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.

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.

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.

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.

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.

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.

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.

Hepatocyte-specific IL11 cis-signaling drives lipotoxicity and underlies the transition from NAFLD to NASH.

IL11 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 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 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.

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.