Antibodies
Commercial antibodies
Adiponectin (AdipoQ, 21613-1-AP, Proteintech), p-AMPK Thr172 (2535, clone 40H9, CST), AMPK (5832, clone D63G4, CST), CD31 (ab222783, clone EPR17260-263, abcam), CD68 (ab125212, abcam), cyclin D1 (55506, clone E3P5S, CST), p-ERK1/2 Thr202/Tyr204 (4370, clone D13.14.4E, CST), ERK1/2 (4695, clone 137F5, CST), GAPDH (2118, clone 14C10, CST), FHL1 (10991-1-AP, Proteintech), GFP (ab290 and ab6673, abcam), p-LKB1 Ser428 (3482, clone C67A3, CST), LKB1 (3047, clone D60C5, CST), p-mTOR Ser2448 (2971, CST), mTOR (2972,CST), p-NF-κB p65 Ser536 (3033, clone 93H1, CST), NF-κB p65 (8242, clone D14E12, CST), p16 (human, ab108349, clone EPR1473, abcam), p16 (mouse, ab232402, clone EPR20418, abcam), p21 (human, ab109520, clone EPR362, abcam), p21 (mouse, ab188224, clone EPR18021, abcam), p-p70S6K Thr389 (9234, clone 108D2, CST), p70S6K (2708, clone 49D7, CST), p-p90RSK Ser380 (11989, clone D3H11, CST), p90RSK (9355, clone 32D7, CST), PDGFRα (AF1062, R&D systems), p-S6 ribosomal protein Ser235/236 (4858, clone D57.2.2E, CST), S6 ribosomal protein (2217, clone 5G10, CST), PCNA (13110, clone D3H8P, CST), PGC1α (ab191838, abcam), SLC10A1 (MBS177905, MyBioSource), SM22α (ab14106, abcam), p-STAT3 Tyr705 (4113, clone M9C6, CST), STAT3 (4904, clone 79D7, CST), UCP1 (72298, clone E9Z2V, CST), anti-rabbit horseradish peroxidase (HRP) (7074, CST), anti-mouse HRP (7076, CST), anti-rabbit Alexa Fluor 488 (ab150077, abcam), anti-goat Alexa Fluor 488 (ab150129, abcam) and anti-rabbit Alexa Fluor 555 (ab150074, abcam). All commercially available antibodies have been validated by their manufacturer as indicated in their respective datasheet and/or website.
Custom-made antibodies
IgG (clone 11E10), anti-IL-11 (clone X203 for western blot and neutralizing studies), anti-IL11RA (clone X209 for neutralizing study) were manufactured by Genovac. The suitability of IgG (11E10) as a control antibody was validated previously29. X203 was validated for neutralization of human and mouse IL-1129,38 and for western blot38,50. X209 was validated previously for neutralization of human and mouse IL11RA38 and for western blot38.
Recombinant proteins
Recombinant human IL-11 (hIL11, Z03108, Genscript).
Chemicals
Bovine serum albumin (BSA, A7906, Sigma), 16% formaldehyde (w/v), methanol-free (28908, Thermo Fisher Scientific), DAPI (D1306, Thermo Fisher Scientific), DMSO (D2650, Sigma), rapamycin, (9904, CST), Triton X-100 (T8787, Sigma), Tween-20 (170-6531, Bio-Rad) and U0126 (9903, CST),
Ethics statements
All experimental protocols involving human subjects (commercial primary human cell lines) were performed in accordance with the ICH Guidelines for Good Clinical Practice. All participants provided written informed consent and ethical approvals have been obtained by the relevant parties as written in the datasheets provided by ScienCell from which primary human cardiac fibroblasts and primary human hepatocytes were commercially sourced.
Animal studies were carried out in compliance with the recommendations in the Guidelines on the Care and Use of Animals for Scientific Purposes of the National Advisory Committee for Laboratory Animal Research (NACLAR). All experimental procedures were approved (SHS/2019/1481 and SHS/2019/1483) and conducted in accordance with the SingHealth Institutional Animal Care and Use Committee (IACUC). Certified veterinarians were responsible for all animal experiment procedures according to the laws governing animal research in Singapore.
Cell culture
Cells 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% confluence, using standard trypsinization techniques. All experiments were carried out at P3, unless otherwise specified. Cells were serum-starved overnight in basal media prior to stimulation with different treatment conditions (in the absence or presence of antibodies or inhibitors) and durations, as outlined in the main text or figure legends. All commercial cell lines were characterized by the company based on their morphology and by using immunofluorescence for cell-specific markers, as detailed in the respective product datasheet and certificate of analysis. Potential biological contaminants for HIV-1, HBV, HCV, mycoplasma, bacteria, yeast and fungi were confirmed negative as outlined in the certificate of analysis.
Primary human cardiac fibroblasts
Primary human cardiac fibroblasts (HCFs) (52-year-old male, 6330, lot 9580, ScienCell) were authenticated by their fibroblast morphology and phenotype, characterized by immunofluorescence staining for fibronectin and vimentin. Cell were grown and maintained in complete fibroblasts medium-2 (2331, ScienCell) supplemented with 5% foetal bovine serum (FBS, 0500, ScienCell), 1% fibroblasts growth supplement-2 (FGS-2, 2382, ScienCell) and 1% penicillin-streptomycin (P/S, 0513, ScienCell). For replicative senescence study, primary HCFs were serially passaged (from P4 to P14) in the absence or presence of a neutralizing IL11RA antibody (X209) or an IgG isotype control (11E10).
Primary human hepatocytes
Primary human hepatocytes were isolated from a 22-week-old foetus (5200, lot 34967, ScienCell) and authenticated by their hepatocyte morphology and phenotype, characterized by positive immunofluorescence for cytokeratin-18 and western blot for albumin. Following recovery from the initial thaw cycle, hepatocytes were seeded at a density 4 × 105 cells per well of a collagen-coated 6-well plate and maintained in hepatocyte medium (5201, ScienCell) which contains 2% FBS and 1% penicillin-streptomycin. Hepatocytes were then used directly for downstream experiments within 48 h of seeding.
Olink proximity extension assay
Human hepatocytes were seeded at a density of 2.5 × 105 cells per well into 6-well plates. The culture supernatants were collected following stimulation with IL-11 (0, 6 and 24 h) and were sent to Olink Proteomics for proximity extension assays using the 92-protein inflammation panel. Zero-hour time points refer to time-matched, unstimulated controls that were cultured and collected in parallel with the other stated time points. In this experiment, IL-11 was added at different times to stimulate cells; for instance, at 15:00 on day 1 for the 24-h time point and at 09:00 on day 2 for the 6-h time point. Supernatants from the unstimulated control, 6 and 24-h time points were collected at the same time. The protein concentrations were expressed as normalized protein expression (NPX; log2 scale) and those proteins with concentrations below the limit of detection were excluded from analysis.
Operetta high throughput phenotyping assay
HCFs (P4, P7, P10 and P14) were seeded in 96-well black CellCarrier plates (PerkinElmer) at a density of 6 × 103 cells per well either untreated or in the presence of IgG or X209. After reaching ~80% confluence, cells were fixed in 4% formaldehyde, and permeabilized with 0.1% Triton X-100. Non-specific sites were blocked with blocking solutions (0.5% BSA and 0.1% Tween-20 in PBS). Cells were incubated overnight (4 °C) with primary antibodies (p16 and p21) at a dilution of 1:500, followed by incubation with the appropriate Alexa Fluor 488 secondary antibodies (1:1,000, 1 h, room temperature). Cells were then counterstained with 1 µg ml−1 DAPI in blocking solution. Antibodies and DAPI were diluted in blocking solutions. Each condition was imaged from duplicated wells and a minimum of seven fields per well using Operetta high-content imaging system 1483 (PerkinElmer). The measurement of p16 and p21 fluorescence intensity per area (normalized to the number of cells) was performed with Columbus 2.9 (PerkinElmer).
Seahorse assay
Primary HCFs were seeded into the Seahorse XF 96-well Cell Culture Microplate (40 × 103 cells per well) and serum-starved overnight prior to stimulations. Seahorse measurements were performed on Seahorse XFe96 Extracellular Flux Analyzer (Agilent). XF Cell Mito Stress Test kit (103015-100, Agilent) and Seahorse XF Mito Fuel Flex Test kit (103260-100, Agilent) were used according to the manufacturer’s protocol to measure the mitochondrial oxygen consumption rate and the percentage of fatty acid oxidation, respectively as described previously51. Seahorse Wave Desktop software (Ver 2.6.3) was used for report generation and data analysis.
Animal models
All mice were housed at 21–24 °C with 40–70% humidity on a 12-h light/dark cycle and provided food and water ad libitum. Our mouse colonies hold specific pathogen free (SPF) status and undergo quarterly and annual tests for common pathogens. The room housing our animals is positive for murine norovirus and Helicobacter, and these particular pathogens are deemed acceptable within our SPF facility. Sample sizes were determined based on the authors’ experience with the preliminary studies and by referencing a healthspan/lifespan study in mice52 to detect a 20% change in phenotype between treatment groups or genotypes with 80% power (α = 0.05). Sample sizes for experiments involving Il11ra1−/− and Il11−/− mice (and their respective wild-type mice) varied depending on animal availability. Mice were randomly allocated to experimental groups on the day of the treatment except for Il11ra1−/− and Il11−/− in which randomization was not applicable. Treatments or genotypes were not disclosed to investigators generating quantitative readouts during data collection but were revealed during the analysis. The mouse strains used in our study are described below.
Il11ra1-deleted mice
Male and female Il11ra1+/+ (wild-type) and Il11ra1−/− mice25 (B6.129S1-Il11ratm1Wehi/J, The Jackson Laboratory) were euthanized at 110 weeks of age for blood and tissue collection; 10–12 weeks old male and female mice of the respective genotypes were used as controls.
Il11-deleted mice
Male and female mice lacking functional alleles for Il11 (Il11−/−), which were generated and characterized previously31,50, and their wild-type counterparts were euthanized at 10–12 weeks of age (young controls) and 104–108 weeks of age (old mice).
Il11-EGFP reporter mice
Young (10-week-old) and old (100-week-old) transgenic mice (C57BL/6 J background) with EGFP knocked into the Il11 gene (Il11-EGFP mice, Cyagen Biosciences)29 were euthanized for immunofluorescence staining studies of liver, gastrocnemius and vWAT. Old wild-type littermates were used as aged negative controls.
In vivo administration of anti-IL-11
Male and female C57BL/6 J mice (Jackson Laboratory) were randomized prior to receiving either no treatment, anti-IL-11 (X203) or IgG (11E10). X203 or 11E10 (40 mg kg−1, every 3 weeks) were administered by intraperitoneal injection, starting from 75 weeks of age for a duration of 25 weeks; mice were then euthanized at 100 weeks of age.
Lifespan studies
Lifespan studies involved two distinct experimental groups (male and female) (1) C57BL/6 J mice (Jackson Laboratory) aged 75 weeks that received monthly injections of either anti-IL-11 (X203) or IgG (11E10) at a dosage of 40 mg kg−1; and (2) wild-type and Il11−/− mice. Mice were inspected daily and medicated for non-life-threatening conditions by an experienced veterinarian, as needed. The principal experimental endpoint was age of death, which was recorded when mice were found dead or at the time of euthanasia if they were deemed severely moribund (or unlikely to survive longer than 48 h) at the time of inspection, according to previously described criteria10. Mice with gross tumours, when present, were monitored for tumour progression and euthanized when tumours developed >1.5 cm in size (at largest dimension) or when tumours become ulcerated, infected or interfered with mobility as permitted by the local SingHealth IACUC. These limits were not exceeded in any of the experiments. Gross examination was conducted at autopsy following natural death or euthanasia of mice to observe and document the presence of visible tumours in the larger body organs and to record any noticeable gross appearances.
GTT and ITT
Mice were fasted for 6 h prior to baseline blood glucose measurement. For GTT, mice were injected intraperitoneally with 20% glucose at 2 mg per g lean mass. For ITT, mice were injected intraperitoneally with recombinant human insulin at 1.2 mU per g body weight. Both glucose and insulin were diluted in sterile DPBS. Blood glucose concentrations were then measured at 15, 30, 60, 75 and 90 min after glucose or insulin administration for GTT or ITT, respectively. Blood was collected via tail snip and Accu-Chek blood glucometer was used for blood glucose measurements.
Echo MRI
Mouse body composition (total body fat and lean mass measurements) was performed 1 day prior to GTT/ITT or euthanasia by Echo MRI analysis using 4in1 Composition Analyzer for live small animals (Echo Medical Systems).
Frailty scoring
Frailty scoring was performed, with observers blinded to treatment, at the start of the experiment or 1–2 days prior to euthanasia using a 27-point frailty scoring system15. Body temperatures were recorded by rectal thermometry using Kimo Thermocouple Thermometer (TK110, Kimo).
Grip strength assessment
A digital grip strength meter (BIO-GS3, BIOSEB) was used to measure full body (4 limbs) and forelimb (both forepaws) grip strengths, as per the manufacturer’s instruction. Mice were allowed to rest for at least 1 h between the two tests. The average of 3 readings of maximal average force exerted by each mouse on the grip strength meter was used for analysis.
Measurement of whole-body metabolic parameters
Whole-body metabolic parameters for IgG and X203-treated (antibodycohort), and wild-type and IL-11-knockout (KO cohort) mice were assessed by open-circuit indirect calorimetry. Animals were single-housed in the PhenoMaster automated home-cage system (TSE Systems) at a temperature of 22°C and in a humidity-controlled environment with a 12-h light/dark cycle. Parameters including oxygen consumption (VO2), carbon dioxide production (VCO2), food intake, and locomotor activity were measured simultaneously at 1-min time intervals. RER was calculated using the VCO2/VO2 ratio. Locomotor activity was divided into horizontal plane locomotor activities, defined as the total number of infrared beam breaks in the x and y axis (counts). Mice were monitored for 5 consecutive overnight periods including an acclimatization period during the first light/dark cycle (day 0–1), which was not used for analysis. For both antibody and KO cohorts, the control (IgG or wild-type) group (n = 10) and intervention (X203 or IL-11-knockout) group (n = 10) were divided equally into two consecutive monitoring sessions. Baseline RER comparison was made using measurements from the second light/dark cycle (day 1–2). Animals were given ad libitum access to food and water except during test phases introduced after day 2 where food access was restricted to assess the resting metabolic rate (measured at thermoneutrality (28°C)) and adaptation to fasting (12 h).
Bomb calorimetry
To measure energy content in mouse stool, bomb calorimetry was performed by the core service at Department of Food Science and Technology, National University of Singapore. All faecal samples were collected and stored at −80 °C prior to measurement. Approximately 0.9 g of faecal samples were placed into a combustion bag in which a cotton thread and benzoic acid tablets were used as combustion aid. The gross calorie content was then determined using the IKA C5003 Control bomb calorimeter on the isoperibolic mode, with C5001 cooling system and oxygen gas supplied. Data was derived as a single point reading (n = 1). The average relative error ranges from 0.07%–0.59%.
Colorimetric and enzyme-linked immunosorbent assays (ELISA)
The levels of ALT, AST, cholesterol and IL-6 in mouse serum were measured using Alanine Transaminase Activity Assay Kit (ab105134, abcam), Aspartate Aminotransferase Activity Assay Kit (ab105135, abcam), Cholesterol Assay Kit (ab65390, Abcam), and Mouse IL-6 Quantikine ELISA Kit (M6000B, R&D Systems), respectively. The levels of triglyceride in mouse livers and serum were measured using Triglyceride Assay Kit (ab65336, Abcam). Total collagen content in mouse livers, gastrocnemius, and vWAT were measured using Quickzyme Total Collagen assay kit (QZBtotco15, Quickzyme Biosciences). The levels of IL-6, IL-8 and IL-11 in equal volumes of cell culture media collected from experiments with primary human cells were quantified using Human IL-8/CXCL8 Quantikine ELISA Kit (D8000C, R&D Systems), Human IL-6 Quantikine ELISA Kit (D6050, R&D Systems), Human IL-11 Quantikine ELISA kit (D1100, R&D Systems). All ELISA and colorimetric assays were performed according to the manufacturer’s protocol. Triglyceride Assay Kit (ab65336, Abcam),
Immunoblotting
Western blots were carried out on total protein extracts from liver, gastrocnemius, and vWAT tissues, which were homogenized in RIPA Lysis and Extraction Buffer (89901, Thermo Fisher Scientific) containing protease and phosphatase inhibitors (A32965 and A32957, Thermo Fisher Scientific). Protein lysates were separated by SDS–PAGE, transferred to PVDF membranes, blocked for 1 h with 3% BSA, and incubated overnight with primary antibodies (1:1,000 in TBST). This study was conducted over six years, and western blots were performed on many tissues, the smallest of which provided limited protein for blotting. To conserve antibody usage and maximize data output, membranes were often cut at the appropriate molecular weight markers and probed with different antibodies. In all instances, equal loading of protein lysates per membrane was ensured. Protein bands were visualized using SuperSignal West Femto Maximum Sensitivity Substrate detection system (34096, Thermo Fisher Scientific) with the appropriate HRP secondary antibodies (1:1,000 in TBST). Raw uncropped blots are provided in Supplementary Fig. 1 and semi-quantitative densitometry analyses are provided in Supplementary Fig. 2.
Quantitative PCR with reverse transcription
Total RNA was extracted from cells or snap-frozen tissues using TRIzol Reagent (15596026, Thermo Fisher Scientific) and RNeasy Mini Kit (74104, Qiagen). PCR amplifications were performed using iScript cDNA Synthesis Kit (1708891, Bio-Rad). Gene expression analysis was performed with QuantiNova SYBR Green PCR Kit (208056, Qiagen) technology using StepOnePlus (Applied Biosystem). Expression data were normalized to GAPDH mRNA expression and fold change was calculated using 2−∆∆Ct method. The primer sequences are provided in the Supplementary Table 8.
Telomere length and mitochondrial copy number quantification
DNA from HCFs (P4 and P14) and snap-frozen liver, gastrocnemius, and vWAT was extracted with the E.Z.N.A. Tissue DNA Kit (D3396-02, Omega Bio-tek) according to the manufacturer’s protocol. Telomere length and mitochondrial copy number for HCFs were evaluated by quantitative PCR with reverse transcription (RT–qPCR) with the Relative Human Telomere Length Quantification qPCR Assay Kit (8908, ScienCell) and Relative Human Mitochondrial DNA copy number Length Quantification qPCR Assay Kit (8938, ScienCell), respectively. Similarly, the telomere length and mitochondrial copy number for mouse tissues were evaluated by RT–qPCR with the Relative Mouse Telomere Length Quantification qPCR Assay Kit (M8908, ScienCell) and Relative Human Mitochondrial DNA copy number Length Quantification qPCR Assay Kit (M8938, ScienCell), respectively.
Histology
Investigators performing histology and analysis were blinded to the genotype and treatment group.
Haematoxylin and eosin staining
Mouse vWAT were fixed in 10% neutral-buffered formalin (NBF) for 48 h, embedded in paraffin, cut into 4-μm sections followed by haematoxylin and eosin staining according to the standard protocol. Lipid droplet areas were quantified by ImageJ (version 1.53t, NIH) with the adipocytes tools plugin (https://github.com/MontpellierRessourcesImagerie/imagej_macros_and_scripts/wiki/Adipocytes-Tools) from 5 randomly selected fields at 200× magnification in vWAT images per mouse, and 5 mice per group were assessed. The mean value of lipid droplet areas per field was plotted for the final data presentation.
Immunohistochemistry
Four-micrometre mouse vWAT sections were dewaxed with histoclear and a gradient ethanol wash, followed by permeabilization using 1% Triton X-100 for 10 min and antigen retrieval process with Reveal Decloaker (RV1000M, Biocare Medical) using a double boiler method at 110 °C for 20 min. Slides were allowed to cool in the container together with the Reveal Decloaker solution for 10 min under running water. Double blocking was achieved with (1) H2O2 for 10 min and (2) 2.5% normal horse serum for 1 h (S-2012, Vector Labs). vWAT sections were incubated overnight at 4 °C with primary antibody (CD68, 1:100 in PBST) and visualized by probing with Horse Anti-Rabbit IgG Polymer Kit (MP-7401, Vector Labs) for 1 h at 37 °C and ImmPACT DAB Peroxidase Substrate Kit (SK-4105, Vector Labs). Haematoxylin (H-3401, Vector Labs) was used to counterstain the nuclei prior to imaging by light microscopy (Olympus IX73).
Immunofluorescence
Young (10-week) and aged (100-week) Il11EGFP/+ and aged wild-type Il11+/+ mice underwent perfusion-fixation with PBS and 4% paraformaldehyde for multi-organ collection at terminal euthanasia. Mouse liver, vWAT and gastrocnemius were further fixed in 4% paraformaldehyde at 4 °C and serial 15–30% sucrose dehydration over 48 h before they were cryo-embedded in OCT medium. 5 µm sections were heat antigen retrieved using Reveal Decloaker (RV1000M, Biocare), permeabilized with 0.5% Triton X-100, and blocked with 5% normal horse serum before probing with primary antibodies diluted in 2.5% normal horse serum at 4 °C overnight. The antibody dilutions used for immunofluorescence studies are as follows: adiponectin, GFP, PDGFRα and SLC10A1 (1:100); CD31, FHL1 and SM22α (1:200). Alexa Fluor-conjugated secondary antibodies (1:300 in 2.5% normal horse serum) were incubated for 2 h at room temperature for visualization. Autofluorescence was quenched with 0.1% Sudan Black B for 20 min. DAPI was included for nuclear staining before mounting and sealed. Photomicrographs were randomly captured by researchers blinded to the strain and age groups.
RNA-seq libraries
Total RNA was isolated from liver, fat and skeletal muscle of mice receiving either IgG or X203 using RNeasy Mini Kit (74104, Qiagen) and quantified using Qubit RNA Broad Range Assay Kit (Q10210, Thermo Fisher Scientific). RNA quality scores (RQS) were assessed using the RNA Assay (CLS960010, PerkinElmer) and DNA 5 K/RNA/CZE HT Chip (760435, PerkinElmer) on a LabChip GX Touch HT Nucleic Acid Analyzer (CLS137031, PerkinElmer). TruSeq Stranded mRNA Library Prep kit (20020594, Illumina) was used to assess transcript abundance following the manufacturer’s instructions. In brief, poly(A) + RNA was purified from 1 µg of total RNA with RQS > 6, fragmented, and used for cDNA conversion, followed by 3′ adenylation, adapter ligation, and PCR amplification. The final libraries were quantified using Qubit DNA Broad Range Assay Kit (Q32853, Thermo Fisher Scientific) according to the manufacturer’s guide. The average fragment size of the final libraries was determined using DNA 1 K/12 K/Hi Sensitivity Assay LabChip (760517, PerkinElmer) and DNA High Sensitivity Reagent Kit (CLS760672, PerkinElmer). Libraries with unique dual indexes were pooled and sequenced on partial lanes targeting ~50 M reads per sample on a HiSeq or a NovaSeq 6000 sequencer (Illumina) using 150-bp paired-end sequencing chemistry.
Data processing and analysis for RNA-seq
Fastq files were generated by demultiplexing raw sequencing files (.bcl) with Illumina’s bcl2fastq v2.20.0.422 with the –no-lane-splitting option. Low quality read removal and adapter trimming was carried out using Trimmomatic V0.36 with the options ILLUMINACLIP:
Statistical analysis
Statistical analyses were performed using GraphPad Prism software (version 10). Datasets were tested for normality with Shapiro–Wilk tests. For normally distributed data, two-tailed Student’s t-tests or one-way ANOVA were used for analysing experimental setups requiring testing of two conditions or more than two conditions, respectively. P values were corrected for multiple testing according to Dunnett (when several experimental groups were compared to a single control group) or Tukey (when several conditions were compared to each other within one experiment) tests. Non-parametric tests (Kruskal–Wallis with Dunn’s correction in place of ANOVA and Mann–Whitney U test in place of two-tailed Student’s t-tests) were conducted for non-normally distributed data. Comparison analysis for two parameters from two different groups were performed by two-way ANOVA and corrected with Sidak’s multiple comparisons. Two-way repeated measures ANOVA (Geisser–Greenhouse correction) with Sidak’s multiple comparisons was applied to temporal sampling in paired subjects for GTT, ITT and body weight. Individual endpoint frailty indices were ranked and compared using two-tailed Mann–Whitney test to compare (1) old Il11−/− versus wild-type females; (2) old Il11−/− versus wild-type males; or (3) IgG versus X203 groups in females, and the Kruskal–Wallis test with Dunn’s multiple comparisons of untreated, IgG and X203 treatment groups in males. The two-population proportions analysis (two-tailed) was used for comparing the difference in the proportion of cancer occurrence and seminal vesicle dilatation between two groups. The criterion for statistical significance was set at P < 0.05. For the lifespan studies, differences in survival between the experimental groups (Il11−/− versus wild-type or X203 versus IgG) were compared using the Kaplan–Meier method implemented in IBM SPSS (release 29.0.1.0), and statistical significance (P value) was assessed by means of the log-rank (Mantel–Cox) test. In addition to the log-rank test (that gives equal weight to all time points), we used the Wilcoxon test (that gives more weight to deaths at early time points), which provided significant results in all comparisons, therefore yielding a similar conclusion to reject the null hypothesis. Both survival comparison methods are non-parametric tests based on the chi-square statistic and provide two-tailed P values. The complete list of exact p-values and terms for supporting statistical information is provided in Supplementary Table 9.
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.