Treatment and prevention of metabolic diseases
11813311 · 2023-11-14
Assignee
Inventors
- Stuart Alexander Cook (Singapore, SG)
- Sebastian Schaefer (Singapore, SG)
- Brijesh Kumar Singh (Singapore, SG)
- Anissa Widjaja (Singapore, SG)
Cpc classification
A61K31/713
HUMAN NECESSITIES
A61K39/3955
HUMAN NECESSITIES
C07K2317/76
CHEMISTRY; METALLURGY
C07K16/2866
CHEMISTRY; METALLURGY
C12N15/1136
CHEMISTRY; METALLURGY
C07K2317/92
CHEMISTRY; METALLURGY
A61K38/1793
HUMAN NECESSITIES
International classification
A61K39/395
HUMAN NECESSITIES
C07K16/24
CHEMISTRY; METALLURGY
C12N15/113
CHEMISTRY; METALLURGY
Abstract
Methods of treating and preventing metabolic disease through inhibiting interleukin 11 (IL-11)-mediated signalling are disclosed, as well as agents for use in such methods.
Claims
1. A method of treating a metabolic disease, comprising administering a therapeutically or prophylactically effective amount of an agent capable of inhibiting interleukin 11 (IL-11)-mediated signalling to a subject; wherein the agent is selected from: (i) an anti-IL-11 antibody antagonist of IL-11-mediated signalling, or an antigen-binding fragment thereof, or (ii) an anti-IL-11Rα antibody antagonist of IL-11-mediated signalling, or an antigen-binding fragment thereof; and wherein the metabolic disease is or comprises: obesity, being overweight, hyperglycaemia, pregnancy-associated hyperglycemia, insulin resistance, pre-diabetes, metabolic syndrome, hyperlipidaemia, hypertriglyceridemia, hypercholesterolemia, pancreatic insufficiency, pancreatitis, acute pancreatitis, chronic pancreatitis, lipotoxicity, or hyperglucagonemia.
2. The method according to claim 1, wherein the agent is an agent capable of preventing or reducing the binding of interleukin 11 (IL-11) to a receptor for interleukin 11 (IL-11R).
3. The method according to claim 1, wherein the method comprises administering the agent to a subject in which expression of interleukin 11 (IL-11) or a receptor for IL-11 (IL-11R) is upregulated.
4. The method according to claim 1, wherein the method comprises administering the agent to a subject in which expression of interleukin 11 (IL-11) or a receptor for interleukin 11 (IL-11R) has been determined to be upregulated.
5. The method according to claim 1, wherein the method comprises determining whether expression of interleukin 11 (IL-11) or a receptor for IL-11 (IL-11R) is upregulated in the subject and administering the agent to a subject in which expression of interleukin 11 (IL-11) or a receptor for IL-11 (IL-11R) is upregulated.
6. The method according to claim 1, wherein the agent is an anti-IL-11 antibody antagonist of IL-11-mediated signalling, or an antigen-binding fragment thereof, comprising: (i) a VH region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 40; HC-CDR2 having the amino acid sequence of SEQ ID NO: 41; HC-CDR3 having the amino acid sequence of SEQ ID NO: 42, and (ii) a VL region incorporating the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 43; LC-CDR2 having the amino acid sequence of SEQ ID NO: 44; LC-CDR3 having the amino acid sequence of SEQ ID NO: 45.
7. The method according to claim 1, wherein the agent is an anti-IL-11 antibody antagonist of IL-11-mediated signalling, or an antigen-binding fragment thereof, comprising: (i) a VH region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 34; HC-CDR2 having the amino acid sequence of SEQ ID NO: 35; HC-CDR3 having the amino acid sequence of SEQ ID NO: 36, and (ii) a VL region incorporating the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 37; LC-CDR2 having the amino acid sequence of SEQ ID NO: 38; LC-CDR3 having the amino acid sequence of SEQ ID NO: 39.
8. The method according to claim 1, wherein the agent is an anti-IL-11Rα antibody antagonist of IL-11-mediated signalling, or an antigen-binding fragment thereof, comprising: (i) a VH region incorporating the following CDRs: HC-CDR1 having the amino acid sequence of SEQ ID NO: 46; HC-CDR2 having the amino acid sequence of SEQ ID NO: 47; HC-CDR3 having the amino acid sequence of SEQ ID NO: 48, and (ii) a VL region incorporating the following CDRs: LC-CDR1 having the amino acid sequence of SEQ ID NO: 49; LC-CDR2 having the amino acid sequence of SEQ ID NO: 50; LC-CDR3 having the amino acid sequence of SEQ ID NO: 51.
Description
BRIEF DESCRIPTION OF THE FIGURES
(1) Embodiments and experiments illustrating the principles of the invention will now be discussed with reference to the accompanying figures.
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EXAMPLES
(52) In the following Examples, the inventors demonstrate that inhibition of IL-11-mediated signalling reduces the severity and reverses the symptoms of a range of metabolic diseases.
Example 1: General Methods for Examples 1 to 4
(53) IL-11-RA-Knockout Mice
(54) Mice lacking functional alleles for Il11rα (Il11rα−/−) were on C576I/6J genetic background (B6.129S1-Il11rαtm1Wehi/J, Jackson's Laboratory).
(55) Treatment with Anti-IL-11 or Anti-IL-11Rα Antibody
(56) Mice were injected intraperitoneally with 10 mg/kg of an antagonist anti-IL-11 antibody, an antagonist anti-IL-11Rα antibody, or an identical amount of isotype-matched IgG control antibody. The anti-IL-11 and anti-IL-11Rα antibodies bind to mouse IL-11 and mouse IL-11Rα respectively, and inhibit IL-11 mediated signalling.
(57) Specifically, the anti-IL-11 antibody used in the present examples is mouse anti-mouse IL-11 IgG X203, which is described e.g. in Ng et al., Sci Transl Med. (2019) 11(511) pii: eaaw1237 (also published as Ng, et al., “IL-11 is a therapeutic target in idiopathic pulmonary fibrosis.” bioRxiv 336537; doi: https://doi.org/10.1101/336537). X203 is also referred to as “Enx203”, and comprises the VH region according to SEQ ID NO:92 of WO 2019/238882 A1 (SEQ ID NO:22 of the present disclosure), and the VL region according to SEQ ID NO:94 of WO 2019/238882 A1 (SEQ ID NO:23 of the present disclosure).
(58) The anti-IL-11Rα antibody used in the present examples is mouse anti-mouse IL-11Rα IgG X209, which is described e.g. in Widjaja et al., Gastroenterology (2019) 157(3):777-792 (also published as Widjaja, et al., “IL-11 neutralising therapies target hepatic stellate cell-induced liver inflammation and fibrosis in NASH.” bioRxiv 470062; doi: https://doi.org/10.1101/470062). X209 is also referred to as “Enx209”, and comprises the VH region according to SEQ ID NO:7 of WO 2019/238884 A1 (SEQ ID NO:24 of the present disclosure), and the VL region according to SEQ ID NO:14 of WO 2019/238884 A1 (SEQ ID NO:25 of the present disclosure).
(59) Diets
(60) Western diet along with fructose (WDF) was used to establish metabolic disorders that closely resemble those in humans during obesity, T2D and NAFLD (Baena et al., Sci Rep (2016) 6: 26149, Machado et al., PLoS One (2015) 10:e0127991).
(61) In order to establish metabolic diseases such as obesity and T2D, mice were fed Western diet (D12079B, Research Diets), supplemented with 15% weight/volume fructose in drinking water (WDF) for 16 weeks, from 12 weeks of age.
(62) High fat methionine choline deficient diet (HFMCD) was used to establish cachexia-like metabolic disorder. In order to establish cachexia weight loss and lean mass loss, C57BL/6N mice were fed with methionine and choline deficient (HFMCD) diet supplemented with 60 kcal % fat (A06071301B, Research Diets).
(63) Control subjects were fed normal chow (NC, Specialty Feeds) and drinking water.
(64) Echo MRI Analysis for Body Composition
(65) Total body fat and lean mass measurements were performed every two weeks by EchoMRI analysis using 4in1 Body Composition Analyzer for Live Small Animals.
(66) Fasting Blood Glucose Measurements
(67) For fasting blood glucose measurements, mice were fasted for 6 hours prior to blood collection (via tail snip), and Accu-Chek blood glucose meter was used to obtain fasting glucose measurements.
(68) Intraperitoneal Glucose Tolerance Test (ipGTT)
(69) For intraperitoneal glucose tolerance tests, mice were fasted for 6 h prior to being subjected to ipGTT. Basal fasting glucose was measured by tail snip using Accu-Chek blood glucose meter. 2 g/kg lean mass glucose was injected intraperitoneally, and glucose measurements were taken every 15 min for 2 hours. The area under the curve (AUC) was calculated, and plotted as bar graphs.
(70) Histology of Pancreas for Islet of Langerhans, Glucagon and Insulin
(71) For histological analysis, pancreas samples were excised and fixed for 24 hours at RT in 4% neutral-buffered formalin (NBF), and stored in 30% sucrose. 5 μm cryosections were stained with either glucagon or insulin antibodies overnight, and visualized with ImmPRESS HRP IgG polymer detection kit (Vector Laboratories) with ImmPACT DAB Peroxidase Substrate (Vector Laboratories) according to standard protocols, and examined by light microscopy.
Example 2: Antagonism of IL-11-Mediated Signalling in Obesity-Related Disorders
(72) To investigate the effect of antagonism of IL-11-mediated signalling on obesity and related disorders like T2D, in vivo experiments were performed using diet-induced mouse models of these metabolic diseases using IL-11 receptor alpha knock out (IL11-RA−/−) mice, or by treatment of mice with antagonist anti-mouse IL-11 antibody, or antagonist anti-mouse IL11-RA antibody.
(73) IL11RA knockout mice fed on normal chow diet (NCD) or WDF displayed an improved metabolic phenotype as compared to wildtype IL11RA-expressing littermates.
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(75) The results suggested that reduction of IL-11 mediated signalling has beneficial effects in metabolism.
(76) The inventors next investigated the effect of an antagonist anti-IL-11 RA antibody or control IgG antibody on mice fed on WDF.
(77) Strikingly, anti-IL-11 RA antibody-treated mice fed on WDF showed significant reduction in body weight when compared to control IgG anybody-treated mice fed on WDF (
(78) Interestingly, an increase in lean mass was also observed in mice treated with anti-IL-11 RA antibody compared to IgG control-treated mice, suggesting that inhibition of IL-11 signalling during WDF-induced metabolic pathogenesis recovered muscle mass. Furthermore, intraperitoneal glucose tolerance test (ipGTT) results showed, along with fasting glucose, significant improvement in glucose tolerance in mice treated with anti-IL-11 RA antibody (
(79) The analysis was extended to the effects on the pancreas. Anti-IL-11 RA antibody-treated mice fed on WDF were unexpectedly found to display remarkable protection against WDF-induced loss of pancreas (
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(81) Moreover, immune-histology of pancreas also revealed increase in glucagon and insulin staining in pancreatic islets along with islet hyperplasia in IgG treated WDF fed mice (
Example 3: Antagonism of IL-11-Mediated Signalling and Cachexia
(82) Anti-IL-11 therapies were assessed for their effects on a mouse model of cachexia.
(83) Feeding mice with a methionine-choline deficient (MCD) diet causes severe non-alcoholic steatohepatitis (NASH), hepatic inflammation and fibrosis, and results in severe and sustained weight loss (up to 30% of body weight after 3 weeks of MCD diet). While mice on an MCD diet have 36% higher metabolic rates than those on normal chow diet (NCD) and have a strong appetite-stimulating milieu (low leptin, low glucose, low TGs/cholesterol, low insulin), they do not increase their food consumption (Rizki et al. J. Lipid Res. (2006) 47:2280-2290). As such, the MCD diet is a well-recognised model of cachexia and has many features in common with cancer-associated cachexia. Steatohepatitis is frequently documented in experimental and human cancer cachexia and plays an important but poorly understood role in wasting syndromes.
(84) Five-week old male mice were fed a methionine- and choline-deficient (MCD) diet with 60 kcal % fat (A06071301B, Research Diets), designated a high fat MCD (HFMCD) diet, which causes more severe NASH than an MCD diet alone. Control mice were fed with normal chow (NC; Specialty Feeds). Mice were intraperitoneally injected twice per week with 10 mg/kg of anti-IL-11 antibody or anti-IL-11 RA antibody, or identical concentration of IgG isotype control one week after they had received HFMCD for the same treatment duration. Body weight was measured weekly.
(85) The results are shown in
(86) To investigate further the effect of inhibition of IL-11-mediated signalling with respect to cachexia, a range of doses of anti-IL-11 therapy were studied in the MCD model. Five-week old male mice were fed on the HFMCD or NC diet as before for one week to induce cachexia, resulting in a ˜15% loss in body weight in MCD mice. After the initial week, mice were intraperitoneally injected twice per week with 0.5, 1, 5 or 10 mg/kg of anti-IL-11 or anti-IL-11 RA antibody. Three antibodies were studied: two anti-IL-11 ((1) and (2)), and one anti-IL-11 RA. 10 mg/kg of IgG isotype antibody was used as a control.
(87) Body weight and food consumption were measured weekly. For food consumption, average food intake was measured (g/mouse/week) in food hoppers from cages (n=3 mice per cage).
(88) The body weight results are shown in
(89) The food consumption results are shown in
(90) Acute disease, e.g. trauma or sepsis, can also be associated with anorexia and cachexia, and so the inventors next investigated the effects of antagonism of IL-11-mediated signalling on anorexia and cachexia in mouse models of acute kidney injury.
(91) Kidney injury was induced by IP injection of folic acid (180 mg/kg) in vehicle (0.3M NaHCO.sub.3) to 10-week old male mice; control mice were administered vehicle alone. Animals were sacrificed 28 days post-injection. Mice were intraperitoneally injected every 3 days with 20 mg/kg of anti-IL-11 antibody, anti-IL-11 RA antibody or identical concentration of IgG isotype control starting from 1 hour before folic acid administration until the mice were sacrificed.
(92) The results are shown in
(93) In a second experiment kidney injury was induced as before by IP injection of folic acid. Mice were only treated with anti-IL-11 antibody or IgG control from 21 days after kidney injury. Animal weight was assessed before and after antibody treatment. Healthy mice that did not receive folic acid were used as a control.
(94) The results are shown in
(95) In further experiments, mice were subjected to unilateral ureter obstruction (UUO)-induced acute kidney injury. UUO surgeries were carried out on 12-week old mice. Briefly, mice were anesthetized by IP injection of ketamine (100 mg/kg)/xylazine (10 mg/kg) and full depth of anaesthesia was accessed with the pedal reflex. Mice were then shaved on the left side of the abdomen. A vertical incision was made through the skin with a scalpel, a second incision was made through the peritoneum to reveal the kidney. Using forceps, the kidney was brought to the surface and the ureter was tied with surgical silk, twice, below the kidney. The ligated kidney was placed gently back into its correct anatomical position and sterile saline was added to replenish loss of fluid. The incisions were then sutured. Animals were post-operatively treated with antibiotic enrofloxacin (15 mg/kg, SC) and analgesic buprenorphine (0.1 mg/kg, SC) for three consecutive days. Mice were sacrificed 10 days post-ligation. Mice were intraperitoneally injected with 20 mg/kg (2×/week) of anti-IL-11 antibody or identical concentration of IgG isotype control from day 4 post-surgery until the mice were sacrificed.
(96) The results are shown in
(97) Thus antagonism of IL-11 mediated signalling is associated with therapeutic recovery of body weight in models of acute disease.
(98) The inventors next investigated the effects of IL-11 overexpression on mouse body weight, via injection of recombinant mouse IL-11 or induction of IL-11 transgene expression.
(99) Recombinant mouse IL-11 (rmIL11) was reconstituted to a concentration of 50 μg ml.sup.−1 in saline. Ten-week-old male wild-type C57BL/6J mice received daily subcutaneous injection of either 100 μg kg.sup.−1 of rmIL11 in saline (n=19) or an identical volume of saline (n=15) for 21 days.
(100) The results are shown in
(101) IL-11 transgenic (IL-11-Tg) mice were created. Heterozygous Rosa26-IL11 mice were crossed with Col1a2-CreER mice to create double heterozygous Col1a2-CreER:Rosa26-IL11 progenies (IL-11-Tg mice) with IL-11 transgene expression in fibroblasts. For Cre-mediated IL-11 transgene induction, IL-11-Tg mice were injected with 50 mg kg.sup.−1 Tamoxifen (Sigma-Aldrich) intraperitoneally at 6 weeks of age for 10 consecutive days and the animals were sacrificed on day 21 (n=14). Rosa26:1111 mice (without Col1a2-CreER allele) were injected with an equivalent dose of tamoxifen for 10 consecutive days as controls (n=10).
(102) The results are shown in
Example 4: Antagonism of IL-11-Mediated Signalling in Mouse Models of Non-Alcoholic Steatohepatitis (NASH)
(103) The inventors investigated the role of IL-11 signalling in the pathogenesis of nonalcoholic steatohepatitis (NASH).
4.1 Methods
(104) Hepatic stellate cells (HSCs) or hepatocytes were stimulated with IL-11 and effects assessed using cellular and high content imaging, immunoblotting, ELISA and invasion assays. Genetic and pharmacological IL-11 gain- or loss-of-function experiments were performed in vitro and in vivo. IL-11 signalling was studied using ERK inhibitors. The effects of anti-IL-11 or anti-IL11RA therapy were assessed in three preclinical NASH models using methionine/choline deficient diets or a Western diet with liquid fructose. Phenotyping was performed using hydroxyproline assay, qPCR, RNA-seq, Western blotting, histology, CyTOF, lipid and metabolic biomarkers.
(105) Animal Experiments
(106) All animal procedures were approved and conducted in accordance with the SingHealth Institutional Animal Care and Use Committee (IACUC). All mice were provided food and water ad libitum.
(107) Mouse Models of NASH
(108) High fat methionine and choline-deficient (HFMCD) diet fed wild-type mice Five week old male C57BL/6N mice were fed with Methionine and Choline deficient diet supplemented with 60 kcal % fat (A060713011316, Research Diets); control mice were fed with normal chow (NC, Specialty Feeds). Durations of diet and antibody therapies are described.
(109) Methionine and Choline-Deficient (MCD) Diet Fed Db/Db Mice
(110) Male BKS.Cg-Dock7m+/+LeprdbJ (db/db) mice on C57BL/6J genetic background were 12 weeks of age and at the hepatic steatosis stage when they were fed methionine- and choline-deficient diet (MCD, A02082002BRi, Research Diets) for 8 weeks; control mice were of the same genotype. Durations of diet and antibody therapies are described.
(111) Western Diet Supplemented with Fructose (WDF) Fed Wild-Type Mice
(112) Ten week old male C57Bl/6J mice were fed Western diet (D12079B, Research Diets), supplemented with 15% weight/volume fructose in drinking water to mimic NAFLD/NASH like humans 17,18, whereas control mice received normal chow and tap water. Durations of diet and antibody therapies are described.
(113) Il11ra-Deleted Mice
(114) Mice lacking functional alleles for Il11ra (Il11ra−/−) were on C57Bl/6J genetic background (66.12951-Il11ratm1Wehi/J, Jackson's Laboratory). Both Il11ra−/− mice and their wild-type littermates (Il11ra+/+) were fed with (1) HFMCD for 10 weeks from 5 weeks of age and (2) WDF for 16 weeks from 12 weeks of age to develop NASH; control mice were fed with NC for the same duration.
(115) In Vivo Administration of Il-11
(116) Ten week old male Col1a1-GFP reporter mice 19 and wild-type C57BL/6J mice received daily subcutaneous injection of either 100 μg/kg of recombinant mouse Il-11 (rmIl-11) or identical volume of saline for 21 days.
(117) In Vivo Administration of Anti-IL-11 or Anti-IL11RA Monoclonal Antibodies
(118) Mice were injected intraperitoneally with either antagonist anti-IL-11 antibody, antagonist anti-IL11RA antibody or an identical amount of IgG isotype control for the treatment durations outlined in the figures.
(119) Fasting Blood Glucose Measurement
(120) Mice were fasted for 6 h prior to blood collection (via tail snip) and Accu-Chek blood glucose meter was used to take fasting glucose measurements.
(121) Cell Culture
(122) Cells (atrial fibroblasts, HSCs and hepatocytes) were grown and maintained at 37° C. and 5% CO2. The growth medium was renewed every 2-3 days and cells were passaged at 80-90% confluence using standard trypsinization techniques. All the experiments were carried out at low cell passage (P1-P2). Cells were serum-starved for 16 h prior to stimulations. Stimulated cells were compared to unstimulated cells that have been grown for the same duration under the same conditions (serum-free media), but without the stimuli.
(123) Primary Human Atrial Fibroblasts
(124) Human atrial fibroblasts were prepared and cultured as described previously 11.
(125) Primary Human Hepatic Stellate Cells (HSCs)
(126) HSCs (5300, ScienCell) were cultured in stellate cells complete media (5301, ScienCell) on poly-L-lysine-coated plates (2 μg/cm2, 0403, ScienCell).
(127) Primary Human Hepatocyte
(128) Human hepatocytes (5200, ScienCell) were grown and maintained in hepatocyte media (5201, ScienCell) supplemented with 2% fetal bovine serum (FBS) and 1% Penicillin-streptomycin.
(129) THP-1
(130) THP-1 (ATCC) were cultured in RPMI 1640 (A1049101, Thermo Fisher) supplemented with 10% FBS and 0.05 mM β-mercaptoethanol. THP-1 cells were differentiated with 10 ng/ml of phorbol 12-myristate 13-acetate (PMA, P1585, Sigma) in RPMI 1640 for 48 h.
(131) Operetta High Throughput Phenotyping Assay
(132) The Operetta assay was performed mostly as described previously 11 with minor modifications described here: HSCs or hepatocytes were seeded in 96-well black CellCarrier plates (PerkinElmer) at a density of 5×10.sup.3 cells per well. Following experimental conditions, cells were fixed in 4% paraformaldehyde (PFA, 28908, Thermo Fisher), permeabilized with 0.1% Triton X-100 (Sigma) and non-specific sites were blocked with 0.5% BSA and 0.1% Tween-20 in PBS. Cells were incubated overnight (4° C.) with primary antibodies (1:500), followed by incubation with the appropriate AlexaFluor 488 secondary antibodies (1:1000). Rhodamine Phalloidin staining (1:1000, R415, Thermo Fisher) was performed by overnight incubation (4° C.). Cells were counterstained with 1 μg/ml DAPI (D1306, Thermo Fisher in blocking solution. Each condition was imaged from duplicated wells and a minimum of 7 fields/well using Operetta high-content imaging system 1483 (PerkinElmer). Cells expressing ACTA2 were quantified using Harmony v3.5.2 (PerkinElmer) and the percentage of activated fibroblasts/total cell number (ACTA2+ve) was determined for each field. The measurement of fluorescence intensity per area (normalized to the number of cells) of Collagen I was performed with Columbus 2.7.1 (PerkinElmer).
(133) Immunofluorescence
(134) Human HSCs and hepatocytes were seeded on 8-well chamber slides (1.5×10.sup.4 cells/well) 24 h before the staining. Cells were fixed in 4% PFA for 20 min, washed with PBS, and non-specific sites were blocked with 5% BSA in PBS for 2 h. Cells were incubated with anti IL11RA or anti IL6R antibody overnight (4° C.), followed by incubation with the appropriate Alexa Fluor 488 secondary antibody for 1 h. Chamber slides were dried in the dark and 5 drops of mounting medium with DAPI were added to the slides for 15 min prior to imaging by fluorescence microscope (Leica).
(135) Mass Cytometry by Time of Flight (CyTOF)
(136) Immune cells were isolated from liver as described previously 20. Liver tissues were minced and digested with 100 μg/ml Collagenase IV and 20 U/ml DNase I, at 37° C. for 1 h. Following digestion, cells were passed through strainer to obtain single cell suspension and subjected to percoll gradient centrifugation for isolation of immune cells. CyTOF staining was performed as previously described 21. Cells were thawed and stained with cisplatin (Fluidigm) to identify live cells, followed by staining with metal-conjugated CD45 antibody, for barcoding purpose. After barcoding, cells were stained with metal-conjugated cell surface antibody (Ly6C). Cells were then fixed with 1.6% PFA, permeabilized with 100% methanol, and subjected to intracellular antibody staining (TGFβ1). Cells were labeled with DNA intercalator before acquisition on Helios mass cytometer (Fluidigm). For analysis, first live single cells were identified, followed by debarcoding to identify individual samples. Manual gating was performed using Flowjo software (Flowjo).
(137) Statistical Analysis
(138) Statistical analyses were performed using GraphPad Prism software (version 6.07). P values were corrected for multiple testing according to Dunnett's (when several experimental groups were compared to one condition), Tukey (when several conditions were compared to each other within one experiment), Sidak (when several conditions from 2 different genotypes were compared to each other). Analysis for two parameters (antibody efficacy across time) for comparison of two different groups were performed by two-way ANOVA. The criterion for statistical significance was P<0.05.
(139) Data Availability
(140) High-throughput sequencing data generated for this study can be downloaded from GSE128940. All other data are in the manuscript or in the supplementary methods.
4.2 Results
(141) Overview
(142) When stimulated with NASH factors HSCs secrete IL-11, which drives an autocrine, ERK-dependent signalling loop required for the HSC-to-myofibroblast transformation. IL-11 is upregulated in human and murine NASH, Il-11 injection causes liver damage, inflammation and fibrosis in mice and Il11ra1 deleted mice are protected from NASH in two preclinical models. Therapeutic antibodies against IL11RA or IL-11 consistently inhibit and reverse fibrosis and steatosis in three murine NASH models. Unexpectedly, IL-11 causes hepatocyte damage and promotes stromal-mediated inflammation and anti-IL-11 therapies reverse NASH-associated hepatotoxicity and hepatitis. Genetic or pharmacologic inhibition of IL-11 signalling in NASH is associated with lower serum triglyceride, cholesterol and glucose.
(143) IL-11 Activates HSCs and Drives Liver Fibrosis
(144) Genome wide RNA-seq analysis revealed that TGFβ1 strongly upregulates IL-11 (14.9-fold, P=3.40×10.sup.−145) in HSCs, which was verified by qPCR, confirmed at the protein level and replicated in experiments using precision cut human liver slices (
(145) To investigate the effect of IL-11 on HSCs, cells were stimulated with either IL-11, TGFβ1 or PDGF. IL-11 activated HSCs to a similar extent as TGFβ1 or PDGF, transforming quiescent HSCs into ACTA2+ve myofibroblasts that secrete collagen and matrix modifying enzymes (
(146) Col1a1-GFP reporter mice.sup.19 treated with recombinant mouse Il-11 (rmIl-11) accumulated GFP-expressing Col1a1.sup.+ve myofibroblasts in the liver, further confirming an effect of Il-11 on HSC-to-myofibroblast transformation in vivo. Subcutaneous administration of rmIl-11 to mice for 21 days also increased hepatic collagen content, expression of key pro-fibrotic and pro-inflammatory genes and serum alanine aminotransferase (ALT) (
(147) Deletion of Il11ra1 Protects Mice from NASH-Associated Inflammation, Hepatotoxicity and Fibrosis and
(148) Lowers Serum Lipids and Glucose Studies were performed in a preclinical model of severe NASH using the high fat methionine- and choline-deficient (HFMCD) diet.sup.16. In this model, Il-11 mRNA was mildly elevated whereas protein levels were highly upregulated, suggesting post-transcriptional regulation of Il-11 expression in the liver (
(149) To evaluate the pathophysiological relevance of increased Il-11 levels in NASH, the inventors used a genetic loss-of-function model: the Il-11 receptor subunit alpha deleted mouse (Il11ra1.sup.−/−).sup.25. Il11ra1.sup.−/− mice on the HFMCD diet were strongly protected from fibrosis and had lesser steatosis and liver damage as compared to controls (
(150) The HFMCD model has early onset steatotic hepatitis followed by fibrosis. However, this model is not obese or insulin resistant. Another NASH model was established, using a Western Diet supplemented with liquid Fructose (WDF).sup.26 that is obese, insulin resistant and hyperglycaemic, mirroring human NASH.sup.18. After 16 weeks of WDF feeding, NASH was established and Il-11 protein was upregulated in the liver (
(151) The primary causes of mortality in NASH are cardiovascular: myocardial infarction, renal failure and stroke.sup.27,28. Biomarkers of cardiovascular risk were measured in Il11ra1.sup.−/− mice after 16 weeks on the WDF diet. As compared to littermate controls, Il11ra1.sup.−/− mice on WDF had lower levels of fasting blood glucose, serum cholesterol and triglycerides (
(152) Neutralising Anti-IL-11 or Anti-IL11RA Antibodies Block HSC Activation
(153) Mice were genetically immunised with IL11RA to generate neutralising anti-IL11RA antibodies. Clones that blocked fibroblast transformation.sup.11 were identified and clone X209 (IgG1.sub.κ, K.sub.D=6 nM) was prioritised. X209 blocked MMP2 secretion from HSCs with an IC.sub.50 of 5.8 pM and has an in vivo half-life of approximately 18 days with good liver uptake (
(154) The inventors found that, in addition to TGFβ1 (
(155) In the Il11ra1.sup.−/− mouse, liver protection on either HFMCD or WDF diets was associated with reduced Erk activation. The inventors found that IL-11 directly activates ERK in HSCs and that all stimuli that induce IL-11 secretion from HSCs also induce ERK activation. X209 abolished ERK phosphorylation and HSC transformation downstream of all factors, including IL-11 itself. ERK inhibitors blocked the IL-11 effect and HSC activation downstream of all NASH triggers, suggesting IL-11 driven ERK phosphorylation is of central importance for HSC transformation (
(156) The published literature on IL-11 in the liver is limited but injection of high dose recombinant human IL-11 into rodents has been associated with protective effects.sup.13,14 and there is confusion as to a role for IL-11 in platelets biology.sup.25. To exclude safety issues, the inventors performed long-term (5 months) high dose (10 mg/kg×2/week) preclinical toxicology studies of X209 and X203 and observed no effect on serum ALT levels or platelets (
(157) Therapeutic Targeting of IL-11 or IL11RA is Effective in Three Preclinical NASH Models
(158) The inventors then tested X209 and X203 therapy in vivo and started antibody administration after six weeks of HFMCD diet when IL-11 is strongly upregulated, collagen has accumulated and steatohepatitis is established (
(159) The inventors also tested anti-IL-11 therapy in twenty week old db/db mice that are obese, diabetic and have steatotic livers when put onto a NASH-inducing methionine-choline-deficient (MCD) diet for eight weeks (
(160) A third model of WDF-induced NASH was used to test effects of anti-IL-11 therapy in the context of obesity, insulin resistance and diabetes.sup.18. Mice were fed WDF for 16 weeks by which time they were obese and insulin resistant with liver steatosis, inflammation and fibrosis. Treatment with anti-IL11RA (X209) therapy was then initiated (
(161) Effects of Combined Metabolic and Anti-IL-11 Interventions on Hepatic Fibrosis
(162) Our data showed that anti-IL-11 therapy reversed fibrosis but did not assess whether this effect was sustained or progressive. Furthermore, combination therapies may be beneficial for reversing fibrosis in NASH′. To address these points, severe liver fibrosis was established using HFMCD for 10 weeks, then converted mice to normal chow, mimicking a robust metabolic intervention, and initiated anti-IL-11 treatments in parallel (
(163) Upon removal of the metabolic stimulus, Erk activation slowly regressed, which was accelerated by X203 or X209-treatment (
(164) Regression of fibrosis is associated with lower TIMP and higher MMP levels, which promotes favorable matrix remodelling.sup.3,32. Consistent with this, X203 or X209 treated mice with severe fibrosis rapidly upregulated Mmp2 and downregulated Timp1 (
(165) Effects of anti-IL-11 therapy on liver health during acute necroinflammation in early-stage NASH The transition from NAFLD to NASH is characterised the development of steatotic hepatitis, inflammation and cell death (necroinflammation). HSCs have a central role in this process through the secretion of pro-inflammatory factors.sup.3,8,37,38. The inventors investigated whether IL-11 affected HSC-driven inflammatory pathways, and found that IL-11 stimulated HSC production of CCL2 whereas IL-11 inhibition blocked CCL2 secretion (
(166) In the HFMCD model of NASH, early inflammation is followed by a fibrotic phase (
(167) Primary human hepatocytes were found to express IL11RA but not IL6R (
(168) RNA-seq analysis was performed to profile the effects of IL-11 therapy during the acute inflammatory phase of HFMCD-induced NASH. Unsupervised analyses showed that antibody treatment almost completely reverses the pathological RNA expression signature induced by the HFMCD diet (
(169) Resident macrophages and infiltrating monocytes are important for NASH pathogenesis and a major source of TGFβ1.sup.39. Inflammatory cell populations were examined in the liver during steatohepatitis and observed fewer immune cells in general in X209-treated livers and a specific reduction in Ly6C.sup.+veTGFβ1.sup.+ve cells (
4.3 Discussion
(170) HSCs are the major source of pro-inflammatory myofibroblasts in the liver.sup.2 and inhibiting and reversing their transformation is a target for NASH therapies. Non-redundant, ERK-dependent IL-11 signalling is shown to be required for HSC transformation, similar to its role for fibroblast activation in heart, kidney and lung.sup.11,12. As such, targeting IL-11 to reverse liver fibrosis may have benefits when compared to therapies against other immune, metabolic or fibrosis factors that often exhibit some level of redundancy. Interestingly, potent metabolic intervention alone had no effect on fibrosis in our experiments and metabolic therapies may have limited effects on reversing fibrosis in NASH.
(171) The inventors discovered an unexpected pro-inflammatory role for IL-11 in the liver and show that HSCs express high levels of IL11 RA whereas immune cells express IL6R instead. The data suggests an indirect effect of IL-11 on immune cells that is mediated via the stroma. Irrespective of using genetic or pharmacologic loss-of-function approaches inhibition of IL-11 mediated signalling was consistently and robustly demonstrated to prevent/reverse inflammation across multiple NASH models. While earlier publications suggest Il-11 may have a protective role in the liver, these studies used extremely high-doses of foreign recombinant human IL-11 in rodents.sup.13,14 that does not stimulate murine Il11ra1.sup.11. The true biological effect of IL-11 at physiological levels is shown to be pro-inflammatory and stromal driven.
(172) Hepatocytes also express IL11 RA and strongly secrete IL-11 upon stimulation with TGFβ1 and IL-11 signalling in hepatocytes induced stress fibre formation and cytotoxicity. The effects of IL-11 on hepatocytes during acute necroinflammation in NASH are profound and therapeutic targeting of IL-11 signalling reversed ALT levels from approximately 700 U/L to normal within three weeks. At later time points in NASH, genetic or therapeutic inhibition of IL-11 also prevents or reverses hepatocyte damage, which requires further study.
(173) Human.sup.40 and mouse.sup.25 knockouts for IL11RA can have a mild skull deformity and exhibit joint laxity but are otherwise healthy and IL-11 appears redundant in adult mammals. This provides compelling genetic safety data for IL-11 as a drug target. On top of this target safety data, the present studies show that there are no adverse effects when IL-11 signalling is neutralized for an extended period of time using high doses of therapeutic antibodies. Furthermore, genetic or pharmacologic inhibition is associated with lower serum triglycerides, cholesterol and fasting glucose. This aspect of IL-11 inhibition is a desirable feature for a potential NASH therapy, as patients with NASH often suffer from cardiovascular diseases.
(174) The inventors have identified an unappreciated and central role for IL-11 in liver pathobiology. Targeting IL-11 signalling with neutralizing antibodies reverses fibrosis, steatosis, hepatocyte death and inflammation across the spectrum of NASH. This novel therapeutic approach is associated with a favorable cardiometabolic profile.
4.4 References to Example 4
(175) 1. Friedman S L, Neuschwander-Tetri B A, Rinella M, et al. Mechanisms of NAFLD development and therapeutic strategies. Nat Med 2018. Available at: http://dx.doi.org/10.1038/s41591-018-0104-9. 2. Mederacke I, Hsu C C, Troeger J S, et al. Fate tracing reveals hepatic stellate cells as dominant contributors to liver fibrosis independent of its aetiology. Nat Commun 2013; 4:2823. 3. Friedman S L. Hepatic stellate cells: protean, multifunctional, and enigmatic cells of the liver. Physiol Rev 2008; 88:125-172. 4. Friedman S L. Molecular Regulation of Hepatic Fibrosis, an Integrated Cellular Response to Tissue Injury. J Biol Chem 2000; 275:2247-2250. 5. Higashi T, Friedman S L, Hoshida Y. Hepatic stellate cells as key target in liver fibrosis. Adv Drug Deliv Rev 2017; 121:27-42. 6. Hellerbrand C, Stefanovic B, Giordano F, et al. The role of TGFβ1 in initiating hepatic stellate cell activation in vivo. J Hepatol 1999; 30:77-87. 7. Tsuchida T, Friedman S L. Mechanisms of hepatic stellate cell activation. Nat Rev Gastroenterol Hepatol 2017; 14:397-411. 8. Kim B-M, Abdelfattah A M, Vasan R, et al. Hepatic stellate cells secrete Ccl5 to induce hepatocyte steatosis. Sci Rep 2018; 8:7499. 9. Banini B A, Sanyal A J. Current and future pharmacologic treatment of nonalcoholic steatohepatitis. Curr Opin Gastroenterol 2017; 33:134-141. 10. Iwaisako K, Jiang C, Zhang M, et al. Origin of myofibroblasts in the fibrotic liver in mice. Proc Natl Acad Sci USA 2014; 111:E3297-305. 11. Schafer S, Viswanathan S, Widjaja A A, et al. IL-11 is a crucial determinant of cardiovascular fibrosis. Nature 2017; 552:110-115. 12. Cook S, Ng B, Dong J, et al. IL-11 is a therapeutic target in idiopathic pulmonary fibrosis. 2018. Available at: http://dx.doi.org/10.1101/336537. 13. Zhu M, Lu B, Cao Q, et al. IL-11 Attenuates Liver Ischemia/Reperfusion Injury (IRI) through STAT3 Signalling Pathway in Mice. PLoS One 2015; 10:e0126296. 14. Yu J, Feng Z, Tan L, et al. Interleukin-11 protects mouse liver from warm ischemia/reperfusion (WI/Rp) injury. Clin Res Hepatol Gastroenterol 2016; 40:562-570. 15. Lawitz E J, Hepburn M J, Casey T J. A pilot study of interleukin-11 in subjects with chronic hepatitis C and advanced liver disease nonresponsive to antiviral therapy. Am J Gastroenterol 2004; 99:2359-2364. 16. Gomes A L, Teijeiro A, Buren S, et al. Metabolic Inflammation-Associated IL-17A Causes Non-alcoholic Steatohepatitis and Hepatocellular Carcinoma. Cancer Cell 2016; 30:161-175. 17. Baena M, Sangüesa G, Flutter N, et al. Liquid fructose in Western-diet-fed mice impairs liver insulin signalling and causes cholesterol and triglyceride loading without changing calorie intake and body weight. J Nutr Biochem 2017; 40:105-115. 18. Machado M V, Michelotti G A, Xie G, et al. Mouse models of diet-induced nonalcoholic steatohepatitis reproduce the heterogeneity of the human disease. PLoS One 2015; 10:e0127991. 19. Yata Y. DNase I-hypersensitive sites enhance al (I) collagen gene expression in hepatic stellate cells. Hepatology 2003; 37:267-276. 20. Sheng J, Ruedl C, Karjalainen K. Most Tissue-Resident Macrophages Except Microglia Are Derived from Fetal Hematopoietic Stem Cells. Immunity 2015; 43:382-393. 21. Chew V, Lai L, Pan L, et al. Delineation of an immunosuppressive gradient in hepatocellular carcinoma using high-dimensional proteomic and transcriptomic analyses. Proc Natl Acad Sci USA 2017; 114:E5900-E5909. 22. Dou C, Liu Z, Tu K, et al. P300 Acetyltransferase Mediates Stiffness-Induced Activation of Hepatic Stellate Cells Into Tumor-Promoting Myofibroblasts. Gastroenterology 2018; 154:2209-2221.e14. 23. Yang C, Zeisberg M, Mosterman B, et al. Liver fibrosis: insights into migration of hepatic stellate cells in response to extracellular matrix and growth factors. Gastroenterology 2003; 124:147-159. 24. Lawan A, Bennett A M. Mitogen-Activated Protein Kinase Regulation in Hepatic Metabolism. Trends Endocrinol Metab 2017; 28:868-878. 25. Nandurkar H H, Robb L, Tarlinton D, et al. Adult mice with targeted mutation of the interleukin-11 receptor (IL11Rα) display normal hematopoiesis. Blood 1997; 90:2148-2159. 26. Stephenson K, Kennedy L, Hargrove L, et al. Updates on Dietary Models of Nonalcoholic Fatty Liver Disease: Current Studies and Insights. Gene Expr 2018; 18:5-17. 27. Simon T G, Bamira D G, Chung R T, et al. Nonalcoholic Steatohepatitis is Associated with Cardiac Remodeling and Dysfunction. Obesity 2017; 25:1313-1316. 28. Yasui K, Sumida Y, Mori Y, et al. Nonalcoholic steatohepatitis and increased risk of chronic kidney disease. Metabolism 2011; 60:735-739. 29. Lau J K C, Zhang X, Yu J. Animal models of non-alcoholic fatty liver disease: current perspectives and recent advances. J Pathol 2017; 241:36-44. 30. Rinella M E, Elias M S, Smolak R R, et al. Mechanisms of hepatic steatosis in mice fed a lipogenic methionine choline-deficient diet. J Lipid Res 2008; 49:1068-1076. 31. Wortham M, He L, Gyamfi M, et al. The Transition from Fatty Liver to NASH Associates with SAMe Depletion in db/db Mice Fed a Methionine Choline-Deficient Diet. Dig Dis Sci 2008; 53:2761-2774. 32. Hemmann S, Graf J, Roderfeld M, et al. Expression of MMPs and TIMPs in liver fibrosis—a systematic review with special emphasis on anti-fibrotic strategies. J Hepatol 2007; 46:955-975. 33. Elsharkawy A M, Oakley F, Mann D A. The role and regulation of hepatic stellate cell apoptosis in reversal of liver fibrosis. Apoptosis 2005; 10:927-939. 34. Krizhanovsky V, Yon M, Dickins R A, et al. Senescence of activated stellate cells limits liver fibrosis. Cell 2008; 134:657-667. 35. Schnabl B, Purbeck C H, Choi Y H, et al. Replicative senescence of activated human hepatic stellate cells is accompanied by a pronounced inflammatory but less fibrogenic phenotype. Hepatology 2003; 37:653-664. 36. Kisseleva T, Cong M, Paik Y, et al. Myofibroblasts revert to an inactive phenotype during regression of liver fibrosis. Proc Natl Acad Sci USA 2012; 109:9448-9453. 37. Seki E, De Minicis S, Osterreicher C H, et al. TLR4 enhances TGF-6 signalling and hepatic fibrosis. Nat Med 2007; 13:1324-1332. 38. Marra F, Valente A J, Pinzani M, et al. Cultured human liver fat-storing cells produce monocyte chemotactic protein-1. Regulation by proinflammatory cytokines. J Clin Invest 1993; 92:1674-1680. 39. Koyama Y, Brenner D A. Liver inflammation and fibrosis. J Clin Invest 2017; 127:55-64. 40. Brischoux-Boucher E, Trimouille A, Baujat G, et al. IL11RA-related Crouzon-like autosomal recessive craniosynostosis in ten new patients: resemblances and differences. Clin Genet 2018. Available at: http://dx.doi.org/10.1111/cge.13409.
4.5 Supplementary Materials
(176) Antibodies
(177) ACTA2 (ab7817, Abcam; IF), ACTA2 (19245, CST; WB), CD45 (103102, Biolegend), Collagen I (ab34710, Abcam), p-ERK1/2 (4370, CST), ERK1/2 (4695, CST), GAPDH (2118, CST), IgG (Aldevron), neutralizing anti-IL-11 (X203, Aldevron), neutralizing anti-IL11RA (X209, Aldevron; in vivo study), IL11RA (ab1250515, Abcam; IF), Ly6C (128039, Biolegend), TGFβ1 (141402, Biolegend), anti-rabbit HRP (7074, CST), anti-mouse HRP (7076, CST), anti-rabbit Alexa Fluor 488 (ab150077, Abcam), anti-mouse Alexa Fluor 488 (ab150113, Abcam).
(178) Recombinant Proteins
(179) Commercial recombinant proteins: Human angiotensin H (A9525, Sigma-Aldrich), human CCL2 (279-MC-050/CF, R&D Systems), human bFGF (233-FB-025, R&D Systems), human (PHC0115, Life Technologies), human PDGF (220-BB-010, R&D Systems), human TGFβ1 (PHP143B, Bio-Rad), and mouse TGFβ1 (7666-MB-005, R&D Systems).
(180) Custom recombinant proteins: Mouse Il-11 (UniProtKB: P47873) were synthesized without the signal peptide. HyperIL-11 (IL11 RA:IL-11 fusion protein), which mimics the trans-signalling complex, was constructed using a fragment of IL11 RA (amino acid residues 1-317; UniProtKB: Q14626) and IL-11 (amino acid residues 22-199, UniProtKB: P20809) with a 20 amino acid linker ((SEQ ID NO: 60) GPAGQSGGGGGSGGGSGGGSV).sup.1. All custom recombinant proteins were synthesized by GenScript using a mammalian expression system.
(181) Chemicals
(182) Hydrogen Peroxide (H.sub.2O.sub.2, 31642, Sigma), PD98059 (9900, CST), U0126 (9930, CST).
(183) Generation of Mouse Monoclonal Antibodies Against IL11RA
(184) Genetic Immunisation and Screening for Specific Binding
(185) A cDNA encoding amino acids 23-422 of human IL11RA was cloned into expression plasmids (Aldevron). Mice were immunised by intradermal application of DNA-coated gold-particles using a hand-held device for particle-bombardment. Cell surface expression on transiently transfected HEK cells was confirmed with anti-tag antibodies recognising a tag added to the N-terminus of the IL11RA protein. Sera were collected after 24 days and a series of immunisations and tested in flow cytometry on HEK293 cells transiently transfected with the aforementioned expression plasmids. The secondary antibody was goat anti-mouse IgG R-phycoerythrin-conjugated antibody (Southern Biotech, #1030-09) at a final concentration of 10 μg ml.sup.−1. Sera were diluted in PBS containing 3% FBS. Interaction of the serum was compared to HEK293 cells transfected with an irrelevant cDNA. Specific reactivity was confirmed in 2 animals and antibody-producing cells were isolated from these animals and fused with mouse myeloma cells (Ag8) according to standard procedures. Supernatant of hybridoma cultures were incubated with HEK cells expressing an IL11RA-flag construct and hybridomas producing antibodies specific for IL11RA were identified by flow cytometry.
(186) Identification of Neutralizing Anti-IL11RA Antibodies
(187) Antibodies that bound to IL11RA-flag cells but not to the negative control were considered specific binders and subsequently tested for anti-fibrotic activity on human and mice atrial fibroblasts as described by Schafer et al.sup.2. Briefly, primary human or mouse fibroblasts were stimulated with human or mouse TGFβ1, respectively (5 ng ml.sup.−1; 24 h) in the presence of the antibody candidates (6 μg ml.sup.−1). TGFβ1 stimulation results in an upregulation of endogenous IL-11, which if neutralized, blocks the pro-fibrotic effect of TGFβ1. The fraction of activated myofibroblasts (ACTA2.sup.+ve cells) was measured on the Operetta platform as described above to estimate the neutralization potential of the antibody candidates. In order to block potential trans-signalling effects, antibodies were also screened in the context of hyperIL-11 stimulation of human fibroblasts (200 μg m1.sup.−1). Three specific and neutralizing anti-IL11RA antibodies were detected, of which X209 was taken forward for in vivo studies. The same procedures were performed to obtain a neutralizing antibody that binds to the ligand IL-11.sup.3.
(188) Bindings Kinetics of X209 to IL11RA
(189) Binding of X209 to human IL11RA was measured on Biacore T200 (GE Healthcare). X209 was immobilized onto an anti-mouse capture chip. Interaction assays were performed with HEPES-buffered saline pH 7.4 containing 0.005% P20 and 0.5% BSA. A concentration range (1.56 nM to 100 nM) of the analyte (human IL11 RA) was injected over X209 and reference surfaces at a flow rate of 40 μl min.sup.−1. Binding to mouse Il11ra1 was confirmed on Octet system (ForteBio) using a similar strategy. All sensograms were aligned and double-referenced.sup.4. Affinity and kinetic constants were determined by fitting the corrected sensorgrams with 1:1 Langmuir model. The equilibrium binding constant K.sub.D was determined by the ratio of k.sub.d/k.sub.a.
(190) X209 IC.sub.50 Measurement.
(191) HSCs were stimulated with TGFβ1(5 ng m1.sup.−1, 24 h) in the presence of IgG (4 μg ml.sup.−1) and varying concentrations of X209 (4 μg ml.sup.−1 to 61 pg ml.sup.−1; 4-fold dilutions). Supernatants were collected and assayed for the amount of secreted MMP2. Dose-response curves were generated by plotting the logarithm of X209 tested concentration (pM) versus corresponding percent inhibition values using least squares (ordinary) fit. The IC.sub.50 value was calculated using log(inhibitor) versus normalized response-variable slope equation.
(192) Blood Pharmacokinetics and Biodistribution
(193) C57BL/6J mice (10-12-weeks old) were retro-orbitally injected (left eye) with 100 μl of freshly radiolabeled .sup.125I-X209 (5 μCi, 2.5 μg) in PBS. Mice were anesthetized with 2% isoflurane and blood were collected at several time points (2, 5, 10, 15, 30 m, 1, 2, 4, 6, 8 h, 1, 2, 3, 7, 14 and 21 days) post injection via submandibular bleeding. For biodistribution studies, blood was collected via cardiac puncture and tissues were harvested at the following time points: 1, 4 h, 1, 3, 7, 14, 21 days post injection. The radioactivity contents were measured using a gamma counter (2480 Wizard2, Perkin Elmer) with decay-corrections (100× dilution of 100 μl dose). The measured radioactivity was normalized to % injected dose/g tissue.
(194) RNA-Seq
(195) Generation of RNA-Seq Libraries
(196) Total RNA was quantified using Qubit RNA high sensitivity assay kit (Thermo Fisher Scientific) and RNA integrity number (RIN) was assessed using the LabChip GX RNA Assay Reagent Kit (Perkin Elmer). TruSeq Stranded mRNA Library Preparation Kit (Illumina) was used to prepare the transcript library according to the manufacturer's protocol. All final libraries were quantified using KAPA library quantification kits (KAPA Biosystems). The quality and average fragment size of the final libraries were determined using LabChip GX DNA High Sensitivity Reagent Kit (Perkin Elmer). Libraries were pooled and sequenced on a NextSeq 500 benchtop sequencer (Illumina) using NextSeq 500 High Output v2 kit and paired-end 75-bp sequencing chemistry.
(197) RNA-Seq Analysis
(198) Stiffness-induced RNA regulation in hepatic stellate cells: Normalized gene expression values were downloaded from Dou et al.sup.5. Lowly expressed genes (FPKM at baseline 2) were removed from the analysis and fold changes were calculated as average FPKM in HSCs on stiff surface divided by average FPKM in HSCs on soft surface. The fold change of RNA expression for upregulated genes (f.c.>1) was plotted and genes were ranked according to their average FPKM value.
(199) TGFβ1 stimulation of human hepatic stellate cells and antibody treatment in HFMCD: Sequenced libraries were demultiplexed using bcl2fastq v2.19.0.316 with the-no-lane-splitting option. Adapter sequences were then trimmed using trimmomatic.sup.6 v0.36 in paired end mode with the options MAXINFO:35:0.5 MINLEN:35. Trimmed reads were aligned to the Homo sapiens GRCh38 using STAR′ v. 2.2.1 with the options—outFilterType BySJout-outFilterMultimapNmax 20-alignSJoverhangMin 8-alignSJDBoverhangMin 1-outFilterMismatchNmax 999-alignIntronMin 20-alignIntronMax 1000000-alignMatesGapMax 1000000 in paired end, single pass mode. Only unique alignments were retained for counting. Counts were calculated at the gene level using the FeatureCounts module from subread.sup.8v. 1.5.1, with the options -O-s 2-J-T 8-p-R-G. The Ensembl release 86 hg38 GTF was used as annotation to prepare STAR indexes and for FeatureCounts.
(200) For the antibody treatment experiments in mouse, libraries were treated as for the human samples, only using mm10 Ensembl release 86 genome and annotation.
(201) Differential expression analyses were performed in R 3.4.1 using the Bioconductor package DESeq2.sup.9 1.18.1, using the Wald test for comparisons and including the variance shrinkage step setting a significance threshold of 0.05.
(202) Gene set enrichment analysis (GSEA) were performed in R 3.4.1 using the fgsea package and the MSigDB Hallmark genesets.sup.10,11, performing 100000 iterations. The “stat” column of the DESeq2 results output was used as ranked input for each enrichment, taking only mouse genes with one-to-one human orthologs.
(203) Enzyme-Linked Immunosorbent Assay (ELISA) and Colorimetric Assays
(204) The levels of IL-11 and MMP-2 in equal volumes of cell culture media were quantified using Human IL-11 Quantikine ELISA kit (D1100, R&D Systems) and Total MMP-2 Quantikine ELISA kit (MMP200, R&D Systems), respectively. Total secreted collagen in the cell culture supernatant was quantified using Sirius red collagen detection kit (9062, Chondrex). Total hydroxyproline content in the livers was measured using Quickzyme Total Collagen assay kit (Quickzyme Biosciences). Mouse serum levels of alanine aminotransferease (ALT), cholesterol, and triglycerides were measured using Alanine Transaminase Activity Assay Kit (ab105134, abcam), Cholesterol Assay Kit (ab65390, abcam), and Triglyceride Assay Kit (ab65336, abcam), respectively. Liver Triglycerides (TG) measurements were performed using triglyceride colorimetric assay kit (Ser. No. 10/010,303, Cayman). All ELISA and colorimetric assays were performed according to the manufacturer's protocol.
(205) Matrigel Invasion Assay
(206) The invasive behavior of human HSCs was assayed using 24-well Boyden chamber invasion assays (Cell Biolabs Inc.). Equal numbers of HSCs in serum-free HSC media were seeded in triplicates onto the ECM-coated matrigel and were allowed to invade towards HSC media containing 0.2% FBS. After 48 h of incubation with stimuli, media was aspirated and non-invasive cells were removed using cotton swabs. The cells that invaded towards the bottom chamber were stained with cell staining solution (Cell Biolabs Inc.) and invasive cells from 5 non-overlapping fields/membrane were imaged and counted under 40× magnification. For antibody inhibition experiments, HSCs were pretreated with X203, X209, or IgG control antibodies for 15 m prior to addition of stimuli.
(207) Precision Cut Liver Slices (PCLS) and Western Blotting of NASH Patient Liver
(208) Briefly, human PCLS were cut and incubated with TGFβ1 for 24 h. ELISA from the supernatant was performed using Human IL-11 DuoSet (DY218, R&D Systems). This CRO also collected liver biopsies from patients undergoing liver resections for cancers where adjacent, non-cancerous tissue was collected for molecular studies. Patients had either no documented intrinsic liver disease (controls) or previously documented alcoholic liver disease, primary biliary cirrhosis, primary sclerosing cholangitis or NASH. For confidentiality reasons no further information was provided for these samples.
(209) Quantitative Polymerase Chain Reaction (qPCR)
(210) Total RNA was extracted from either the snap-frozen liver tissues or HSCs lysate using Trizol (Invitrogen) followed by RNeasy column (Qiagen) purification. The cDNAs were synthesized with iScript™ cDNA synthesis kit (Bio-Rad) according to manufacturer's instructions. Gene expression analysis was performed on duplicate samples with either TaqMan (Applied Biosystems) or fast SYBR green (Qiagen) technology using StepOnePlus™ (Applied Biosystem) over 40 cycles. Expression data were normalized to GAPDH mRNA expression and fold change was calculated using 2.sup.−ΔΔCt method. The sequences of specific TaqMan probes and SYBR green primers are available upon request.
(211) Immunoblotting
(212) Western blots were carried out on total protein extracts from HSCs and liver tissues. Both cells and frozen tissues were homogenized in radioimmunoprecipitation assay (RIPA) buffer containing protease and phosphatase inhibitors (Thermo Scientifics), followed by centrifugation to clear the lysate. Protein concentrations were determined by Bradford assay (Bio-Rad). Equal amount of protein lysates were separated by SDS-PAGE, transferred to PVDF membrane, and subjected to immunoblot analysis for the indicated primary antibodies. Proteins were visualized using the ECL detection system (Pierce) with the appropriate secondary antibodies.
(213) Histology Liver tissues were fixed for 48 h at RT in 10% neutral-buffered formalin (NBF), dehydrated, embedded in paraffin blocks and sectioned at 7 μm. Sections stained with Masson's Trichrome were examined by light microscopy. Each histology experiment was repeated independently with similar results from n=3/group Images of the sections were captured and blue-stained fibrotic areas were semi-quantitatively determined with Image-J software (color deconvolution-Masson Trichrome) from 4 sections/liver. Treatment and genotypes were not disclosed to investigators performing the histology and generating semi-quantitative readouts.
REFERENCES TO SUPPLEMENTARY MATERIALS
(214) 1. Dams-Kozlowska H, Gryska K, Kwiatkowska-Borowczyk E, et al. A designer hyper interleukin 11 (H11) is a biologically active cytokine. BMC Biotechnol 2012; 12:8. 2. Schafer S, Viswanathan S, Widjaja A A, et al. IL-11 is a crucial determinant of cardiovascular fibrosis. Nature 2017; 552:110-115. 3. Cook S, Ng B, Dong J, et al. IL-11 is a therapeutic target in idiopathic pulmonary fibrosis. 2018. Available at: http://dx.doi.org/10.1101/336537. 4. Myszka D G. Improving biosensor analysis. J Mol Recognit 1999; 12:279-284. 5. Dou C, Liu Z, Tu K, et al. P300 Acetyltransferase Mediates Stiffness-Induced Activation of Hepatic Stellate Cells Into Tumor-Promoting Myofibroblasts. Gastroenterology 2018; 154:2209-2221.e14. 6. Bolger A M, Lohse M, Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 2014; 30:2114-2120. 7. Dobin A, Davis C A, Schlesinger F, et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 2013; 29:15-21. 8. Liao Y, Smyth G K, Shi W. The Subread aligner: fast, accurate and scalable read mapping by seed-and-vote. Nucleic Acids Res 2013; 41:e108. 9. Love M I, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol 2014; 15:550. 10. Liberzon A, Subramanian A, Pinchback R, et al. Molecular signatures database (MSigDB) 3.0. Bioinformatics 2011; 27:1739-1740. 11. Subramanian A, Tamayo P, Mootha V K, et al. Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci USA 2005; 102:15545-15550.
Example 5: Autocrine IL11 Cis-Signaling in Hepatocytes is an Initiating Nexus Between Lipotoxicity and Non-Alcoholic Steatohepatitis (NASH)
5.1 Overview
(215) IL11 signaling is important in non-alcoholic steatohepatitis (NASH). In the present Example, the inventors show that lipid-laden hepatocytes secrete IL11, which acts through an autocrine cis-signaling loop to cause lipoapoptosis. While IL6 protects hepatocytes, IL11 causes hepatocyte death through activation of non-canonical signaling pathways and upregulation of NOX4 and reactive oxygen species. In two preclinical models, hepatocyte-specific deletion of Il11ra1 protected mice from all aspects of NASH. In addition, restoration of IL11 cis-signaling in hepatocytes only in global Il11ra1 knock out mice reconstituted steatosis and inflammation. No evidence was found to support the existence of IL6 or IL11 trans-signaling. The inventors conclude that hepatocyte lipotoxicity stimulates IL11 secretion leading to hepatocyte death that is followed by fibrosis and inflammation. These data outline a new, hepatocyte-specific mechanism for the transition from non-alcoholic fatty liver disease to NASH.
5.2 Introduction
(216) Interleukin 11 (IL11) is a key fibrogenic factor (Ng et al., 2019; Schafer et al., 2017) that is elevated in fibrotic precision-cut liver slices across species (Bigaeva et al., 2019). IL11 has been shown to have negative effects on hepatocyte function after toxic liver insult (Widjaja et al.) and, directly or indirectly, contributes to nonalcoholic steatohepatitis (NASH) pathologies (Widjaja et al., 2019). At the other end of the spectrum, a number of earlier publications suggested that IL11 is protective in mouse models of ischemic-, infective- or toxin-induced liver damage (Bozza et al., 1999; Maeshima et al., 2004; Nishina et al., 2012; Trepicchio et al., 2001; Yu et al., 2016; Zhu et al., 2015). However, it is now apparent that the recombinant human IL11 (rhIL11) reagent used in earlier studies is ineffective in the mouse (Widjaja et al.).
(217) IL11 is a member of the interleukin 6 (IL6) cytokine family and, like IL6, binds to its membrane-bound alpha receptor (IL11 RA) and glycoprotein 130 (gp130) to signal in cis. IL6 itself has been linked to liver function and publications suggest an overall beneficial effect (Klein et al., 2005; Kroy et al., 2010; Matthews et al., 2010; Schmidt-Arras and Rose-John, 2016; Wuestefeld et al., 2003). However, it is also thought that IL6 can bind to soluble IL6 receptor (sIL6R) and signal in trans, which is considered maladaptive (Schmidt-Arras and Rose-John, 2016). It is possible that IL11, like IL6, signals in a pathogenic mode in trans but experiments to date have found no evidence for this in tumors or reproductive tissues (Agthe et al., 2017; Balic et al., 2017).
(218) The factors underlying the transition from non-alcoholic fatty liver disease (NAFLD) to NASH are multifactorial but lipid loading of hepatocytes is centrally important (Friedman et al., 2018). Certain lipid species are toxic for hepatocytes and this lipotoxicity stimulates cytokine release causing hepatocyte death and paracrine activation of hepatic stellate cells (HSCs) and immune cells (Farrell et al., 2018; Friedman et al., 2018). Lipotoxicity, such as that due to palmitate (Kakisaka et al., 2012), is an early event in NASH and represents a linkage between diet, NAFLD and NASH. While genetic or pharmacological inhibition of IL6 cis-signaling worsens steatosis phenotypes (Kroy et al., 2010; Matthews et al., 2010; Yamaguchi et al., 2010), a role for IL11 in hepatic lipotoxicity has not been described.
(219) In the present Example, a range of in vitro and in vivo approaches were used to address key questions regarding IL11 in hepatocyte biology, NAFLD and NASH: (1) Defining the true role of IL11 cis- and trans-signaling in human hepatocytes, (2) examining whether lipotoxicity is related to IL11 activity in hepatocytes, (3) establishing whether IL11 or IL6 trans-signaling contributes to NASH, (4) dissecting the inter-relationship between IL11 cis-signaling in hepatocytes and the development of steatosis, hepatocyte death, inflammation, and fibrosis. These studies reveal unexpected aspects of IL6 and IL11 biology and demonstrate an unambiguous pathogenic effect of lipotoxicity-activated, autocrine IL11 cis-signaling in hepatocytes that initiates the transition from NAFLD to NASH.
5.3 Results
5.3.1 Synthetic IL11 Trans-Signaling Constructs Cause Hepatocyte Death
(220) The inventors first assessed the expression levels of IL6R, IL11 RA and gp130 in primary human hepatocytes by flow cytometry. Robust expression of IL11 RA and gp130 was observed in the large majority of cells (92.6% and 91.9%, respectively) but only few hepatocytes (3.0%) expressed IL6R, and at low levels (
(221) Given the lack of IL6R expression by human hepatocytes the inventors employed a synthetic IL6 trans-signaling construct (hyperIL6) to activate IL6 signaling in these cells and compared this with a synthetic IL11 trans-signaling complex (hyperIL11). HyperIL11, like IL11 itself (Widjaja et al.), activated ERK and JNK in a dose-dependent manner (2.5 ng/ml to 20 ng/ml). By contrast, IL6 trans-signaling did not activate non-canonical signaling pathways but instead dose-dependently induced STAT3 activation (
(222) HyperIL11 caused a dose-dependent increase in alanine transaminase (ALT) in the media of primary human hepatocyte cell cultures whereas hyperIL6 (20 ng/ml) was found to have a significant, albeit limited, cytoprotective effect (fold change (FC)=0.9; P=0.0468) (
(223) The inventors then performed experiments in order to detect IL11 trans-signaling in the absence of the artificial protein complexes hyperIL6 or hyperIL11. Cells were stimulated with IL11 in the presence of either soluble gp130 (sgp130, to inhibit putative trans-signaling) or soluble IL11 RA (sIL11RA, to potentiate putative trans-signaling). IL11-induced hepatocyte death and signaling were unaffected by sgp130 or sIL11 RA (
5.3.2 IL11 Cis-Signaling Underlies Lipotoxicity in Hepatocytes
(224) In order to address the role of IL11 in fatty liver disease, the inventors modelled hepatocyte lipotoxicity, viewed as an initiating pathology for NASH and related to cytokine release from damaged hepatocytes (Friedman et al., 2018). Hepatocytes were loaded with palmitate:BSA mixture at a ratio of 6:1 using a concentration of saturated fatty acids (sFSs) seen in the serum of NAFLD patients (Kleinfeld et al., 1996). Compared to control, sFA loaded cells secreted greater amounts of IL11 (FC=28, P<0.0001) and also IL6, CCL2 and CCL5 (
(225) To test if IL11 secretion from lipid loaded hepatocytes was functionally related to lipotoxicity in a cis- or trans-signaling manner, cells were incubated either with anti-IL11 RA antibody (X209) or sgp130. X209 reduced the secretion of all cytokines, including IL11 itself, whereas sgp130 had no effect (
(226) The inventors next examined signaling events. Lipotoxicity is strongly associated with activation of JNK, which drives caspase-3 activation and lipoapoptosis. Accordingly, palmitate loaded hepatocytes displayed JNK activation and caspase-3 cleavage, as well as ERK phosphorylation (
5.3.3 No Evidence for IL11 or IL6 Trans-Signaling in Two NASH Models
(227) The inventors then investigated whether trans-signaling underlies NASH in vivo using two preclinical mouse NASH models: The Western Diet supplemented with fructose (WDF) model and the methionine- and choline-deficient high fat diet (HFMCD) model. The WDF model is associated with obesity, hyperlipidemia and insulin resistance and seen as translatable to common forms of human NASH, as in diabetic patients. The HFMCD model stimulates rapid onset NASH, specifically driven by hepatocyte lipotoxicity, which is associated with weight loss in the absence of insulin resistance. Lipotoxicity is common to both models whereas obesity and insulin resistance are not.
(228) Three weeks prior to starting either the WDF and HFMCD diet, mice were injected with an AAV8 virus encoding either albumin promoter-driven sgp130 (AAV8-Alb-sgp130), which contains the whole extracellular domain of mouse gp130 protein (amino acid 1 to 617), or albumin promoter alone (AAV8-Alb-Null) (
(229) After 16 weeks of WDF, IL11 levels were strongly upregulated in the liver and the periphery but IL6 expression was not affected (
(230) In a second set of experiments NASH was induced using the HFMCD diet (
(231) At the signaling level, both WDF and HFMCD diets stimulated ERK and JNK activation, consistent with elevated IL11 cis-signaling (
5.3.4 Hepatocyte-Specific IL11 Cis-Signaling is Required to Initiate NASH
(232) While no evidence was found to support IL11 trans-signaling in NASH models, overall the data suggested increased IL11 effects in hepatocytes, presumed in cis. To test this premise, the inventors administered AAV8-Alb-Cre to Il11ra.sub.loxP/loxP mice to delete Il11ra1 in hepatocytes only (CKO mice). CKO mice were then fed either normal chow (NC), HFMCD diet or WDF (
(233) In addition to rapidly stimulating lipotoxicity-driven NASH (Stephenson et al., 2018) the HFMCD diet causes weight loss (Stephenson et al., 2018). Surprisingly, weight loss in mice on the HFMCD diet was initially limited and later reversed in CKO mice (
(234) By gross morphology, histology and quantitative triglyceride analysis, the CKO mice on either HFMCD or WDF diet were robustly protected from steatosis (
(235) Liver fibrosis was greatly reduced in CKO mice on either NASH diet as compared to controls (reduction: HFMCD, 87%; WDF, 64%; P<0.001 for both) (
5.3.5 Reconstitution of Hepatocyte-Specific IL11 Cis-Signaling in IL11ra1 Null Mice Restores Steatohepatitis but not Liver Fibrosis
(236) In vivo gain-of-function experiments were employed to complement loss-of-function experiments using the CKO mice. The inventors investigated whether restoring IL11 cis- or trans-signaling specifically in hepatocytes in mice with global Il11ra1 deletion (Il11ra1−/− knockouts (KOs)) resulted in disease. KO mice were injected with AAV8 encoding either the full length, membrane bound Il11ra1 (mbIl11ra1; to reconstitute cis-signaling) or a secreted/soluble form of Il11ra1 (sIl1ra1, which constitutes the extracellular portion of Il11ra1; to enable trans-signaling) or a control construct and the animals were then fed with NC, HFMCD diet or WDF (
(237) KO mice injected with AAV8-Alb-mbIl11ra1 re-expressed IL11RA1 on hepatocytes and KO mice injected with AAV8-Alb-sIl11ra1 displayed increased expression of sIL11RA1 in both the liver and the circulation (
(238) Restoration of IL11 cis-signaling in KO mice using mbIl11ra1 recapitulated hepatic steatosis and inflammation that was evident from gross morphology to molecular patterns of gene expression and signaling (
5.4 Discussion
(239) Metabolic liver disease commonly occurs in the context of obesity and type 2 diabetes and manifests initially as NAFLD that can progress to NASH (Friedman et al., 2018; Sanyal, 2019). A key underlying pathology in progression to NASH is “substrate overload”, whereby an abundance of metabolites overrun the hepatocyte's ability to process fat, causing lipotoxicity. Cytokines are key NASH factors secreted from lipotoxic hepatocytes (Friedman et al., 2018) and here the inventors establish IL11 as an important component of the lipotoxic milieu and a driver of NAFLD-to-NASH transition.
(240) A large body of evidence supports the idea that IL6 signaling in the liver is beneficial (Kroy et al., 2010; Schmidt-Arras and Rose-John, 2016; Yamaguchi et al., 2010). However, a pathogenic role for IL6 trans-signaling in hepatic steatosis has been proposed (Kammoun et al., 2017; Wieckowska et al., 2008). The inventors found using synthetic constructs that hyperIL11, initiating IL11 trans-signaling, is cytotoxic, whereas hyperIL6 is protective in hepatocytes. However, there was no evidence for trans-signaling in a biologically relevant context in vitro or in vivo, using both gain- and loss-of-function. This suggests that IL6 family member trans-signaling plays no role in NASH, which is in agreement with previous studies outside the liver (Agthe et al., 2017; Balic et al., 2017). The relevance of these findings for other diseases is unclear and clinical trials are underway targeting trans-signaling in ulcerative colitis (Kang et al., 2019). Previous studies have suggested that IL6R is expressed in hepatocytes (Schmidt-Arras and Rose-John, 2016) and so it was surprising that primary human hepatocytes were found to express very little/no IL6R. This may reflect a strong reliance on transformed hepatocyte-like cells (e.g. HepG2) in earlier studies.
(241) Here the inventors show the critical importance of IL11 cis-signaling in hepatocytes for NASH. This effect was established using both hepatocyte-specific loss-of-function on a wildtype genetic background and also hepatocyte-specific gain-of-function on an Il11ra1 null background. This overturns the suggestion in the literature that IL11 is protective for hepatocytes based on the use of rhIL11, ineffective in the mouse, in murine models of liver disease (Maeshima et al., 2004; Nishina et al., 2012; Trepicchio et al., 2001; Zhu et al., 2015). Importantly, while restoration of hepatocyte-specific IL11 cis-signaling causes steatohepatitis in KO mice, fibrosis is not restored whereas it was prevented in the CKO. This demonstrates that IL11 cis-signaling in HSCs is required for liver fibrosis and places hepatocyte dysfunction upstream of HSC activation.
(242) The inventors propose a mechanistic model for NASH whereby lipid loaded hepatocytes secrete IL11 leading to autocrine cell death, paracrine activation of HSCs and secondary inflammatory cell activation and infiltration (
5.5 Materials and Methods for Example 5
5.5.1 AAV8 Vectors
(243) All Adeno-associated virus serotype 8 (AAV8) vectors were synthesized by Vector Biolabs. AAV8 vector carrying a mouse membrane-bound Il11ra1 cDNA (NCBI accession number: BC069984), a mouse soluble Il11ra1 cDNA, and a mouse soluble gp130 cDNA driven by Albumin (Alb) promoter is referred to as AAV8-Alb-mbIl11ra1, AAV8-Alb-sIl11ra1, and AAV8-Alb-sgp130, respectively. AAV8-Alb-sgp130 and AAV8-Alb-sIl11ra1 were constructed by removing the transmembrane and cytoplasmic regions of mouse gp130 sequence (NCBI accession number: BC058679) and mouse Il11ra1 sequence, respectively. AAV8-Null vector was used as vector control. To specifically delete Il11ra1 in Albumin-expressing cells, AAV8-Alb-iCre vector was injected to mice homozygous for LoxP-flanked Il11ra1 alleles (Il11ra1.sub.loxP/loxP mice).
5.5.2 Antibodies
(244) Albumin (ab207327, Abcam), Alexa Fluor 488 secondary antibody (ab150077, Abcam), Cleaved Caspase-3 (9664, CST), Caspase3 (9662, CST) p-ERK1/2 (4370, CST), ERK1/2 (4695, CST), GAPDH (2118, CST), gp130 (PA5-28932, Thermo Fisher), IL6 (AF506, R&D systems), IL6R (flow cytometry, ab222101, Abcam), IL6R (for immunofluorescence staining, MA1-80456, Thermo Fisher), IL11 (Aldevron), IL11 RA (flow cytometry and immunofluorescence staining, ab125015, Abcam), IL11 RA (western blot, 130920, Santa Cruz), p-JNK (4668, CST), JNK (9258, CST), p-STAT3 (4113, CST), STAT3 (4904, CST), mouse HRP (7076, CST), rabbit HRP (7074, CST), rat HRP (31470, Santa Cruz).
5.5.3 Recombinant Proteins
(245) Commercial recombinant proteins: Human hyperlL6 (IL6R:IL6 fusion protein, 8954-SR, R&D systems), human soluble gp130 Fc (671-GP-100, R&D systems), human IL11 RA (8895-MR-050, R&D systems). Custom recombinant proteins: Human IL11 (UniProtKB:P20809, Genscript). Human hyperIL11 (IL11 RA:IL11 fusion protein), which mimics the trans-signalling complex, was constructed using a fragment of IL11RA (amino acid residues 1-317; UniProtKB: Q14626) and IL11 (amino acid residues 22-199, UniProtKB: P20809) with a 20 amino acid linker (SEQ ID NO:20) (Schafer et al., 2017).
5.5.4 Chemicals
(246) Palmitate (P5585, Sigma), Paraformaldehyde (PFA, 28908; Thermo Fisher), phorbol 12-myristate 13-acetate (PMA, P1585, Sigma), Triton X-100 (T8787, Sigma), and 4′,6-diamidino-2-phenylindole (D1306; Thermo Fisher).
5.5.5 Primary Human Hepatocytes Culture
(247) Primary human hepatocytes (5200, ScienCell) were maintained in hepatocyte medium (520, ScienCell) supplemented with 2% fetal bovine serum, 1% Penicillin-streptomycin at 37° C. and 5% CO2. Hepatocytes (P2-P3) were serum-starved overnight unless otherwise specified in the methods prior to 24 hours stimulation with different doses of various recombinant proteins as described.
5.5.6 THP-1 Culture
(248) THP-1 (ATCC) were cultured in RPMI 1640 (A1049101, Thermo Fisher) supplemented with 10% FBS and 0.05 mM β-mercaptoethanol. THP-1 cells were differentiated with 10 ng/ml of PMA in RPMI 1640 for 48 hours.
5.5.7 Palmitate (Saturated Fatty Acid) Treatment In Vitro
(249) Palmitate:BSA conjugated solution in the ratio of 6:1 was prepared as described earlier (Alsabeeh et al., 2018). Palmitate (0.5 mM) conjugated in fatty acids free BSA was used to treated cells as described in figure legends; 0.5% BSA solution was used as control.
5.5.8 Flow Cytometry
(250) For surface IL11RA, IL6R, and gp130 analysis, primary human hepatocytes and THP-1 cells were stained with IL11RA, IL6R, or gp130 antibody and the corresponding Alexa Fluor 488 secondary antibody. Cell death analysis was performed by staining primary human hepatocytes with Dead Cell Apoptosis Kit with Annexin V FITC and PI (V13242, Thermo Fisher). PI+ve cells were then quantified with the flow cytometer (Fortessa, BD Biosciences) and analyzed with FlowJo version X software (TreeStar).
5.5.9 Immunofluorescence
(251) Primary human hepatocytes were seeded on 8-well chamber slides (1.5×10.sup.4 cells/well) 24 hours before the staining. Cells were fixed in 4% PFA for 20 minutes, washed with PBS, and non-specific sites were blocked with 5% BSA in PBS for 2 hours. Cells were incubated with IL11RA, IL6R, gp130, or Albumin antibody overnight (4° C.), followed by incubation with the appropriate Alexa Fluor 488 secondary antibody for 1 hour. Chamber slides were dried in the dark and 5 drops of mounting medium with DAPI were added to the slides for 15 minutes prior to imaging by fluorescence microscope (Leica).
5.5.10 Oil Red O Staining
(252) Primary human hepatocytes were seeded on 8-well chamber slides (1×10.sup.4 cells/well) Following 24 hours of palmitate treatment, cells were fixed in 10% PFA for 30 minutes, washed with distilled water, and incubated with 60% (v/v) isopropyl alcohol for 5 minutes. Cells were then stained with Oil Red O Solution for 30 minutes and washed with distilled water prior to imaging with bright field microscope (BX53, Olympus). The lipid droplets were identified by their red staining.
5.5.11 Reactive Oxygen Species (ROS) Detection
(253) Primary human hepatocytes were seeded on 8-well chamber slides (1×10.sup.4 cells/well). For this experiment, cells were not serum-starved prior to palmitate treatment. 24 hours following palmitate stimulation, cells were washed, incubated with 25 μM of DCFDA solution (ab113851, Abcam) for 45 minutes at 37° C. in the dark, and rinsed with dilution buffer according to the manufacturer's protocol. Live cells with positive DCF staining were imaged with filter set appropriate for fluorescein (FITC) using a fluorescence microscope (Leica).
5.5.12 Animal Models
(254) Animal experiments were performed under the guidelines of SingHealth Institutional Animal Care and Use Committee (IACUC). Mice were maintained in SPF environment and provided with food and water ad libitum.
(255) Mouse Models of Metabolic Liver Disease
(256) HFMCD
(257) 6-8 weeks old C57BL/6N, Il11ra1−/− mice, and Il11ra1.sub.loxP/loxP and their respective control were fed with methionine- and choline-deficient diet supplemented with 60 kcal % fat (HFMCD, A06071301B16, Research Diets) for 4 weeks. Control mice received normal chow (NC, Specialty Feeds).
(258) WDF
(259) 6-8 weeks old C57BL/6N, Il11ra1−/− mice, and Il11ra1.sub.loxP/loxP and their respective control were fed western diet (D12079B, Research Diets) supplemented with 15% weight/volume fructose in drinking water (WDF) for 16 weeks. Control mice received NC and tap water.
(260) Il11ra1-Deleted Mice (KO)
(261) 6-8-week old male Il11ra1−/− mice (B6.129S1-Il11ratm1Wehi/J, Jackson's Laboratory) were intravenously injected with 4×10.sup.11 genome copies (gc) of AAV8-Alb-mbIl11ra1 or AAV8-Alb-sIl11ra1 virus to induce hepatocyte specific expression of mouse Il11ra1 or soluble Il11ra1, respectively. As controls, both Il11ra1−/− mice and their wildtype littermates (Il11ra1+/+) were intravenously injected with 4×10.sup.11 gc AAV8-Alb-Null virus. 3 weeks after virus injection, mice were fed with HFMCD, WDF, or NC. Durations of diet are described.
(262) In Vivo Administration of Soluble Gp130
(263) 6-8-week old male C57BL/6N mice (InVivos) were injected with 4×10.sup.11 gc AAV8-Alb-sgp130 virus to induce hepatocyte specific expression of soluble gp130; control mice were injected with 4×10.sup.11 gc AAV8-Alb-Null virus. 3 weeks following virus administration, mice were fed with HFMCD, WDF, or NC for durations that are described.
(264) Il11ra-Floxed Mice (CKO)
(265) Il11ra-floxed mice, in which exons 4 to 7 of the Il11ra1 gene were flanked by loxP sites, were created using CRISPR/Cas9 system as previously described (Ng et al.). To induce the specific deletion of Il11ra1 in hepatocytes, 6-8-week old male homozygous Il11ra1-floxed mice were intravenously injected with AAV8-Alb-Cre virus (4×10.sup.11 gc); a similar amount of AAV8-Alb-Null virus were injected into homozygous Il11ra1-floxed mice as controls. The AAV8-injected mice were allowed to recover for three weeks prior to HFMCD, WDF, or NC feeding. Knockdown efficiency was determined by Western blotting of hepatic IL11RA.
5.5.13 RNA-Sequencing (RNA-Seq) and Ribosome Profiling (Ribo-Seq)
(266) RNA-seq and Ribo-Seq library preparations were performed as previously described (Chothani et al., 2019).
(267) Generation of RNA-Seq Libraries
(268) Total RNA was extracted from human hepatocytes using RNeasy columns (Qiagen). RNA was quantified using a Qubit RNA High-Sensitivity Assay kit (Life Technologies) and its quality was assessed on the basis of their RNA integrity number using the LabChip GX RNA Assay Reagent Kit (Perkin Elmer). TruSeq Stranded mRNA Library Preparation kit (Illumina) was used to measure transcript abundance following standard instructions from the manufacturer.
(269) Generation of Ribo-Seq Libraries
(270) Hepatocytes were grown to 90% confluence in a 10 cm culture dish and lysed in 1 mL cold lysis buffer (formulation as in TruSeq® Ribo Profile Mammalian Kit, RPHMR12126, Illumina) supplemented with 0.1 mg/mL cycloheximide. Homogenized and cleared lysates were then footprinted with Truseq Nuclease (Illumina) according to the manufacturer's instructions. Ribosomes were purified using Illustra Sephacryl S400 columns (GE Healthcare), and the protected RNA fragments were extracted with a standard phenol:chloroform:isoamylalcohol technique. Following ribosomal RNA removal (Mammalian RiboZero Magnetic Gold, Illumina), sequencing libraries were then prepared out of the footprinted RNA by using TruSeq® Ribo Profile Mammalian Kit according to the manufacturer's protocol.
(271) The final RNA-seq and ribosome profiling libraries were quantified using KAPA library quantification kits (KAPA Biosystems) on a StepOnePlus Real-Time PCR system (Applied Biosystems) according to the manufacturer's protocol. The quality and average fragment size of the final libraries were determined using a LabChip GX DNA High Sensitivity Reagent Kit (Perkin Elmer). Libraries with unique indexes were pooled and sequenced on a NextSeq 500 benchtop sequencer (Illumina) using NextSeq 500 High Output v2 kit and paired-end 75-bp sequencing chemistry.
(272) Data Processing and Analyses for RNA-Sequencing and Ribosome Profiling
(273) Raw sequencing data were demultiplexed with bcl2fastq V2.19.0.316 and the adaptors were trimmed using Trimmomatic (Bolger et al., 2014) V0.36, retaining reads longer than 20 nt post-clipping. Ribo-seq reads were aligned using bowtie (Langmead et al., 2009) to known mtRNA, rRNA and tRNA sequences (RNACentral(The RNAcentral Consortium, 2017), release 5.0) and only unaligned reads were retained as Ribosome protected fragments (RPFs). Alignment to the human genome (hg38) was carried out using STAR (Dobin et al., 2012). Gene expression was quantified on the CDS (coding sequence) regions for Ribo-seq and exonic regions for RNA-seq using uniquely mapped reads (Ensembl database release GRCh38 v86) with feature counts (Liao et al., 2014). TPM was calculated and visualized using boxplot to compare baseline expression of IL11RA (ENSG00000137070), IL6R (ENSG00000160712), and gp130 (ENSG00000134352). Read coverage using Ribo-seq and RNA-seq reads for IL11RA, IL6R and gp130 was visualized using Gviz R package (Hahne and Ivanek, 2016) with strand specific alignment files.
5.5.14 Colorimetric Assays
(274) Alanine Aminotransferase (ALT) activity in the cell culture supernatant and mouse serum was measured using ALT Activity Assay Kit (ab105134, Abcam). Liver Glutathione (GSH) levels were measured using Glutathione Colorimetric Detection Kit (EIAGSHC, Thermo Fisher). Total hydroxyproline content in mouse livers was measured using Quickzyme Total Collagen assay kit (QZBtotco15, Quickzyme Biosciences). The levels of serum and liver triglycerides were measured using Triglyceride Assay Kit (ab65336, Abcam). Mouse serum levels of Aspartate Aminotransferase (AST) and cholesterol were measured using AST Assay Kit (ab105135, Abcam) and Cholesterol Assay Kit (ab65390; Abcam), respectively. All colorimetric assays were performed according to the manufacturer's protocol.
5.5.15 Enzyme-Linked Immunosorbent Assay (ELISA)
(275) The levels of gp130 in mouse serum were quantified using Mouse gp130 DuoSet ELISA (DY468, R&D systems) according to the manufacturer's protocol.
5.5.16 RT-qPCR
(276) Total RNA was extracted from snap-frozen liver tissues using Trizol (Invitrogen) and RNeasy Mini Kit (Qiagen). PCR amplifications were performed using iScript cDNA Synthesis Kit (Biorad). Gene expression was analyzed in duplicate by TaqMan (Applied Biosystems) or SYBR green (Qiagen) technology using StepOnePlus (Applied Biosystem) over 40 cycles. Expression data were normalized to GAPDH mRNA expression and fold change was calculated using 2-ΔΔCt method. The sequences of specific TaqMan probes and SYBR green primers are available upon request.
5.5.17 Immunoblotting
(277) Western blots were carried out on total protein extracts from hepatocytes and liver tissues. Hepatocyte and liver tissue lysates were homogenized in RIPA Lysis and Extraction Buffer (89901, Thermo Scientific) containing protease and phosphatase inhibitors (Roche). Protein lysates were separated by SDS-PAGE and transferred to PVDF membranes. Protein bands were visualized using the ECL detection system (Pierce) with the appropriate secondary antibodies: anti-rabbit HRP or anti-mouse HRP.
5.5.18 Liver Tissue Processing and Histological Analysis
(278) Liver samples were fixed in 10% neutral formalin, paraffinized, cut into 5-μm sections, stained with hematoxylin and eosin (H&E) according to standard protocol, and examined by light microscopy.
5.5.19 Statistical Analysis
(279) All statistical analyses were performed using Graph Pad Prism software (version 6.07). P values were corrected for multiple testing according to Dunnett's (when several experimental groups were compared to one condition), Tukey (when several conditions were compared to each other within one experiment), Sidak (when several conditions from 2 different genotypes were compared to each other). Analysis for two parameters for comparison of two different groups were performed by two-way ANOVA. The criterion for statistical significance was set at P<0.05.
5.6 References to Example 5
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Example 6: Dissecting Fibro-Inflammatory Mechanisms in Pancreatitis
(284) Chronic pancreatitis is an aetiologically heterogeneous fibro-inflammatory syndrome, which leads to exocrine and endocrine pancreatic insufficiency.
(285) Pancreatic stellate cells (PSC) exist in two states and are the predominant fibrogenic cell type involved in pancreatic injury. PSCs are part of a wider retinoid-storing cellular network in the body, including cells in liver parenchyma (HSC). IL-11 has recently been shown to have a key role in HSC transformation, a defining pathology in NASH (Widjaja et al., Gastroenterology (2019) 157(3): 777-792).
(286) Single-cell RNA sequencing (scRNA-seq) analysis of pancreatic tissue from Mus musculus (from the Tabula muris Consortium data—Schaum et al., Nature (2018) 562: 367-372) reveals that PSCs (and ductal cells) display high expression of Il11ra1 but not Il6ra—see
(287) PSC activation was show to be triggered in vitro by treatment with IL-11, and was inhibited by treatment with an anti-IL-11 RA antibody antagonist of IL-11 mediated signalling, irrespective of the stimulus for PSC activation (i.e. TGFβ1, IL-11, bFGF, CTGF, PDGF or ET-1)—see
(288) Transgenic mice having inducible, fibroblast-specific expression of IL-11 develop pancreatic fibrosis—see
(289) In a pancreatic duct ligation (PDL) model of pancreatic injury, treatment with an anti-IL-11 RA antibody antagonist of IL-11 mediated signalling reduced pancreatic fibrosis and inhibited the reduction in pancreatic tissue associated with PDL—see