IL-11 is a crucial determinant of cardiovascular fibrosis |...

来自 : 发布时间：2024-05-09

AbstractFibrosis is a common pathology in cardiovascular disease1. In the heart, fibrosis causes mechanical and electrical dysfunction1,2 and in the kidney, it predicts the onset of renal failure3. Transforming growth factor 尾1 (TGF尾1) is the principal pro-fibrotic factor4,5, but its inhibition is associated with side effects due to its pleiotropic roles6,7. We hypothesized that downstream effectors of TGF尾1 in fibroblasts could be attractive therapeutic targets and lack upstream toxicity. Here we show, using integrated imaging鈥揼enomics analyses of primary human fibroblasts, that upregulation of interleukin-11 (IL-11) is the dominant transcriptional response to TGF尾1 exposure and required for its pro-fibrotic effect. IL-11 and its receptor (IL11RA) are expressed specifically in fibroblasts, in which they drive non-canonical, ERK-dependent autocrine signalling that is required for fibrogenic protein synthesis. In mice, fibroblast-specific Il11 transgene expression or Il-11 injection causes heart and kidney fibrosis and organ failure, whereas genetic deletion of Il11ra1 protects against disease. Therefore, inhibition of IL-11 prevents fibroblast activation across organs and species in response to a range of important pro-fibrotic stimuli. These results reveal a central role of IL-11 in fibrosis and we propose that inhibition of IL-11 is a potential therapeutic strategy to treat fibrotic diseases. Subscription info for Chinese customersWe have a dedicated website for our Chinese customers. Please go to naturechina.com to subscribe to this journal.Go to naturechina.comRent or Buy articleGet time limited or full article access on ReadCube.from$8.99Rent or BuyAll prices are NET prices. References1Rockey, D. C., Bell, P. D. Hill, J. A. Fibrosis鈥攁 common pathway to organ injury and failure. N. Engl. J. Med. 372, 1138鈥?149 (2015)CAS聽 Article聽Google Scholar聽 2Burstein, B. Nattel, S. 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Biol. 1130, 233鈥?43 (2014)CAS聽 Article聽Google Scholar聽 Download referencesAcknowledgementsWe thank all patients for taking part in this research, which was performed with approval from the SingHealth Centralised IRB Review Board (CIRB; 2013/103/C). The research was supported by the National Medical Research Council (NMRC) Singapore STaR award (S.A.C.) (NMRC/STaR/0011/2012), the NMRC Centre Grant to the National Heart Centre Singapore (NHCS), Goh Foundation, Tanoto Foundation, NHLBI 5R01HL080494 (J.G.S., C.E.S.), HHMI (C.E.S.) and a grant from the Fondation Leducq (N.H., J.G.S., C.E.S., S.C.). We thank I. Kamer and R. Plehm, Max-Delbr眉ck-Center for Molecular Medicine (MDC), for expert technical help with telemetry blood pressure measurements.Author informationAuthor notesSebastian Schafer, Sivakumar Viswanathan and Anissa A. Widjaja: These authors contributed equally to this work.AffiliationsNational Heart Centre Singapore, SingaporeSebastian Schafer,聽Wei-Wen Lim,聽Benjamin Ng,聽Kingsley Chow,聽Jessie Tan,聽Lei Ye,聽Chee Jian Pua,聽Nicole T. G. Zhen,聽Chen Xie,聽Shiqi Lim,聽See L. Lim,聽Jia L. Soon,聽Victor T. T. Chao,聽Yeow L. Chua,聽Teing E. Tan,聽Yee J. Loh,聽Muhammad H. Jamal,聽Kim K. Ong,聽Kim C. Chua,聽Boon-Hean Ong,聽Mathew J. Chakaramakkil,聽Kenny Y. K. Sin聽 聽Stuart A. CookDuke鈥揘ational University of Singapore Medical School, SingaporeSebastian Schafer,聽Sivakumar Viswanathan,聽Anissa A. Widjaja,聽Aida Moreno-Moral,聽Ester Khin,聽Sonia P. Chothani,聽Owen J. L. Rackham,聽Nicole S. J. Ko,聽Norliza E. Sahib,聽Mao Wang,聽Enrico Petretto,聽Kristmundur Sigmundsson,聽Jia L. Soon,聽Victor T. T. Chao,聽Kenny Y. K. Sin聽 聽Stuart A. CookDepartment of Genetics, Harvard Medical School, Boston, 02115, Massachusetts, USADaniel M. DeLaughter,聽Hiroko Wakimoto,聽Jonathan G. Seidman聽 聽Christine E. SeidmanCardiovascular and Metabolic Sciences, Max Delbr眉ck Center for Molecular Medicine in the Helmholtz Association (MDC), Robert-Rossle Strasse 10, Berlin, 13125, GermanyGiannino Patone,聽Henrike Maatz,聽Kathrin Saar,聽Susanne Blachut,聽Sabine Schmidt,聽Sebastiaan van Heesch聽 聽Norbert HubnerInflammation Division, Walter and Eliza Hall Institute of Medical Research, Parkville, 3052, Victoria, AustraliaTracy PutoczkiDepartment of Medical Biology, The University of Melbourne, Parkville, 3050, Victoria, AustraliaTracy PutoczkiSkaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, California, USANuno Guimar茫es-Camboa聽 聽Sylvia M. EvansDepartment of Medicine, University of California at San Diego, La Jolla, 92093, California, USASylvia M. EvansDepartment of Pharmacology, University of California at San Diego, La Jolla, 92093, California, USASylvia M. EvansKandang Kerbau Women鈥檚 and Children鈥檚 Hospital, SingaporeYee J. Loh聽 聽Kim K. OngDivision of Cardiovascular Medicine, Brigham and Women鈥檚 Hospital, Boston, 02115, Massachusettes, USAChristine E. SeidmanHoward Hughes Medical Institute, Chevy Chase, 20815, Maryland, USAChristine E. SeidmanDZHK (German Centre for Cardiovascular Research), partner site, Berlin, GermanyNorbert HubnerCharit茅-Universit盲tsmedizin, Berlin, GermanyNorbert HubnerBerlin Institute of Health (BIH), Berlin, GermanyNorbert HubnerNational Heart and Lung Institute, Imperial College London, London, UKStuart A. CookMRC-London Institute of Medical Sciences, Hammersmith Hospital Campus, Du Cane Road, London, W12 0NN, UKStuart A. CookAuthorsSebastian SchaferView author publicationsYou can also search for this author in PubMed聽Google ScholarSivakumar ViswanathanView author publicationsYou can also search for this author in PubMed聽Google ScholarAnissa A. WidjajaView author publicationsYou can also search for this author in PubMed聽Google ScholarWei-Wen LimView author publicationsYou can also search for this author in PubMed聽Google ScholarAida Moreno-MoralView author publicationsYou can also search for this author in PubMed聽Google ScholarDaniel M. DeLaughterView author publicationsYou can also search for this author in PubMed聽Google ScholarBenjamin NgView author publicationsYou can also search for this author in PubMed聽Google ScholarGiannino PatoneView author publicationsYou can also search for this author in PubMed聽Google ScholarKingsley ChowView author publicationsYou can also search for this author in PubMed聽Google ScholarEster KhinView author publicationsYou can also search for this author in PubMed聽Google ScholarJessie TanView author publicationsYou can also search for this author in PubMed聽Google ScholarSonia P. ChothaniView author publicationsYou can also search for this author in PubMed聽Google ScholarLei YeView author publicationsYou can also search for this author in PubMed聽Google ScholarOwen J. L. RackhamView author publicationsYou can also search for this author in PubMed聽Google ScholarNicole S. J. KoView author publicationsYou can also search for this author in PubMed聽Google ScholarNorliza E. SahibView author publicationsYou can also search for this author in PubMed聽Google ScholarChee Jian PuaView author publicationsYou can also search for this author in PubMed聽Google ScholarNicole T. G. ZhenView author publicationsYou can also search for this author in PubMed聽Google ScholarChen XieView author publicationsYou can also search for this author in PubMed聽Google ScholarMao WangView author publicationsYou can also search for this author in PubMed聽Google ScholarHenrike MaatzView author publicationsYou can also search for this author in PubMed聽Google ScholarShiqi LimView author publicationsYou can also search for this author in PubMed聽Google ScholarKathrin SaarView author publicationsYou can also search for this author in PubMed聽Google ScholarSusanne BlachutView author publicationsYou can also search for this author in PubMed聽Google ScholarEnrico PetrettoView author publicationsYou can also search for this author in PubMed聽Google ScholarSabine SchmidtView author publicationsYou can also search for this author in PubMed聽Google ScholarTracy PutoczkiView author publicationsYou can also search for this author in PubMed聽Google ScholarNuno Guimar茫es-CamboaView author publicationsYou can also search for this author in PubMed聽Google ScholarHiroko WakimotoView author publicationsYou can also search for this author in PubMed聽Google ScholarSebastiaan van HeeschView author publicationsYou can also search for this author in PubMed聽Google ScholarKristmundur SigmundssonView author publicationsYou can also search for this author in PubMed聽Google ScholarSee L. LimView author publicationsYou can also search for this author in PubMed聽Google ScholarJia L. SoonView author publicationsYou can also search for this author in PubMed聽Google ScholarVictor T. T. ChaoView author publicationsYou can also search for this author in PubMed聽Google ScholarYeow L. ChuaView author publicationsYou can also search for this author in PubMed聽Google ScholarTeing E. TanView author publicationsYou can also search for this author in PubMed聽Google ScholarSylvia M. EvansView author publicationsYou can also search for this author in PubMed聽Google ScholarYee J. LohView author publicationsYou can also search for this author in PubMed聽Google ScholarMuhammad H. JamalView author publicationsYou can also search for this author in PubMed聽Google ScholarKim K. OngView author publicationsYou can also search for this author in PubMed聽Google ScholarKim C. ChuaView author publicationsYou can also search for this author in PubMed聽Google ScholarBoon-Hean OngView author publicationsYou can also search for this author in PubMed聽Google ScholarMathew J. ChakaramakkilView author publicationsYou can also search for this author in PubMed聽Google ScholarJonathan G. SeidmanView author publicationsYou can also search for this author in PubMed聽Google ScholarChristine E. SeidmanView author publicationsYou can also search for this author in PubMed聽Google ScholarNorbert HubnerView author publicationsYou can also search for this author in PubMed聽Google ScholarKenny Y. K. SinView author publicationsYou can also search for this author in PubMed聽Google ScholarStuart A. CookView author publicationsYou can also search for this author in PubMed聽Google ScholarContributionsS.A.C. conceived, managed and arranged funding for the project. Wet lab experiments (cell culture, cell biology, molecular biology, RNA-seq) were carried out by S.V., A.A.W., W.-W.L., B.N., G.P., J.T., L.Y., N.E.S., C.J.P., C.X., M.W., S.L., K.Sa., S.B., S.Schm., T.P., N.G.-C., H.W., S.v.H. and K.Si. Single-cell studies were carried out by D.M.D., J.G.S. and C.E.S. In vivo gain-of-function and loss-of-function mouse experiments were performed by A.A.W., W.-W.L., B.N., J.T., E.K., L.Y., N.S.J.K., N.T.G.Z., D.M.D., G.P. and H.M. Data were analysed by S.Scha., S.V., A.A.W., A.M.-M., K.C., S.P.C., O.J.L.R., K.Sa., E.P., S.M.E., J.G.S., C.E.S. and N.H. Patient-based studies were carried out by S.L.L., J.L.S., V.T.T.C., Y.L.C., T.E.T., Y.J.L., M.H.J., K.K.O., K.C.C., B.-H.O., M.J.C. and K.Y.K.S. S.Scha., S.V., A.A.W. and S.A.C. designed experiments and prepared the manuscript with input from co-authors.Corresponding authorCorrespondence to Stuart A. Cook.Ethics declarations Competing interests S.A.C. and S.Scha. are co-inventors of the patent application (WO2017103108) 鈥楾reatment of fibrosis鈥? S.A.C. and S.Scha. are co-founders and shareholders of Enleofen Bio PTE LTD, a company that develops therapeutics based on findings described in this manuscript. Additional informationReviewer Information Nature thanks S. Friedman and the other anonymous reviewer(s) for their contribution to the peer review of this work.Publisher\'s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Extended data figures and tablesExtended Data Figure 1 RNA-seq of fibrotic cardiac fibroblasts.a, Mapped RNA-seq reads for human fibroblasts (n鈥?鈥?4 biologically independent samples). We aimed to generate at least 60 million reads per sample to assess RNA expression with and without stimulation of primary cardiac fibroblasts with TGF尾1. Fibroblasts were derived from the atria of 84 individuals, resulting in a total of 168 RNA-seq datasets for unstimulated and stimulated cells. After mapping, we used only reads that map to one unique location in the genome to estimate gene expression levels. b, Distribution of mean gene expression across all samples for each gene. We found 12,081 genes to be expressed at log2(TPM鈥?鈥?)鈥?gt;鈥?. Genes below this cut-off were not considered in subsequent analyses. c, TPM distribution for each sample after filtering (n鈥?鈥?4). d, Ward clustering of Euclidian distance of RNA-seq samples. Sample gene expression tends to cluster mostly by genotype, indicating a strong genetic effect, beyond the effects of TGF尾1 stimulation.Extended Data Figure 2 Characterization the TGF尾1-regulated gene, Il-11.a, We measured the amount of activated fibroblasts (ACTA2+ cells) using the Operetta High-content imaging platform and IL-11 transcript levels by RNA-seq (n鈥?鈥?4 biologically independent samples). The increase in IL-11 expression correlated strongly with fibroblast activation in the cohort (蟻鈥?鈥?.47, 95% confidence interval鈥?鈥?.28鈥?.62). ST, stimulated cells (5鈥塶g ml鈭?, 24鈥塰); BL, baseline cells (unstimulated, 24鈥塰). b, TGF尾1 (5鈥塶g ml鈭?, 24鈥塰) significantly upregulates IL11 RNA (8.5-fold, P鈥?鈥?鈥壝椻€?0鈭?18) in human atrial fibroblasts according to RNA-seq analysis (n鈥?鈥?4 biologically independent samples). P values were calculated with DEseq2. c, The change in gene expression was confirmed at the protein level using an ELISA assay to measure IL-11 protein in the supernatant of cardiac fibroblasts (n鈥?鈥? biologically independent samples). fc, fold change. Two-tailed Student鈥檚 t-test. d, RNA-seq-based expression differences in IL11 transcript between unstimulated and TGF尾1-stimulated cardiac fibroblasts were confirmed via RT鈥搎PCR. e, f, The JSD was calculated for all protein-coding genes expressed in activated fibroblasts. Low JSD scores indicate that a gene is highly expressed in stimulated cardiac fibroblasts and lowly expressed in healthy tissues (e; GTEx) or unstimulated, primary cell lines (f; FANTOM). g, h, IL11 RNA expression in TGF尾1-stimulated and unstimulated cardiac fibroblasts (n鈥?鈥?4 biologically independent samples) compared to GTEx (g; tissues from n鈥?鈥?,723 biologically independent samples) and FANTOM (h; cell types from n鈥?鈥?85 biologically independent samples) databases. g, RNA expression of IL11 in TGF尾1-stimulated cardiac fibroblasts (red) and healthy tissues. h, RNA expression of IL11 in TGF尾1-stimulated cardiac fibroblasts (red) and primary cells. b, c, g, h, Box-and-whisker plots show median (middle line), 25th鈥?5th percentiles (box) and 10th鈥?0th percentiles (whiskers).Extended Data Figure 3 Single-cell RNA-seq of fibrotic mouse heart.Single-cell RNA-seq analysis of cells isolated from PlnR9C/+ and wild-type adult left ventricles shown in Fig. 1. a, Cell types were defined according to indicated marker genes. b, Cells were clustered and cell type was determined using the Seurat R package (see Methods). c, Upregulation of Il-11 in the heart in the PlnR9C/+ fibrosis model was confirmed using western blotting. All mice were 18 weeks old and male. d, Subsequently, 1,263 fibroblasts from PlnR9C/+ and wild-type mice were re-clustered using Seurat. WNT signalling, downstream of TGF尾1 in cardiac fibroblast activation, target genes were used in this analysis of the four subsequent clusters of fibroblasts; Il-11+ cells were primarily found in clusters 0 and 1 (16 out of 18 Il-11-expressing cells). Clusters 0 and 1 were also enriched for PlnR9C/+ cells compared to wild-type. Il11ra1 was expressed in all clusters. Cardiac cells were sequenced from n鈥?鈥? mouse, experiment was repeated one time with similar results.Extended Data Figure 4 IL-11 activates fibroblasts and is required for the pro-fibrotic effect of TGF尾1.a, b, High-resolution fluorescence imaging after TGF尾1 or IL-11 treatment (5鈥塶g ml鈭?, 24鈥塰) of primary cardiac fibroblasts. Immunostaining of nuclei (DAPI, blue), ACTA2 (red) and F-actin (phalloidin, green) indicated that both TGF尾1 and IL-11 activate fibroblast stress fibre formation and increase the number of myofibroblasts in vitro to similar levels. Experiment was repeated four times with similar results. c, Automated quantification of fluorescence (Operetta assay n鈥?鈥? measurements per n鈥?鈥? independent experiments) of primary atrial fibroblasts reveals significant fibroblast activation and ECM production induced by both TGF尾1 and IL-11 (5鈥塶g ml鈭?, 24鈥塰). d, In addition, TGF尾1 effects can be reduced with an anti-IL-11 antibody (2鈥壩糶 ml鈭?). c, d, Collagen secretion in the supernatant (n鈥?鈥? independent experiments) was assessed with Sirius Red. e, Mouse primary fibroblasts were incubated for 24鈥塰 with indicated concentrations of recombinant human or mouse IL-11. Fibroblast activation was monitored using the Operetta High-Content Imaging platform and immunostaining for ACTA2. rhIL-11 was found to inefficiently activate mouse fibroblasts (rmIL-11, n鈥?鈥?, rhIL-11, n鈥?鈥? biologically independent samples) compared to rmIL-11; this occurred for rhIL-11 treatment with rhIL-11 from two separate suppliers. f, g, MMP-2 (f) and TIMP-1 (g) concentration in the supernatant (ELISA) of cardiac fibroblasts (n鈥?鈥? biologically independent samples) without stimulus (鈭?, with TGF尾1 or IL-11 (5鈥塶g ml鈭?, 24鈥塰). h, Il-11-neutralizing antibodies (anti-IL-11, 2鈥壩糶 ml鈭?) block the increase in MMP-2 and TIMP-1 protein. i, In vitro monolayer scratch wound assay of cardiac fibroblasts. Wound closure was compared between stimulated (TGF尾1 or IL-11; 5鈥塶g ml鈭?, 24鈥塰) and unstimulated cardiac fibroblasts (n鈥?鈥? biologically independent samples) after 24鈥塰. j, Cardiac fibroblasts (n鈥?鈥? biologically independent samples) were seeded in collagen gel and the contraction was monitored. The area of contraction is compared between stimulated (TGF尾1 or IL-11; 5鈥塶g ml鈭?) and unstimulated groups after 72鈥塰. k, Trans-well migration assay. After 24鈥塰 of stimulation (TGF尾1 or IL-11; 5鈥塶g ml鈭?), cardiac fibroblasts (n鈥?鈥? biologically independent samples) that crossed the membrane towards either a TGF尾1- or IL-11-containing compartment were colourimetrically quantified and compared to data from unstimulated cells. l鈥?b>n, Cardiac fibroblasts were incubated with TGF尾1 (5鈥塶g ml鈭?, 24鈥塰) and indicated amounts of IL11RA:gp130 decoy receptors (l; 33 amino acid (aa) or 50 aa linker peptide), anti-IL11RA antibody (m; 2鈥壩糶 ml鈭?) or siRNA pools against IL-11 or IL11RA (n). l鈥?b>n, Fibroblast activation was monitored via immunostaining for ACTA2 on the Operetta platform. decoy receptors (l): n鈥?鈥? measurements per n鈥?鈥? independent experiments; anti-IL11RA (m): n鈥?鈥? measurements per n鈥?鈥? independent experiments; siRNA (n): Operetta assay n鈥?鈥? measurements per n鈥?鈥?0 independent experiments. o, Human renal fibroblasts were incubated with TGF尾1 or IL-11 (5鈥塶g ml鈭?, 24鈥塰) in the presence or absence of anti-IL-11 or an IgG control antibodies (2鈥壩糶 ml鈭? each) for 24鈥塰. ECM was assessed using the Operetta platform by staining for collagen I. Fluorescence was normalized to non-stimulated cells (black). p, These results were confirmed with Sirius red assay of the total collagen in the supernatant. q, rmIl-11 stimulation (5鈥塶g ml鈭?, 24鈥塰) also activated mouse cardiac and renal fibroblasts. Myofibroblasts and ECM were assessed using the Operetta platform by staining for ACTA2, collagen I or POSTN. Fluorescence was normalized to non-stimulated cells (black). o鈥?b>q, These experiments were repeated three times with similar results. r, Cardiac fibroblasts analysed on the Operetta high-content imaging platform with immunostaining of ACTA2 after 24鈥塰 incubation without stimulus, TGF尾1 (5鈥塶g ml鈭?, 24h) or TGF尾1 and IL-6-neutralizing antibody (2鈥壩糶 ml鈭?, 24h). Automated quantification of fluorescence (Operetta assay n鈥?鈥? measurements per n鈥?鈥? independent experiments) shows no significant decrease in fibroblast activation using anti-IL-6 antibodies. Data are mean and circles show individual values (e) or mean鈥壜扁€塻.d. and circles show individual values (c, d bottom right, f鈥?b>h, k); box-and-whisker plots (c, d, l鈥?b>n, r) show median (middle line), 25th鈥?5th percentiles (box) and 10th鈥?0th percentiles (whiskers). Two-tailed Dunnett鈥檚 test (c, f, g, i鈥?b>k), two-tailed Student鈥檚 t-test (d, h, r) or two-tailed, Sidak-corrected Student鈥檚 t-test (l鈥?b>n). *P鈥?lt;鈥?.05; **P鈥?lt;鈥?.01; ***P鈥?lt;鈥?.001; ****P鈥?lt;鈥?.0001.Extended Data Figure 5 IL-11 drives fibrogenic protein expression via non-canonical ERK signalling.a, Genome-wide RNA expression differences of cardiac fibroblasts in response to IL-11 (n鈥?鈥? biologically independent samples, 5鈥塶g ml鈭?, 24鈥塰). Red indicates differentially expressed genes according to DEseq2. Fibrosis gene RNA is not increased by IL-11 treatment. b鈥?b>e, RT鈥搎PCR experiments for RNA expression of ACTA2 (b), POSTN (c), MMP2 (d) and TIMP1 (e) in response to IL-11 treatment (5鈥塶g ml鈭?, 24鈥塰) compared to unstimulated cells. IL-11 does not significantly upregulate these genes at the RNA level in cardiac fibroblasts (n鈥?鈥? biologically independent samples). f, Sirius red assay reveals significant increase in collagen protein. g, ELISA reveals increase in MMP-2 protein in the supernatant of the samples (shown in Fig. 2b, n鈥?鈥? biologically independent samples) that lack a change in RNA transcripts. h, Concentration of IL11RA (ELISA) in the supernatant of cardiac fibroblasts (n鈥?鈥? biologically independent samples) after TGF尾1 stimulation (5鈥塶g ml鈭?, 24鈥塰). i, Cardiac fibroblasts were incubated with increasing concentrations of a fusion protein consisting of IL-11 and IL11RA connected with a linker peptide that recapitulates the features of the IL-11 trans-signalling complex. Concentrations as low as 200 pg ml鈭? significantly activated cardiac fibroblasts as measured using a high-content imaging platform and staining for ACTA2 expression (Operetta assay n鈥?鈥? measurements per n鈥?鈥? independent experiments). j, k, rhIL-11 (j) and hyperIL-11 (k) were added at indicated concentrations and subsequently measured using a commercially available IL-11 ELISA (n鈥?鈥? independent experiment). The ELISA did not detect rhIL-11 or hyperIL-11. We note that the reactivity to rhIL-11 was variable dependent on batch and provider and rhIL-11 was sometimes detected. However, in all experiments presented in the main figures, we confirmed that the rhIL-11 used was not detectable by the ELISA by additional measurements. This ELISA reliably detected native IL-11 secreted by human fibroblasts. l, Cardiac fibroblasts (n鈥?鈥? biologically independent samples) were incubated (8鈥塰) with hyperIL-11 (0.2鈥塶g ml鈭?) in the presence of absence of the inhibitor of protein translation, cyclohexamide (CHX, 5鈥壩糶 ml鈭?), or protein secretion, brefeldin A (BFA, 1鈥壩糶 ml鈭?). Both inhibitors block the increase in IL-11 protein in the supernatant in response to hyperIL-11 treatment. m, Western blot and ELISA of IL-11 in cardiac fibroblasts after hyperIL-11, the inhibitor of the Golgi secretory pathway BFA and/or the translation inhibitor CHX treatment shows de novo protein synthesis and canonical secretion of IL-11 after stimulation. n, ELISA (n鈥?鈥? biologically independent samples) and RT鈥搎PCR (n鈥?鈥? biologically independent samples) assays show an increase in endogenous IL-11 protein but not RNA over time after rhIL-11 (5鈥塶g ml鈭?) treatment. o, ERK signalling pathway activation by TGF尾1 and IL-11. Western blots show activation of the non-canonical MEK鈥揈RK鈥揜SK cascade in response to IL-11 stimulation of human cardiac fibroblasts. Here the response was greatest at 15 min in the two patients analysed, but more prolonged ERK activation was also seen in additional experiments. p, Downstream substrates of ERK, such as eIF4E, were also phosphorylated by rhIL-11. q, TGF尾1 also activates the ERK pathway. The time course and degree of activation was variable between patients. r, Collagen secretion (Sirius red) from cardiac fibroblasts (control, n鈥?鈥?; TGF尾1, n鈥?鈥?; TGF尾1 +U0126, n鈥?鈥?; TGF尾1 +PD98059, n鈥?鈥?; IL-11, n鈥?鈥?; IL-11 +U0126, n鈥?鈥?; IL-11 +PD98059, n鈥?鈥? biologically independent samples) induced by TGF尾1 or IL-11 (5鈥塶g ml鈭?, 24鈥塰) is reduced by two separate MEK inhibitors (10鈥壩糓). s, Western blot of total protein levels of key signalling molecules in fibroblasts after 24鈥塰 stimulation with AngII (100 nM), CTGF (50鈥塶g ml鈭?), EDN1 (250鈥塶g ml鈭?), bFGF (10鈥塶g ml鈭?), IL-13 (100鈥塶g ml鈭?), OSM (100鈥塶g ml鈭?), PDGF (200鈥塶g ml鈭?) and TGF尾1 (5鈥塶g ml鈭?). The corresponding activated protein levels are shown in Fig. 2e. t, siRNA treatment of TGF尾1-stimulated cardiac fibroblasts. RT鈥搎PCR shows SMAD-dependent upregulation of IL11 RNA (control, n鈥?鈥?; TGF尾1, n鈥?鈥?; siTGFB1R, n鈥?鈥?; siSMAD2, n鈥?鈥?; siSMAD3, n鈥?鈥? biologically independent samples). Data are mean鈥壜扁€塻.d. (b鈥?b>h, l, n, r, t); box-and-whisker plots (i) show median (middle line), 25th鈥?5th percentiles (box) and 10th鈥?0th percentiles (whiskers). Two-tailed Student鈥檚 t-test (b鈥?b>h), two-tailed Dunnett鈥檚 test (i, r) or Sidak-corrected, two-tailed Student鈥檚 t-test (l, t) or one-way ANOVA (n). *P鈥?lt;鈥?.05; **P鈥?lt;鈥?.01; ***P鈥?lt;鈥?.001; ****P鈥?lt;鈥?.0001.Extended Data Figure 6 IL-11 is required for the pro-fibrotic effects of multiple stimuli.a鈥?b>h, Cardiac fibroblasts were incubated for 24鈥塰 with TGF尾1 (a; 5鈥塶g ml鈭?), CTGF (b; 50鈥塶g ml鈭?), PDGF (c; 200鈥塶g ml鈭?), IL-13 (d; 100鈥塶g ml鈭?), AngII (e; 100 nM), OSM (f; 100鈥塶g ml鈭?), EDN1 (g; 250鈥塶g ml鈭?) or bFGF (h; 10鈥塶g ml鈭?) in the presence or absence of an IL-11-neutralizing antibody (IL-11ab) or IgG control (2鈥壩糶 ml鈭?). Cells were stained for ACTA2, collagen I and POSTN to monitor the amount of myofibroblasts and ECM production. High-content imaging and quantification of fluorescence (Operetta assay n鈥?鈥? measurements per n鈥?鈥? independent experiments for each condition and cellular phenotype) revealed that anti-IL-11 antibodies significantly reduce the pro-fibrotic effect of these stimuli on myofibroblast ratio and ECM production. Two-tailed Dunnett鈥檚 test. Box-and-whisker plots show median (middle line), 25th鈥?5th percentiles (box) and 10th鈥?0th percentiles (whiskers).Extended Data Figure 7 Il-11 acts post-transcriptionally and causes fibrosis in vivo.a, RNA-seq fold change in TGF尾1-regulated genes in Il11ra1+/+ cardiac fibroblasts (n鈥?鈥? biologically independent samples) compared to Il11ra1鈭?鈭?/i> cardiac fibroblasts (n鈥?鈥? biologically independent samples) after TGF尾1 stimulation (5鈥塶g ml鈭?, 24鈥塰) are highly correlated. Spearman鈥檚 correlation shows that RNA levels of fibrosis genes are upregulated equally in both genotypes. b, Wild-type and knockout fibroblasts (n鈥?鈥? biologically independent samples) were incubated with TGF尾1 (5鈥塶g ml鈭?, 24鈥塰) and RNA-seq was performed to detect differentially expressed genes using DEseq2. All genes regulated by TGF尾1 in wild-type cells are plotted with decreasing 鈭抣og2(Padjusted). The P value of the same genes in stimulated Il11ra1鈭?鈭?/i> cells are plotted to the right. A similar P-value distribution suggests that TGF尾1-driven RNA expression changes are still present in the absence of IL-11 signalling showing that loss of Il11ra1 does not influence the TGF尾1-driven transcriptional response. c, d, Primary atrial fibroblasts were prepared from Il11ra1+/+ (c) or Il11ra1鈭?鈭?/i> (d) mice were incubated for 24鈥塰 without stimulus or with TGF尾1 (5鈥塶g ml鈭?), IL-11 (5鈥塶g ml鈭?) or AngII (100鈥塶M). Cells were stained with antibodies against ACTA2, collagen I or POSTN. Images were taken at low magnification (10脳) on the Operetta imaging platform. As shown, fibroblasts from knockout mice do not respond to pro-fibrotic stimuli at the level of pro-fibrotic protein expression. This experiment was repeated four times with similar results. e, Circulating markers of inflammation after rmIl-11 injection (100鈥壩糶 kg鈭? per day, three weeks; n鈥?鈥?4 biologically independent samples). f, Circulating levels of Tgf尾1 (ELISA) after rmIl-11 injection (control, n鈥?鈥?; Il-11 injection, n鈥?鈥?2 biologically independent samples). g, Collagen content (HPA assay) in atrium (control, n鈥?鈥?; rmIl-11, n鈥?鈥?0 biologically independent samples) after rmIL-11 treatment. h, The area indicative for collagen deposition was assessed over several fields in n鈥?鈥? biologically independent samples and compared between samples from rmIl-11-treated and control mice. i, Representative histological images of the heart and kidney after rmIl-11 injection indicate increased collagen content according to Masson鈥檚 trichrome staining. This experiment was repeated three times with similar results. j, RNA expression (RT鈥搎PCR) of fibrosis genes in heart (n鈥?鈥?2 biologically independent samples) and kidney (n鈥?鈥?1 biologically independent samples) after rmIl-11 treatment compared to control. k, RNA expression (RT鈥搎PCR) of fibrosis genes in heart (control, n鈥?鈥?; Il-11-Tg, n鈥?鈥? biologically independent samples) and kidney (control, n鈥?鈥?; Il-11-Tg, n鈥?鈥? biologically independent samples) of tamoxifen-treated Il-11-Tg, Col1a2鈥揅reER and control mice. l, Cardiac fibroblasts were incubated with TGF尾1 or IL-11 (5鈥塶g ml鈭?) and EdU (10鈥壩糓 ml鈭?, 24鈥塰), which was used to detect replicating DNA by fluorescence by automated quantification of images (Operetta assay n鈥?鈥? measurements per n鈥?鈥? independent experiments). This analysis reveals a significant increase in fibroblast proliferation (EdU+ ells) induced by both TGF尾1 and IL-11. The percentage of EdU+ cells was normalized to the average detected in non-stimulated cells. m, Cells were incubated with TGF尾1 (5鈥塶g ml鈭?, 24鈥塰) and either an IgG control or anti-IL-11 antibody (2鈥壩糶 ml鈭?, 24鈥塰). High-content imaging (Operetta assay n鈥?鈥? measurements per n鈥?鈥? independent experiments) and quantification of proliferating cells show that anti-IL-11 antibodies significantly reduce the effects of TGF尾1 on fibroblast proliferation. The percentage of EdU+ ells was normalized to the average detected in cells stimulated with TGF尾1 and IgG control. n, Western blots show an increase in Il-11 protein expression in the heart and kidney after tamoxifen treatment in Il-11-Tg, Col1a2鈥揅reER mice. o, Il-11 transgenic mice were crossed with a Col1a2-promoter, tamoxifen-inducible Cre mouse strain (Il-11-Tg). Six-week-old mice were treated with tamoxifen (1鈥塵g鈥塸er day, 10 consecutive days) to induce Cre-mediated recombination. Likewise, wild-type littermates were injected with tamoxifen for 10 consecutive days as controls. The mice (control creatinine, n鈥?鈥?; Il-11-Tg creatinine, n鈥?鈥?; control urea, n鈥?鈥?; Il-11-Tg urea, n鈥?鈥?; control Tgf尾1, n鈥?鈥?; Il-11-Tg Tgf尾1, n鈥?鈥? biologically independent samples) were euthanized 14 days after cessation of tamoxifen administration. Serum urea and creatinine increased and indicated renal impairment. We also observed an increase in circulating Tgf尾1 levels. p, Collagen content (HPA assay) in atrium (control, n鈥?鈥?1; Il-11-Tg, n鈥?鈥? biologically independent samples) from tamoxifen-treated or control Il-11-Tg mice. Data are mean鈥壜扁€塻.d. (e, f, h, k, o); box-and-whisker plots (g, j, l, m, p) show median (middle line), 25th鈥?5th percentiles (box) and 10th鈥?0th percentiles (whiskers). Sidak-corrected, two-tailed Student鈥檚 t-test (e, j, k) or two-tailed Student鈥檚 t-test (f鈥?b>h, m, o, p) or Dunnett鈥檚 test (l). *P鈥?lt;鈥?.05; **P鈥?lt;鈥?.01; ***P鈥?lt;鈥?.001; ****P鈥?lt;鈥?.0001.Extended Data Figure 8 Il-11 inhibition does not alter blood pressure after AngII treatment.Il11ra1+/+ wild-type (n鈥?鈥? biologically independent samples) and Il11ra1鈭?鈭?/i> knockout (n鈥?鈥? biologically independent samples) mice were injected with AngII (2鈥塵g鈥塳g鈭? per day, 28 days). a, b, Systolic (a) and diastolic (b) blood pressure was measured by in vivo telemetry for one week before and four weeks after the AngII infusion. Systolic (c) and diastolic (d) blood pressure for individual mice. AngII resulted in an increase in blood pressure as expected. The genotype did not have a significant effect on blood pressure. e, Aortic root or arch velocity of the blood. There was no significant difference in the degree of aortic constriction in the TAC model between genotypes (n鈥?鈥? biologically independent samples). Two-sided Mann鈥揥hitney U-test (e). n.s., not significant. Data are mean鈥壜扁€塻.d. (a, b, e).Extended Data Figure 9 Reduction in collagen deposition in Il11ra1鈭?鈭?/i> animals is independent of p38 MAPK signalling.Il11ra1+/+ mice (control, n鈥?鈥?3; AngII, n鈥?鈥?0 biologically independent samples) and Il11ra1鈭?鈭?/i> mice (control, n鈥?鈥?; AngII, n鈥?鈥? biologically independent samples) were injected with AngII (100鈥壩糶 kg鈭? per day, three weeks). a, HPA assay of ventricular tissue shows a decrease in collagen deposition in Il11ra1鈭?鈭?/i> mice after AngII infusion. b, Indexed heart weight of wild-type (control, n鈥?鈥?7; AngII, n鈥?鈥?7 biologically independent samples) and Il11ra1鈭?鈭?/i> (control, n鈥?鈥?; AngII, n鈥?鈥? biologically independent samples) mice after AngII injection. c, Indexed heart weight of Il11ra1+/+ (control, n鈥?鈥?; TAC, n鈥?鈥? biologically independent samples) and Il11ra1鈭?鈭?/i> (control, n鈥?鈥?; TAC, n鈥?鈥? biologically independent samples) mice after TAC. d, Kidney weight of Il11ra1+/+ (control, n鈥?鈥?; folate, n鈥?鈥? biologically independent samples) and Il11ra1鈭?鈭?/i> (control, n鈥?鈥?; folate, n鈥?鈥? biologically independent samples) mice three weeks after folate injection (180鈥塵g鈥塳g鈭?). a鈥?b>d, Sidak-corrected, two-tailed Student鈥檚 t-test. Data are mean鈥壜扁€塻.d. e鈥?b>g, Western blot of p38 MAPK signalling in tissues of Il11ra1+/+ and Il11ra1鈭?鈭?/i> mice after AngII infusion (e), TAC (f) or folate treatment (g). h, Schematic showing the proposed role of IL-11 fibroblasts. An autocrine loop of IL-11 signalling is required to feed-forward changes in pro-fibrotic mRNA abundances to the protein level by activating translational processes that are ERK-dependent. Blocking this loop limits fibrosis caused by multiple upstream stimuli and fibrosis in preclinical models of heart and kidney disease.Extended Data Table 1 84 patients undergoing coronary artery bypass grafting donated right atrial biopsiesFull size tableSupplementary information Supplementary FigureThis file contains source data for all western blot experiments. (PDF 575 kb) Life Sciences Reporting Summary (PDF 91 kb) Supplementary Table 1Detailed information about the quality of each RNA sample, RNA-seq library and sample information about each individual that has contributed primary cells for the therapeutic target discovery high-content imaging screening and transcriptome profiling. (XLSX 71 kb) Supplementary Table 2Therapeutic Target Screen results: 1) Differentially expressed genes between TGFB stimulated fibroblasts and non-stimulated fibroblasts, 2) Spearman correlation (SPcor) between delta of fibroblasts expression (stimulated/non-stimulated) and delta of SMA, 3) Jensen鈥揝hannon divergence (JSD) between of each gene across all GTEx tissues and FANTOM primary cell types (see more details in methods), 4) Average expression levels (transcripts per million, TPM) in TGFB1 stimulated and non-stimulated (baseline only) fibroblasts. Log2 fold change, shrunken Log2-fold changes computed by DESeq2 package. BH adj.P, Benjamini-Hochberg (BH) adjusted p-value. (XLSX 1759 kb) Supplementary Table 3Gene Ontology database gene set enrichment analysis (GSEA) results for the stimulated versus baseline fibroblasts (GSEA computed by ranking all the genes by DESeq output statistic). Only terms enriched with FDR 0.05 are presented. NES denotes normalized enrichment score. (XLSX 191 kb)PowerPoint slides Takashi Nishina, Yutaka Deguchi, Daisuke Ohshima, Wakami Takeda, Masato Ohtsuka, Shigeyuki Shichino, Satoshi Ueha, Soh Yamazaki, Mika Kawauchi, Eri Nakamura, Chiharu Nishiyama, Yuko Kojima, Satomi Adachi-Akahane, Mizuho Hasegawa, Mizuho Nakayama, Masanobu Oshima, Hideo Yagita, Kazutoshi Shibuya, Tetuo Mikami, Naohiro Inohara, Kouji Matsushima, Norihiro Tada Hiroyasu Nakano Nature Communications (2021) Jinrui Dong, Sivakumar Viswanathan, Eleonora Adami, Brijesh K. Singh, Sonia P. Chothani, Benjamin Ng, Wei Wen Lim, Jin Zhou, Madhulika Tripathi, Nicole S. J. Ko, Shamini G. Shekeran, Jessie Tan, Sze Yun Lim, Mao Wang, Pei Min Lio, Paul M. Yen, Sebastian Schafer, Stuart A. Cook Anissa A. Widjaja Nature Communications (2021) Ulrich Blache, Stefania L. Wunderli, Amro A. Hussien, Tino Stauber, Gabriel Fl眉ckiger, Maja Bollhalder, Barbara Nieder枚st, Sandro F. Fucentese Jess G. Snedeker Scientific Reports (2021) CommentsBy submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate. Editorial SummaryProtein behind cardiovascular fibrosisFibrosis鈥攖he overproduction of fibrous connective tissue鈥攊s a feature of many diseases and can contribute to pathology by causing scarring, thickening of tissue and interference with normal organ function. In the heart, fibrosis can cause mechanical and electrical dysfunction. Stuart Cook and colleagues identify a protein that has a crucial role in cardiac fibrosis: the cytokine IL-11. They find that, in primary human cardiac fibroblasts, transcription of IL-11 is a dominant response to transforming growth factor beta (TGF尾) exposure and that it is required for the pro-fibrotic effect of TGF尾. Loss of IL-11 reduced fibrosis in three preclinical models of cardiovascular fibrosis, leading the authors to propose IL-11 as a therapeutic target. Sign up for the Nature Briefing newsletter 鈥?what matters in science, free to your inbox daily.