Nonspecific Inhibition of IL6 Family Cytokine Signalling by Soluble gp130.

IL6 is a proinflammatory cytokine that binds to membrane-bound IL6 receptor (IL6R) or soluble IL6R to signal via gp130 in cis or trans, respectively. We tested the hypothesis that sgp130Fc, which is believed to be a selective IL6 trans-signalling inhibitor, is in fact a non-specific inhibitor of gp130 signalling. In human cancer and primary cells, sgp130Fc inhibited IL6, IL11, OSM and CT1 cis-signalling. The IC50 values of sgp130Fc for IL6 and OSM cis-signalling were markedly (20- to 200-fold) lower than the concentrations of sgp130Fc used in mouse studies and clinical trials. sgp130 inhibited IL6 and OSM signalling in the presence of an ADAM10/17 inhibitor and the absence of soluble IL6R or OSMR, with effects that were indistinguishable from those of a gp130 neutralising antibody. These data show that sgp130Fc does not exclusively block IL6 trans-signalling and reveal instead that broad inhibition of gp130 signalling likely underlies its therapeutic effects. This proposes global or modular inhibition of gp130 as a therapeutic approach for treating human disease.

Cardiomyocyte-Restricted Expression of IL11 Causes Cardiac Fibrosis, Inflammation, and Dysfunction.

Cardiac fibrosis is a common pathological process in heart disease, representing a therapeutic target. Transforming growth factor ß (TGFß) is the canonical driver of cardiac fibrosis and was recently shown to be dependent on interleukin 11 (IL11) for its profibrotic effects in fibroblasts. In the opposite direction, recombinant human IL11 has been reported as anti-fibrotic and anti-inflammatory in the mouse heart. In this study, we determined the effects of IL11 expression in cardiomyocytes on cardiac pathobiology and function. We used the Cre-loxP system to generate a tamoxifen-inducible mouse with cardiomyocyte-restricted murine Il11 expression. Using protein assays, bulk RNA-sequencing, and in vivo imaging, we analyzed the effects of IL11 on myocardial fibrosis, inflammation, and cardiac function, challenging previous reports suggesting the cardioprotective potential of IL11. TGFß stimulation of cardiomyocytes caused Il11 upregulation. Compared to wild-type controls, Il11-expressing hearts demonstrated severe cardiac fibrosis and inflammation that was associated with the upregulation of cytokines, chemokines, complement factors, and increased inflammatory cells. IL11 expression also activated a program of endothelial-to-mesenchymal transition and resulted in left ventricular dysfunction. Our data define species-matched IL11 as strongly profibrotic and proinflammatory when secreted from cardiomyocytes and further establish IL11 as a disease factor.

Interleukin-11 drives human and mouse alcohol-related liver disease.

OBJECTIVE: Alcoholic hepatitis (AH) reflects acute exacerbation of alcoholic liver disease (ALD) and is a growing healthcare burden worldwide. Interleukin-11 (IL-11) is a profibrotic, proinflammatory cytokine with increasingly recognised toxicities in parenchymal and epithelial cells. We explored IL-11 serum levels and their prognostic value in patients suffering from AH and cirrhosis of various aetiology and experimental ALD. DESIGN: IL-11 serum concentration and tissue expression was determined in a cohort comprising 50 patients with AH, 110 patients with cirrhosis and 19 healthy volunteers. Findings were replicated in an independent patient cohort (n=186). Primary human hepatocytes exposed to ethanol were studied in vitro. Ethanol-fed wildtype mice were treated with a neutralising murine IL-11 receptor-antibody (anti-IL11RA) and examined for severity signs and markers of ALD. RESULTS: IL-11 serum concentration and hepatic expression increased with severity of liver disease, mostly pronounced in AH. In a multivariate Cox-regression, a serum level above 6.4 pg/mL was a model of end-stage liver disease independent risk factor for transplant-free survival in patients with compensated and decompensated cirrhosis. In mice, severity of alcohol-induced liver inflammation correlated with enhanced hepatic IL-11 and IL11RA expression. In vitro and in vivo, anti-IL11RA reduced pathogenic signalling pathways (extracellular signal-regulated kinases, c-Jun N-terminal kinase, NADPH oxidase 4) and protected hepatocytes and murine livers from ethanol-induced inflammation and injury. CONCLUSION: Pathogenic IL-11 signalling in hepatocytes plays a crucial role in the pathogenesis of ALD and could serve as an independent prognostic factor for transplant-free survival. Blocking IL-11 signalling might be a therapeutic option in human ALD, particularly AH.

Targeting endogenous kidney regeneration using anti-IL11 therapy in acute and chronic models of kidney disease.

The kidney has large regenerative capacity, but this is compromised when kidney damage is excessive and renal tubular epithelial cells (TECs) undergo SNAI1-driven growth arrest. Here we investigate the role of IL11 in TECs, kidney injury and renal repair. IL11 stimulation of TECs induces ERK- and p90RSK-mediated GSK3ß inactivation, SNAI1 upregulation and pro-inflammatory gene expression. Mice with acute kidney injury upregulate IL11 in TECs leading to SNAI1 expression and kidney dysfunction, which is not seen in Il11 deleted mice or in mice administered a neutralizing IL11 antibody in either preemptive or treatment modes. In acute kidney injury, anti-TGFß reduces renal fibrosis but exacerbates inflammation and tubule damage whereas anti-IL11 reduces all pathologies. Mice with TEC-specific deletion of Il11ra1 have reduced pathogenic signaling and are protected from renal injury-induced inflammation, fibrosis, and failure. In a model of chronic kidney disease, anti-IL11 therapy promotes TEC proliferation and parenchymal regeneration, reverses fibroinflammation and restores renal mass and function. These data highlight IL11-induced mesenchymal transition of injured TECs as an important renal pathology and suggest IL11 as a therapeutic target for restoring stalled endogenous regeneration in the diseased kidney.

Spermidine-mediated hypusination of translation factor EIF5A improves mitochondrial fatty acid oxidation and prevents non-alcoholic steatohepatitis progression.

Spermidine is a natural polyamine that has health benefits and extends life span in several species. Deoxyhypusine synthase (DHPS) and deoxyhypusine hydroxylase (DOHH) are key enzymes that utilize spermidine to catalyze the post-translational hypusination of the translation factor EIF5A (EIF5AH). Here, we have found that hepatic DOHH mRNA expression is decreased in patients and mice with non-alcoholic steatohepatitis (NASH), and hepatic cells treated with fatty acids. The mouse and cell culture models of NASH have concomitant decreases in Eif5aH and mitochondrial protein synthesis which leads to lower mitochondrial activity and fatty acid ß-oxidation. Spermidine treatment restores EIF5AH, partially restores protein synthesis and mitochondrial function in NASH, and prevents NASH progression in vivo. Thus, the disrupted DHPS-DOHH-EIF5AH pathway during NASH represents a therapeutic target to increase hepatic protein synthesis and mitochondrial fatty acid oxidation (FAO) and prevent NASH progression.

IL11 stimulates ERK/P90RSK to inhibit LKB1/AMPK and activate mTOR initiating a mesenchymal program in stromal, epithelial, and cancer cells.

IL11 initiates fibroblast activation but also causes epithelial cell dysfunction. The mechanisms underlying these processes are not known. We report that IL11-stimulated ERK/P90RSK activity causes the phosphorylation of LKB1 at S325 and S428, leading to its inactivation. This inhibits AMPK and activates mTOR across cell types. In stromal cells, IL11-stimulated ERK activity inhibits LKB1/AMPK which is associated with mTOR activation, ⍺SMA expression, and myofibroblast transformation. In hepatocytes and epithelial cells, IL11/ERK activity inhibits LKB1/AMPK leading to mTOR activation, SNAI1 expression, and cell dysfunction. Across cells, IL11-induced phenotypes were inhibited by metformin stimulated AMPK activation. In mice, genetic or pharmacologic manipulation of IL11 activity revealed a critical role of IL11/ERK signaling for LKB1/AMPK inhibition and mTOR activation in fatty liver disease. These data identify the IL11/mTOR axis as a signaling commonality in stromal, epithelial, and cancer cells and reveal a shared IL11-driven mesenchymal program across cell types.

IL11 Stimulates IL33 Expression and Proinflammatory Fibroblast Activation across Tissues.

Interleukin 11 (IL11) is upregulated in inflammatory conditions, where it is mostly believed to have anti-inflammatory activity. However, recent studies suggest instead that IL11 promotes inflammation by activating fibroblasts. Here, we assessed whether IL11 is pro- or anti-inflammatory in fibroblasts. Primary cultures of human kidney, lung or skin fibroblasts were stimulated with IL11 that resulted in the transient phosphorylation of signal transducer and activator of transcription 3 (STAT3) and the sustained activation of extracellular signal-regulated protein kinases (ERK). RNA sequencing over a time course of IL11 stimulation revealed a robust but short-lived transcriptional response that was enriched for gene set hallmarks of inflammation and characterized by the upregulation of SERPINB2, TNFRSF18, Interleukin 33 (IL33), CCL20, IL1RL1, CXCL3/5/8, ICAM1 and IL11 itself. IL33 was the most upregulated signaling factor (38-fold, p = 9.8 × 10-5), and IL1RL1, its cognate receptor, was similarly increased (18-fold, p = 1.1 × 10-34). In proteomic studies, IL11 triggered a proinflammatory secretome with the notable upregulation of IL8, IL6, MCP1, CCL20 and CXCL1/5/6, which are important chemotaxins for neutrophils, monocytes, and lymphocytes. IL11 induced IL33 expression across fibroblast types, and the inhibition of STAT3 but not of MEK/ERK prevented this. These data establish IL11 as pro-inflammatory with specific importance for priming the IL33 alarmin response in inflammatory fibroblasts across tissues.

Hepatocyte Specific gp130 Signalling Underlies APAP Induced Liver Injury.

N-acetyl-p-aminophenol (APAP)-induced liver damage is associated with upregulation of Interleukin-11 (IL11), which is thought to stimulate IL6ST (gp130)-mediated STAT3 activity in hepatocytes, as a compensatory response. However, recent studies have found IL11/IL11RA/gp130 signaling to be hepatotoxic. To investigate further the role of IL11 and gp130 in APAP liver injury, we generated two new mouse strains with conditional knockout (CKO) of either Il11 (CKOIl11) or gp130 (CKOgp130) in adult hepatocytes. Following APAP, as compared to controls, CKOgp130 mice had lesser liver damage with lower serum Alanine Transaminase (ALT) and Aspartate Aminotransferase (AST), greatly reduced serum IL11 levels (90% lower), and lesser centrilobular necrosis. Livers from APAP-injured CKOgp130 mice had lesser ERK, JNK, NOX4 activation and increased markers of regeneration (PCNA, Cyclin D1, Ki67). Experiments were repeated in CKOIl11 mice that, as compared to wild-type mice, had lower APAP-induced ALT/AST, reduced centrilobular necrosis and undetectable IL11 in serum. As seen with CKOgp130 mice, APAP-treated CKOIl11 mice had lesser ERK/JNK/NOX4 activation and greater features of regeneration. Both CKOgp130 and CKOIl11 mice had normal APAP metabolism. After APAP, CKOgp130 and CKOIl11 mice had reduced Il6, Ccl2, Ccl5, Il1ß, and Tnfα expression. These studies exclude IL11 upregulation as compensatory and establish autocrine, self-amplifying, gp130-dependent IL11 secretion from damaged hepatocytes as toxic and anti-regenerative.

Vitamin B12 and folate decrease inflammation and fibrosis in NASH by preventing syntaxin 17 homocysteinylation.

BACKGROUND & AIMS: Several recent clinical studies have shown that serum homocysteine (Hcy) levels are positively correlated, while vitamin B12 (B12) and folate levels are negative correlated, with non-alcoholic steatohepatitis (NASH) severity. However, it is not known whether hyperhomocysteinemia (HHcy) plays a pathogenic role in NASH. METHODS: We examined the effects of HHcy on NASH progression, metabolism, and autophagy in dietary and genetic mouse models, patients, and primates. We employed vitamin B12 (B12) and folate (Fol) to reverse NASH features in mice and cell culture. RESULTS: Serum Hcy correlated with hepatic inflammation and fibrosis in NASH. Elevated hepatic Hcy induced and exacerbated NASH. Gene expression of hepatic Hcy-metabolizing enzymes was downregulated in NASH. Surprisingly, we found increased homocysteinylation (Hcy-lation) and ubiquitination of multiple hepatic proteins in NASH including the key autophagosome/lysosome fusion protein, Syntaxin 17 (Stx17). This protein was Hcy-lated and ubiquitinated, and its degradation led to a block in autophagy. Genetic manipulation of Stx17 revealed its critical role in regulating autophagy, inflammation and fibrosis during HHcy. Remarkably, dietary B12/Fol, which promotes enzymatic conversion of Hcy to methionine, decreased HHcy and hepatic Hcy-lated protein levels, restored Stx17 expression and autophagy, stimulated ß -oxidation of fatty acids, and improved hepatic histology in mice with pre-established NASH. CONCLUSIONS: HHcy plays a key role in the pathogenesis of NASH via Stx17 homocysteinylation. B12/folate also may represent a novel first-line therapy for NASH. LAY SUMMARY: The incidence of non-alcoholic steatohepatitis, for which there are no approved pharmacological therapies, is increasing, posing a significant healthcare challenge. Herein, based on studies in mice, primates and humans, we found that dietary supplementation with vitamin B12 and folate could have therapeutic potential for the prevention or treatment of non-alcoholic steatohepatitis.

A high-resolution map of human RNA translation.

Translated small open reading frames (smORFs) can have important regulatory roles and encode microproteins, yet their genome-wide identification has been challenging. We determined the ribosome locations across six primary human cell types and five tissues and detected 7,767 smORFs with translational profiles matching those of known proteins. The human genome was found to contain highly cell-type- and tissue-specific smORFs and a subset that encodes highly conserved amino acid sequences. Changes in the translational efficiency of upstream-encoded smORFs (uORFs) and the corresponding main ORFs predominantly occur in the same direction. Integration with 456 mass-spectrometry datasets confirms the presence of 603 small peptides at the protein level in humans and provides insights into the subcellular localization of these small proteins. This study provides a comprehensive atlas of high-confidence translated smORFs derived from primary human cells and tissues in order to provide a more complete understanding of the translated human genome.

IL11 Activates Pancreatic Stellate Cells and Causes Pancreatic Inflammation, Fibrosis and Atrophy in a Mouse Model of Pancreatitis.

Interleukin-11 (IL11) is important for fibrosis and inflammation, but its role in the pancreas is unclear. In pancreatitis, fibrosis, inflammation and organ dysfunction are associated with pancreatic stellate cell (PSC)-to-myofibroblast transformation. Here, we show that IL11 stimulation of PSCs, which specifically express IL11RA in the pancreas, results in transient STAT3 phosphorylation, sustained ERK activation and PSC activation. In contrast, IL6 stimulation of PSCs caused sustained STAT3 phosphorylation but did not result in ERK activation or PSC transformation. Pancreatitis factors, including TGFß, CTGF and PDGF, induced IL11 secretion from PSCs and a neutralising IL11RA antibody prevented PSC activation by these stimuli. This revealed an important ERK-dependent role for autocrine IL11 activity in PSCs. In mice, IL11 was increased in the pancreas after pancreatic duct ligation, and in humans, IL11 and IL11RA levels were elevated in chronic pancreatitis. Following pancreatic duct ligation, administration of anti-IL11RA to mice reduced pathologic (ERK, STAT, NF-κB) signalling, pancreatic atrophy, fibrosis and pro-inflammatory cytokine (TNFα, IL6 and IL1ß) levels. This is the first description of IL11-mediated activation of PSCs, and the data suggest IL11 as a stromal therapeutic target in pancreatitis.

Thyroid Hormone Decreases Hepatic Steatosis, Inflammation, and Fibrosis in a Dietary Mouse Model of Nonalcoholic Steatohepatitis.

Background: Nonalcoholic steatohepatitis (NASH) is characterized by hepatic steatosis, lobular inflammation, and fibrosis. Thyroid hormone (TH) reduces steatosis; however, the therapeutic effect of TH on NASH-associated inflammation and fibrosis is not known. This study examined the therapeutic effect of TH on hepatic inflammation and fibrosis during NASH and investigated THs molecular actions on autophagy and mitochondrial biogenesis. Methods: HepG2-TRß cells were treated with bovine serum albumin-conjugated palmitic acid (PA) to mimic lipotoxic conditions in vitro. Mice with NASH were established by feeding C57BL/6J mice Western diet with 15% fructose in drinking water for 16 weeks. These mice were administered triiodothyronine (T3)/thyroxine (T4) supplemented in drinking water for the next eight weeks. Results: In cultured HepG2-TRß cells, TH treatment increased mitochondrial respiration and fatty acid oxidation under basal and PA-treated conditions, as well as decreased lipopolysaccharides and PA-stimulated inflammatory and fibrotic responses. In a dietary mouse model of NASH, TH administration decreased hepatic triglyceride content (3.19 ± 0.68 vs. 8.04 ± 0.42 mM/g liver) and hydroxyproline (1.44 ± 0.07 vs. 2.58 ± 0.30 mg/g liver) when compared with mice with untreated NASH. Metabolomics profiling of lipid metabolites showed that mice with NASH had increased triacylglycerol, diacylglycerol, monoacylglycerol, and hepatic cholesterol esters species, and these lipid species were decreased by TH treatment. Mice with NASH also showed decreased autophagic degradation as evidenced by decreased transcription Factor EB and lysosomal protease expression, and accumulation of LC3B-II and p62. TH treatment restored the level of lysosomal proteins and resolved the accumulation of LC3B-II and p62. Impaired mitochondrial biogenesis was also restored by TH. The simultaneous restoration of autophagy and mitochondrial biogenesis by TH increased ß-oxidation of fatty acids. Additionally, the elevated oxidative stress and inflammasome activation in NASH liver were also decreased by TH. Conclusions: In a mouse model of NASH, TH restored autophagy and mitochondrial biogenesis to increase ß-oxidation of fatty acids and to reduce lipotoxicity, oxidative stress, hepatic inflammation, and fibrosis. Activating thyroid hormone receptor in the liver may represent an effective strategy for NASH treatment.

A Neutralizing IL-11 Antibody Improves Renal Function and Increases Lifespan in a Mouse Model of Alport Syndrome.

BACKGROUND: Alport syndrome is a genetic disorder characterized by a defective glomerular basement membrane, tubulointerstitial fibrosis, inflammation, and progressive renal failure. IL-11 was recently implicated in fibrotic kidney disease, but its role in Alport syndrome is unknown. METHODS: We determined IL-11 expression by molecular analyses and in an Alport syndrome mouse model. We assessed the effects of a neutralizing IL-11 antibody (×203) versus an IgG control in Col4a3-/- mice (lacking the gene encoding a type IV collagen component) on renal tubule damage, function, fibrosis, and inflammation. Effects of ×203, the IgG control, an angiotensin-converting enzyme (ACE) inhibitor (ramipril), or ramipril+X203 on lifespan were also studied. RESULTS: In Col4a3-/- mice, as kidney failure advanced, renal IL-11 levels increased, and IL-11 expression localized to tubular epithelial cells. The IL-11 receptor (IL-11RA1) is expressed in tubular epithelial cells and podocytes and is upregulated in tubular epithelial cells of Col4a3-/- mice. Administration of ×203 reduced albuminuria, improved renal function, and preserved podocyte numbers and levels of key podocyte proteins that are reduced in Col4a3-/- mice; these effects were accompanied by reduced fibrosis and inflammation, attenuation of epithelial-to-mesenchymal transition, and increased expression of regenerative markers. X203 attenuated pathogenic ERK and STAT3 pathways, which were activated in Col4a3-/- mice. The median lifespan of Col4a3-/- mice was prolonged 22% by ramipril, 44% with ×203, and 99% with ramipril+X203. CONCLUSIONS: In an Alport syndrome mouse model, renal IL-11 is upregulated, and neutralization of IL-11 reduces epithelial-to-mesenchymal transition, fibrosis, and inflammation while improving renal function. Anti-IL-11 combined with ACE inhibition synergistically extends lifespan. This suggests that a therapeutic approach targeting IL-11 holds promise for progressive kidney disease in Alport syndrome.

Inhibition of IL11 Signaling Reduces Aortic Pathology in Murine Marfan Syndrome.

BACKGROUND: Marfan syndrome (MFS) is associated with TGF (transforming growth factor) ß-stimulated ERK (extracellular signal-regulated kinase) activity in vascular smooth muscle cells (VSMCs), which adopt a mixed synthetic/contractile phenotype. In VSMCs, TGFß induces IL (interleukin) 11) that stimulates ERK-dependent secretion of collagens and MMPs (matrix metalloproteinases). Here, we examined the role of IL11 in the MFS aorta. METHODS: We used echocardiography, histology, immunostaining, and biochemical methods to study aortic anatomy, physiology, and molecular endophenotypes in Fbn1C1041G/+ mice, an established murine model of MFS (mMFS). mMFS mice were crossed to an IL11-tagged EGFP (enhanced green fluorescent protein; Il11EGFP/+) reporter strain or to a strain deleted for the IL11 receptor (Il11ra1-/-). In therapeutic studies, mMFS were administered an X209 (neutralizing antibody against IL11RA [IL11 receptor subunit alpha]) or IgG for 20 weeks and imaged longitudinally. RESULTS: IL11 mRNA and protein were elevated in the aortas of mMFS mice, as compared to controls. mMFS mice crossed to Il11EGFP/+ mice had increased IL11 expression in VSMCs, notably in the aortic root and ascending aorta. As compared to the mMFS parental strain, double mutant mMFS:Il11ra1-/- mice had reduced aortic dilatation and exhibited lesser fibrosis, inflammation, elastin breaks, and VSMC loss, which was associated with reduced aortic COL1A1 (collagen type I alpha 1 chain), IL11, MMP2/9, and phospho-ERK expression. To explore therapeutic targeting of IL11 signaling in MFS, we administered either a neutralizing antibody against IL11RA (X209) or an IgG control. After 20 weeks of antibody administration, as compared to IgG, mMFS mice receiving X209 had reduced thoracic and abdominal aortic dilation as well as lesser fibrosis, inflammation, elastin breaks, and VSMC loss. By immunoblotting, X209 was shown to reduce aortic COL1A1, IL11, MMP2/9, and phospho-ERK expression. CONCLUSIONS: In MFS, IL11 is upregulated in aortic VSMCs to cause ERK-related thoracic aortic dilatation, inflammation, and fibrosis. Therapeutic inhibition of IL11, imminent in clinical trials, might be considered as a new approach in MFS.

Molecular Dissection of Pro-Fibrotic IL11 Signaling in Cardiac and Pulmonary Fibroblasts.

In fibroblasts, TGFß1 stimulates IL11 upregulation that leads to an autocrine loop of IL11-dependent pro-fibrotic protein translation. The signaling pathways downstream of IL11, which acts via IL6ST, are contentious with both STAT3 and ERK implicated. Here we dissect IL11 signaling in fibroblasts and study IL11-dependent protein synthesis pathways in the context of approved anti-fibrotic drug mechanisms of action. We show that IL11-induced ERK activation drives fibrogenesis and while STAT3 phosphorylation (pSTAT3) is also seen, this appears unrelated to fibroblast activation. Ironically, recombinant human IL11, which has been used extensively in mouse experiments to infer STAT3 activity downstream of IL11, increases pSTAT3 in Il11ra1 null mouse fibroblasts. Unexpectedly, inhibition of STAT3 was found to induce severe proteotoxic ER stress, generalized fibroblast dysfunction and cell death. In contrast, inhibition of ERK prevented fibroblast activation in the absence of ER stress. IL11 stimulated an axis of ERK/mTOR/P70RSK protein translation and its selectivity for Collagen 1 synthesis was ascribed to an EPRS-regulated, ribosome stalling mechanism. Surprisingly, the anti-fibrotic drug nintedanib caused dose-dependent ER stress and lesser pSTAT3 expression. Pirfenidone had no effect on ER stress whereas anti-IL11 specifically inhibited the ERK/mTOR axis while reducing ER stress. These studies define the translation-specific signaling pathways downstream of IL11, intersect immune and metabolic signaling and reveal unappreciated effects of nintedanib.

Critical Conditions for Studying Interleukin-11 Signaling In Vitro and Avoiding Experimental Artefacts.

Interleukin (IL) 11 is a member of the IL6 family of cytokines which require the ubiquitous gp130 receptor to activate canonical (JAK/STAT) and non-canonical (e.g., ERK) signaling pathways. The IL11 cytokine is upregulated in a number of fibro-inflammatory diseases and cancer, where it binds the cognate IL11 receptor alpha subunit (IL11RA) to form a hexameric IL11:IL11RA:gp130 signaling complex. The specific IL11RA receptor is highly expressed on cells of the stromal and parenchymal niche but expressed at low levels on immune cells, highly passaged cells, or transformed cell lines. Consequently, primary cells such as hepatic stellate cells, fibroblasts, and hepatocytes are ideal experimental systems to study IL11 signaling in vitro. In contrast to immortalized cell lines, primary cells better display relevant cellular physiology and pathobiology. This collection of protocols details experimental and culturing conditions for primary cells that preserve meaningful cellular states and physiological responses ex vivo in conventional 2D cell culture systems. Readouts of cellular activity are chosen carefully to capture the non-canonical, post-transcriptional activity of IL11 signaling. Our data suggest that cell type, cell culture conditions, passage number, concentrations of stimuli, timing, and other factors have major implications for studies of IL11 signaling. In vitro experiments with primary cell material need to be planned and executed with great caution. Otherwise, physiologically relevant mechanisms may become dysfunctional and reproducible experimental artefacts can obscure our view of true cytokine biology. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Expansion of primary human hepatic stellate cells (HSCs) and human renal proximal tubular epithelial cells (HRPTEpiCs) Basic Protocol 2: Expansion of primary human lung fibroblasts (HLFs) Alternate Protocol 1: Isolation and expansion of primary mouse lung fibroblasts Support Protocol 1: Freezing and thawing of primary cells Support Protocol 2: Operetta high-content imaging-based phenotyping Support Protocol 3: Colorimetric assay of solubilized collagen Support Protocol 4: Quantification of fibrosis marker secretion Support Protocol 5: Western blotting studies of IL11 signaling in HSCs, HLFs, and HRPTEpiCs Basic Protocol 3: IL11 stimulation of primary human hepatocytes Alternate Protocol 2: IL11 stimulation of primary mouse hepatocytes Support Protocol 6: Alanine transaminase (ALT) secretion by human and mouse hepatocytes.

The pro-regenerative effects of hyperIL6 in drug-induced liver injury are unexpectedly due to competitive inhibition of IL11 signaling.

It is generally accepted that IL6-mediated STAT3 signaling in hepatocytes, mediated via glycoprotein 130 (gp130; IL6ST), is beneficial and that the synthetic IL6:IL6ST fusion protein (HyperIL6) promotes liver regeneration. Recently, autocrine IL11 activity that also acts via IL6ST but uses ERK rather than STAT3 to signal, was found to be hepatotoxic. Here we examined whether the beneficial effects of HyperIL6 could reflect unappreciated competitive inhibition of IL11-dependent IL6ST signaling. In human and mouse hepatocytes, HyperIL6 reduced N-acetyl-p-aminophenol (APAP)-induced cell death independent of STAT3 activation and instead, dose-dependently, inhibited IL11-related signaling and toxicities. In mice, expression of HyperIl6 reduced ERK activation and promoted STAT3-independent hepatic regeneration (PCNA, Cyclin D1, Ki67) following administration of either IL11 or APAP. Inhibition of putative intrinsic IL6 trans-signaling had no effect on liver regeneration in mice. Following APAP, mice deleted for Il11 exhibited spontaneous liver repair but HyperIl6, despite robustly activating STAT3, had no effect on liver regeneration in this strain. These data show that synthetic IL6ST binding proteins such as HyperIL6 can have unexpected, on-target effects and suggest IL11, not IL6, as important for liver regeneration.

Similarities and differences between IL11 and IL11RA1 knockout mice for lung fibro-inflammation, fertility and craniosynostosis.

Loss of function (LOF) in IL11RA infers IL11 signaling as important for fertility, fibrosis, inflammation and incompletely penetrant craniosynostosis. The impact of LOF in IL11 has not been characterized. We generated IL11 knockout (Il11-/-) mice that are born in expected ratios and have normal hematological profiles. Lung fibroblasts from Il11-/- mice are resistant to pro-fibrotic stimulation with TGFß1. Following bleomycin-induced lung injury, Il11-/- mice are protected from pulmonary fibrosis and exhibit lesser ERK, STAT3 and NF-kB activation, reduced Il1b, Timp1, Ccl2 and diminished IL6 expression, both at baseline and after injury: placing Il11 activity upstream of IL6 in this model. Il11-/- female mice are infertile. Unlike Il11ra1-/- mice, Il11-/- mice do not have craniosynostosis, have normal long bone mass and reduced body weights. These data further establish the role of IL11 signaling in lung fibrosis while suggesting that bone development abnormalities can be associated with mutation of IL11RA but not IL11, which may have implications for therapeutic targeting of IL11 signaling.

Redefining IL11 as a regeneration-limiting hepatotoxin and therapeutic target in acetaminophen-induced liver injury.

Acetaminophen (N-acetyl-p-aminophenol; APAP) toxicity is a common cause of liver damage. In the mouse model of APAP-induced liver injury (AILI), interleukin 11 (IL11) is highly up-regulated and administration of recombinant human IL11 (rhIL11) has been shown to be protective. Here, we demonstrate that the beneficial effect of rhIL11 in the mouse model of AILI is due to its inhibition of endogenous mouse IL11 activity. Our results show that species-matched IL11 behaves like a hepatotoxin. IL11 secreted from APAP-damaged human and mouse hepatocytes triggered an autocrine loop of NADPH oxidase 4 (NOX4)-dependent cell death, which occurred downstream of APAP-initiated mitochondrial dysfunction. Hepatocyte-specific deletion of Il11 receptor subunit alpha chain 1 (Il11ra1) in adult mice protected against AILI despite normal APAP metabolism and glutathione (GSH) depletion. Mice with germline deletion of Il11 were also protected from AILI, and deletion of Il1ra1 or Il11 was associated with reduced c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) activation and quickly restored GSH concentrations. Administration of a neutralizing IL11RA antibody reduced AILI in mice across genetic backgrounds and promoted survival when administered up to 10 hours after APAP. Inhibition of IL11 signaling was associated with the up-regulation of markers of liver regenerations: cyclins and proliferating cell nuclear antigen (PCNA) as well as with phosphorylation of retinoblastoma protein (RB) 24 hours after AILI. Our data suggest that species-matched IL11 is a hepatotoxin and that IL11 signaling might be an effective therapeutic target for APAP-induced liver damage.