Treatment of hepatotoxicity with IL-11 antibody
11319368 · 2022-05-03
Assignee
Inventors
- Stuart Alexander Cook (Singapore, SG)
- Sebastian Schaefer (Singapore, SG)
- Anissa Widjaja (Singapore, SG)
Cpc classification
C07K16/2866
CHEMISTRY; METALLURGY
A61K31/198
HUMAN NECESSITIES
A61K38/1793
HUMAN NECESSITIES
C07K2317/76
CHEMISTRY; METALLURGY
A61K31/198
HUMAN NECESSITIES
A61K2300/00
HUMAN NECESSITIES
A61K2300/00
HUMAN NECESSITIES
C12N15/1136
CHEMISTRY; METALLURGY
A61P1/16
HUMAN NECESSITIES
International classification
A61P1/16
HUMAN NECESSITIES
C07K16/24
CHEMISTRY; METALLURGY
C07K16/28
CHEMISTRY; METALLURGY
C12N15/113
CHEMISTRY; METALLURGY
Abstract
Methods of treating and preventing hepatotoxicity 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 acetaminophen (APAP) induced liver injury, the method comprising administering a therapeutically effective amount of an anti-IL-11 antibody or an antigen-binding fragment thereof which is an antagonist of the IL-11 mediated signaling to a subject.
2. The method according to claim 1, wherein the anti-IL-11 antibody or an antigen-binding fragment thereof which is an antagonist of the IL-11 mediated signaling is 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 2, wherein the interleukin 11 receptor is or comprises IL-11Rα.
4. The method according to claim 1, wherein the method of treating APAP-induced liver injury further comprises treatment with N-acetylcysteine.
5. The method according to claim 1, wherein the method of treating comprises administering the anti-IL-11 antibody or an antigen-binding fragment thereof which is an antagonist of the IL-11 mediated signaling 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 method of treating comprises administering the anti-IL-11 antibody or an antigen-binding fragment thereof which is an antagonist of the IL-11 mediated signaling 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.
7. The method according to claim 1, wherein the method of treating 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 anti-IL-11 antibody or an antigen-binding fragment thereof which is an antagonist of the IL-11 mediated signaling to a subject in which expression of interleukin 11 (IL-11) or a receptor for IL-11 (IL-11R) is upregulated.
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
(34) In the following examples, the inventors demonstrate that IL-11 directly impairs hepatocyte survival and that anti-IL-11 therapy can ameliorate hepatotoxicity. The inventors demonstrate the ability of IL-11 antagonist administered prior to DILI to protect against hepatocyte death and preserve liver function, and also show that IL-11 antagonist administered after DILI can reverse symptoms of liver damage and restore liver function.
Example 1: Effect of IL-11 on Hepatocytes
(35) To investigate the effect of IL-11 on hepatocytes, experiments were performed with primary human hepatocytes in cell culture.
(36) Human hepatocytes (5200, ScienCell) were grown and maintained at 37° C. and 5% CO.sub.2. Hepatocyte medium (5201, ScienCell) supplemented with 2% fetal bovine serum and 1% Penicillin-streptomycin was renewed every 2-3 days and cells were passaged at 80% confluence using standard trypsinization techniques. All the experiments were carried out at low cell passage (P2-P3) and cells were serum-starved for 16 hours prior to respective stimulations (24 hours) that were performed in serum-free hepatocyte media. Stimulated cells were compared to unstimulated cells that have been grown for the same duration under the same conditions (serum-free hepatocyte media), but without the stimuli.
(37) IL-11Rα expression from human hepatocytes was determined by immunofluorescence staining. Human hepatocytes were seeded on 8-well chamber slides (1.5×104 cells per 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 blocking buffer (5% BSA in PBS) for 2 hours. Cells were incubated with anti-IL11Rα antibody [EPR5446](ab125015, Abcam, 1:100) overnight (4C), followed by incubation with Goat Anti-Rabbit IgG H&L (Alexa Fluor 488) (ab150077, Abcam, 1:200) 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). Negative control sample was exposed to the same procedure excluding anti-IL11Rα antibody incubation step.
(38) IL-11 mediated hepatocyte cell death was measured by determining the levels of alanine transaminase (ALT) in hepatocyte supernatant after treatment with a range of IL-11 doses (0.019-10 ng/ml). ALT levels were measured using ALT Activity Assay Kit (ab105134, Abcam) according to the manufacturer's protocol. Concurrent number of stress fibres 24 h after IL-11 stimulation was detected by rhodamine-phalloidin staining.
(39) The effect of reactive oxygen species (ROS; stimulated by hydrogen peroxide (0.2 mM H.sub.2O.sub.2, 24 hours, 31642, Sigma)) on primary human hepatocytes was investigated.
(40) The results are shown in
(41) Primary human hepatocytes were found to highly express the IL-11Rα receptor (1A). IL-11 stimulation was found to induce dose-dependent hepatocyte cell death as evidenced by a progressive increase in alanine aminotransferase (ALT) over the physiologically relevant dose range (1B). In addition, over the dose range IL-11 progressively stimulated an increase in actin stress fibres in hepatocytes (1B; micrographs from across the dose range), which reflects a partial epithelial-to-mesenchymal transformation of hepatocytes that is known to cause hepatocyte dysfunction (Grant Rowe et al. Molecular and Cellular Biology 2011; 31 (12): 2392-2403).
(42) As APAP is known to induce liver injury in ROS-dependent manner, we stimulated human hepatocytes with H.sub.2O.sub.2 and found that IL-11 was upregulated by 10-fold in the supernatant (1C). Hence, IL-11 directly causes hepatocyte cell death and drives hepatocyte to dysfunctional partial epithelial-mesenchymal cell transition (EMT) state that is known to limit the regenerative capacity of the liver (Grant Rowe et al. supra).
Example 2: Effect of Anti-IL-11 Therapy on Hepatotoxicity
(43) A mouse model of acetaminophen (APAP)-induced liver injury was employed to investigate the effect of anti-IL-11 therapy on hepatotoxicity.
(44) This 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, unless during the starvation period.
(45) Briefly, 12-14 weeks old male mice were starved and intraperitoneally (IP) injected with 10 mg/kg of anti-IL-11Rα antibody or IgG isotype control 16 hours prior to APAP (A3035, Sigma) injection (IP, 400 mg/kg). Mice were sacrificed 24 hours post-APAP administration.
(46) The levels of IL-11 in mouse serum and hepatocyte supernatant were quantified using Mouse IL-11 DuoSet (DY418 and DY008, R&D Systems) and Human IL-11 Quantikine ELISA kit D1100, R&D Systems), respectively, according to the manufacturer's protocol.
(47) Liver samples were excised and fixed for 48 hours at room temperature in 10% neutral-buffered formalin (NBF), dehydrated, embedded in paraffin blocks and sectioned at 7 μm. Sections were stained with Hematoxylin&Eosin (H&E) according to standard protocol and examined by light microscopy.
(48) The results are shown in
(49) As shown above, marked elevation of IL-11 was found in the serum following APAP toxicity (2B), mean±SD, control, n=2; APAP, n=3. Mice receiving a single dose of anti-IL11Rα antibody therapy were found to have significantly lower ALT levels (55% lower compared to IgG control; 2C), i.e. markedly reduced the extent of liver damage. Anti-IL-11 therapy was also found to prevent APAP-induced loss in liver mass, which reflects destruction of liver cells, as compared to 24% loss of liver mass with IgG control antibody (liver index; 2D). (E) Liver histology by Hematoxylin&Eosin (H&E) staining showed severe centrilobular necrosis in IgG-treated mice, a typical histological feature of APAP toxicity, which was found to be reduced with anti-IL11Rα therapy.
(50) The mobility and activity of the mice treated with IgG control or anti-IL-11Rα antibody was observed at 24 hours post-APAP treatment. Control IgG-treated mice were found to be static/moribund with visible features of ill health (e.g. piloerection, hunched posture), whereas mice treated with anti-IL-11Rα antibody had normal mobility and activity.
(51) Hence inhibiting IL-11 signalling by blocking IL-11Rα prevents hepatotoxicity in the accepted, translational model of APAP-induced liver injury (DILI).
Example 3: Antagonism of IL-11 Mediated Signalling Protects Hepatocytes Against Drug-Induced Cell Death
(52) The effects of antagonism of IL-11 mediated signalling on hepatocyte viability was analysed in vitro.
(53) Human hepatocytes (5200, ScienCell) cultured at 37° C. and 5% CO.sub.2 in hepatocyte medium (5201, ScienCell) supplemented with 2% fetal bovine serum and 1% Penicillin-streptomycin. Medium was replaced every 2-3 days, and cells were passaged at 80% confluence using standard trypsinization techniques. All experiments were carried out at low cell passage (P2-P3). Cells were serum-starved for 16 hours prior to their used in experiments, by culture in serum-free hepatocyte medium.
(54) In a first experiment, hepatocytes were treated with APAP (A3035, Sigma) at a final concentration of 20 mM for 24 hours, in the absence (baseline, BL) or presence of antagonist anti-IL11Rα antibody (X209, 2 μg/ml) or isotype-matched IgG control antibody (IgG, 2 μg/ml).
(55) Hepatocytes were then stained using the FITC Annexin V/Dead Cell Apoptosis Kit (V13242, Thermo Fisher) according to the manufacturer's instructions, and Annexin V-FITC/PI-stained cells were analysed by flow cytometry using a BD LSRFortessa flow cytometer (BD Bioscience). 10,000 cells were analyzed per sample. Data was analyzed using FlowJo version 7 software.
(56) The results are shown in
(57) In a separate experiment, hepatocytes were treated with APAP (A3035, Sigma) at a final concentration of 10 mM for 24 hours, in the absence (baseline, BL) or presence of antagonist anti-IL11Rα antibody (X209, 2 μg/ml) or isotype-matched IgG control antibody (IgG, 2 μg/ml).
(58) Protein extracts were prepared from the hepatocytes using 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 amounts of protein lysates were separated by SDS-PAGE, transferred to PVDF membrane, and subjected to immunoblot analysis for the indicated primary antibodies (ERK, pERK, pJNK). Proteins were visualized using the ECL detection system (Pierce) with the appropriate secondary antibodies.
(59) The results are shown in
Example 4: Antagonism of IL-11 Mediated Signalling Protects Against Drug-Induced Liver Injury
(60) A severe APAP overdose (400 mg/kg) or an equivalent volume of saline was administered to 12-14 weeks old male mice by IP injection, 16 hours after IP administration of 20 mg/kg of antagonist anti-IL11Rα antibody (X209) or isotype-matched IgG control antibody.
(61) 24 hours after APAP administration, mice were euthanized. Serum alanine aminotransferase (ALT) levels were measured using ALT Activity Assay Kit (ab105134, Abcam) according to the manufacturer's instructions, and livers were harvested, fixed for 48 h at room temperature in 10% neutral-buffered formalin (NBF), dehydrated, embedded in paraffin blocks and sectioned at 7 μm. Sections were stained with Hematoxylin&Eosin (H&E) according to standard protocol and examined by light microscopy.
(62) The results are shown in
(63) In a further experiment, a severe APAP overdose (400 mg/kg) was administered to 12-14 weeks old male mice by IP injection, 16 hours after IP administration of 20 mg/kg of antagonist anti-IL11 antibody (X203) or isotype-matched IgG control antibody.
(64) 24 hours after APAP administration, the levels of alanine transaminase (ALT) and aspartate aminotransferase (AST) in mouse serum were measured using ALT Activity (ab105134, Abcam) and AST (ab105135, Abcam) Assay Kits according to the manufacturer's protocol.
(65) The results are shown in
Example 5: Antagonism of IL-11 Mediated Signalling after Drug-Induced Liver Injury Reverse Symptoms of Liver Damage and Restores Liver Function
(66) A severe APAP overdose (400 mg/kg) or an equivalent volume of saline was administered to 12-14 weeks old male mice by IP injection, and 10 hours later mice were administered IP with 20 mg/kg of antagonist anti-IL11Rα antibody (X209), isotype-matched IgG control antibody, or untreated.
(67) Mice were euthanized at 24, 36 and 48 hours. Serum ALT levels were analysed as described in Example 4.
(68) Livers were harvested, and fixed as described in Example 4, and digital photographs were taken.
(69) The results are shown in
(70) Western blots were also performed on protein extracts prepared from the livers of the mice. Liver tissue was homogenized in radioimmunoprecipitation assay (RIPA) buffer containing protease and phosphatase inhibitors (Thermo Scientifics), and lysates were subsequently separated by SDS-PAGE and analysed by western blot as described in Example 3.
(71) The results are shown in
(72) In further experiments, a lethal APAP overdose (550 mg/kg) or an equivalent volume of saline was administered to 12-14 weeks old male mice by IP injection, and 10 hours later mice were administered IP with 20 mg/kg of antagonist anti-IL11Rα antibody (X209), isotype-matched IgG control antibody, or untreated.
(73) Survival of mice was monitored for 8 days after APAP/saline administration, and the results are shown in
(74) Mice were euthanized at 24 hours and 192 hours (8 days). Serum ALT levels were analysed as described in Example 4. Livers were harvested, and fixed as described in Example 4, and digital photographs were taken.
(75) The results are shown in
(76) The ability of treatment with antagonist of IL-11 mediated signalling administered 10 hours after hepatotoxic insult to reverse DILI-associated hepatotoxicity and prevent death of subjects administered a severe/lethal APAP overdose was a truly remarkable result. 10 hours after overdose in mice is thought to be equivalent to about 24 hours after overdose in humans.
(77) The results identify antagonism of IL-11 mediated signalling as an extremely promising therapeutic strategy for reducing liver injury and associated morbidity/mortality following hepatotoxic insult.
Example 6: Antagonism of IL-11 Mediated Signalling after Drug-Induced Liver Injury Reverse Symptoms of Liver Damage and Restores Liver Function
6.1 Overview
(78) Acetaminophen (APAP) overdose is a leading cause of liver failure. In the mouse model of APAP-induced liver injury (AILI), the administration of recombinant human interleukin 11 (rhIL11) is protective.
(79) The present disclosure shows that the beneficial effect of rhIL11 in mouse AILI is due to an unexpected inhibitory effect of foreign rhIL11 on endogenous mouse IL11 activity. Contrary to the accepted paradigm, IL11 is secreted by damaged hepatocytes to drive apoptosis and inhibit liver regeneration.
(80) Mice with hepatocyte-specific Il11 expression spontaneously develop liver damage whereas those with Il11ra1 deletion are robustly protected from AILI. Neutralizing anti-IL11R antibodies administered to moribund mice 10 hours post lethal APAP overdose results in 90% survival.
(81) The data of the present disclosure overturn a misconception, indicate a disease mechanism and identify a therapeutic target.
6.2 Introduction
(82) Acetaminophen (N-acetyl-p-aminophenol, APAP) is an over-the-counter analgesic that is commonly taken as an overdose (OD) leading to APAP-induced liver injury (AILI), a major cause of acute liver failure (1). The antioxidant N-acetyl cysteine (NAC) is beneficial for patients presenting early (2), but there is no drug-based treatment beyond eight hours post-OD and death can ensue if liver transplantation is not possible (3, 4).
(83) In hepatocytes, APAP is metabolized to N-Acetyl-p-benzochinonimin (NAPQI) which depletes cellular glutathione (GSH) levels and damages mitochondrial proteins leading to reactive oxygen species (ROS) production and JNK activation (5). ROS-related JNK activation results in a combination of necrotic, apoptotic and other forms of hepatocyte cell death causing liver failure (1, 6, 7). JNK and ASK1 inhibitors have partial protective effects against AILI in mouse models, but this has not translated to the clinic (8, 9). Liver regeneration has fascinated humans since the stories of Prometheus and can be truly profound, as seen after partial hepatic resection in rodents and humans (10, 11). However, in the setting of AILI, liver regeneration is persistently suppressed resulting in permanent injury and patient mortality. Targeting the pathways that hinder the liver's extraordinary regenerative capacity may trigger natural regeneration, which could be particularly useful in AILI (12, 13).
(84) Interleukin 11 (IL11) is a scarcely studied cytokine that is of critical importance for myofibroblast activation and fibrosis of the heart, kidney, lung, and liver (14-16). It is established that IL11 is secreted from injured hepatocytes and Il11 can be detected at high levels in the serum of the mouse model of AILI, where its expression is considered compensatory and cytoprotective (17). In keeping with this paradigm, administration of recombinant human IL11 (rhIL11) is effective in treating the mouse model of AILI and also protects against liver ischemia, endotoxemia or inflammation (17-22). As recently as 2016, rhIL11 has been proposed as a treatment for patients with AILI (23).
(85) In studies of liver fibrosis the inventors made the unexpected observation that, in the context of some models of fibro-inflammatory liver disease, IL11 may be detrimental for hepatocyte function (14). This apparent discrepancy with the previous literature prompted the inventors to look in more detail at the effects of IL11 on hepatocytes independent of fibrosis, and they chose to do so in the mouse model of AILI, where Il11 is largely upregulated (17).
6.3 IL11 Drives APAP-Induced Hepatocyte Cell Death
(86) As reported previously (17), AILI was confirmed to be characterized by elevated IL11 serum levels in injured mice (
(87) Having identified the source of Il11 upregulation during AILI in vivo, the inventors conducted in vitro experiments to study underlying mechanisms. Exposure of primary human hepatocytes to APAP resulted in the dose-dependent secretion of IL11 (
6.4 Species-Specific Effects of Recombinant Human IL11
(88) rhIL11 is consistently reported to be protective in rodent models of liver damage (17-20, 23), yet the results described in Example 6.3 suggested rhIL11 has the exact opposite effect on human hepatocytes in vitro (
(89) This showed that the role of IL11 signaling in hepatocyte death is conserved across species, but that recombinant IL11 protein has species-specific effects and does not activate the pathway in foreign species. This hypothesis was tested in vivo by injecting either rmIL11 or rhIL11 into mice (
(90) To follow up on the potential protective effect of rhIL11 in the mouse, a protocol similar to the AILI study of 2001 (20) was performed, where rhIL11 was injected into the mouse after APAP OD (
(91) The therapeutic effect of rhIL11 was accompanied by a reduction in hepatic ERK and JNK activation (
(92) Using surface plasmon resonance (SPR), rhIL11 was found to bind to mouse interleukin 11 receptor alpha chain 1 (mIL11Rα1) with a KD of 72 nM, which is slightly stronger than the rmIL11:mIL11Rα1 interaction (94 nM) and close to that reported previously for rhIL11:hIL11Rα (50 nM), which was reconfirmed (
6.5 Hepatocyte-Specific Expression of Il11 Causes Spontaneous Liver Failure
(93) To test the effects of endogenous mouse IL11 secreted from hepatocytes in vivo, an Il11 transgene was expressed specifically in hepatocytes by injecting Rosa26.sup.Il11/+ mice (15, 16) with AAV8 virus encoding an albumin promoter-driven Cre construct (Il11-Tg mice,
6.6 IL11 Stimulates NOX4-Mediated Reactive Oxygen Species Production
(94) IL11 signaling is required for APAP-driven JNK activation in vitro (
(95) In fibroblasts, the expression of NOX4, an NADPH oxidase, and source of ROS, is strongly associated with IL11 expression (15, 25), and hepatocyte-specific Nox4 deletion prevents pathological activation of JNK (26). Therefore, the inventors investigated the relationship between IL11, NOX4, and ROS in greater detail. In Il11-Tg mice, hepatic Nox4 expression was upregulated (
(96) APAP stimulation also resulted in NOX4 upregulation in hepatocytes, coincident with depletion in hepatocyte GSH levels, which was blocked with the anti-IL11Rα antibody X209 (
6.7 Hepatocyte-Specific Deletion of Il11Ra1 Prevents APAP-Induced Liver Failure
(97) To delete Il11ra1 specifically in adult mouse hepatocytes Il11ra1 conditional knockouts (CKOs) were created by injecting AAV8-ALB-Cre virus to mice homozygous for LoxP-flanked Il11ra1 alleles, along with wildtype controls. Three weeks after viral infection, control mice and CKOs were administered APAP (400 mg kg.sup.−1) (
(98) Strikingly, CKO mice had 99% and 95% lower ALT and AST levels, respectively, as compared to controls and GSH levels that were similar to baseline. Both groups had similar levels of APAP and APAP-Glutathione (APAP metabolite) in the serum and thus Il11ra1 deletion does not impact APAP metabolism (
6.8 Effects of Anti-IL11Rα Administration Early During APAP-Induced Liver Injury
(99) The inventors next tested if therapeutic inhibition of IL11 signaling was effective in mitigating AILI by administering anti-Il11Rα (X209) antibody (14). Initially, a preventive treatment was performed by injecting X209 or control antibody (10 mg kg.sup.−1) 16 h prior to APAP. This approach reduced serum markers of liver damage by over 70%, largely restored hepatic GSH levels, and limited histological evidence of centrilobular necrosis (
(100) Next, anti-IL11Rα therapy was administered in a therapeutically-relevant mode by giving antibody 3 h after APAP, a time point by which APAP metabolism and toxicity is established and after which most interventions have no effect in the mouse model of AILI (
(101) Lastly, it was determined whether inhibiting IL11 signaling had added value when given in combination with the current standard of care, NAC, 3 h after APAP dosing (
6.9 Liver Regeneration with Anti-IL11Rα Therapy
(102) For patients presenting to the emergency room 8 h or later after APAP OD there is no effective treatment. This prompted us to test anti-IL11Rα 10 h after APAP (400 mg kg.sup.−1) administration to mice (
(103) Interventions promoting liver regeneration, which has very large potential, may provide a new means of treating AILI (12). The status of genes important for liver regeneration was therefore assessed (10). Inhibition of IL11 signaling was associated with a robust signature of regeneration with strong upregulation of PCNA, Cyclin D1/D3/E1, and phosphorylation of RB, as seen during regeneration following partial hepatectomy (10). EdU injection and histological analyses showed very large numbers of nuclei with evidence of recent DNA synthesis in X209-treated mice as compared to controls (
(104) Finally, X209 (20 mg kg.sup.−1) 10 h after a higher and lethal acetaminophen dose (550 mg kg.sup.−1) at a time point when mice were moribund and livers undergoing fulminant necroinflammation (
6.10 Discussion
(105) APAP OD is common with up to 50,000 individuals attending emergency departments every year in the UK, some who develop liver failure requiring transplantation (1). Here, IL11, which has previously been reported as protective against APAP-induced liver failure (17, 20), liver ischemia (18, 21), endotoxemia (22) and inflammation (19), is shown to actually be hepatotoxic and of central importance for liver failure following APAP OD.
(106) The observation that endogenous IL11 is hepatotoxic is most surprising as over 30 publications have reported cytoprotective and/or anti-inflammatory effects of rhIL11 in rodent models of human disease (Tables 1 and 2). rhIL11 is shown to be a competitive inhibitor of mouse IL11 binding to IL11Rα1, which overturns previous understanding of the role of IL11 in AILI and liver disease more generally. This also implies that anti-IL11 therapies may be effective in additional diseases where rhIL11 had protective effects in mouse models such as rheumatoid arthritis (27) and colitis (28), among others (Table 2). Based on the erroneous assumption that rhIL11 effects in mice embodied beneficial IL11 gain-of-function, a number of clinical trials using rhIL11 were performed in patients (Table 3).
(107) TABLE-US-00002 TABLE 1 List of publications showing protective effects of recombinant human IL11 (rhIL11) in rodent models of liver injury Yu et al. 2016. “Interleukin-11 Protects Mouse Liver In vivo administration of rhIL11 (500 μg/kg, IV) prior to WI/Rp from Warm Ischemia/reperfusion (WI/Rp) Injury.” injury protects mouse livers. In vitro, pre-treatment with rhIL11 (2 Clinics and Research in Hepatology and μg/mL, 12 hours) reduces murine hepatocyte apoptosis due to Gastroenterology 40 (5): 562-70 hypoxia/reperfusion. Zhu et al. 2015. “IL-11 Attenuates Liver Hepatoprotective effects of rhIL11 in mice subjected to a single Ischemia/Reperfusion Injury (IRI) through STAT3 injection of rhIL11 (500 μg/kg, IP) one hour prior to IRI. In vitro, Signaling Pathway in Mice.” PloS One 10 (5): murine hepatocytes were treated with 1 μg/ml of rhIL11. e012629. Nishina et al. 2012. “Interleukin-11 Links Oxidative Administration of rhIL11 receptor superagonist, (N.sub.T-3N, Stress and Compensatory Proliferation.” Science 500 μg/kg) 2 hours prior to acetaminophen (APAP) injection Signaling 5 (207): ra5. reduces acute liver injury in mice. Maeshima et al. 2004. “A Protective Role of The authors conclude that rhIL11 (150 μg/kg, IP) plays a Interleukin 11 on Hepatic Injury in Acute significant protective role in LPS-induced hepatic injury (acute Endotoxemia.” Shock 21 (2): 134-38. endotoxemia) in rats. Trepicchio et al. 2001. “Protective Effect of rhIL-11 in The authors indicate a protective role of rhIL11 (250 or a Murine Model of Acetaminophen-Induced 500 μg/kg, SC) against acetaminophen-induced liver damage, in Hepatotoxicity.” Toxicologic Pathology 29 (2): 242- which rhIL11 was injected to mice 2 hours before 249. acetaminophen administration. Bozza et al. 1999. “Interleukin-11 Reduces T-Cell- Administration of rhIL11 (50-500 μg/kg, IP) 2 hours prior to Dependent Experimental Liver Injury in Mice.” Concanavalin A-induced T-cell-mediated hepatotoxicity reduces Hepatology 30 (6): 1441-47. liver necrosis and enhanced survival in mice.
(108) TABLE-US-00003 TABLE 2 List of publications showing protective and/or anti inflammatory effects of rhIL11 in other rodent disease models Bowel Gibson et al. 2010. “Interleukin-11 Reduces The authors report that administration of rhIL11 (5 μg/kg, TLR4-Induced Colitis in TLR2-Deficient Mice IP) ameliorates infection colitis and is cytoprotective in TLR2- and Restores Intestinal STAT3 Signaling.” deficient mice. Gastroenterology 139 (4): 1277-88. Boerma et al. 2007. “Local Administration of The authors conclude that IL11 ameliorates early intestinal Interleukin-11 Ameliorates Intestinal Radiation radiation injury, in which rats were given daily injections of Injury in Rats.” Cancer Research 67 (19): 9501- rhIL11 (2 mg/kg/d) from 2 days prior until 2 weeks after 6. irradiation. Opal et al. 2003. “Orally Administered The authors suggest that IL11 maintains epithelial cell integrity Recombinant Human Interleukin-11 Is during cytoreductive chemotherapy by cyclophosphamide based Protective in Experimental Neutropenic Sepsis.” on effects observed in rats receiving daily oral administration of The Journal of Infectious Diseases 187 (1): 70- rhIL11 (0.5 mg/kg/day), starting from 1 day before the first dose 76. of cyclophosphamide for a total of 12 days. Ropeleski et al. 2003. “Interleukin-11-Induced The authors conclude that IL11 confers epithelial-specific Heat Shock Protein 25 Confers Intestinal cytoprotection during intestinal epithelial injury. Rat, mouse and Epithelial-Specific Cytoprotection from Oxidant canine cell lines (IEC-18, YAMC, NIH3T3, MDCK-HR) were Stress.” Gastroenterology 124 (5): 1358-68. stimulated with high (50-100 ng/ml) levels of rhIL11. Greenwood-Van Meerveld et al 2000. The authors conclude that during intestinal inflammation IL11 “Recombinant Human Interleukin-11 Modulates acts as a modulator of epithelial transport or as an anti- Ion Transport and Mucosal Inflammation in the inflammatory cytokine based on effects of rhIL11 on rat mucosal Small Intestine and Colon.” Laboratory sheets (10-10,000 ng/ml) and in rats (33 μg/kg, alternate days for Investigation; a Journal of Technical Methods 1 or 2 weeks). and Pathology 80 (8): 1269-80. Du et al 1997. “Protective Effects of Interleukin- Administration of rhIL11 (250 μg/kg/day) for 3 days prior to and 11 in a Murine Model of Ischemic Bowel for 7 days post bowel ischemia induction confers a protective Necrosis.” American Journal of Physiology- effect against ischemic bowel necrosis and the authors suggest Gastrointestinal and Liver Physiology. its use as a treatment for gastrointestinal mucosal diseases. Orazi et al. 1996. “Interleukin-11 Prevents Administration of rhIL11 (250 μg/kg) promotes recovery from Apoptosis and Accelerates Recovery of Small chemotherapy and radiation-induced damage to the mice small Intestinal Mucosa in Mice Treated with intestinal mucosa. Combined Chemotherapy and Radiation.” Laboratory Investigation; a Journal of Technical Methods and Pathology 75 (1): 33-42. Potten et al 1996. “Protection of the Small RhIL11 (100 μg/kg, SC), administered to mice prior to and after Intestinal Clonogenic Stem Cells from cytotoxic exposure, protects clonogenic cells in intestinal crypts Radiation-Induced Damage by Pretreatment and increases murine survival times following radiation with Interleukin 11 Also Increases Murine exposure. Survival Time.” Stem Cells. 1996 14(4): 452-9. Qiu et al. 1996. “Protection by Recombinant The authors describe protective effects of rhIL11 in Human Interleukin-11 against Experimental trinitrobenzene sulfonic acid-induced colitis in rats. Rats were TNB-Induced Colitis in Rats.” Digestive injected daily with rhIL11 (100, 300, or 1000 μg/kg, SC) 3 days Diseases and Sciences 41 (8): 1625-30. before, or daily for 3-7-14 days after TNB administration. Du et al. 1994. “A Bone Marrow Stromal- Administration of rhIL11 (250 μg/kg/d, SC) promotes recovery of Derived Growth Factor, Interleukin-11, small intestinal mucosa following combination radiation and Stimulates Recovery of Small Intestinal Mucosal chemotherapy in mice. Cells after Cytoablative Therapy.” Blood 83 (1): 33-37. Heart Tamura et al. 2018. “The Cardioprotective Effect Administration of rhIL11 (18 μg/ml, IV, 10 minutes prior to heart of Interleukin-11 against Ischemia-Reperfusion collection) preserves heart function and lower apoptosis index in Injury in a Heart Donor Model.” rat following ex vivo model of cold ischemia. Annals of Cardiothoracic Surgery 7 (1): 99-105. Obana et al. 2012. “Therapeutic Administration Administration of rhIL11 (20 μg/kg, IV at the start of reperfusion) of IL-11 Exhibits the Postconditioning Effects prevents adverse cardiac remodeling and apoptosis after against Ischemia-Reperfusion Injury via STAT3 ischemia reperfusion injury-induced acute myocardial infarction in the Heart.” American Journal of Physiology. in mice Heart and Circulatory Physiology 303 (5): H569-77. Obana et al. 2010. “Therapeutic Activation of Administration of rhIL11 (8 μg/kg, IV) 24 hours following left Signal Transducer and Activator of Transcription coronary artery ligation-induced myocardiac infarction (MI) and 3 by Interleukin-11 Ameliorates Cardiac Fibrosis then consecutively every 24 hours for 4 days reduces post-MI after Myocardial Infarction.” Circulation 121 (5): scar volume in mice. 684-91. Kimura et al. 2007. “Identification of Cardiac The authors conclude that IL11 is a cardioprotective based on Myocytes as the Target of Interleukin 11, a effects of rhIL11 (8 μg/kg) administered to mouse 15 hours prior Cardioprotective Cytokine.” Cytokine 38 (2): 107- to cardiac ischemia-reperfusion 115 Immune Bozza et al. 2001. “Interleukin-11 Modulates The authors state that IL11 acts directly on activated murine System Th1/Th2 Cytokine Production from Activated CD4+ve T-cells and modulates, not represses, the immune CD4 T Cells.” Journal of Interferon & Cytokine response following stimulation with rhIL11 (1-500 ng/ml). Research 21(1): 21-30. Opal et al. 2000. “Recombinant Human Daily administration of rhIL11 (150 mg/kg, IV) for 7 days prior to Interleukin-11 Has Anti-inflammatory Actions Listeria infection reduces interferon-γ levels. Interestingly, the Yet Does Not Exacerbate Systemic Listeria authors stated that inflammatory markers IL-6/IFN-γ trend down Infection.” The Journal of Infectious Diseases after anti-IL11mAb (10 mg/kg) treatment. 181(2): 754-756 Hill et al. 1998. “Interleukin-11 Promotes T Cell The authors conclude that IL11 prevents Graft-vs-Host-Disease Polarization and Prevents Acute Graft-versus- (GVHD) via T Cell polarization, based on experiments in which a Host Disease after Allogeneic Bone Marrow high dose of rhIL11 (250 μg/kg, SC, twice daily) was injected into Transplantation.” The Journal of Clinical a murine model of GVHD. Investigation 102(1): 115-23. Sonis et al. 1997. “Mitigating Effects of Administration of rhIL11 (50-100 μg/animal/day, SC) protects Interleukin 11 on Consecutive Courses of 5- from 5-fluorouracil-induced ulcerative mucositis in hamsters. Fluorouracil-Induced Ulcerative Mucositis in Hamsters.” Cytokine 9 (8): 605-12. Trepicchio et al. 1997. “IL-11 Regulates The authors conclude that IL11 inhibits the secretion of pro- Macrophage Effector Function through the inflammatory cytokines by macrophages; murine primary Inhibition of Nuclear Factor-kappaB.” Journal of macrophages were treated with rhIL11 (10-100 ng/ml). Immunology 159 (11): 5661-70. Trepicchio et al. 1996. “Recombinant Human IL- The authors report that IL11 reduces levels of TNF-α, IL-1β and 11 Attenuates the Inflammatory Response IFN-γ in the serum of LPS-treated mice and in LPS-stimulated through down-Regulation of Proinflammatory macrophage media. Mice and murine macrophages were treated Cytokine Release and Nitric Oxide Production.” with rhIL11 (500 μg/kg or 10-100 ng/ml, respectively). Journal of Immunology 157 (8): 3627-34. Joint Anguita et al. 1999. “Selective Anti-Inflammatory Administration of rhIL11(0.1-2 μg/mouse/day, 5 days/week for 3 Action of Interleukin-11 in Murine Lyme weeks) reduces arthritis, but not carditis, in Borrelia burgdorferi- Disease: Arthritis Decreases While Carditis infected mice (a murine model of Lyme disease). Persists.” The Journal of Infectious Diseases 179 (3): 734-37. Walmsley et al. 1998. “An Anti-Inflammatory Daily administration of rhIL11 (0.3-100 μg/mouse/day, IP, 10 Role for Interleukin-11 in Established Murine days) reduces inflammation in a murine model of collagen- Collagen-Induced Arthritis.” Immunology 95 (1): induced arthritis. 31-37. Kidney Lee et al. 2012. “Interleukin-11 Protects against The authors conclude that IL11 is renoprotective based on pre- Renal Ischemia and Reperfusion Injury.” treatment (10 minutes prior to IR) and post-treatment (30-60 American Journal of Physiology. Renal minutes following IR) effects of rhIL11 and PEGylated rhIL11 Physiology 303 (8): F1216-24. (100-1000 μg/kg, IP) in mice. Stangou et al. 2011. “Effect of IL-11 on Administration of rhIL11 (800-1360 μg/kg, IP) 2 hours prior to Glomerular Expression of TGF-Beta and nephrotoxic nephritis and then once daily for 6 days suppresses Extracellular Matrix in Nephrotoxic Nephritis in ECM deposition in rats. Wistar Kyoto Rats.” Journal of Nephrology 24 (1): 106-111. Lung Sheridan et al 1999. “Interleukin-11 Attenuates The authors conclude that rhIL11 (200 mg/kg, IP) exerts an anti- Pulmonary Inflammation and Vasomotor inflammatory activity that protects against LPS-induced lung Dysfunction in Endotoxin-Induced Lung Injury.” injury and lethality in rats The American Journal of Physiology 277 (5): L861-67. Waxman et al. 1998. “Targeted Lung The authors conclude that IL11 protects from hyperoxic-induced Expression of Interleukin-11 Enhances Murine lung injury, based on the effects of lung-specific human IL11 Tolerance of 100% Oxygen and Diminishes overexpression in mice. Hyperoxia-Induced DNA Fragmentation.” J. Clin. Invest. 101(9): 1970-1982
(109) TABLE-US-00004 TABLE 3 List of publications from clinical trials where rhIL11 was administered to patients, based mainly on an inferred protective effect of rhIL11 use in rodent models of disease. Herrlinger et al. 2006. “Randomized, Double Blind RhIL11 (1 mg, weekly for 12 weeks, SC) was administered to 51 Controlled Trial of Subcutaneous Recombinant patients with active Crohn's disease and found to be significantly Human Interleukin-11 versus Prednisolone in Active inferior as compared to prednisolone treatment. Crohn's Disease.” The American Journal of Gastroenterology 101 (4): 793-797. Lawitz et al. 2004. “A Pilot Study of Interleukin-11 in RhIL11 (5 μg/kg, daily for 12 weeks, SC) was administered to 20 Subjects with Chronic Hepatitis C and Advanced patients with chronic Hepatitis C and late stage liver disease. Liver Disease Nonresponsive to Antiviral Therapy.” Lower serum ALT was observed by study end. The most The American Journal of Gastroenterology 99 (12): common side effect is oedema in lower extremities, which was 2359-64. observed in all subjects. Sands et al. 2002. “Randomized, Controlled Trial of RhIL11 (15 μg/kg, weekly for 6 weeks, SC) was administered to Recombinant Human Interleukin-11 in Patients with 49 patients with Crohn's disease. A greater proportion of patients Active Crohn's Disease.” Alimentary Pharmacology & receiving rhIL11 achieved remission compared to placebo. Side Therapeutics 16 (3): 399-406. effects including oedema were observed. Moreland et al. 2001. “Results of a Phase-I/II Administration of up to 15 μg/kg rhIL11 weekly for 12 weeks (SC) Randomized, Masked, Placebo-Controlled Trial of in rheumatoid arthritis patients is safe but no therapeutic benefit Recombinant Human Interleukin-11 (rhIL-11) in the was observed. In addition, mild adverse effect (erythema Treatment of Subjects with Active Rheumatoid with/without induration) at the injection site was seen in 60.6% of Arthritis.” Arthritis Research 3 (4): 247-252. patients receiving rhIL11. Trepicchio et al. 1999. “Interleukin-11 Therapy Patients with extensive psoriasis were treated with 2.5 or 5 mg/kg Selectively Downregulates Type I Cytokine of rhIL11 (daily for 8 weeks, SC). A response (RNA expression Proinflammatory Pathways in Psoriasis Lesions.” of inflammatory markers) was observed in a subset (n = 7) of 12 The Journal of Clinical Investigation 104 (11): 1527-1537. patients; the other 5 patients were nonresponsive and no improvement was observed.
(110) The inventors propose a refined mechanism for APAP toxicity whereby NAPQI damaged mitochondria produce ROS that stimulates IL11-dependent NOX4 upregulation and further sustained ROS production (
6.11 Materials and Methods for Example 6
(111) Antibodies
(112) Cleaved Caspase 3 (9664, CST), Caspase 3 (9662, CST), Cyclin D1 (55506, CST), Cyclin D3 (2936, CST), Cyclin E1 (20808, CST), p-ERK1/2 (4370, CST), ERK1/2 (4695, CST), GAPDH (2118, CST), GFP (ab6673, Abcam), IgG (Aldevron), p-JNK (4668, CST), JNK (9258, CST), neutralizing anti-IL11Rα (X209, Aldevron; in vivo study), IL11Rα (130920, Santa Cruz; WB), NOX4 (110-58849, Novus Biologicals), PCNA (13110, CST), p-RB (8516, CST), RB (9313, CST), anti-rabbit HRP (7074, CST), anti-mouse HRP (7076, CST), anti-rabbit Alexa Fluor 488 (ab150077, Abcam), anti-rabbit Alexa Fluor 647 (ab150079, Abcam), anti-mouse Alexa Fluor 488 (ab150113, Abcam), anti-goat Alexa Fluor 488 (ab150129, Abcam).
(113) Recombinant Proteins
(114) Recombinant human IL11 (rhIL11, UniProtKB:P20809, Genscript), recombinant mouse IL11 (rmIL11, UniProtKB: P47873, Genscript), human IL11Rα (10252-H08H, SinoBiological), mouse IL11Rα (50075-M08H, SinoBiological).
(115) Chemical
(116) Acetaminophen (APAP, A3035, Sigma), DAPI (D1306, ThermoFisher Scientifics), D-Luciferin (L6882, Sigma), GKT-137831 (17764, Cayman Chemical), N-Acetyl-L-Csyteine (NAC, A7250, Sigma).
(117) Reagents for LC-MS/MS
(118) Reference Standard acetaminophen (APAP, P0300000, Sigma), internal standard (IS) acetaminophen-d4 (APAP-D4, A161222, Toronto Research Chemicals), IS acetaminophen glutathione (APAP GLUT, A161223, Toronto Research Chemicals), Acetronitrile (900667, Sigma), Ammonium formate (A115-50, Sigma), Formic acid (F0507, Sigma), mouse serum (IGMSCD1 SER50ML, i-DNA Biotechnology). All chemicals, reagents and solvents were of LC-MS grade quality.
(119) Animal Models
(120) 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, except in the fasting period, during which only water was provided ad libitum.
(121) Mouse Models of Acetaminophen Poisoning
(122) Prior to APAP, 12-14 weeks old male mice (C57BL6/NTAC, unless otherwise specified) were fasted overnight. Mice were then given a severe (400 mg kg.sup.−1) or lethal (550 mg kg.sup.−1) dose of APAP by intraperitoneal (IP) administration. Mice were administered anti-IL11Rα (X209) or IgG isotype control antibody at different times and doses, as described above or in the figure legends. Mice were euthanized at various time points post APAP, from 10 h to 8 days, as described above or in the figure legends.
(123) Il11-Luciferase mice
(124) The mouse Il11 gene consists of 5 exons, with the ATG start codon in exon 1 and TGA stop codon in exon 5. Three transcripts of mouse Il11 have been identified (ENSMUSG00000004371): transcript Il11-201 is the longest and encodes a 199aa pro-peptide, whereas transcripts Il11-202 and Il11-203 contain an alternative first exon, and are both predicted to encode a shorter 140 aa isoform that lacks the signal peptide. Using the CRISPR/Cas9 technique, a Kozak-Luciferase-WPRE-polyA sequence was introduced to replace the ATG start codon in exon 1 of Il11-201 (ENSMUST00000094892.11), resulting in translational disruption of this specific transcript. Single guide RNAs (sgRNAs) with recognition sites in exon 1 along with Cas9 and the targeting construct containing a Kozak-Luciferase-WPRE-polyA sequence were microinjected into fertilized zygotes and subsequently transferred into pseudopregnant mice (Shanghai Model Organisms Center, Inc). Insertion of the luciferase cassette into the Il11 gene locus was verified by sequencing. Mutant Il11-Luciferase offsprings were generated on a C57BL/6 background and identified by genotyping to detect the insertion of the luciferase construct in exon 1, using primers which amplify a 818 bp region corresponding to the wildtype Il11 allele (5′-GGAGGGAGGGGACGCCAATGACC-3′ (SEQ ID NO:22) and 5′-TCTGCCTCCCCTGCCTGTTTCTCG-3′ (SEQ ID NO:23)), and a second set of primers that amplifies a 928 bp region corresponding to the targeted allele containing the luciferase construct (5′-AATTCCGTGGTGTTGTCG-3′ (SEQ ID NO:24) and 5′-TCTGCCTCCCCTGCCTGTTTCTCG-3′ (SEQ ID NO:25)).
(125) Heterozygous Il11-Luciferase were subjected to APAP-induced liver injury as described above. After 24 h, mice were injected intraperitoneally with 150 mg kg.sup.−1 of D-Luciferin in PBS and bioluminescence images of the liver were subsequently acquired using the IVIS Lumina System (Perkin Elmer), according to the manufacturer's instructions.
(126) Il11-EGFP Mice
(127) Transgenic mice with EGFP constitutively knocked-in to the Il11 gene were generated by Cyagen Biosciences Inc. Briefly, knockin mice were generated to contain a 2A-EGFP cassette inserted into exon 5, which replaces the TGA stop codon sequence, and translation of the targeted transcript would give rise to full-length IL11 pro-peptide and EGFP separated by a 2A self cleaving peptide linker. The targeting vector homology arms of Il11 gene, containing a Neo cassette inserted into intron 4 (flanked by SDA: self-deletion anchor sites) and a 2A-EGFP cassette inserted into exon 5, were generated by PCR using BAC clones from the C57BL/6 library. C57BL/6 ES cells were used for gene targeting and successfully targeted clones were injected into C57BL/6 albino embryos, which were then re-implanted into CD-1 pseudo-pregnant females. Founder animals were identified by their coat color and germline transmission was confirmed by breeding with C57BL/6 females and subsequent genotyping of the offsprings. Genotyping primers were designed to amplify selected regions of intron 4, spanning the Neo cassette SDA sites, according to the following primer sequences: (5′-GAAATGAGAGCCTAGAGTCCAGAG-3′ (SEQ ID NO:26) and 5′-GAGGCTTGGAAGAATGCACAATTA-3′ (SEQ ID NO:27)).
(128) Hepatocyte-Specific Il11 Overexpressing Mice (Il11-Tg)
(129) Mice in which mouse Il11 cDNA was introduced into the Rosa26 locus under the control of loxP-Stop-loxP sites to allow for cell-type specific overexpression of Il11 following Cre recombinase-mediated excision have previously been described (15). These animals are made available at The Jackson Laboratory (C57BL/6N-Gt(ROSA)26Sortm1(CAG-Il11)Cook/J). To induce the specific expression of Il11 in hepatocytes, heterozygous Il11-Rosa26 mice were injected intravenously (IV) with either 4×10.sup.11 genome copies in PBS/mouse (VectorBiolabs) of AAV8-ALB-Null (Control) AAV8-ALB-Cre (Il11-Tg). Livers and serum were assessed after three weeks.
(130) Hepatocyte-Specific Il11Ra1 Deleted Mice
(131) Il11ra1-floxed mice were recently generated and validated, in which exons 4 to 7 of the Il11ra1 gene were flanked by loxP sites, allowing for the spatial and temporal deletion of Il11ra1 upon Cre recombinase-mediated excision (Ng et al., Sci Transl Med. (2019) 11(511) pii: eaaw1237). To induce the specific deletion of Il11 ra1 in hepatocytes, homozygous Il11ra1-floxed mice were IV injected with AAV8-ALB-Cre virus (4×10.sup.11 genome copies in PBS/mouse, VectorBiolabs) via the tail vein. A similar amount of AAV8-ALB-Null virus were injected into homozygous Il11ra1-floxed mice as controls. The AAV8 treated mice were allowed to recover for three weeks prior to APAP injury. Knockdown efficiency was determined by Western blotting of hepatic IL11Rα.
(132) Cell Culture
(133) Both primary human and mouse hepatocytes were grown and maintained at 37° C. and 5% CO.sub.2. The growth medium was renewed every 2-3 days and cells were passaged at 80% confluence, using standard trypsinization techniques. All the experiments were carried out at low cell passage (P1-P3). Stimulated cells were compared to unstimulated cells that have been grown for the same duration under the same conditions, but without the stimuli.
(134) Primary Human Hepatocytes
(135) Human hepatocytes (5200, ScienCell) were maintained in hepatocyte medium (520, ScienCell) supplemented with 2% fetal bovine serum, 1% Penicillin-streptomycin. Cells were serum-starved for 16 h prior to respective stimulations, as described above or in the figure legends, that were performed in serum-free hepatocyte media for 24 h.
(136) Primary Mouse Hepatocytes
(137) Mouse hepatocytes (ABC-TC3928, AcceGen Biotech) were maintained in mouse hepatocyte medium (ABC-TM3928, AcceGen Biotech) supplemented with 1% Penicillin-streptomycin. Cells were stimulated with different treatment conditions for 24 h, as described above or in the figure legends.
(138) siRNA Transfection
(139) Primary human hepatocytes were seeded at 60-70% confluency in 6-well plate, 16 h before transfection. Cells were transfected with 50 nM of NOX4 siRNA (ON-TARGETplus SMARTpool siRNA, L-010194-00-0005, Dharmacon) or control siRNA (D-001810-10-05, Dharmacon) for 24 h at 37° C. in OptiMEM (31985070, Thermo Fisher) containing Lipofectamine RNAiMAX Transfection Reagent (13778150, Thermo Fisher). Transfected cells were then stimulated with rhIL11 for 24 h. Knockdown efficiency was determined by immunoblotting of NOX4.
(140) Flow Cytometry
(141) Primary human hepatocytes (5×105) were stained using FITC Annexin V/Dead Cell Apoptosis Kit (V13242, Thermo Fisher), according to the manufacturer's instructions. PI.sup.+ve cells were quantified with the flow cytometer (Fortessa, BD Biosciences) and analyzed with FlowJo version 7 software (TreeStar).
(142) Colorimetric Assays
(143) The levels of alanine transaminase (ALT) or aspartate aminotransferase (AST) in mouse serum and hepatocyte supernatant were measured using ALT Activity (ab105134, Abcam) or AST (ab105135, Abcam) Assay Kits. Liver glutathione sulfhydryl (GSH) measurements were performed using Glutathione Colorimetric Detection Kit (EIAGSHC, Thermo Fisher). All colorimetric assays were performed according to the manufacturer's protocol.
(144) Enzyme-Linked Immunosorbent Assay (ELISA)
(145) The levels of IL11 in mouse serum and hepatocyte supernatant were quantified using Mouse IL-11 DuoSet (DY418 and DY008, R&D Systems) and Human IL11 Quantikine ELISA kit D1100, R&D Systems), respectively, according to the manufacturer's protocol.
(146) Competitive ELISA
(147) Mouse IL11Rα (1 μg ml.sup.−1 in PBS) was coated on a 96-well plate (overnight at 4° C.) and then blocked with blocking buffer (1% BSA in PBS containing 0.05% Tween20). Biotinylated mouse Il11 was prepared using Lightning-Link Rapid Biotin type A kit (Expedeon) according to the manufacturer's instructions. RhIL11 or rmIl11 was two-fold serially diluted in blocking buffer (starting at 5 μg ml.sup.−1) and mixed with 0.01 μg ml.sup.−1 biotinylated mouse IL11. The mixture of biotinylated mouse IL11 and either rhIL11 or rmIL11 was added into the coated plate and incubated for 1 h at RT. Color development was performed by adding Streptavidin-HRP (1:1000 in blocking buffer) and TMB chromogen solution (002023, ThermoFisher Scientific).
(148) Immunoblotting
(149) Western blots were carried out from hepatocyte and liver tissue lysates. Hepatocytes and tissues were homogenized in radioimmunoprecipitation assay (RIPA) buffer containing protease and phosphatase inhibitors (Thermo Fisher), followed by centrifugation to clear the lysate. Protein concentrations were determined by Bradford assay (Bio-Rad). Equal amounts 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.
(150) Quantitative Polymerase Chain Reaction (qPCR)
(151) Total RNA was extracted from either the snap-frozen liver tissues or hepatocyte lysates using Trizol (Invitrogen) followed by RNeasy column (Qiagen) purification. 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.
(152) Surface Plasmon Resonance (SPR)
(153) SPR measurements were performed on a BIAcore T200 (GE Healthcare) at 25° C. Buffers were degassed and filter-sterilized through 0.2 μm filters prior to use. RhIL11 or rmIl11 was immobilized onto a carboxymethylated dextran (CM5) sensor chip using standard amine coupling chemistry. For kinetic analysis, a concentration series (3.125 nM to 100 nM) of human IL11Rα or mouse Il11 rα was injected over the rhIL11, rmIl11 and reference surfaces at a flow rate of 40 μl min.sup.−1. All the analytes were dissolved in HBS-EP+(BR100669, GE Healthcare) containing 1 mg ml.sup.−1 BSA. The association and dissociation were measured for 150s and 200s respectively. After each analyte injection, the surface was regenerated by a 30 s injection of Glycine pH2.5, followed by a 5 min stabilisation period. All sensorgrams were aligned and double-referenced. Affinity and kinetic constants were determined by fitting the corrected sensorgrams with the 1:1 Langmuir model using BIAevaluation v3.0 software (GE Healthcare). The equilibrium binding constant KD was determined by the ratio of the binding rate constants kd/ka.
(154) Histology
(155) Hematoxylin&Eosin (H&E) Staining
(156) Livers were fixed for 48 h at room temperature (RT) in 10% neutral-buffered formalin (NBF), dehydrated, embedded in paraffin blocks and sectioned at 7 μm. Sections were stained with H&E according to standard protocol and examined by light microscopy.
(157) EdU Staining
(158) Livers were rinsed in cold PBS and patted dry with a lint free paper and cryo-molded in OCT compound (4583, Tissue-Tek®). After the OCT compound is frozen, liver specimens were wrapped in aluminium foil and stored in −80° C. Cryo-embedded livers were cryosectioned (−20° C.) at 7 μm thickness and allowed to dry on the slides for 1 h before proceeding to EdU detection using Baseclick's EdU IV Imaging Kit 488L (BCK488-IV-IM-L) according to the manufacturer's protocol.
(159) Immunofluorescence Staining
(160) Livers were processed and frozen as mentioned above (EdU staining section). Frozen liver tissues were sectioned at 7 μm at −20° C. and left to dry for 1 h (RT). Liver sections were fixed in cold acetone for 15 min prior to brief PBS washes, permeabilized with 0.1% TritonX-100 (T8787, Sigma), and blocked with 2.5% normal goat serum (S-1012, Vector Labs) for 1 h (RT). Liver sections were incubated with GFP (1:500) and Caspase 3 (1:1000) primary antibodies overnight (4° C.), followed by incubation with the appropriate Alexa Fluor 488/647 secondary antibodies (1:250) for 1 h (RT). DAPI was used to stain the nuclei prior to imaging by fluorescence microscope (Leica).
(161) LC-MS/MS
(162) Mouse serum samples (20 μL), calibrators and QCs were transferred into a deep well 96-well plate, then spiked with 50 μL of 10 μg l.sup.−1 of APAP-D4 heavy isotope standards. After treating with 360 μL of ice-cold Acetonitrile containing 0.1% Formic acid, the plate was mixed (1000 rpm min.sup.−1, 10 min), followed by centrifugation (2270 g, 50 min, 4° C.). 140 μL of the supernatant was carefully transferred to a 96-microwell plate and loaded into the auto-sampler for analysis by LC-MS/MS. Ion counts were then normalized against that of the heavy isotope standard, before using the standard curve for quantification. Liquid chromatographic (LC) separation of the biomarkers was carried out on an Agilent 1290 Infinity II LC system (Agilent Technologies) with PEEK coated SeQuant®ZIC®-cHILIC 3 mm, 100 Å 100×2.1 mm HPLC column (Merck Pte Ltd) maintained at 40° C. The organic solvent was Acetonitrile containing 0.1% Formic acid (Solvent A) and the aqueous solvent was 20 mM Ammonium Formate pH 4.0 (Solvent B). A linear LC gradient on Binary Pump A (G7120A, Agilent Technologies) was set up with percentage of Solvent B as follows: 10% at 0 min, 70% at 9 min, 70% at 11 min, and 10% between 11.1 and 11.5 min, with a flow rate of 0.4 ml min.sup.−1. The column was further equilibrated for 11.5 min with 10% Solvent B. An additional high speed pump, Binary Pump B, together with a Quick-Change valve head, 2-position/10-port, 1,300 bar (5067-4240, Agilent), were utilized to reduce the cycle times by automated alternating column regeneration. Percentage of Solvent B on BinaryPump B was maintained at 10% with a flow rate of 0.3 ml min.sup.−1. For mass detection, the LC eluent is connected to an Agilent 6495 Triple Quadrupole MS system (G6495A, Agilent Technologies) operated with the electrospray source in either positive or negative ionization mode. The electrospray ionization source conditions were as follows: capillary voltage of 4.0 kV, nozzle voltage of 500 V, iFunnel parameter high/low pressure RF of 90 V, nebulizer pressure of 60 psi, gas temperature of 290° C., sheath gas temperature of 350° C., Nebulizer was 35 psi, and sheath gas flow of 12 l min.sup.−1. The multiple reaction monitoring (MRM) conditions used for APAP and APAP-D4 were 152.1.fwdarw.110 with Collision Energy (CE) of 16 eV, Collision Accelerator Voltage (CAV) of 5 V and 156.fwdarw.114 with CE of 8 eV and CAV of 5 V, respectively. The MRM used for APAP-Glutathione was 457.1.fwdarw.140 with Collision Energy (CE) of 42 eV, Collision Accelerator Voltage (CAV) of 5 V.
(163) Calibration and Linearity
(164) Nine-point calibration curves were obtained by fortifying drug-free mouse serum with working solutions of APAP and APAP-Glutathione. The final concentrations of APAP were 0.32, 0.46, 2.6, 5.2, 10.3, 20.6, 41.3, 82.5 and 330 mg l.sup.− (low QC: 1.29 mg l.sup.−1; high QC: 165 mg l.sup.−1). The final concentrations of APAP-Glutathione were 0.244, 0.49, 0.98, 1.95, 3.91, 7.81, 15.6, 62.5, 125 and 250 mg l.sup.−1 (low QC: 1.95 mg l.sup.−1; high QC of 31.3 mg l.sup.−1). Standard curves corresponded to peak area ratios of each analyte to IS using weighted linear least-squares regression (1/x2) for APAP and (1/x) for APAP-Glutathione, the linearity coefficients of determination (r2) were 0.97807145 and 0.99655914, respectively. The precision and accuracy of the assay in the mice serum samples were determined as described previously (32).
(165) Statistical Analysis
(166) 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 for comparison of two different groups were performed by two-way ANOVA. Survival curves were analyzed by Gehan-Breslow-Wilcoxon test. The criterion for statistical significance was P<0.05.
6.12 References to Example 6
(167) 1. W. Bernal, J. Wendon, Acute liver failure. N. Engl. J. Med. 370 (2014), pp. 1170-1171. 2. W. M. Lee, L. S. Hynan, L. Rossaro, R. J. Fontana, R. T. Stravitz, A. M. Larson, T. J. Davern 2nd, N. G. Murray, T. McCashland, J. S. Reisch, P. R. Robuck, Acute Liver Failure Study Group, Intravenous N-acetylcysteine improves transplant-free survival in early stage non-acetaminophen acute liver failure. Gastroenterology. 137, 856-64, 864.e1 (2009). 3. H. Jaeschke, Acetaminophen: Dose-Dependent Drug Hepatotoxicity and Acute Liver Failure in Patients. Dig. Dis. 33, 464-471 (2015). 4. A. L. Chiew, C. Gluud, J. Brok, N. A. Buckley, Interventions for paracetamol (acetaminophen) overdose. Cochrane Database Syst. Rev. 2, CD003328 (2018). 5. S. Win, T. A. Than, J. Zhang, C. Oo, R. W. M. Min, N. Kaplowitz, New insights into the role and mechanism of c-Jun-N-terminal kinase signaling in the pathobiology of liver diseases. Hepatology. 67, 2013-2024 (2018). 6. H. Zhang, J. Cook, J. Nickel, R. Yu, K. Stecker, K. Myers, N. M. Dean, Reduction of liver Fas expression by an antisense oligonucleotide protects mice from fulminant hepatitis. Nat. Biotechnol. 18, 862-867 (2000). 7. R. F. Schwabe, T. Luedde, Apoptosis and necroptosis in the liver: a matter of life and death. Nat. Rev. Gastroenterol. Hepatol. 15, 738-752 (2018). 8. B. K. Gunawan, Z. Liu, D. Han, N. Hanawa, W. A. Gaarde, N. Kaplowitz, c-Jun N-Terminal Kinase Plays a Major Role in Murine Acetaminophen Hepatotoxicity. Gastroenterology. 131 (2006), pp. 165-178. 9. Y. Xie, A. Ramachandran, D. G. Breckenridge, J. T. Liles, M. Lebofsky, A. Farhood, H. Jaeschke, Inhibitor of apoptosis signal-regulating kinase 1 protects against acetaminophen-induced liver injury. Toxicol. Appl. Pharmacol. 286, 1-9 (2015). 10. S. Sekiya, A. Suzuki, Glycogen synthase kinase 3 β-dependent Snail degradation directs hepatocyte proliferation in normal liver regeneration. Proc. Natl. Acad. Sci. U.S.A 108, 11175-11180 (2011). 11. A. Marcos, R. A. Fisher, J. M. Ham, M. L. Shiffman, A. J. Sanyal, V. A. Luketic, R. K. Sterling, A. S. Fulcher, M. P. Posner, Liver regeneration and function in donor and recipient after right lobe adult to adult living donor liver transplantation. Transplantation. 69, 1375-1379 (2000). 12. B. Bhushan, U. Apte, Liver Regeneration after Acetaminophen Hepatotoxicity: Mechanisms and Therapeutic Opportunities. Am. J. Pathol. 189, 719-729 (2019). 13. G. K. Michalopoulos, Hepatostat: Liver regeneration and normal liver tissue maintenance. Hepatology. 65, 1384-1392 (2017). 14. A. A. Widjaja, B. K. Singh, E. Adami, S. Viswanathan, J. Dong, G. A. D'Agostino, B. Ng, W. W. Lim, J. Tan, B. S. Paleja, M. Tripathi, S. Y. Lim, S. G. Shekeran, S. P. Chothani, A. Rabes, M. Sombetzki, E. Bruinstroop, L. P. Min, R. A. Sinha, S. Albani, P. M. Yen, S. Schafer, S. A. Cook, Inhibiting Interleukin 11 Signaling Reduces Hepatocyte Death and Liver Fibrosis, Inflammation, and Steatosis in Mouse Models of Non-Alcoholic Steatohepatitis. Gastroenterology (2019), doi:10.1053/j.gastro.2019.05.002. 15. S. Schafer, S. Viswanathan, A. A. Widjaja, W.-W. Lim, A. Moreno-Moral, D. M. DeLaughter, B. Ng, G. Patone, K. Chow, E. Khin, J. Tan, S. P. Chothani, L. Ye, O. J. L. Rackham, N. S. J. Ko, N. E. Sahib, C. J. Pua, N. T. G. Zhen, C. Xie, M. Wang, H. Maatz, S. Lim, K. Saar, S. Blachut, E. Petretto, S. Schmidt, T. Putoczki, N. Guimarães-Camboa, H. Wakimoto, S. van Heesch, K. Sigmundsson, S. L. Lim, J. L. Soon, V. T. T. Chao, Y. L. Chua, T. E. Tan, S. M. Evans, Y. J. Loh, M. H. Jamal, K. K. Ong, K. C. Chua, B.-H. Ong, M. J. Chakaramakkil, J. G. Seidman, C. E. Seidman, N. Hubner, K. Y. K. Sin, S. A. Cook, IL-11 is a crucial determinant of cardiovascular fibrosis. Nature. 552, 110-115 (2017). 16. S. Cook, B. Ng, J. Dong, S. Viswanathan, G. DAgostino, A. Widjaja, W.-W. Lim, N. Ko, J. Tan, S. Chothani, B. Huang, C. Xie, A.-M. Chacko, N. Guimaraes-Camboa, S. Evans, A. Byrne, T. Maher, J. Liang, P. Noble, S. Schafer, IL-11 is a therapeutic target in idiopathic pulmonary fibrosis (2018), doi:10.1101/336537. 17. T. Nishina, S. Komazawa-Sakon, S. Yanaka, X. Piao, D.-M. Zheng, J.-H. Piao, Y. Kojima, S. Yamashina, E. Sano, T. Putoczki, T. Doi, T. Ueno, J. Ezaki, H. Ushio, M. Ernst, K. Tsumoto, K. Okumura, H. Nakano, Interleukin-11 links oxidative stress and compensatory proliferation. Sci. Signal. 5, ra5 (2012). 18. M. Zhu, B. Lu, Q. Cao, Z. Wu, Z. Xu, W. Li, X. Yao, F. Liu, IL-11 Attenuates Liver Ischemia/Reperfusion Injury (IRI) through STAT3 Signaling Pathway in Mice. PLoS One. 10, e0126296 (2015). 19. M. Bozza, J. L. Bliss, R. Maylor, J. Erickson, L. Donnelly, P. Bouchard, A. J. Dorner, W. L. Trepicchio, Interleukin-11 reduces T-cell-dependent experimental liver injury in mice. Hepatology. 30, 1441-1447 (1999). 20. W. L. Trepicchio, M. Bozza, P. Bouchard, A. J. Dorner, Protective effect of rhIL-11 in a murine model of acetaminophen-induced hepatotoxicity. Toxicol. Pathol. 29, 242-249 (2001). 21. J. Yu, Z. Feng, L. Tan, L. Pu, L. Kong, Interleukin-11 protects mouse liver from warm ischemia/reperfusion (WI/Rp) injury. Clin. Res. Hepatol. Gastroenterol. 40, 562-570 (2016). 22. K. Maeshima, T. Takahashi, K. Nakahira, H. Shimizu, H. Fujii, H. Katayama, M. Yokoyama, K. Morita, R. Akagi, S. Sassa, A protective role of interleukin 11 on hepatic injury in acute endotoxemia. Shock. 21, 134-138 (2004). 23. H. Mühl, STAT3, a key parameter of cytokine-driven tissue protection during sterile inflammation—the case of experimental acetaminophen (Paracetamol)-induced liver damage. Front. Immunol. 7, 163 (2016). 24. K. Schleinkofer, A. Dingley, I. Tacken, M. Federwisch, G. Mu»ller-Newen, P. C. Heinrich, P. Vusio, Y. Jacques, A. Gro»tzinger, Identification of the Domain in the Human Interleukin-11 Receptorthat Mediates Ligand Binding. available online at http://www.idealibrary.com on J. Mol. Biol. 306, 263-274 (2001). 25. C. P. Denton, V. H. Ong, S. Xu, H. Chen-Harris, Z. Modrusan, R. Lafyatis, D. Khanna, A. Jahreis, J. Siegel, T. Sornasse, Therapeutic interleukin-6 blockade reverses transforming growth factor-beta pathway activation in dermal fibroblasts: insights from the faSScinate clinical trial in systemic sclerosis. Ann. Rheum. Dis. 77, 1362-1371 (2018). 26. A. Bettaieb, J. X. Jiang, Y. Sasaki, T.-I. Chao, Z. Kiss, X. Chen, J. Tian, M. Katsuyama, C. Yabe-Nishimura, Y. Xi, C. Szyndralewiez, K. Schröder, A. Shah, R. P. Brandes, F. G. Haj, N. J. Török, Hepatocyte Nicotinamide Adenine Dinucleotide Phosphate Reduced Oxidase 4 Regulates Stress Signaling, Fibrosis, and Insulin Sensitivity During Development of Steatohepatitis in Mice. Gastroenterology. 149, 468-80.e10 (2015). 27. M. Walmsley, D. M. Butler, L. Marinova-Mutafchieva, M. Feldmann, An anti-inflammatory role for interleukin-11 in established murine collagen-induced arthritis. Immunology. 95, 31-37 (1998). 28. B. S. Qiu, C. J. Pfeiffer, J. C. Keith, Protection by recombinant human interleukin-11 against experimental TNB-induced colitis in rats. Digestive Diseases and Sciences. 41 (1996), pp. 1625-1630. 29. T. V. A. Murray, X. Dong, G. J. Sawyer, A. Caldwell, J. Halket, R. Sherwood, A. Quaglia, T. Dew, N. Anilkumar, S. Burr, R. K. Mistry, D. Martin, K. Schroder, R. P. Brandes, R. D. Hughes, A. M. Shah, A. C. Brewer, NADPH oxidase 4 regulates homocysteine metabolism and protects against acetaminophen-induced liver damage in mice. Free Radic. Biol. Med. 89, 918-930 (2015). 30. L. Hecker, R. Vittal, T. Jones, R. Jagirdar, T. R. Luckhardt, J. C. Horowitz, S. Pennathur, F. J. Martinez, V. J. Thannickal, NADPH oxidase-4 mediates myofibroblast activation and fibrogenic responses to lung injury. Nat. Med. 15, 1077-1081 (2009). 31. P. J. Wermuth, F. A. Mendoza, S. A. Jimenez, Abrogation of transforming growth factor-β-induced tissue fibrosis in mice with a global genetic deletion of Nox4. Lab. Invest. 99, 470-482 (2019). 32. T. Gicquel, J. Aubert, S. Lepage, B. Fromenty, I. Morel, Quantitative Analysis of Acetaminophen and its Primary Metabolites in Small Plasma Volumes by Liquid Chromatography-Tandem Mass Spectrometry.
(168) Journal of Analytical Toxicology. 37 (2013), pp. 110-116.
Example 7: IL-11 and IL-6 Receptor Expression and Signalling in Primary Human Hepatocytes
7.1 Introduction
(169) IL11 is a member of the interleukin 6 (IL6) cytokine family and, like IL6, binds to its membrane-bound alpha receptor (IL11Rα) 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).
7.2 Results
(170) The inventors first assessed the expression levels of IL6R, IL11Rα and gp130 in primary human hepatocytes by flow cytometry. Robust expression of IL11Rα 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 (
(171) 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 (see Example 6), 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 (
(172) 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) (
(173) 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 IL11Rα (sIL11Rα, to potentiate putative trans-signaling). IL11-induced hepatocyte death and signaling were unaffected by sgp130 or sIL11Rα (
7.3 Materials and Methods for Example 7
(174) Antibodies
(175) Albumin (ab207327, Abcam), Alexa Fluor 488 secondary antibody (ab150077, Abcam), p-ERK1/2 (4370, CST), ERK1/2 (4695, 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), IL11Rα (flow cytometry and immunofluorescence staining, ab125015, Abcam), IL11Rα (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).
(176) Recombinant Proteins
(177) Commercial recombinant proteins: Human hyperIL6 (IL6R:IL6 fusion protein, 8954-SR, R&D systems), human soluble gp130 Fc (671-GP-100, R&D systems), human IL11Rα (8895-MR-050, R&D systems). Custom recombinant proteins: Human IL11 (UniProtKB:P20809, Genscript). Human hyperIL11 (IL11Rα:IL11 fusion protein), which mimics the trans-signalling complex, was constructed using a fragment of IL11Rα (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).
(178) Chemicals
(179) 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).
(180) Primary Human Hepatocyte Culture
(181) 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% CO.sub.2. 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 outlined in the main text and/or figure legends.
(182) THP-1 Culture
(183) 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.
(184) Flow Cytometry
(185) For surface IL11Rα, IL6R, and gp130 analysis, primary human hepatocytes and THP-1 cells were stained with IL11Rα, 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).
(186) Immunofluorescence
(187) Primary human hepatocytes were seeded on 8-well chamber slides (1.5×104 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 IL11Rα, 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).
(188) RNA-Sequencing (RNA-Seq) and Ribosome Profiling (Ribo-Seq)
(189) RNA-seq and Ribo-Seq library preparations were performed as previously described (Chothani et al., 2019).
(190) Generation of RNA-Seq Libraries
(191) 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.
(192) Generation of Ribo-Seq Libraries
(193) 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. 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.
(194) 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.
(195) Data Processing and Analyses for RNA-Sequencing and Ribosome Profiling
(196) 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 IL11Rα (ENSG00000137070), IL6R (ENSG00000160712), and gp130 (ENSG00000134352). Read coverage using Ribo-seq and RNA-seq reads for IL11Rα, IL6R and gp130 was visualized using Gviz R package (Hahne and Ivanek, 2016) with strand specific alignment files.
(197) Colorimetric Assays
(198) Alanine Aminotransferase (ALT) activity in the cell culture supernatant was measured using ALT Activity Assay Kit (ab105134, Abcam) according to the manufacturer's protocol.
(199) Immunoblotting
(200) Western blots were carried out on total protein extracts from hepatocytes. Hepatocyte 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.
(201) Statistical Analysis
(202) All statistical analyses were performed using GraphPad Prism software (version 6.07). P values were corrected for multiple testing according to Tukey when several conditions were compared to each other within one experiment. The criterion for statistical significance was set at P<0.05.
7.4 References to Example 7
(203) Agthe, M., Garbers, Y., Putoczki, T., and Garbers, C. (2017). Interleukin-11 classic but not trans-signaling is essential for fertility in mice. Placenta 57, 13-16. Balic, J. J., Garbers, C., Rose-John, S., Yu, L., and Jenkins, B. J. (2017). Interleukin-11-driven gastric tumourigenesis is independent of trans-signalling. Cytokine 92, 118-123. Bolger, A. M., Lohse, M., and Usadel, B. (2014). Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30, 2114-2120. Chothani, S., Schafer, S., Adami, E., Viswanathan, S., Widjaja, A. A., Langley, S. R., Tan, J., Wang, M., Quaife, N. M., Jian Pua, C., et al. (2019). Widespread Translational Control of Fibrosis in the Human Heart by RNA-Binding Proteins. Circulation 140, 937-951. Dobin, A., Davis, C. A., Schlesinger, F., Drenkow, J., Zaleski, C., Jha, S., Batut, P., Chaisson, M., and Gingeras, T. R. (2012). STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15-21. Hahne, F., and Ivanek, R. (2016). Visualizing Genomic Data Using Gviz and Bioconductor. In Statistical Genomics, (Humana Press, New York, N.Y.), pp. 335-351. Klein, C., Wüstefeld, T., Assmus, U., Roskams, T., Rose-John, S., Müller, M., Manns, M. P., Ernst, M., and Trautwein, C. (2005). The IL-6-gp130-STAT3 pathway in hepatocytes triggers liver protection in T cell-mediated liver injury. J. Clin. Invest. 115, 860-869. Kroy, D. C., Beraza, N., Tschaharganeh, D. F., Sander, L. E., Erschfeld, S., Giebeler, A., Liedtke, C., Wasmuth, H. E., Trautwein, C., and Streetz, K. L. (2010). Lack of interleukin-6/glycoprotein 130/signal transducers and activators of transcription-3 signaling in hepatocytes predisposes to liver steatosis and injury in mice. Hepatology 51, 463-473. Langmead, B., Trapnell, C., Pop, M., and Salzberg, S. L. (2009). Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 10, R25. Liao, Y., Smyth, G. K., and Shi, W. (2014). featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics 30, 923-930. Matthews, V. B., Allen, T. L., Risis, S., Chan, M. H. S., Henstridge, D. C., Watson, N., Zaffino, L. A., Babb, J. R., Boon, J., Meikle, P. J., et al. (2010). Interleukin-6-deficient mice develop hepatic inflammation and systemic insulin resistance. Diabetologia 53, 2431-2441. Schafer, S., Viswanathan, S., Widjaja, A. A., Lim, W.-W., Moreno-Moral, A., DeLaughter, D. M., Ng, B., Patone, G., Chow, K., Khin, E., et al. (2017). IL-11 is a crucial determinant of cardiovascular fibrosis. Nature 552, 110-115. Schmidt-Arras, D., and Rose-John, S. (2016). IL-6 pathway in the liver: From physiopathology to therapy. J. Hepatol. 64, 1403-1415. The RNAcentral Consortium (2017). RNAcentral: a comprehensive database of non-coding RNA sequences. Nucleic Acids Res. 45, D128-D134. Wuestefeld, T., Klein, C., Streetz, K. L., Betz, U., Lauber, J., Buer, J., Manns, M. P., Müller, W., and Trautwein, C. (2003). Interleukin-6/glycoprotein 130-dependent pathways are protective during liver regeneration. J. Biol. Chem. 278, 11281-11288.