of the same procedure are applied to genes unaffected by Top1 depletion (gray). The displayed data are the result of defining promoters as (−1000, +1) relative to the transcriptional start site. The top three TFs and examples of insignificant TFs are shown. (C) Basal state meta-analysis of RNAPII (POL) occupancy, DNase hypersensitivity, and H3K27ac occupancy at the promoters of genes designated as either Top1 affected (N = 84) or Top1 nonaffected (N = 296) after infection. Data sets used are from ENCODE (see materials and methods). (D) Basal state meta-analysis of CpG island occupancy at the promoters of genes designated as either Top1 affected or Top1 nonaffected after infection.
Fig. 5
Fig. 5. Top1 regulates LPS-induced inflammation in vitro and in vivo
(A and B) Gene expression in A549 (A) or RAW 264.7 (B) cells, left untreated (−) or treated with 0.5 μM CPT, 100 nM TPTor DMSO, in the presence of LPS stimulation or not (UT). (C to H) C57BL/6J mice left untreated or treated with CPT in response to LPS-induced septic shock. (C) Survival curve. (D) Serum titers of indicated cytokines at 4 hours after LPS injection. [(E) to (H)] Ninety minutes after LPS injection spleens were harvested to perform transcriptional analysis of indicated inflammatory genes (E) and to determine cell viability and activation by flow cytometry [(F) to (H)]. (F) Gating strategy. (G) Histograms comparing the incorporation of a live/dead dye after gating on R1, R2, R3, and R4. (H) CD69 expression after gating on R3. *P < 0.05, **P < 0.005, ***P < 0.0005 (Student's t test with Holm-Bonferroni sequential correction) for [(A), (B), (D), (E)] or log rank test (C). Data are means ± SD from three independent experiments [(A) to (C)] with n = 11 (LPS) and n = 12 (LPS + CPT) individual mice, or two independent experiments [(D) to (H)] with n = 6 (LPS) and n = 7 CPT (LPS + CPT) individual mice.
Fig. 6
Fig. 6. Top1 inhibition blocks lethal inflammation in vivo
(A to C) Survival curves of C57BL/6J mice left untreated or treated with CPT in response to S. aureus infection (A), PR8–S. aureus co-infection (B), or D-GalN/LPS injection (C). Mice were treated with CPT 3, 24, and 48 hours after S. aureus infection (A); 12, 24, and 36 hours after PR8 infection (B); or 2 hours and 30 min after D-GalN/LPS injection (C). **P < 0.005, ***P < 0.0005 (log rank test). Data are from three independent experiments (A) with n = 8 to 12 individual mice, and two independent experiments [(B) and (C)] with n = 5 to 9 individual mice.
Fig. 7
Fig. 7. Suppression of Ebola virus induced inflammation by Top1 inhibitors
THP-1 cells were mock-treated or infected with wild-type (WT) Ebola virus (Zaire-Mayinga strain) in the presence of 0.5 μM CPT, 100 nM TPT, or DMSO. Bar graphs show the relative expression of selected genes. Data are means ± SD from three independent experiments. *P < 0.05, **P < 0.005 (Student's t test with Holm-Bonferroni sequential correction).
See this image and copyright information in PMC

Comment in

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Janeway CA, Jr., Medzhitov R. Innate immune recognition. Annu. Rev. Immunol. 2002;20:197–216. doi: 10.1146/annurev.immunol.20.083001.084359; pmid: 11861602. - PubMed
    1. Medzhitov R. Approaching the asymptote: 20 years later. Immunity. 2009;30:766–775. doi: 10.1016/j.immuni.2009.06.004; pmid: 19538928. - PubMed
    1. Beutler B, et al. Genetic analysis of resistance to viral infection. Nat. Rev. Immunol. 2007;7:753–766. doi: 10.1038/nri2174; pmid: 17893693. - PubMed
    1. Schoggins JW, et al. A diverse range of gene products are effectors of the type I interferon antiviral response. Nature. 2011;472:481–485. doi: 10.1038/nature09907; pmid: 21478870. - PMC - PubMed
    1. Crow YJ. Type I interferonopathies: Mendelian type I interferon up-regulation. Curr. Opin. Immunol. 2015;32:7–12. doi: 10.1016/j.coi.2014.10.005; pmid: 25463593. - PubMed

Publication types

MeSH terms