LETTER
`
`doi:10.1038/nature24676
`
`IL-11 is a crucial determinant of cardiovascular
`fibrosis
`
`Sebastian Schafer1,2*, Sivakumar Viswanathan2*, Anissa A. Widjaja2*, Wei-Wen Lim1, Aida Moreno-Moral2,
`Daniel M. DeLaughter3, Benjamin Ng1, Giannino Patone4, Kingsley Chow1, Ester Khin2, Jessie Tan1, Sonia P. Chothani2,
`Lei Ye1, Owen J. L. Rackham2, Nicole S. J. Ko2, Norliza E. Sahib2, Chee Jian Pua1, Nicole T. G. Zhen1, Chen Xie1, Mao Wang2,
`Henrike Maatz4, Shiqi Lim1, Kathrin Saar4, Susanne Blachut4, Enrico Petretto2, Sabine Schmidt4, Tracy Putoczki5,6,
`Nuno Guimarães-Camboa7, Hiroko Wakimoto3, Sebastiaan van Heesch4, Kristmundur Sigmundsson2, See L. Lim1,
`Jia L. Soon1,2, Victor T. T. Chao1,2, Yeow L. Chua1, Teing E. Tan1, Sylvia M. Evans7,8,9, Yee J. Loh1,10, Muhammad H. Jamal1,
`Kim K. Ong1,10, Kim C. Chua1, Boon-Hean Ong1, Mathew J. Chakaramakkil1, Jonathan G. Seidman3, Christine E. Seidman3,11,12,
`Norbert Hubner4,13,14,15, Kenny Y. K. Sin1,2 & Stuart A. Cook1,2,16,17
`
`Fibrosis 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 β1 (TGFβ1) 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 TGFβ1 in
`fibroblasts could be attractive therapeutic targets and lack upstream
`toxicity. Here we show, using integrated imaging–genomics analyses
`of primary human fibroblasts, that upregulation of interleukin-11
`(IL-11) is the dominant transcriptional response to TGFβ1 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.
`Trans-differentiation of fibroblasts into activated myofibroblasts,
`which express α -smooth muscle actin (ACTA2) and secrete extracellular
`matrix (ECM) proteins, is a defining feature of fibrosis8. We automated
`the quantification of myofibroblasts and ECM production in primary
`human cardiac fibroblast cultures (n = 84; Extended Data Table 1
`and Supplementary Table 1), and performed RNA sequencing (RNA-
`seq) on paired unstimulated and TGFβ 1-stimulated samples (Extended
`Data Fig. 1). Genes were ranked on the basis of the magnitude and
`significance of their differential expression and their correlation with
`myofibroblasts. Typical fibrosis genes such as COMP and NOX49 were
`among the most-upregulated genes (Fig. 1a and Supplementary Table 2).
`Gene set enrichment analysis showed upregulation of genes important
`for protein secretion (Supplementary Table 3).
`Notably, IL11 expression showed the most positive correlation with
`myofibroblast numbers (r = 0.47, Padjusted = 6.44 × 10−6; Spearman’s
`correlation) (Fig. 1a) and upregulation of IL11 expression (8.5-fold,
`
`Padjusted = 6 × 10−218; DESeq210) defined the dominant transcriptional
`response of cardiac fibroblasts to TGFβ 1. IL11 expression is highly
`specific to fibroblasts, especially when stimulated, but is undetectable
`in most healthy human tissues and cells11,12 (Extended Data Fig. 2). To
`further explore the biological context of IL-11, we examined the expres-
`sion of its receptor (IL11RA) compared to the receptor of IL-6 (IL6R),
`a close family member, across 512 cell lines12. IL6R was present at high
`levels in immune cells, whereas IL11RA was most highly expressed in
`fibroblasts (Fig. 1b).
`To investigate Il11 expression in vivo, we performed single-cell
`RNA-seq of hearts from a PlnR9C/+ mouse, which has a cardiac fibrosis
`phenotype13, and a wild-type control mouse (Fig. 1c and Extended
`Data Fig. 3). Both Il11 (P = 5.6 × 10−8) and Il11ra1 (P = 2.2 × 10−16)
`were enriched in fibroblasts and Il11 was highly expressed in fibroblast
`subpopulations that had transcriptional features of TGFβ 1 activation
`or ECM production (Fig. 1d). Il11-expressing cells were most common
`in fibrotic PlnR9C/+ hearts and we confirmed Il-11 protein upregulation
`in this model (Extended Data Fig. 3).
`IL11 has been linked to haematopoiesis14 and tumorigenesis15,
`among other roles. In contrast to a previous cardiac study16, we found
`that recombinant human IL-11 (rhIL-11) is strongly pro-fibrotic in
`cardiac fibroblasts, increasing myofibroblasts and ECM production,
`motility, contraction and invasion (Fig. 1e and Extended Data Fig. 4).
`To better understand the apparent contradiction between our data
`and the previous work in which rhIL-11 was used in mouse models16,
`we tested whether rhIL-11 could activate mouse cardiac fibroblasts.
`rhIL-11 was mostly ineffective in mouse cardiac fibroblasts, whereas
`recombinant mouse Il-11 (rmIl-11) activated mouse cardiac and renal
`fibroblasts (Extended Data Fig. 4). rhIL-11 strongly activated human
`renal fibroblasts.
`Our findings implicate a pro-fibrotic role for IL-11 downstream
`of TGFβ 1. In the presence of neutralizing anti-IL-11 antibodies, the
`pro-fibrotic effects of TGFβ 1 were greatly diminished across a wide
`range of fibrosis assays (Fig. 1f–i and Extended Data Fig. 4). We made
`ligand traps by fusing IL11RA and gp130 (IL11RA:gp130) and these
`traps inhibited the pro-fibrotic effects of TGFβ 1 in a dose- dependent
`manner. The specificity of IL-11 inhibition was further confirmed
`
`1National Heart Centre Singapore, Singapore. 2Duke–National University of Singapore Medical School, Singapore. 3Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115,
`USA. 4Cardiovascular and Metabolic Sciences, Max Delbrück Center for Molecular Medicine in the Helmholtz Association (MDC), Robert-Rossle Strasse 10, 13125 Berlin, Germany. 5Inflammation
`Division, Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia. 6Department of Medical Biology, The University of Melbourne, Parkville, Victoria 3050, Australia.
`7Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, California, USA. 8Department of Medicine, University of California at San Diego, La Jolla,
`California 92093, USA. 9Department of Pharmacology, University of California at San Diego, La Jolla, California 92093, USA. 10Kandang Kerbau Women’s and Children’s Hospital, Singapore.
`11Division of Cardiovascular Medicine, Brigham and Women’s Hospital, Boston, Massachusettes 02115, USA. 12Howard Hughes Medical Institute, Chevy Chase, Maryland 20815, USA. 13DZHK
`(German Centre for Cardiovascular Research), partner site, Berlin, Germany. 14Charité-Universitätsmedizin, Berlin, Germany. 15Berlin Institute of Health (BIH), Berlin, Germany. 16National Heart
`and Lung Institute, Imperial College London, London, UK. 17MRC-London Institute of Medical Sciences, Hammersmith Hospital Campus, Du Cane Road, London, W12 0NN, UK.
`*These authors contributed equally to this work.
`
`1 1 0 | N A T U R E | V O L 5 5 2 | 7 D E C E M B E R 2 0 1 7
`
`© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.
`
`Lassen - Exhibit 1066, p. 1
`
`

`

`Fibroblasts
`Osteoblasts
`Adipocytes
`Melanocytes
`Mesenchymal stem cells
`
`CD14+ monocytes
`CD14+CD16+ monocytes
`
`200
`IL6R expression (TPM)
`
`400
`
`TGF 1
`
`Proliferating
`
`b
`
`50
`
`25
`
`0
`0
`
`IL11RA expression (TPM)
`
`RASL11B
`
`XYLT1
`
`IL11
`
`COMP
`
`1
`2
`3
`ACTA2 correlation
`
`–0.5 0
`
`0.5
`
`4
`
`5
`
`6
`
`7
`
`8
`9
`10
`11
`Genomic position
`
`12
`
`14 15 16
`
`17
`
`19 20
`X
`–log2 (Padjusted)
`200
`400
`600
`
`ECM
`production
`
`20
`
`10
`
`0
`
`–10
`
`–20
`
`d
`
`tSNE2
`
`Endothelial
`cells
`
`Vessel
`endothelial
`
`Epicardial
`
`Smooth
`muscle
`
`Macrophages
`
`Proliferating
`(cid:31)broblasts
`
`Cardiomyocytes
`Red
`blood cells
`
`Fibroblasts
`
`10
`
`02468
`
`40
`
`20
`
`0
`
`–20
`
`a
`
`Fold change in RNA expression
`
`c
`
`tSNE2
`
`25
`
`Heart
`50
`
`–30
`
`IL-11-expressing cells
`–20
`–10
`
`Quiescent
`
`120
`
`110
`
`100
`
`90
`
`80
`
`g
`
`Gel contraction (mm2)
`
`i
`
`1.5
`
`1.0
`
`0.5
`
`scratch closure
`Fold change in
`
`TGF 1
`+
`IgG
`
`TGF 1
`+
`anti-IL-11
`
`f
`
`ACTA2
`
`Collagen I
`
`POSTN
`
`–40
`
`e
`
`Lymphocytes
`IL-11-expressing cells
`–25
`
`0
`tSNE1
`
`Control
`
`TGF 1
`
`IL-11
`
`ACTA2
`
`Collagen I
`
`POSTN
`
`Cardiac (cid:31)broblasts
`20
`
`10
`
`**
`
`0
`tSNE1
`h
`
`1.0
`
`0.5
`
`cell migration
`Fold change in
`
`*
`
`TGF 1
`+
`IgG
`
`TGF 1
`+
`anti-IL-11
`
`TGF 1
`+
`IgG
`
`TGF 1
`+
`anti-IL-11
`
`**
`
`200 m
`
`Figure 1 | Fibrosis target discovery platform identifies IL-11. a, RNA-
`seq of primary cardiac fibroblasts (n = 84 biologically independent
`samples) with or without TGFβ 1 treatment (5 ng ml−1, 24 h) and
`Spearman’s correlation of expression changes with fibroblast activation
`(Supplementary Table 2). DEseq210 fold change in expression and false-
`discovery rate (FDR)-adjusted P values are shown. b, RNA expression in
`transcripts per million (TPM) of IL11RA and IL6R across 512 cell lines
`from the FANTOM repository12. c, Single-cell resolution of cardiac
`Il11 expression (more than 0 reads per cell). t-distributed stochastic
`neighbour embedding (tSNE) analysis28 clusters cell types of the heart.
`Il11 expression is highly enriched in fibroblasts. χ2 test (P = 5.7 × 10−8).
`d, tSNE analysis of fibroblasts alone shows highest Il-11 expression in
`ECM-secreting and TGFβ 1-activated fibroblasts. χ2 test (P = 0.033).
`
`200 m
`
`TGF 1
`TGF 1
`+
`+
`IgG
`anti-IL-11
`c, d, Cardiac cells were sequenced from n = 1 mouse, the experiment was
`repeated once with similar results. e, f, Representative images (chosen
`from 42 per condition) of cardiac fibroblasts immunostained for ACTA2,
`collagen I or periostin (POSTN) after a 24-h incubation without stimulus
`(control), TGFβ 1 or IL-11 (5 ng ml−1) (e) or with TGFβ 1 (5 ng ml−1) and
`an anti-IL-11 neutralizing antibody or an IgG control (2 μ g ml−1) (f).
`g, Cardiac fibroblasts were seeded in collagen gel and the area of
`contraction determined (n = 3 biologically independent samples) after
`72 h. h, Trans-well migration assay (colourimetrically quantified, n = 3
`biologically independent samples, 24 h). i, Scratch assay of wound closure
`in a monolayer of cardiac fibroblasts (n = 5 biologically independent
`samples) after 24 h. g–i, Two-tailed Student’s t-test; data are mean ± s.d.;
`* P < 0.05; * * P < 0.01.
`
`through inhibition of IL11RA using receptor-blocking antibodies or
`by short interfering RNA (siRNA), all of which attenuated the effects
`of TGFβ 1. By contrast, anti-IL-6 antibodies had no effect on TGFβ 1-
`induced cardiac fibroblast activation (Extended Data Fig. 4).
`We next investigated the consequences of IL-11 signalling in cardiac
`fibroblasts using RNA-seq. Surprisingly, the effect of IL-11 on the
` transcriptome was negligible, whereas TGFβ 1-driven transcriptional
`regulation in cardiac fibroblasts from the same patients remained
` profound (Fig. 2a, b). We repeated the experiment using cardiac fibro-
`blasts from multiple patients and consistently observed very little
`effect of IL-11 on mRNA levels but, in the cell culture supernatants of
`identical samples, we reproducibly documented pro-fibrotic protein
` secretion. Therefore, the effects of IL-11 on cardiac fibroblasts are
`mainly at the protein level and are unrelated to transcriptional changes
`(Extended Data Fig. 5). This differs from effects of IL-11 on cancer cells,
`in which JAK–STAT signalling is involved17.
`IL-11 can bind to free IL11RA, which is shed from cardiac fibro-
`blast membranes (Extended Data Fig. 5) and signal in trans in cells
`
`that express GP130 (also known as IL6STP1)18. We generated an
`IL11RA:IL-11 fusion protein (hyperIL-11), which mimics the trans-
`signalling complex19. Concentrations of hyperIL-11 as low as
`0.2 ng ml−1 activated cardiac fibroblasts (Extended Data Fig. 5),
` supporting a role for IL-11 trans-signalling in fibrosis.
`We found that both IL11 and IL11RA are expressed in fibro-
`blasts, which implies that an autocrine signalling loop exists. Using
`hyperIL-11, which is not detected in an IL-11 enzyme-linked immuno-
`sorbent assay (ELISA), we confirmed the existence of autocrine IL-11
`signalling in cardiac fibroblasts. This autocrine, feed-forward loop of
`hyperIL-11-induced IL-11 secretion is dependent on de novo IL-11
`protein synthesis and secretion (Fig. 2c and Extended Data Fig. 5), but
`is independent of IL11 RNA levels. In separate experiments, rhIL-11
`(not detected by IL-11 ELISA) cis-signalling also strongly induced
`endogenous IL-11 secretion, and this also occurred in the absence of
`changes in IL11 mRNA levels (Extended Data Fig. 5).
`TGFβ 1 activation of non-canonical ERK signalling in fibroblasts
`is important for fibrosis4,5 and we observed that IL-11 also activated
`
`7 D E C E M B E R 2 0 1 7 | V O L 5 5 2 | N A T U R E | 1 1 1
`
`Letter reSeArCH
`
`© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.
`
`Lassen - Exhibit 1066, p. 2
`
`

`

`IL11 stimulation
`COL1A1 RNA fc: 1.03
`COL1A2 RNA fc: 1.02
`COL3A1 RNA fc: 1.12
`
`2
`
`1 0
`
`–1
`
`–2
`
` log2(fold change in RNA)
`
`TGF 1 stimulation
`
`b
`
`2
`
`1 0
`
`–1
`
`–2
`
` log2(fold change in RNA)
`
`a
`
`POSTN RNA fc: 1.01
`ACTA2 RNA fc: 1.00
`MMP2 RNA fc: 1.01
`103
`105
`1
`10
`Normalized mean counts
`
`Unstim ulated
`
`
`
`
`
`AngII
`
`CTGFED N1bFGFIL-11IL-13
`
`O S M
`
`
`
`PD GFTGF 1
`
`p-ERK1/2
`p-P38
`p-STAT3
`p-SMAD2
`
`p-AKT
`
`p-mTOR
`
`GAPDH
`
`Stimulant
`
`Stimulant
`+
`anti-IL-11
`
`Stimulant
`+
`IgG
`
`g T
`
`GF 1
`
`1
`
`105
`103
`10
`Normalized mean counts
`
`– + –+
`– – + +
`
`BFA
`Hyper-IL-11
`
`Intracellular IL-11
`GAPDH
`
`25 kDa
`
`35 kDa
`
`d
`ACTA2+
`
`Collagen I
`
`MEK inhibitors
`
`e
`
`40 kDa
`40 kDa
`100 kDa
`
`70 kDa
`70 kDa
`
`260 kDa
`
`–
`+
`+
`–
`
`–
`+
`–
`+
`
`35 kDa
`
`1.5×
`
`Protein (P = 4 × 10-4)
`RNA (P = 0.90)
`
`0
`
`28
`1
`2
`24
`48
`After hyper-IL-11 treatment (h)
`
`72
`
`POSTN
`TGF 1
`+
`+
`–
`+
`–
`–
`+
`–
`IL-11
`–
`+
`–
`–
`U0126
`+
`–
`–
`–
`PD98059
`Fluorescent signal:
`1×
`
`IL-11 protein
`IL11 RNA
`
`****
`
`****
`
`****
`
`30
`
`20
`
`****
`
`****
`
`c
`
`80
`
`60
`
`40
`
`20
`
`Fold change in IL-11
`
`f
`
`PDGF
`
`AngII
`
`EDN1
`
`CTGF
`
`IL-13
`
`OSM
`
`IL11 RNA expressiont
`
`Fold change in
`
`10
`
`TGF 1
`****
`
`****
`
`****
`
`POSTN
`
`Collagen I
`
`ACTA2+
`
`POSTN
`
`Collagen I
`
`ACTA2+
`
`POSTN
`
`Collagen I
`
`ACTA2+
`
`NS
`
`bFGF
`
`NS
`
`NS
`
`Unstimulated
`
`Fluorescent signal:
`1×
`1.5×
`Il11ra1+/+
`Il11ra1–/–
`fibroblasts in response to various pro-fibrotic stimuli (see also Extended
`Data Fig. 5). f, ELISA (supernatant, n = 3 biologically independent
`samples) and RT–qPCR (n = 2 biologically independent samples) of
`IL-11 expression in cardiac fibroblasts after 24 h stimulation with AngII
`(100 nM), CTGF (50 ng ml−1), EDN1 (250 ng ml−1), bFGF (10 ng ml−1),
`IL-13 (100 ng ml−1), OSM (100 ng ml−1), PDGF (200 ng ml−1) and TGFβ 1
`(5 ng ml−1). Two-tailed Dunnett’s test; Data are mean ± s.d. g, Cardiac
`fibroblasts were incubated with pro-fibrotic cytokines (24 h) and cardiac
`fibroblast activation was reduced by anti-IL-11 antibodies (2 μ g ml−1;
`Extended Data Fig. 6). h, Pro-fibrotic proteins (Operetta assay n = 7
`measurements per n = 2 independent experiments) are not upregulated
`in cardiac fibroblasts from Il11ra1−/− mice in response to TGFβ 1, IL-11
`(5 ng ml−1) or AngII (100 nM, 24 h). Experiments were repeated twice
`with similar results. Two-tailed Dunnett’s test; box-and-whisker plots
`show median (middle line), 25th–75th percentiles (box) and 10th–90th
`percentiles (whiskers); * * * * P < 0.0001; NS, not significant.
`
`****
`
`****
`
`****
`
`2468
`
`change in expression
`
`IL-11 protein fold
`
`AngII
`
`CTGF
`
`EDN1
`
`IL-13
`
`OSM PDGF
`
`bFGF
`****
`
`3
`
`2
`
`1
`
`Fold change in collagen I
`
`protein expression
`
`NS
`
`NS
`
`NS
`
`****
`
`****
`
`****
`
`Unstimulated
`
`2
`
`1
`
`h
`
`ACTA2+ cells
`
`1234
`
`Fold change in POSTN
`
`protein expression
`
`****
`
`****
`
`NS
`
`NS
`
`NS
`
`Unstimulated
`
`TGF 1
`IL-11
`AngII
`Il11ra1+/+
`Il11ra1–/–
`Il11ra1+/+
`Il11ra1–/–
`Figure 2 | Non-canonical IL-11 signalling drives fibrogenic protein
`synthesis. a, b, RNA-seq of primary cardiac fibroblasts in response
`to TGFβ 1 (a) or IL-11 (b) (n = 6 biologically independent samples,
`5 ng ml−1, 24 h). Red indicates significantly differentially expressed
`genes (FDR ≤ 0.05, DEseq210). RNA expression of genes associated with
`fibrosis is not increased by IL-11 treatment. fc, fold change. c, ELISA and
`quantitative PCR with reverse transcription (RT–qPCR) assays of IL-11
`expression (n = 3 biologically independent samples) after hyperIL-11
`treatment (0.2 ng ml−1). Benjamini–Hochberg corrected one-way
`ANOVA; data are mean ± s.d. Inset, western blot of cardiac fibroblast
`lysates after hyperIL-11 stimulation and brefeldin A (BFA, 1 μ g ml−1)
`treatment indicates canonical secretion of IL-11. d, Cardiac fibroblasts
`were incubated with TGFβ 1, IL-11 (5 ng ml−1) and MEK inhibitors
`U0126 or PD98059 (10 μ M, 24 h). ACTA2+ cells and ECM production
`was assessed and normalized to non-stimulated cells. e, Western blots of
`phosphorylated protein (p-) expression of signalling pathways in cardiac
`
`ERK in cardiac fibroblasts and that both TGFβ 1 and IL-11 required
`ERK to induce pro-fibrotic phenotypes (Fig. 2d and Extended Data
`Fig. 5). Because the effects of IL-11 on pro-fibrotic gene expression are
`post-transcriptional, we suggest that this phenomenon may be driven,
`in part, by activation of ERK and its downstream substrates (Extended
`Data Fig. 5).
`We therefore investigated the activation of STAT, SMAD, ERK and
`kinases that are important for protein synthesis in cardiac fibroblasts
`in response to stimulation by a range of pro-fibrotic factors. These
`factors included established stimuli for cardiac fibrosis (endothelin-1
`(END1), angiotensin II (AngII) and PDGF) and other key pro-fibrotic
`cytokines (OSM, bFGF, CTGF and IL13)1,5. IL-11 activation of STAT in
`cardiac fibroblasts was negligible, consistent with its lack of transcrip-
`tional effects in this cell type. As expected, TGFβ 1 activated SMAD,
`but the only pathway that was consistently activated by all stimuli was
`the ERK pathway (Fig. 2e). All pro-fibrotic stimuli that were tested
`induced changes in IL-11 protein levels but not mRNA; only TGFβ 1
`increased SMAD-dependent IL11 transcription (Fig. 2f and Extended
`Data Fig. 5). Remarkably, as seen with TGFβ 1, fibroblast activation in
`
`response to all pro-fibrotic stimuli that were tested was dependent on
`IL-11 signalling (Fig. 2g and Extended Data Fig. 6).
`We studied the fibrotic response of cardiac fibroblasts from
`Il11ra1−/− mice20. TGFβ 1-induced transcriptional regulation in
`Il11ra1−/− cardiac fibroblasts was maintained and similar to that of
`Il11ra1+/+ (wild-type) cardiac fibroblasts (R2 = 0.94, P < 2.2 × 10−16,
`Spearman’s correlation; Extended Data Fig. 7). However, protein-based
`assays showed that cardiac fibroblasts from Il11ra1−/− mice did not
`increase synthesis of ECM proteins or become myofibroblasts upon
`stimulation (Fig. 2h and Extended Data Fig. 7), again demonstrating
`the effect of IL-11 at the protein level.
`We investigated the role of Il-11 in a mouse model of myocardial
`infarction, but rather than injecting rhIL-11, as had been done in the
`previous study16, we administered rmIl-11. We measured epicardial
`activation, a defining feature of active fibrosis in myocardial infarction21
`and found that rmIl-11 robustly stimulated fibroblasts in the epicardium
`(Fig. 3a–c) and caused ventricular impairment (Fig. 3d, e). We then
`tested the effects of rmIl-11 injection in healthy mice using regimens
`similar to those used for rhIL-11 in patients with cancer22. rmIL-11
`
`1 1 2 | N A T U R E | V O L 5 5 2 | 7 D E C E M B E R 2 0 1 7
`
`reSeArCH
`
`Letter
`
`© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.
`
`Lassen - Exhibit 1066, p. 3
`
`

`

`***
`
`*
`
`**
`
`Sham
`
`MI
`+
`PBS
`
`MI
`+
`rmIl-11
`
`****
`***
`
`**
`
`80
`
`60
`
`40
`
`20
`
`60
`
`40
`
`20
`
`d
`
`Ejection fraction (%)
`
`e
`
`ESV (µl)
`
`Distal
`
`*
`
`**
`
`246
`
`b
`
`epicardial thickness
`
`Fold change in
`
`Sham
`
`MI
`+
`PBS
`
`MI
`+
`rmIl-11
`
`Proximal
`
`c
`
`*
`
`**
`
`epicardial thickness
`
`Fold change in
`
`10
`
`5
`
`a
`
`Epicardium
`
`Sham
`
`MI + PBS
`
`MI + rmIl-11
`
`Masson’s trichrome
`
`ACTA2
`
`50 m
`
`Col1a1–GFP epicardium
`
`Col1a1–GFP renal cortex
`
`f
`
`Control
`
`Sham
`
`MI
`+
`PBS
`
`MI
`+
`rmIl-11
`
`Sham
`
`MI
`+
`PBS
`
`MI
`+
`rmIl-11
`
`g
`Control
`
`Fold change in renal function markers
`Urea
`Creatinine
`
`rmIl-11
`injection
`
`****
`**
`
`2.0
`
`0.5
`
`1.0
`
`1.5
`
`**
`
`***
`
`Ventricle
`
`****
`
`2
`
`1
`
`123
`
`i
`
`HPA collagen ( g ml–1)
`
`k
`
`HPA collagen ( g ml–1)
`
`*
`
`Control rmIl-11
`Injection
`
`80
`
`70
`
`60
`
`50
`
`h
`
`Ejection fraction (%)
`
`DAPI GFP
`
`GFP
`
`DAPI GFP
`
`GFP
`
`50 µm
`
`50 µm
`
`Epicardium
`
`Renal cortex
`
`Control
`rmIl-11
`injection
`Kidney
`
`****
`
`Control
`Il-11-Tg
`Kidney
`
`1234
`
`6
`
`4
`
`2
`
`rmIl-11
`injection
`
`j
`
`Control
`
`Il-11-Tg
`
`Ventricle
`
`*
`
`80
`
`70
`
`60
`
`50
`
`l
`
`Ejection fraction (%)
`
`Masson’s trichrome
`
`ACTA2
`
`Masson’s trichrome
`
`ACTA2
`
`50 µm
`
`50 µm
`
`Figure 3 | Il-11 causes cardiovascular fibrosis and organ failure.
`a, Representative histological images of the epicardium with Masson’s
`trichrome staining and ACTA2 immunostaining in mice with
`myocardial infarction (MI) treated with rmIL-11 or PBS. b, c, Epicardial
`thickness of regions distal (b) or proximal (c) to the region of myocardial
`infarction. a–c, Sham, n = 5; myocardial infarction, n = 6; myocardial
`infarction + rmIl-11, n = 8 biologically independent samples.
`d, e, Echocardiography show a decrease in ejection fraction (d) and
`increase in end-systolic volume (ESV) (e) after myocardial infarction
`and rmIl-11 treatment (sham, n = 3; myocardial infarction, myocardial
`infarction + rmIl-11, n = 5 biologically independent samples). b–e, two-
`tailed, Holm–Sidak-corrected Student’s t-test; Data are mean ± s.d.
`f, Representative histological images (chosen from control, n = 3;
`rmIl-11 injection, n = 4 biologically independent samples) of tissues
`from a Col1a1–GFP-reporter mouse after rmIL-11 injection (100 μ g kg−1
`per day, three weeks). g, Serum urea and creatinine levels after rmIL-11
`injection in Col1a1–GFP reporter mice23 resulted in specific activa-
`tion of fibroblasts in the epicardium and renal interstitium (Fig. 3f).
`rmIl-11-treated mice had high circulating levels of TGFβ 1, but not
`of other pro-inflammatory factors and features of cardiac and renal
`impairment along with cardiovascular fibrosis (Fig. 3g–i and Extended
`Data Fig. 7).
`To investigate the effects of autocrine Il-11 signalling in fibroblasts
`in vivo, we generated rmIl-11-transgenic mice, which we crossed
`with inducible Col1a2–Cre mice (Il-11-Tg; see Methods). Il-11 was
`
`Control
`Il-11-Tg
`injection (control urea, n = 8; control creatinine, n = 7; rmIl-11, n = 12
`biologically independent samples). h, Reduced ejection fraction
`(echocardiography) in rmIl-11-treated mice. i, Hydroxyproline assay
`(HPA) quantifies cardiac and renal collagen content after rmIL-11
`treatment. h, i, Control, n = 8; rmIl-11, n = 11 biologically independent
`samples. j, Representative histological images of Masson’s trichrome
`staining and ACTA2 immunostaining in the epicardium Il-11-Tg mice.
`k, HPA indicates cardiac and renal collagen content in Il-11-Tg mice.
`j, k, Control, n = 12; Il-11-Tg, n = 4 biologically independent samples.
`i, k, Two-tailed Student’s t-test; box-and-whisker plots show median
`(middle line), 25th–75th percentiles (box) and 10th–90th percentiles
`(whiskers). l, Reduction in ejection fraction (echocardiography) in
`Il-11-Tg mice (control, n = 6; Il-11-Tg, n = 4 biologically independent
`samples). g, h, l, Two-tailed Student’s t-test; data are mean ± s.d.;
`* P < 0.05; * * P < 0.01; * * * P < 0.001; * * * * P < 0.0001.
`
`expressed after induction with tamoxifen and within two weeks, there
`was widespread activation of cardiac and renal fibroblasts and accu-
`mulation of collagen (Fig. 3j, k). This was accompanied by a reduction
`in cardiac function (Fig. 3l), increased serum TGFβ 1 and an increase
`in serum urea and creatinine, which are biomarkers of renal failure
`(Extended Data Fig. 7).
`Il-11 expression was found to be increased in three preclinical models
`of cardiovascular fibrosis (Fig. 4a, e, i). Therefore, using knockout and
`wild-type mice we determined whether inhibition of Il-11 could reduce
`
`7 D E C E M B E R 2 0 1 7 | V O L 5 5 2 | N A T U R E | 1 1 3
`
`Letter reSeArCH
`
`© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.
`
`Lassen - Exhibit 1066, p. 4
`
`

`

`Heart
`
`Control
`
`AngII
`
`Il-11
`
`GAPDH
`
`P = 1 × 10–4
`
`P = 0.0262
`
`NS
`
`b
`
`Il11ra1+/+
`
`Il11ra1–/–
`
`Control
`
`AngII
`
`Control
`
`AngII
`
`100µm
`
`Il11ra1+/+
`AngII
`Control
`
`Il11ra1–/–
`AngII
`Control
`
`p-ERK1/2
`
`ERK1/2
`
`Il11ra1+/+
`
`Il11ra1–/–
`
`d
`
`40 kDa
`
`40 kDa
`
`Control
`AngII
`
`f
`
`Il-11
`
`GAPDH
`
`Il11ra1+/+
`
`Il11ra1–/–
`
`Heart
`
`Sham
`
`TAC
`
`P < 1 × 10–4
`P = 5 × 10–3
`
`Sham
`
`TAC
`
`Sham
`
`TAC
`
`a
`
`25 kDa
`
`35 kDa
`
`1.5
`
`1.0
`
`0.5
`
`c
`
`Fold change in HPA collagen
`
`e
`
`25 kDa
`
`35 kDa
`g
`
`4
`
`P = 2 × 10–3
`
`Il11ra1+/+
`
`Il11ra1–/–
`
`Kidney
`Control
`
`Folate
`
`P < 1 × 10–4
`
`P = 0.0022
`
`NS
`
`Sham
`TAC
`
`j
`
`Il-11
`
`GAPDH
`
`Control
`Folate
`
`Fold change in HPA collagen
`
`2
`
`i
`
`25 kDa
`
`35 kDa
`
`3
`
`2
`
`1
`
`k
`
`Fold change in HPA collagen
`
`Il11ra1+/+
`Il11ra1–/–
`Figure 4 | Inhibition of Il-11 reduces cardiovascular fibrosis.
`a, Western blot of cardiac Il-11 after AngIl infusion (2 mg kg−1 per day for
`28 days). b, c, Representative histological images (b; Masson’s trichrome
`staining) and collagen content (c) in the atrium of Il11ra1+/+ (control,
`n = 12; AngII, n = 9 biologically independent samples) and Il11ra1−/−
`(control, n = 5; AngII, n = 7 biologically independent samples) mice.
`d, Western blot of cardiac ERK activation after AngII infusion. e, Western
`blot of cardiac Il-11 after transverse aortic constriction (TAC).
`f, g, Representative histological images (f; Masson’s trichrome staining) and
`collagen content. g; HPA of hearts from Il11ra1+/+ (control, n = 4; TAC:
`
`fibrosis in these models. After either AngII infusion or transverse aortic
`constriction, less fibrosis occurred in the hearts of knockout mice com-
`pared to wild-type mice (Fig. 4b, c, f, g). This effect was independent
`of loading conditions (Extended Data Fig. 8). Similarly, after folate-
`induced kidney damage, knockout mice had reduced renal fibrosis
`(Fig. 4j, k). Deletion of Il11ra1 signalling resulted in reduced ERK
`signalling across all models tested, whereas p38 signalling was unaf-
`fected (Fig. 4d, h, l and Extended Data Fig. 9).
`IL-11 was discovered owing to its ability to sustain an IL-6-dependent
`haematopoetic cell line when secreted from fibroblastic cells24, but was
`later found to be redundant for haematopoiesis20. Here we show that
`IL11 is a crucial fibrosis gene acting downstream of TGFβ 1 and many
`other pro-fibrotic factors. We believe that the importance of IL-11 in
`fibroblasts may have gone unnoticed because its effects are apparent only
`at the post-transcriptional level (Extended Data Fig. 9). We highlight
`that IL11RA is not only expressed in fibroblasts but also in other cells
`and that IL-11 signalling may therefore be important in other cell types.
`
`1 1 4 | N A T U R E | V O L 5 5 2 | 7 D E C E M B E R 2 0 1 7
`
`n = 6 biologically independent samples) and Il11ra1−/− (control, n = 6;
`TAC, n = 6 biologically independent samples) mice after TAC or sham
`operations. h, Western blot of cardiac ERK activation after TAC. i, Western
`blot of renal Il-11 after folate treatment (180 mg kg−1). j, k, Representative
`histological images (j; Masson’s trichrome staining) and collagen content
`(k; HPA assay) of kidneys from Il11ra1+/+ (control, n = 5; folate, n = 9
`biologically independent samples) and Il11ra1−/− (control, n = 6;
`folate, n = 5 biologically independent samples) mice. c, g, k, Two-tailed,
`Sidak-corrected Student’s t-test; data are mean ± s.d. NS, not significant.
`l, Western blot of renal ERK activation after folate treatment.
`
`We note that rhIL-11 has been given to patients with myocardial
` infarction25 and it is possible that the use of rhIL-11 in cancer patients22
`causes fibrosis-related side effects. We therefore suggest that the use of
`rhIL-11 in humans should be reviewed. IL11 is highly upregulated in
`fibroblasts from patients with idiopathic pulmonary fibrosis or systemic
`sclerosis, by a 100-fold and 30-fold, respectively26; this suggests a role
`for IL-11 in fibrotic human disease beyond the cardiovascular system.
`IL-11 inhibitors may be particularly effective in treating fibrosis, because
`they would target a nodal point of pro-fibrotic signalling. Because of these
`results, and target safety data from human27 and mouse knockouts20, we
`propose that IL-11 is a potential therapeutic target.
`
`Online Content Methods, along with any additional Extended Data display items and
`Source Data, are available in the online version of the paper; references unique to
`these sections appear only in the online paper.
`
`received 22 March; accepted 2 November 2017.
`Published online 13 November 2017.
`
`h
`
`40 kDa
`
`40 kDa
`
`Il11ra1+/+
`TAC
`Sham
`
`Il11ra1–/–
`TAC
`Sham
`
`p-ERK1/2
`
`ERK1/2
`
`Il11ra1+/+
`
`Il11ra1–/–
`
`Control
`
`Folate
`
`Control
`
`Folate
`
`1mm
`
`Il11ra1+/+
`Folate
`Control
`
`Il11ra1–/–
`Folate
`Control
`
`l
`
`40 kDa
`
`40 kDa
`
`p-ERK1/2
`
`ERK1/2
`
`reSeArCH
`
`Letter
`
`© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.
`
`Lassen - Exhibit 1066, p. 5
`
`

`

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