Hepatocyte-specific IL11 cis-signaling drives lipotoxicity and underlies the transition from NAFLD to NASH AbstractIL11 is important for fibrosis in non-alcoholic steatohepatitis (NASH) but its role beyond the stroma in liver disease is unclear. Here, we investigate the role of IL11 in hepatocyte lipotoxicity. Hepatocytes highly聽express IL11RA and secrete IL11 in response to lipid loading. Autocrine IL11 activity causes hepatocyte death through NOX4-derived ROS, activation of ERK, JNK and caspase-3, impaired mitochondrial function and reduced fatty acid oxidation. Paracrine IL11 activity stimulates hepatic stellate cells and causes fibrosis. In mouse models of NASH, hepatocyte-specific deletion of Il11ra1 protects against liver steatosis, fibrosis and inflammation while reducing serum glucose, cholesterol and triglyceride levels and limiting obesity. In mice deleted for Il11ra1, restoration of IL11 cis-signaling in hepatocytes reconstitutes steatosis and inflammation聽but not fibrosis. We found no evidence for the existence of IL6 or IL11 trans-signaling in hepatocytes or NASH. These data show that IL11 modulates hepatocyte metabolism and suggests a mechanism for NAFLD to NASH transition. IntroductionInterleukin 11 (IL11) is a fibrogenic factor1,2,3,4 that is elevated in fibrotic precision-cut liver slices across species5. IL11 has recently been shown to have negative effects on hepatocyte function after toxic liver insult6 and, directly or indirectly, contributes to nonalcoholic steatohepatitis (NASH) pathologies7. At the other end of the spectrum, a number of earlier publications suggest that IL11 is protective in mouse models of ischemic-, infective-, or toxin-induced liver damage8,9,10,11,12,13. However, it is now apparent that the recombinant human IL11 (rhIL11) reagent used in these earlier studies has unexpected effects in the mouse6 and the question as to the true biological effect of IL11 in the liver, specifically in hepatocytes, remains open.IL11 is a member of the interleukin 6 (IL6) cytokine family and binds to its cognate alpha receptor (IL11RA) and then to glycoprotein 130 (gp130) to signal in cis. IL6 itself has been linked to liver function and publications suggest an overall beneficial effect14,15,16,17,18,19. Aside from cis-signaling, IL6 can also bind to soluble IL6 receptor (sIL6R) and signal in trans. IL6 trans-signaling is considered maladaptive in the context of metabolic and autoimmune disease but, somewhat paradoxically, beneficial for liver regeneration16. It is possible that IL11, like IL6, also signals in trans but experiments to date have found no evidence for this in tumors or reproductive tissues20,21.The factors underlying the transition from nonalcoholic fatty liver disease (NAFLD) to NASH are multifactorial but lipid loading of hepatocytes is of central importance22. Certain lipid species are toxic for hepatocytes and lipotoxicity leads to cytokine release causing hepatocyte death along with activation of hepatic stellate cells (HSCs) and immune cells22,23. Lipotoxicity, such as that due to palmitate24, is an early event in NASH and represents a linkage between diet, NAFLD, and NASH. While genetic or pharmacological inhibition of IL6 cis-signaling worsens steatosis phenotypes17,18,25, a role for IL11 in hepatic lipotoxicity has not been described.In the current study, we used a range of in vitro and in vivo approaches to address key questions regarding IL11 in hepatocyte biology, NAFLD, and NASH: (1) Defining the role of IL11 signaling in human hepatocytes, (2) examining whether lipotoxicity is related to IL11 activity in hepatocytes, (3) establishing whether IL11 (or IL6) trans-signaling contributes to NASH, (4) dissecting the inter-relationship between IL11 cis-signaling in hepatocytes and the development of steatosis, hepatocyte death, inflammation, and fibrosis. These studies demonstrate a detrimental effect of lipotoxicity-associated IL11 signaling in hepatocytes that appears to be apical pathology in the aetiology of NASH.ResultsHigh levels of IL11RA expression in primary human hepatocytesWe first assessed the expression levels of IL6R and IL11RA in healthy human or mouse liver by immunohistochemistry. In both human and mouse liver sections there was limited staining of IL6R but robust expression of IL11RA, which appeared mostly localized to hepatocytes (Fig.聽1a; Supplementary Fig.聽2a). This is consistent with staining data from the human protein atlas using two additional antibodies (CAB032830 and HPA036652; https://www.proteinatlas.org). Interestingly, as compared to control livers, IL11RA expression was increased in liver biopsies from patients with NASH and also in livers from mice with NASH on a Western Diet supplemented with fructose (Supplementary Fig.聽2b and c).Fig. 1: IL11RA is highly expressed in hepatocytes and IL11 cis-signaling is hepatotoxic.a Immunohistochemistry staining of IL11RA and IL6R in healthy human liver sections (scale bars, 20鈥壜祄, n鈥?鈥? independent experiment, due to limited amount of human liver section). b Flow cytometry forward scatter (FSC) plots of IL11RA, IL6R, and gp130 staining and fluorescence intensity plots of IL11RA and IL6R staining on hepatocytes and THP-1. c Abundance of IL11RA1 and IL6R reads in hepatocytes at baseline based on RNA-seq (left) and Ribo-seq (right) (transcripts per million, TPM) (n鈥?鈥?). d, e Read coverage of d IL11RA1 and e IL6R transcripts based on RNA-seq (gray) and Ribo-seq (red) of primary human hepatocytes (n鈥?鈥?). f Western blots showing ERK, JNK, and STAT3 activation status and g ALT secretion (n鈥?鈥?) by hepatocytes following a dose range stimulation of either hyperIL11 or hyperIL6. h ALT levels in the supernatants of hepatocytes stimulated with hyperIL11 alone or in the presence of increasing amounts of soluble gp130 (sgp130) (n鈥?鈥?). i, j Western blots of hepatocyte lysates showing i phosphorylated ERK and JNK and their respective total expression in response to hyperIL11 stimulation alone or with sgp130 and j phospho-STAT3 and total STAT3 in response to hyperIL6 stimulation with and without sgp130. k Representative FSC plots of propidium Iodide (PI) staining of IL11-stimulated hepatocytes in the presence of sgp130 or soluble IL11RA (sIL11RA). l Western blots showing phospho-ERK, phospho-JNK, cleaved caspase-3, and their respective total expression, NOX4, and GAPDH in hepatocytes in response to IL11 stimulation alone or in the presence of sgp130 or sIL11RA. i, j, l Representative data of n鈥?鈥? independent experiments. b鈥?b>l Primary human hepatocytes; f鈥?b>l 24鈥塰 stimulation; hyperIL11, hyperIL6, IL11 (20鈥塶g/ml), sgp130, sIL11RA (1鈥壜礸/ml). c, g, h Data are shown as box-and-whisker with median (middle line), 25th鈥?5th percentiles (box), and min鈥搈ax values (whiskers). g, h One-way ANOVA with Dunnett鈥檚 correction. Source data are provided as a Source data file.Full size imageFlow cytometry studies confirmed that both IL11RA and gp130 are聽highly expressed in primary human hepatocytes whereas IL6R was expressed at much lower levels (Fig.聽1b; Supplementary Fig.聽3a). In contrast, IL6R was highly聽expressed on immune cells where IL11RA expression was low that is聽in keeping with previous data showing reciprocal expression of these two聽receptors on different cells (Fig.聽1b)1,19. RNA-seq and Ribo-seq studies confirmed IL11RA and gp130 transcripts to be highly expressed and actively translated in hepatocytes, whereas IL6R transcripts were low and there was little eveidence of聽IL6R translation (Fig.聽1c鈥揺; Supplementary Fig.聽3b and c). Immunofluorescence staining of primary human and mouse hepatocytes and some of the most commonly used hepatocyte-like cell lines (HepG2 and AML12) revealed that all of these cells consistently had high IL11RA but low IL6R expression (Supplementary Fig.聽3d). Overall, these data show strong co-expression of both IL11RA and gp130 in hepatocytes across species.IL11 cis-signaling causes hepatocyte deathIn hepatocytes, IL11 activated non-canonical signaling pathways (ERK and JNK) in a dose-dependent manner (2.5鈥?0鈥塶g/ml), while IL6 activated STAT3 (Supplementary Fig.聽4a). To compare IL6 and IL11 signaling, while circumventing potential complexities associated with different levels of IL11RA or IL6R expression, we used a synthetic IL6 trans-signaling construct (hyperIL6) and compared this with a synthetic IL11 trans-signaling complex (hyperIL11). HyperIL11, like IL11 itself, dose-dependently activated ERK and JNK. Similarly, IL6 trans-signaling dose-dependently induced STAT3 phosphorylation, as seen with IL6 itself, but did not activate ERK or JNK (Fig.聽1f). Thus, IL11 or IL6 (cis and trans) signaling results in activation of different intracellular pathways in hepatocytes.HyperIL11, like IL116, caused a dose-dependent increase in alanine transaminase (ALT) in the media of primary human hepatocyte cell cultures whereas hyperIL6 (20鈥塶g/ml) had a significant, albeit limited, protective effect (ALT fold change (FC)鈥?鈥?.9; P鈥?鈥?.0468) (Fig.聽1g). Soluble gp130 (sgp130) is a selective inhibitor of trans-signaling complexes acting through gp13016. Consistent with its reported decoy effects, sgp130 blocked the activation of signaling pathways downstream of both hyperIL11 (ERK/ JNK) and hyperIL6 (STAT3) as well as inhibited the hepatotoxic effects of hyperIL11 (Fig.聽1h鈥搄).We next probed for the existence of IL11 trans-signaling in a physiological context, in the absence of preformed, synthetic/alien protein complexes. We stimulated cells with IL11 in the presence of either soluble gp130 (sgp130, to inhibit putative trans-signaling) or soluble IL11RA (sIL11RA, to potentiate putative trans-signaling). IL11-induced caspase-3 activation, NOX4 upregulation, ERK and JNK signaling, and hepatocyte cell death were unaffected by sgp130 or sIL11RA (1鈥壜礸/ml) (Fig.聽1k and l; Supplementary Fig.聽4b). Furthermore, IL11 dose-dependently caused hepatocyte cell death, which was unaffected by the addition of sgp130 or sIL11RA (Supplementary Fig.聽4c). Reciprocally, increasing doses of sgp130 or sIL11RA had no effect on ALT release from IL11-stimulated hepatocytes (Supplementary Fig.聽4d). These data argue against the existence of IL11 trans-signaling in hepatocytes.Reactive oxygen species and caspases are implicated, together or alone, in lipotoxic cell death26. To probe the mechanisms underlying IL11-regulated hepatocyte death we inhibited NOX4 with GKT-13781 or DPI and caspases with Z-VAD-FMK. NOX4 inhibitors reduced IL11-induced ERK and JNK activation and robustly protected hepatocytes from IL11-induced cell death (Supplementary Fig.聽4e and f). Pan-caspase inhibition, while protective, was not as effective as NOX4 inhibition in preventing cell death and did not reduce either NOX4 induction or ERK activation (Supplementary Fig.聽4g and h). This places NOX4 activity upstream of late-stage (24鈥塰) ERK and caspase-3 activation in IL11-stimulated hepatocytes and suggests that apoptotic cell death is only one mode of cell death in this context.IL11 cis-signaling underlies lipotoxicity in hepatocytesTo begin to examine the role of IL11 in fatty liver disease, we modeled hepatocyte lipotoxicity, widely viewed as an initiating pathology for NASH and related to cytokine release22. To do so, we loaded hepatocytes with palmitate using a concentration of saturated fatty acids seen in the serum of NAFLD patients27. Palmitate-loaded hepatocytes secreted large amounts of IL11 (28-fold higher than control, P鈥?lt;鈥?.0001) (Fig.聽2a), produced more IL6, CCL2, and CCL5 (Fig.聽2b鈥揹), and exhibited cell death and ALT release (Fig.聽2e鈥揼).Fig. 2: IL11 drives NASH phenotypes through autocrine effects in lipotoxic hepatocytes and paracrine activity in hepatic stellate cells.a鈥?b>l Data for palmitate (0.5鈥塵M) loading experiment on primary human hepatocytes (24鈥塰) in the presence of either IgG (2鈥壜礸/ml), anti-IL11RA (X209, 2鈥壜礸/ml), or sgp130 (1鈥壜礸/ml). a IL11, b IL6, c CCL2, and d CCL5 protein secretion levels as measured by ELISA of supernatants (n鈥?鈥?). e Representative FSC plots and f quantification of PI+ve hepatocytes stimulated with palmitate (n鈥?鈥?). g ALT levels in supernatants (n鈥?鈥?). h Total and reduced hepatocyte glutathione (GSH) levels (n鈥?鈥?). i Representative fluorescence images of DCFDA (2鈥?7鈥?dichlorofluorescein diacetate) staining for ROS detection (scale bars, 100鈥壜祄) (n鈥?鈥? independent experiments). j Western blots of phospho-ERK, ERK, phospho-JNK, JNK, cleaved caspase-3, caspase-3, NOX4, and GAPDH. Data from two independent biological experiments are shown. k Percentage of fatty acid oxidation by Seahorse assay (n鈥?鈥?0). l Representative fluorescence images (scale bars, 100鈥壜祄) of ACTA2+ve cells and Collagen I immunostaining for experiment shown in Supplementary Fig.聽5j (n鈥?鈥? independent experiments, 14 measurements per condition per experiment). a鈥?b>d, f, g Mean鈥壜扁€塖D; h, k data are shown as box-and-whisker with median (middle line), 25th鈥?5th percentiles (box), and min鈥搈ax values (whiskers). a鈥?b>d, f鈥?b>h, k One-way ANOVA with Tukey鈥檚 correction. Source data are provided as a Source data file.Full size imageTo test if IL11 secretion from lipid-laden hepatocytes was mechanistically related to lipotoxicity we incubated cells with neutralizing anti-IL11RA antibody (X209)7 or sgp130. X209 reduced the secretion of all cytokines, including IL11 itself, whereas sgp130 had no effect (Fig.聽2a鈥揼). This suggests the importance of autocrine loop of IL11 cis-signaling for hepatocyte lipotoxicity. Using hyperIL11 stimulation, which is not detected by IL11 enzyme-linked immunosorbent assay1, we then established the existence of feed-forward autocrine IL11 signaling in hepatocytes (Supplementary Fig.聽5a). The production of reactive oxygen species (ROS) from damaged mitochondria is important for lipotoxicity23 and ROS from NOX4 are also pertinent28. Consistent with IL11 cis-driven effects on ROS in palmitate-loaded hepatocytes, we found that X209, but not sgp130, partially restored total and reduced glutathione (GSH) levels, and this was accompanied by reduction in ROS (Fig.聽2h and i; Supplementary Fig.聽5b).Lipotoxicity is strongly associated with activation of JNK, which contributes to caspase-3 activation, lipoapoptosis, and dysfunctional mitochondria function. Accordingly, palmitate-loaded hepatocytes exhibited an increase in JNK activation along with ERK phosphorylation, caspase-3 cleavage, and NOX4 upregulation (Fig.聽2j). Treatment of lipotoxic cells with Z-VAD-FMK prevented caspase-3 cleavage, as expected, and reduced hepatocyte death (Supplementary Fig.聽5c and d). Notably, inhibition of IL11 signaling with X209 reduced palmitate-induced NOX4 upregulation along with JNK, ERK, and caspase-3 activation (Fig.聽2j). While STAT3 was activated by palmitate loading, this effect appears unrelated to IL11-driven lipoapoptosis as it was unaffected by X209 (Fig.聽2j).Lipid loading of hepatocytes was confirmed by Oil Red O staining (Supplementary Fig.聽5e) and quantitative analysis revealed a reduction in hepatocyte triglyceride (TG) levels with X209 treatment (Supplementary Fig.聽5f). Inhibition of IL11 signaling also resulted in increased mitochondrial O2 consumption rates, and maximal and spare respiratory capacity levels (Supplementary Fig.聽5g and h). This effect was mediated by IL11 cis-signaling as sgp130 had no effect (Supplementary Fig.聽5g and h). The fact that TGs were reduced suggested increased beta-oxidation may play a role29. We tested for this and found that inhibition of IL11 cis-signaling improved fatty acid oxidation in lipid-laden hepatocytes (Fig.聽2k).In the context of fatty liver disease, hepatocytes release factors to activate HSCs and activate/recruit immune cells, which are key events in the progression from compensated steatosis to NASH. As IL11 is secreted by lipotoxic hepatocytes (Fig.聽2a), it could in theory act in paracrine on HSCs to drive HSC-to-myofibroblast transformation7. We cultured HSCs with conditioned media from either control or palmitate-treated hepatocytes and found that media from lipotoxic hepatocytes strongly induced ACTA2 and Collagen expression in HSCs (Fig.聽2l; Supplementary Fig.聽5i and j). Addition of X209 to the conditioned media blocked ACTA2 and Collagen (Fig.聽2l; Supplementary Fig.聽5i and j). These data demonstrate that lipotoxic hepatocytes release IL11 that acts in a paracrine fashion to activate IL11 signaling in HSCs.No evidence for IL11 or IL6 trans-signaling in two NASH modelsWe then tested whether trans-signaling underlies NASH in vivo using two preclinical mouse NASH models: The Western Diet supplemented with fructose (WDF) model and the methionine- and choline-deficient high fat diet (HFMCD) model. The WDF model is associated with obesity, hyperlipidemia, high glucose levels, and insulin resistance and is seen as translatable to common forms of human NASH, as in diabetic patients. The HFMCD model stimulates rapid onset NASH, specifically driven by hepatocyte lipotoxicity, that is associated with weight loss in the absence of insulin resistance. Lipotoxicity is common to both models whereas obesity and insulin resistance are not.Three weeks prior to starting either the WDF or HFMCD diet, mice were injected with an AAV8 virus encoding either albumin promoter-driven sgp130 (AAV8-Alb-sgp130), which contains the whole extracellular domain of mouse gp130 protein (amino acid 1鈥?17), or albumin promoter alone (AAV8-Alb-Null) (Fig.聽3a; Supplementary Fig.聽6a, 7a). AAV8-Alb-sgp130 administration induced high levels of sgp130 in the liver, which was also detectable in the peripheral circulation, suitable for both local and systemic inhibition of putative IL6 or IL11 trans-signaling (Fig.聽3b; Supplementary Figs.聽6b, 7b and c).Fig. 3: Inhibition of IL6 family cytokine trans-signaling has no effect on NASH or metabolic phenotypes in mice on Western Diet supplemented with fructose.a Schematic of WDF feeding in mice with hepatocyte-specific expression of sgp130 for data shown in (b鈥?b>p). Three weeks following AAV8-Alb-Null or AAV8-Alb-sgp130 virus injection, mice were fed WDF for 16 weeks. b Western blots showing hepatic levels of sgp130, IL11, IL6, and GAPDH as internal control (n鈥?鈥? mice/group). c Serum IL11 levels. d Serum IL6 levels. e Representative gross anatomy, H E-stained (scale bars, 50鈥壜祄), and Masson鈥檚 Trichrome (scale bars, 100鈥壜祄) images of livers. Representative dataset from n鈥?鈥? mice/group is shown for gross anatomy; representative dataset from n鈥?鈥? mice/group is shown for H E-stained and Masson鈥檚 Trichrome images. f Liver weight. g Hepatic triglycerides content. h Serum ALT levels. i Serum AST levels. j Hepatic collagen levels. k Fasting blood glucose levels. l Serum triglycerides levels. m Serum cholesterol levels. n Hepatic GSH content. o Hepatic pro-inflammatory and fibrotic genes expression heatmap (values are shown in Supplementary Fig.聽6d and e). p Western blots of hepatic phospho-ERK, ERK, phospho-JNK, JNK, phospho-STAT3, and STAT3 (n鈥?鈥? mice/group). c, d, f鈥?b>o n鈥?鈥? mice/group. c, d, f鈥?b>n Data are shown as box-and-whisker with median (middle line), 25th鈥?5th percentiles (box), and min鈥搈ax values (whiskers); one-way ANOVA with Tukey鈥檚 correction. Source data are provided as a Source data file.Full size imageAfter 16 weeks of WDF, IL11 levels were strongly upregulated in the liver and the periphery but IL6 expression was unaffected (Fig.聽3b鈥揹). Mice on WDF became obese (Supplementary Fig.聽6c), an approximate twofold increase in liver mass and developed severe steatosis and fibrosis by gross morphology, histology, and quantitative analysis of liver triglycerides (Fig.聽3e鈥揼). These phenotypes were unaffected by high levels of sgp130 expression (Fig.聽3b鈥揼). Similarly, mice on WDF had elevated levels of ALT, AST, collagen, and peripheral cardiovascular risk factors (fasting blood glucose, serum triglycerides, and serum cholesterol), along with depleted levels of GSH but none of these parameters were affected by sgp130 (Fig.聽3h鈥搉). Livers from mice on WDF showed increased expression of pro-inflammatory and fibrosis genes and this signature was unaffected by sgp130-mediated inhibition of putative trans-signaling (Fig.聽3o; Supplementary Fig.聽6d and e).In a second set of experiments we induced NASH using the HFMCD diet (Supplementary Fig.聽7a). HFMCD diet increased IL11 levels in liver and serum, whereas IL6 levels were slightly lower in the liver albeit mildly increased in the periphery (Supplementary Fig.聽7b, d and e). Mice on HFMCD diet developed rapid and profound steatosis by gross morphology, histology, and molecular assays, which was unaltered by sgp130 expression (Supplementary Fig.聽7f and g). Hepatocyte damage markers (ALT and AST) and collagen expression were elevated and GSH levels were depleted by HFMCD diet, irrespective of sgp130 expression (Supplementary Fig.聽7f, h鈥搆). Similarly, the HFMCD diet was associated with dysregulated expression of inflammation and fibrosis genes and these molecular phenotypes were unaffected by sgp130 expression (Supplementary Fig.聽7l and m).At the signaling level, both WDF and HFMCD diets stimulated ERK and JNK activation, consistent with elevated IL11 cis-signaling (Fig.聽3p; Supplementary Fig.聽7n). In contrast, phospho-STAT3 levels in the liver were not elevated by WDF (Fig.聽3p) and were mildly increased in mice on the HFMCD diet (Supplementary Fig.聽7n). In all instances, there was no effect of sgp130 on diet-induced signaling events. Overall, these data suggest that neither IL6 nor IL11 trans-signaling plays a role in NASH, which is consistent with other studies where IL6 family trans-signaling has not been detected20,21,30,31.Hepatocyte-specific IL11 cis-signaling is required to initiate NASHWhile we found no evidence to support IL11 trans-signaling in NASH models, our in vitro data showed evidence of pathological IL11 cis-signaling in lipotoxic hepatocytes. To test the effects of IL11 cis-signaling in heptocytes in vivo, we administered AAV8-Alb-Cre to Il11ra1loxP/loxP mice to delete Il11ra1 specifically in hepatocytes (CKO mice). CKO mice were then fed either normal chow (NC), HFMCD diet or WDF (Figs.聽4a, 5a). Liver IL11RA protein was greatly diminished in the CKOs following AAV8-Alb-Cre injection, showing the model to be effective and suggesting that hepatocytes are the largest hepatic reservoir of IL11RA (Figs.聽4b, 5b). Both WT and CKO mice had similar levels of serum IL11 after 4 weeks of HFMCD and 16 weeks of WDF (Supplementary Figs.聽8a, 9a).Fig. 4: Hepatocyte-specific inhibition of IL11 cis-signaling protects mice against HFMCD diet-induced NASH.a Schematic of HFMCD feeding regimen for AAV8-Alb-Cre injected Il11ra1loxP/loxP (conditional knockout; CKO) mice for experiments shown in (b鈥?b>k). Il11ra1loxP/loxP mice were intravenously injected with either AAV8-Alb-Null or AAV8-Alb-Cre to delete Il11ra1 specifically in hepatocytes 3 weeks prior to the start of HFMCD diet. b Western blots of hepatic IL11RA and GAPDH (n鈥?鈥? mice/group). c Body weight (shown as a percentage (%) of initial body weight). d Representative gross anatomy, H E-stained (scale bars, 50鈥壜祄), and Masson鈥檚 Trichrome (scale bars, 100鈥壜祄) images of livers. Representative dataset from n鈥?鈥? mice/group is shown for gross anatomy; representative dataset from n鈥?鈥? mice/group is shown for H E-stained and Masson鈥檚 Trichrome images. e Hepatic triglycerides content. f Serum ALT levels. g Serum AST levels. h Hepatic GSH content. i Hepatic collagen levels. j Heatmap showing hepatic mRNA expression of pro-inflammatory markers (Tnf伪, Ccl2, Ccl5) and fibrotic markers (Col1a1, Col1a2, Col3a1, Acta2). Values are shown in Supplementary Fig.聽8c and d. k Western blots showing hepatic ERK and JNK activation status (n鈥?鈥? mice/group). c, e鈥?b>j NCD (n鈥?鈥? mice/group), HFMCD (n鈥?鈥? mice/group). c Data are shown as mean鈥壜扁€塖D, two-way ANOVA with Tukey鈥檚 correction, statistical significance (P values) are shown for comparison between WT HFMCD and CKO HFMCD; e鈥?b>i data are shown as box-and-whisker with median (middle line), 25th鈥?5th percentiles (box), and min鈥搈ax values (whiskers); two-way ANOVA with Tukey鈥檚 correction. Source data are provided as a Source data file.Full size imageFig. 5: Hepatocyte-specific inhibition of IL11 cis-signaling protects mice against WDF-induced obesity and NASH.a Schematic of WDF-fed control and CKO mice for data shown in (b鈥?b>m). Three weeks following AAV8-Alb-Null or AAV8-Alb-Cre virus injection, CKO mice were fed WDF for 16 weeks. b Western blots showing hepatic levels of IL11RA and GAPDH (n鈥?鈥? mice/group). c Body weight (shown as a percentage (%) of initial body weight). d Fat mass. e Representative gross anatomy, H E-stained (scale bars, 50鈥壜祄), and Masson鈥檚 Trichrome (scale bars, 100鈥壜祄) images of livers. Representative dataset from n鈥?鈥?/group is shown for gross anatomy; representative dataset from n鈥?鈥? mice/group is shown for H E-stained and Masson鈥檚 Trichrome images. f Hepatic triglycerides content. g Liver weight. h Serum ALT levels. i Serum AST levels. j Hepatic GSH content. k Hepatic collagen levels. l Hepatic pro-inflammatory and fibrotic genes expression on heatmap (values are shown in Supplementary Fig.聽9c and d). m Western blots showing activation status of hepatic ERK and JNK (n鈥?鈥? mice/group). c, d, f鈥?b>l n鈥?鈥? mice/group. c, d Data are shown as mean鈥壜扁€塖D, two-way ANOVA with Tukey鈥檚 correction, statistical significance (P values) are shown for comparison between WT WDF and CKO WDF; f鈥?b>k data are shown as box-and-whisker with median (middle line), 25th鈥?5th percentiles (box), and min鈥搈ax values (whiskers); two-way ANOVA with Tukey鈥檚 correction. Source data are provided as a Source data file.Full size imageIn addition to rapidly stimulating lipotoxicity-driven NASH, the HFMCD diet causes weight loss32. Surprisingly, weight loss in mice on the HFMCD diet was initially limited and later reversed in CKO mice (Fig.聽4c; Supplementary Fig.聽8b). Mice on WDF gained weight and fat mass throughout the experimental period, as expected. However, and equally surprising, these obesity phenotypes were mitigated in CKO mice (Fig.聽5c and d; Supplementary Fig.聽9b). These data suggest that inhibition of IL11 signaling is permissive for weight homeostasis, with context-specific anti-cachectic or anti-obesity effects, which requires further study.By gross morphology, histology and quantitative triglyceride analysis, the CKO mice on either HFMCD or WDF diet were protected from steatosis (Figs.聽4d and e, 5e, and f) and those on WDF had less liver mass (Fig.聽5g). Liver damage markers were markedly reduced in CKO mice fed with either HFMCD diet (reduction: ALT, 99%; AST, 97%; P鈥?lt;鈥?.0001 for both) or WDF (reduction: ALT, 98%; AST, 98%; P鈥?lt;鈥?.0001 for both) and found to be comparable to NC control levels (Figs.聽4f and g, 5h and i). In both models, GSH levels were diminished in WT mice on both NASH diets but normalized in CKOs (Figs.聽4h, 5j).Liver fibrosis was greatly reduced in CKO mice on either NASH diet as compared to WT (reduction: HFMCD, 87%; WDF, 64%) (Figs.聽4d, 4i, 5e, 5k). Upregulation of pro-inflammatory and fibrosis genes in mice on either the HFMCD or WDF diets was also diminished in the CKOs (Figs.聽4j, 5l; Supplementary Figs.聽8c and d, 9c and d). This suggests that transformation of HSCs to myofibroblasts and activation of immune cells are, in part, secondary to upstream, IL11-driven events in hepatocytes that are consistent with the paracrine effects we detected in vitro (Fig.聽2l; Supplementary Fig.聽5i and j). At the signaling level, both HFMCD diet and WDF resulted in elevated ERK and JNK phosphorylation. This was prevented in CKO mice, consistent with inhibition of IL11 signaling in hepatocytes (Figs.聽4k, 5m).Mice on WDF are known to develop hyperglycemia, hypertriglyceridemia, and hypercholesterolemia, all of which were improved in the CKOs (Supplementary Fig.聽9e鈥揼). Furthermore, as compared to controls, CKOs on WDF had elevated serum levels of 尾-hydroxybutyrate, a peripheral marker of liver fatty acid oxidation and ketone production (Supplementary Fig.聽9h). Overall these data suggest improvement of liver metabolism in CKOs, in keeping with our finding that inhibition of IL11 signaling promotes fatty acid oxidation in lipotoxic hepatocytes (Fig.聽2k).Reconstitution of IL11 cis-signaling in hepatocytes in IL11ra1 null mice restores steatohepatitis but not liver fibrosisTo complement our loss-of-function experiments using the CKO mice we employed in vivo gain-of-function experiments. To do so, we assessed whether restoring IL11 cis- or trans-signaling specifically in hepatocytes in mice with global Il11ra1 deletion (Il11ra1鈭?鈭?/i> knockouts (KOs)) resulted in disease. KO mice were injected with AAV8 encoding either the full-length, membrane-bound Il11ra1 (mbIl11ra1; to reconstitute cis-signaling) or a secreted/soluble form of Il11ra1 (sIl11ra1, which constitutes the extracellular portion of Il11ra1; to enable trans-signaling) or a control construct, and the animals were then fed with NC, HFMCD diet, or WDF (Fig.聽6a; Supplementary Fig.聽10a, 11a).Fig. 6: Hepatocyte-specific IL11 cis-signaling but not IL11 trans-signaling drives steatohepatitis in mice on WDF.a Schematic showing WDF feeding regimen of Il11ra1+/+ (WT) and Il11ra1鈭?鈭?/i> (KO) mice for experiments shown in (b鈥?b>n). AAV8-Alb-Null, AAV8-Alb-mbIl11ra1 (full-length membrane-bound Il11ra1), and AAV8-Alb-sIl11ra1 (soluble form of Il11ra1)-injected KO mice were given 16 weeks of WDF feeding, three weeks following virus administration. b Western blots showing hepatic levels of IL11RA and GAPDH (n鈥?鈥? mice/group). c Representative gross anatomy, H E-stained (scale bars, 50鈥壜祄) and Masson鈥檚 Trichrome (scale bars, 100鈥壜祄) images of livers. Representative dataset from n鈥?鈥? mice/group is shown for gross anatomy; representative dataset from n鈥?鈥? mice/group is shown for H E-stained and Masson鈥檚 Trichrome images. d Liver weight. e Hepatic triglycerides content. f Serum ALT levels. g Serum AST levels. h Hepatic GSH content. i Hepatic collagen content. j Hepatic pro-inflammatory and fibrotic genes expression heatmap (values are shown in Supplementary Fig.聽10c and d). k Western blots showing activation status of hepatic ERK and JNK (n鈥?鈥? mice/group). l Fasting blood glucose levels. m Serum triglycerides levels. n Serum cholesterol levels. d鈥?b>j, l鈥?b>n n鈥?鈥? mice/group. d鈥?b>i, l鈥?b>n Data are shown as box-and-whisker with median (middle line), 25th鈥?5th percentiles (box), and min鈥搈ax values (whiskers); one-way ANOVA with Tukey鈥檚 correction. Source data are provided as a Source data file.Full size imageKO mice injected with AAV8-Alb-mbIl11ra1 re-expressed IL11RA1 on hepatocytes and KO mice injected with AAV8-Alb-sIl11ra1 had increased expression of sIL11RA1 in both the liver and the periphery (Fig.聽6b; Supplementary Fig.聽10b, 11b and c). As expected, WT mice receiving control AAV8 constructs (AAV8-Alb-Null) on NC had normal livers and developed steatosis, inflammation, liver damage, and liver fibrosis when on either HFMCD diet or WDF (Fig.聽6c鈥搄; Supplementary Fig.聽10c and d, 11d鈥搆). KO mice injected with control virus and fed either HFMCD or WDF diets were protected from NASH phenotypes, although protection from NASH with germline global deletion of Il11ra1 was not as strong as seen in the CKOs (Figs.聽4d鈥搄, 5c鈥搇, 6c鈥搄; Supplementary Fig.聽8c and d, 9b鈥揼, 10c and d, 11d鈥搆).Restoration of IL11 cis-signaling in KO mice using mbIl11ra1 recapitulated hepatic steatosis and inflammation that was evident from gross morphology to molecular patterns of gene expression and signaling (Fig.聽6c鈥揾, j and k; Supplementary Fig.聽10c, 11d鈥揾, 11j, 11l). However, hepatic collagen content and fibrotic gene expression were not restored (Fig.聽6c, i and j; Supplementary Fig.聽10d, 11d, 11i, 11k) presumably because聽IL11 cis-signaling in HSCs, important for HSC-to-myofibroblast transformation7, was unaffected by the albumin-driven Il11ra1 expression (i.e., HSCs remain deleted for Il11ra1 in this model).In contrast, expression of the sIL11RA in hepatocytes of KOs, which would theoretically activate trans-signaling as IL11 levels are already聽elevated, had no effect despite very high IL11RA levels (Fig.聽6b; Supplementary Fig.聽10b, 11b and c) and mice remained protected from NASH (Fig.聽6c鈥搄; Supplementary Fig.聽10c, 10d, 11d鈥搆). Signaling changes were consistent in that mIL11RA expression restored pathological聽ERK and JNK activation in KOs on either diet, whereas sIL11RA1 did not (Fig.聽6k; Supplementary Fig.聽11l). Similarly, in the WDF model, restoration of hepatocyte-specific IL11 cis-signaling (mIL11RA) in KO mice caused hyperglycemia, hypertriglyceridemia, and hypercholesterolemia but expression of sIl11ra1 did not (Fig.聽6l鈥搉).DiscussionMetabolic liver disease commonly occurs in the context of obesity and type 2 diabetes and manifests initially as NAFLD that can progress to NASH22,33. A key underlying pathology in the progression to NASH is 鈥渟ubstrate overload鈥? whereby a nutritional abundance overruns the hepatocyte鈥檚 ability to process fat, causing lipotoxicity. Cytokines are key NASH factors secreted from lipotoxic hepatocytes22 and here we establish IL11 as an important component of the lipotoxic milieu and a driver of the NAFLD-to-NASH transition.A large body of evidence supports the idea that IL6 cis- and trans-signaling in the liver is beneficial16,17,19,25. However, at the same time, a pathogenic role for IL6 trans-signaling in hepatic steatosis has also been proposed30,34. Using man-made, artificial protein constructs we found that IL11 trans-signaling is toxic in hepatocytes, whereas hyperIL6 appears protective. However, we found no evidence to support a meaningful role for IL6 or IL11 trans-signaling in a biologically relevant context either in vitro or in vivo, using both gain- and loss-of-function approaches.聽Notably, sgp130, a therapeutic agent that inhibits聽IL6 trans-signaling, had聽no effect on lipotoxicity, NAFLD or NASH.聽 Thus, we suggest that IL6 family member trans-signaling has no role in hepatocytes or NASH, which is in agreement with studies outside the liver20,21.Our data show a central importance of IL11 cis-signaling in hepatocytes for multiple NASH pathologies. This effect was established using both hepatocyte-specific loss-of-function on a wild-type genetic background and also hepatocyte-specific gain-of-function on an Il11ra1 null background. This overturns the suggestion in the literature that IL11 is protective for hepatocytes based on the use of recombinant human IL11 in murine models of liver disease8,10,11,12. While restoration of IL11 cis-signaling in hepatocytes causes steatohepatitis in mice globally deleted for Il11ra1, lipotoxicity-associated fibrosis was still prevented. In contrast, hepatocyte-specific Il11ra1 deletion protected mice from both steatohepatitis and fibrosis. This places hepatocyte dysfunction upstream of HSC activation, which is consistent with the observed聽paracrine effects of IL11 from lipotoxic hepatocytes.We repeatedly documented metabolically advantageous effects associated with the inhibition of IL11 signaling. In vitro, inhibition of IL11 improved mitochondrial function and increased beta-oxidation along with a reduction of intracellular triglycerides and ROS production. The effects on ROS are likely complex as IL11 induces NOX4 but also impacts mitochondria function and perhaps also the endoplasmic reticulum, directly or indirectly. Interestingly, inhibition of NOX4 was more effective than caspase inhibition in reducing IL11-stimulated cell death. Thus IL11-induced NOX4/ROS lies upstream of late-stage ERK and caspase activation in hepatocytes and apoptosis is not the only form of cell death in this context. In vivo, deletion of Il11ra1 in hepatocytes limited WDF-induced fat accumulation and body weight gain while reducing serum glucose, triglyceride, and cholesterol levels. This was associated with lower liver fat, lesser hepatic oxidative stress, and increased serum levels of beta-hydroxybutyrate, thought metabolically beneficial in itself35.Our studies have limitations and pose questions. The published literature suggests IL6R is highly expressed in hepatocytes16 and it was surprising that primary human hepatocytes express very little IL6R. The differential activation of ERK/JNK by IL11 as compared to STAT3 by IL6 at 24聽h聽in hepatocytes was also notable but the underlying mechanisms are unknown. While we show consistent effects of IL11 inhibition on pro-inflammatory factors we did not specifically address effects on immune cells themselves. Metabolic effects appear closely related to IL11-mediated NOX4-derived ROS but ROS from other sources also likely contributes. Temporal relationships between ERK and NOX4 activation require further evaluation. We found a beneficial effect of hepatocyte-specific IL11 inhibition on reducing fat deposition and body weight gain in mice on an obesogenic NASH diet, which while notable was not studied in depth. We surmise that IL11 hepatocyte biology is a nascent field and that these various matters require further study.In conclusion, we propose a model for lipotoxicity-driven NAFLD-to-NASH transitions whereby lipid-laden hepatocytes secrete IL11 leading to autocrine hepatocyte metabolic dysfunction and cell death along with paracrine activation of neighbouring聽HSCs and other cells (Fig.聽7). We rule out IL6 or IL11 trans-signaling as relevant for hepatocyte biology or liver pathology in lipotoxicity. We suggest that inhibiting IL11 signaling in hepatocytes targets an initiating nexus for diet-induced steatohepatitis that impacts subsequent liver fibrosis and inflammation. Hence, therapeutic inhibition of IL11-induced lipotoxicity may be beneficial in metabolic liver diseases, such as NASH.Fig. 7: Proposed mechanism for IL11 in lipotoxicity-driven NASH transition.Excessive lipid accumulation in hepatocytes stimulates IL11 protein secretion and autocrine IL11 activity, which upregulates NOX4 and increases reactive oxygen species production. Subsequently, hepatocyte mitochondrial oxidative capacity and fatty acid metabolism impaired and steatosis established. ERK, JNK, and caspase-3 become activated and this leads to lipoapoptosis, along with other forms of cell death. IL11 also acts in paracrine to drive hepatic stellate cell-to-myofibroblast transformation and fibrosis. Cytokines and chemokines released from lipotoxic hepatocytes and HSCs activate and recruit immune cells causing inflammation.Full size imageMethodsEthics statementsAll experimental protocols involving human subjects (commercial primary human cell lines and human liver sections) have been performed in accordance with the ICH Guidelines for Good Clinical Practice. As written in their respective datasheets, ethical approvals have been obtained by the relevant parties and all participants gave written informed consent: commercial human liver sections (by Abcam); liver sections from healthy control and NASH patients (by Fibrofind); primary human hepatocytes and hepatic stellate cells (by ScienCell); HepG2 and THP-1 (by ATCC).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/2014/0925 and SHS/2019/1482) and conducted in accordance with the SingHealth Institutional Animal Care and Use Committee.AAV8 vectorsAll Adeno-associated virus serotype 8 (AAV8) vectors used in this study were synthesized by Vector Biolabs. AAV8 vector carrying a mouse membrane-bound Il11ra1 cDNA (NCBI accession number: BC069984), a mouse soluble Il11ra1 cDNA, and a mouse soluble gp130 cDNA driven by Albumin (Alb) promoter is referred to as AAV8-Alb-mbIl11ra1, AAV8-Alb-sIl11ra1, and AAV8-Alb-sgp130, respectively. AAV8-Alb-sgp130 and AAV8-Alb-sIl11ra1 were constructed by removing the transmembrane and cytoplasmic regions of mouse gp130 sequence (NCBI accession number: BC058679) and mouse Il11ra1 sequence, respectively. AAV8-Null vector was used as vector control. To specifically delete Il11ra1 in Albumin-expressing cells, AAV8-Alb-iCre vector was injected to mice homozygous for LoxP-flanked Il11ra1 alleles (Il11ra1loxP/loxP mice).AntibodiesACTA2 (ab7817, Abcam, 1:1000 for western blot and 1:500 for operetta assay), Albumin (ab207327, Abcam, 1:100 for IF and flow cytometry), Cleaved Caspase-3 (9664, CST, 1:1000), Caspase-3 (9662, CST, 1:1000), Collagen I (ab34710, Abcam, 1:500), phospho-ERK1/2 (4370, CST, 1:1000), ERK1/2 (4695, CST, 1:1000), GAPDH (2118, CST, 1:1000), gp130 (human, PA5-28932, Thermo Fisher, 1:100), gp130 (mouse, PA5-99526, Thermo Fisher, 1:100), gp130 (extracellular, PA5-77476, Thermo Fisher, 1:1000), IgG (11E10, Aldevron), IL6 (AF506, R D Systems, 1:1000), IL6R (flow cytometry, ab222101, Abcam, 1:100), IL6R (human, IHC and IF, MA1-80456, Thermo Fisher, 1:100), IL6R (mouse, ab83053, Abcam, 1:100), IL11 (X203, Aldevron), IL11RA (inhibition study, X209, Aldevron), IL11RA (IHC, IF, flow cytometry, ab125015, Abcam, 1:100), IL11RA (western blot, sc-130920, Santa Cruz, 1:1000), phospho-JNK (4668, CST, 1:1000), JNK (9252, CST, 1:1000), NOX4 (MA5-32090, Invitrogen, 1:1000), phospho-STAT3 (4113, CST, 1:1000), STAT3 (4904, CST, 1:1000), mouse Alexa Fluor 488 secondary antibody (ab150113, Abcam, 1:200), mouse HRP (7076, CST, 1:2000), rabbit Alexa Fluor 488 secondary antibody (ab150077, Abcam, 1:200), rabbit HRP (7074, CST, 1:2000), rat Alexa Fluor 488 secondary antibody (ab150157, Abcam, 1:200), rat HRP (31470, Santa Cruz, 1:800).Recombinant proteinsCommercial recombinant proteins: Human hyperIL6 (IL6R:IL6 fusion protein, 8954-SR, R D Systems), human IL6 (206-IL-010, R D Systems), human soluble gp130 Fc (671-GP-100, R D Systems), human IL11RA (8895-MR-050, R D Systems).Custom recombinant proteins: Human IL11 (UniProtKB:P20809, Genscript). Human hyperIL11 (IL11RA:IL11 fusion protein), which mimics the trans-signaling complex, was constructed using a fragment of IL11RA (amino acid residues 1鈥?17; UniProtKB: Q14626) and IL11 (amino acid residues 22鈥?99, UniProtKB: P20809) with a 20 amino acid linker (GPAGQSGGGGGSGGGSGGGSV)1.Chemicals4鈥?6-diamidino-2-phenylindole (DAPI, D1306, Thermo Fisher), diphenyleneiodonium chloride (DPI, 141310, Abcam), GKT-137831 (17764, Cayman Chemical), palmitate (P5585, Sigma), paraformaldehyde (PFA, 28908; Thermo Fisher), phorbol 12-myristate 13-acetate (PMA, P1585, Sigma), Triton X-100 (T8787, Sigma), and Z-VAD-FMK (FMK001, Sigma).Cell cultureAll the experiments performed with primary human hepatocytes, primary adult mouse hepatocytes, and primary adult human hepatic stellate cells were carried out at low cell passage (鈮3). Primary human hepatocytes culture Primary human hepatocytes (5200, ScienCell) were maintained in hepatocyte medium (5201, ScienCell) supplemented with 2% fetal bovine serum, 1% penicillin-streptomycin at 37鈥壜癈 and 5% CO2. Hepatocytes were serum-starved overnight unless otherwise specified in the methods prior to 24鈥塰 stimulation with different doses of various recombinant proteins as outlined in the main text and/or figure legends. Primary adult mouse hepatocytes culture Mouse hepatocytes (ABC-TC3928, AcceGen Biotech) were maintained in mouse hepatocyte medium (ABC-TM3928, AcceGen Biotech) supplemented with 1% penicillin-streptomycin. Primary adult human hepatic stellate cells HSCs (5300, ScienCell) were cultured in stellate cells complete media (5301, ScienCell) on poly-L-lysine-coated plates (2鈥壜礸/cm2, 0403, ScienCell). HSCs were serum-starved overnight prior to 24鈥塰 stimulation with conditioned media from BSA or palmitate-stimulated hepatocyte (24鈥塰) in the presence of various recombinant proteins as outlined in the main text and/or figure legends. HepG2 culture HepG2 (ATCC) were cultured in Eagle鈥檚 minimum essential medium (30-2003, ATCC) supplemented with 10% FBS. AML12 culture AML12 (ATCC) were cultured in DMEM:F12 medium (30-2006, ATCC) supplemented with 10% FBS, 10鈥壜礸/ml insulin, 5.5鈥壜礸/ml transferrin, 5鈥塶g/ml selenium, and 40鈥塶g/ml dexamethasone. THP-1 culture THP-1 (ATCC) were cultured in RPMI 1640 (A1049101, Thermo Fisher) supplemented with 10% FBS and 0.05鈥塵M 尾-mercaptoethanol. THP-1 cells were differentiated with 10鈥塶g/ml of PMA in RPMI 1640 for 48鈥塰. Palmitate (saturated fatty acid) treatment in vitroPalmitate (0.5鈥塵M) conjugated in fatty acids free BSA in the ratio of 6:1 was used to treat cells as described in figure legends; 0.5% BSA solution was used as control.Flow cytometryFor surface IL11RA, IL6R, and gp130 analysis, primary human hepatocytes and THP-1 cells were stained with IL11RA, IL6R, or gp130 antibody, and the corresponding Alexa Fluor 488 secondary antibody. Omission of primary antibody staining was used as negative control. 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): the preliminary FSC/SSC gates of the starting cell population was 10,000 events. Debris (SSC鈥怉 vs FSC鈥怉) and doublets (FSC鈥怘 vs FSC鈥怉) were excluded. Boundaries between 鈥減ositive鈥?and 鈥渘egative鈥?staining were set at 103 for PI staining. A figure exemplifying the gating strategy is provided in the Supplementary Fig.聽1.Immunofluorescence (IF)Primary human hepatocytes were seeded on 8-well chamber slides (1.5鈥壝椻€?04鈥塩ells/well) 24鈥塰 before the staining. Cells were fixed in 4% PFA for 20鈥塵in, washed with PBS, and non-specific sites were blocked with 5% BSA in PBS for 2鈥塰. Cells were incubated with IL11RA, IL6R, gp130, or Albumin antibody overnight (4鈥壜癈), followed by incubation with the appropriate Alexa Fluor 488 secondary antibody for 1鈥塰 (RT). Negative control cells (鈭? were only stained with the secondary antibody. Chamber slides were dried in the dark and 5 drops of mounting medium with DAPI were added to the slides for 15鈥塵in prior to imaging by fluorescence microscope (Leica).Operetta high throughput phenotyping assayHSCs were seeded in 96-well black CellCarrier plates (PerkinElmer) at a density of 5鈥壝椻€?03 cells per well. Following simulations, cells were fixed in 4% PFA (Thermo Fisher), permeabilized with 0.1% Triton X-100 (Sigma), and non-specific sites were blocked with 0.5% BSA and 0.1% Tween-20 in PBS. Cells were incubated overnight (4鈥壜癈) with primary antibodies (1:500), followed by incubation with the appropriate Alexa Fluor 488 secondary antibodies (1:1000). Cells were counterstained with 1鈥壜礸/ml DAPI (D1306, Thermo Fisher in blocking solution. Each condition was imaged from duplicated wells and a minimum of 7 fields/well using Operetta high-content imaging system 1483 (PerkinElmer). Cells expressing ACTA2 were quantified using Harmony v3.5.2 (PerkinElmer) and the percentage of activated fibroblasts/total cell number (ACTA2+ve) was determined for each field. The measurement of fluorescence intensity per area (normalized to the number of cells) of Collagen I was performed with Columbus 2.7.1 (PerkinElmer).Oil Red O stainingPrimary human hepatocytes were seeded on 8-well chamber slides (1.5鈥壝椻€?04 cells/well) Following 24鈥塰 of palmitate treatment, cells were fixed in 10% PFA for 30鈥塵in, washed with distilled water, and incubated with 60% (v/v) isopropyl alcohol for 5鈥塵in. Cells were then stained with Oil Red O Solution (O0625, Sigma) for 30鈥塵in and washed with distilled water prior to imaging with a bright field microscope (BX53, Olympus). The lipid droplets were identified by their red staining.Reactive oxygen species (ROS) detectionPrimary human hepatocytes were seeded on 8-well chamber slides (1.5鈥壝椻€?04鈥塩ells/well). For this experiment, cells were not serum-starved prior to palmitate treatment. Twenty-four hours following palmitate stimulation, cells were washed, incubated with 25鈥壜礛 of DCFDA solution (ab113851, Abcam) for 45鈥塵in at 37鈥壜癈 in the dark, and rinsed with the dilution buffer according to the manufacturer鈥檚 protocol. Live cells with positive DCF staining were imaged with a filter set appropriate for fluorescein (FITC) using a fluorescence microscope (Leica).Seahorse assayPrimary human hepatocytes were seeded into the Seahorse XF Cell Culture Microplate (1鈥壝椻€?04鈥塩ells/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) was used to measure the mitochondrial oxygen consumption rate as per the manufacturer鈥檚 protocol. Briefly, stimulation media were removed and replaced with 180鈥壩糽 of Mitostress assay medium at 37鈥壜癈. Oligomycin (1鈥壩糓; ATP synthase inhibitor) was injected following basal OCR measurements followed by injection of FCCP (1鈥壩糓; an uncoupling agent that collapses the proton gradient and disrupts the mitochondrial membrane potential), and finally by injection of a mixture of rotenone (1鈥壩糓; a complex I inhibitor) and antimycin A (1鈥壩糓; a complex III inhibitor). The percentage of fatty acid oxidation analysis was performed by using Seahorse XF Mito Fuel Flex Test kit (103260-100). Acute injections of CPT1 alpha inhibitor Etomoxir (4鈥壜礛) was used to inhibit mitochondrial FAO, whereas BPTES (3鈥壜礛) and UK5099 (2鈥壜礛) were used to inhibit mitochondrial glutamine and glucose oxidation, respectively, to inhibit 100% mitochondrial fuel oxidation. Seahorse Wave Desktop software was used for report generation and data analysis.RNA-sequencing (RNA-seq) and ribosome profiling (Ribo-seq)Generation of RNA-seq librariesTotal 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 (PerkinElmer). TruSeq Stranded mRNA Library Preparation kit (Illumina) was used to measure transcript abundance following standard instructions from the manufacturer.Generation of Ribo-seq librariesHepatocytes were grown to 90% confluence in a 10鈥塩m culture dish and lysed in 1鈥塵l cold lysis buffer (formulation as in TruSeq庐 Ribo Profile Mammalian Kit, RPHMR12126, Illumina) supplemented with 0.1鈥塵g/ml cycloheximide. Homogenized and cleared lysates were then footprinted with Truseq Nuclease (Illumina) according to the manufacturer鈥檚 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庐 Ribo Profile Mammalian Kit according to the manufacturer鈥檚 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鈥檚 protocol. The quality and average fragment size of the final libraries were determined using a LabChip GX DNA High Sensitivity Reagent Kit (PerkinElmer). Libraries with unique indexes were pooled and sequenced on a NextSeq 500 benchtop sequencer (Illumina) using NextSeq 500 High Output v2 kit and single-end 75-bp sequencing chemistry.Data processing and analyses for RNA-sequencing and ribosome profilingRaw sequencing data were demultiplexed with bcl2fastq V2.19.0.316 and the adaptors were trimmed using Trimmomatic36 V0.36, retaining reads longer than 20鈥塶t post-clipping. Ribo-seq reads were aligned using bowtie37 to known mtRNA, rRNA, and tRNA sequences (RNACentral38, release 5.0) and only unaligned reads were retained as Ribosome protected fragments (RPFs). Alignment to the human genome (hg38) was carried out using STAR39. 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 counts40. TPM was calculated and visualized using boxplot to compare baseline expression of IL11RA (ENSG00000137070), IL6R (ENSG00000160712), and gp130 (ENSG00000134352). Read coverage using Ribo-seq and RNA-seq reads for IL11RA, IL6R, and gp130 was visualized using Gviz R package41 with strand-specific alignment files.Animal modelsMice were housed in temperatures of 21鈥?4鈥壜癈 with 40鈥?0% humidity on a 12鈥塰 light/12鈥塰 dark cycle and provided with food and water ad libitum.Mouse models of metabolic liver disease HFMCD 6鈥?-week-old C57BL/6N, Il11ra1鈭?鈭?/i> mice, and Il11ra1loxP/loxP and their respective control were fed with methionine- and choline-deficient diet supplemented with 60鈥塳cal% fat (HFMCD, A06071301B, Research Diets) for 4 weeks. Control mice received normal chow (NC, Specialty Feeds). WDF 6鈥?-week-old C57BL/6N, Il11ra1鈭?鈭?/i> mice, and Il11ra1loxP/loxP and their respective control were fed western diet (D12079B, Research Diets) supplemented with 15% weight/volume fructose in drinking water (WDF) for 16 weeks. Control mice received NC and tap water. Il11ra1-deleted mice (KO)Six- to eight-week-old male Il11ra1鈭?鈭?/i> mice (B6.129S1-Il11ratm1Wehi/J, Jackson鈥檚 Laboratory) were intravenously injected with 4鈥壝椻€?011 genome copies (gc) of AAV8-Alb-mbIl11ra1 or AAV8-Alb-sIl11ra1 virus to induce hepatocyte-specific expression of mouse Il11ra1 or soluble Il11ra1, respectively. As controls, both Il11ra1鈭?鈭?/i> mice and their wild-type littermates (Il11ra1+/+) were intravenously injected with 4鈥壝椻€?011鈥塯c AAV8-Alb-Null virus. Three weeks after virus injection, mice were fed with HFMCD, WDF, or NC. Durations of diet are outlined in the main text and/or figure legends.In vivo administration of soluble gp130Six- to eight-week-old male C57BL/6N mice (InVivos) were injected with 4鈥壝椻€?011鈥塯c AAV8-Alb-sgp130 virus to induce hepatocyte-specific expression of soluble gp130; control mice were injected with 4鈥壝椻€?011鈥塯c AAV8-Alb-Null virus. Three weeks following virus administration, mice were fed with HFMCD, WDF, or NC for durations that are outlined in the main text and/or figure legends. Il11ra-floxed mice (CKO)Il11ra-floxed mice, in which exons 4鈥? of the Il11ra1 gene were flanked by loxP sites, were created using the CRISPR/Cas9 system as previously described42. To induce the specific deletion of Il11ra1 in hepatocytes, 6鈥?-week-old male homozygous Il11ra1-floxed mice were intravenously injected with AAV8-Alb-Cre virus (4鈥壝椻€?011鈥塯c); a similar amount of AAV8-Alb-Null virus were injected into homozygous Il11ra1-floxed mice as controls. The AAV8-injected mice were allowed to recover for 3 weeks prior to HFMCD, WDF, or NC feeding. Knockdown efficiency was determined by western blotting of hepatic IL11RA.RT-qPCRTotal RNA was extracted from snap-frozen liver tissues using Trizol (Invitrogen) and RNeasy Mini Kit (Qiagen). PCR amplifications were performed using iScript cDNA Synthesis Kit (Bio-Rad). Gene expression was analyzed in duplicate by SYBR green (Qiagen) technology using StepOnePlus (Applied Biosystems) over 40 cycles. Expression data were normalized to GAPDH mRNA expression and fold change was calculated using 2鈭掆垎鈭咰t method. The primer sequences are listed in Supplementary Table聽1.ImmunoblottingWestern blots were carried out on total protein extracts from hepatocytes and liver tissues. Hepatocyte and liver tissue lysates were homogenized in RIPA Lysis and Extraction Buffer (89901, Thermo Scientific) containing protease and phosphatase inhibitors (Roche). Protein lysates were separated by SDS-PAGE and transferred to PVDF membranes. Protein bands were visualized using the ECL detection system (Pierce) with the appropriate secondary antibodies: anti-rabbit HRP, anti-mouse HRP, or anti-rat HRP. Uncropped western blot images are provided in Source data file.Colorimetric assaysAlanine aminotransferase (ALT) activity in the cell culture supernatant and mouse serum was measured using ALT Activity Assay Kit (ab105134, Abcam). Cellular and liver glutathione (GSH) levels were measured using Glutathione Colorimetric Detection Kit (EIAGSHC, Thermo Fisher). Total hydroxyproline content in mouse livers was measured using Quickzyme Total Collagen assay kit (QZBtotco15, Quickzyme Biosciences). The levels of triglycerides in hepatocyte lysates and in mouse serum and livers were measured using Triglyceride Assay Kit (ab65336, Abcam). Mouse serum levels of aspartate aminotransferase (AST), cholesterol, and 尾-hydroxybutyrate were measured using AST Assay Kit (ab105135, Abcam), Cholesterol Assay Kit (ab65390; Abcam), and beta-hydroxybutyrate (Ketone body) Colorimetric Assay Kit (700190; Cayman Chemicals), respectively. All colorimetric assays were performed according to the manufacturer鈥檚 protocol.Enzyme-linked immunosorbent assay (ELISA)The levels of IL11, IL6, CCL2, and CCL5 in equal volumes of cell culture media were quantified using Human IL11 Quantikine ELISA kit (D1100; R D Systems), Human IL-6 Quantikine ELISA Kit (D6050; R D Systems), Human CCL2/MCP-1 Quantikine ELISA Kit (DCP00; R D Systems), and Human CCL5/RANTES Quantikine ELISA Kit (DRN00B; R D Systems), respectively. The levels of IL11, IL6, gp130, and IL11RA in mouse serum were quantified using Mouse IL11 DuoSet ELISA (DY418; R D Systems), Mouse IL6 Quantikine ELISA Kit (M6000B; R D Systems), Mouse Interleukin 11 Receptor Alpha (IL11Ra) ELISA Kit (MBS452535; MyBioSource), and Mouse gp130 DuoSet ELISA (DY468, R D Systems), respectively. All ELISA assays were performed according to the manufacturer鈥檚 protocol.Liver tissue processing and histological analysis Immunohistochemistry (IHC) Comparison of IL11RA and IL6R expression in healthy human liver (ab4348, Abcam) and in mouse liver: mouse livers were fixed in 10% neutral-buffered formalin (NBF), paraffinized, cut into 7-渭m sections. Both human and mouse liver tissue sections were incubated with primary antibodies overnight and visualized using an ImmPRESS HRP anti-rabbit IgG polymer detection kit (MP-7401, Vector Laboratories) with ImmPACT DAB Peroxidase Substrate (SK-4105, Vector Laboratories). Comparison of IL11RA expression in the human livers from healthy control and patients suffering from NASH: these studies were outsourced to a company (Fibrofind). Briefly, paraffin embedded blocks of human livers were cut into slides and stained with IL11RA antibody (NBP2-32671, Novus Biologicals) or control. Analysis was performed on liver sections from three healthy individuals as control (NHL20, NHL60, NHL67; 1 section/code) and on liver sections from 2 NASH patients (TLPAT5, TLPAT14; 1 section/code). Due to data protection policies, no further information is available on samples or patients. H E and Masson鈥檚 Trichrome staining Mouse liver samples were processed and sectioned as mentioned above, followed by hematoxylin and eosin (H E) or Masson鈥檚 Trichrome staining according to standard protocol. Statistical analysisAll statistical analyses were performed using GraphPad Prism software (version 6.07). 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Fibroblast-specific IL11 signaling is required for lung fibrosis and inflammation. https://doi.org/10.1101/801852 (2019).Download referencesAcknowledgementsThis research is supported by the National Medical Research Council (NMRC), Singapore STaR awards (NMRC/STaR/0029/2017), NMRC Center Grant to the NHCS, MOH鈥怌IRG18nov鈥?002, Goh Foundation, Tanoto Foundation. A.A.W. is supported by NMRC/OFYIRG/0053/2017. P.M.Y. is supported by NMRC/CIRG/1457/2016. The authors would like to acknowledge the technical support of B.L. George and S. Lim. We thank Fibrofind for helping with IL11RA staining in patients with NASH.Author informationAuthor notesThese authors jointly supervised this work: Stuart A. Cook, Anissa A. Widjaja.AffiliationsCardiovascular and Metabolic Disorders Program, Duke-National University of Singapore Medical School, Singapore, SingaporeJinrui Dong,聽Sivakumar Viswanathan,聽Eleonora Adami,聽Brijesh K. Singh,聽Sonia P. Chothani,聽Benjamin Ng,聽Jin Zhou,聽Madhulika Tripathi,聽Nicole S. J. Ko,聽Shamini G. Shekeran,聽Jessie Tan,聽Mao Wang,聽Paul M. Yen,聽Sebastian Schafer,聽Stuart A. Cook聽 聽Anissa A. WidjajaNational Heart Research Institute Singapore, National Heart Centre Singapore, Singapore, SingaporeBenjamin Ng,聽Wei Wen Lim,聽Jessie Tan,聽Sze Yun Lim,聽Pei Min Lio,聽Sebastian Schafer聽 聽Stuart A. CookNational Heart and Lung Institute, Imperial College London, London, UKStuart A. CookMRC-London Institute of Medical Sciences, Hammersmith Hospital Campus, London, UKStuart A. CookAuthorsJinrui DongView author publicationsYou can also search for this author in PubMed聽Google ScholarSivakumar ViswanathanView author publicationsYou can also search for this author in PubMed聽Google ScholarEleonora AdamiView author publicationsYou can also search for this author in PubMed聽Google ScholarBrijesh K. SinghView author publicationsYou can also search for this author in PubMed聽Google ScholarSonia P. ChothaniView author publicationsYou can also search for this author in PubMed聽Google ScholarBenjamin NgView author publicationsYou can also search for this author in PubMed聽Google ScholarWei Wen LimView author publicationsYou can also search for this author in PubMed聽Google ScholarJin ZhouView author publicationsYou can also search for this author in PubMed聽Google ScholarMadhulika TripathiView author publicationsYou can also search for this author in PubMed聽Google ScholarNicole S. J. KoView author publicationsYou can also search for this author in PubMed聽Google ScholarShamini G. ShekeranView author publicationsYou can also search for this author in PubMed聽Google ScholarJessie TanView author publicationsYou can also search for this author in PubMed聽Google ScholarSze Yun LimView author publicationsYou can also search for this author in PubMed聽Google ScholarMao WangView author publicationsYou can also search for this author in PubMed聽Google ScholarPei Min LioView author publicationsYou can also search for this author in PubMed聽Google ScholarPaul M. YenView author publicationsYou can also search for this author in PubMed聽Google ScholarSebastian SchaferView author publicationsYou can also search for this author in PubMed聽Google ScholarStuart A. CookView author publicationsYou can also search for this author in PubMed聽Google ScholarAnissa A. WidjajaView author publicationsYou can also search for this author in PubMed聽Google ScholarContributionsS.A.C. and A.A.W. conceived and designed the study. J.D., S.V., E.A., B.N., W.W.L., B.K.S., J.T., M.W., and A.A.W. performed in vitro cell culture, cell biology, and molecular biology experiments. J.D., J.Z., M.T., S.G.S., and N.S.J.K. performed in vivo studies. S.G.S. and S.Y.L. performed histology analysis. J.D., S.P.C., P.M.Y., S.S., S.A.C., and A.A.W. analyzed the data. L.P.M. and E.A. performed RNA and Ribo sequencing. J.D., E.A., S.A.C., and A.A.W. prepared the manuscript with input from co-authors.Corresponding authorsCorrespondence to Stuart A. Cook or Anissa A. Widjaja.Ethics declarations Competing interests S.A.C., S.S., A.A.W., B.N., W.W.L., and B.K.S. are co-inventors on a number of patent applications relating to the role of IL11 in human diseases that include the published patents: WO2017103108, WO2017103108 A2, WO 2018/109174 A2, WO 2018/109170 A2. S.A.C. and S.S. are co-founders and shareholders of Enleofen Bio PTE LTD, a company (which S.A.C. is a director of) that developed anti-IL11 therapeutics, which were acquired for further development by Boehringer Ingelheim. All other authors declare non-competing interests. Additional informationPeer review information Nature Communications thanks Natalia Nieto and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.Publisher鈥檚 note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary information CommentsBy submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate. Sign up for the Nature Briefing newsletter 鈥?what matters in science, free to your inbox daily.