Cytokines of the interleukin-6 family as emerging targets in inflammatory bowel disease.

INTRODUCTION: Inflammatory bowel disease (IBD) is an umbrella term that includes different chronic inflammatory diseases of the gastrointestinal tract, most commonly Crohn's disease and ulcerative colitis. IBD affects more than 6 million people worldwide and constitutes not only a debilitating disease for the patients, but also a significant factor for society due to costs for health care and reduced working capacity. Despite the introduction of biologicals for the treatment of IBD, the identification of novel targets that could lead to novel therapeutics is still needed. AREAS COVERED: In this review, we summarize current knowledge about the interleukin-6 family of cytokines as potential therapeutic targets for improving the therapy of patients with IBD. We discuss cytokines like IL-6 itself for which therapeutics such as inhibitory monoclonal antibodies have already entered the clinics, but also focus on other family members whose therapeutic potential has not been explored yet. EXPERT OPINION: The different cytokines of the IL-6 family offer multiple therapeutic targets that can potentially be used to treat patients with inflammatory bowel disease, but unwanted side effects like inhibition of epithelial regeneration have to be considered.

Molecular characterization of the craniosynostosis-associated interleukin-11 receptor variants p.T306_S308dup and p.E364_V368del.

Interleukin-11 (IL-11) is a member of the IL-6 family of cytokines and is an important factor for bone homeostasis. IL-11 binds to and signals via the membrane-bound IL-11 receptor (IL-11R, classic signaling) or soluble forms of the IL-11R (sIL-11R, trans-signaling). Mutations in the IL11RA gene, which encodes the IL-11R, are associated with craniosynostosis, a human condition in which one or several of the sutures close prematurely, resulting in malformation of the skull. The biological mechanisms of how mutations within the IL-11R are linked to craniosynostosis are mostly unexplored. In this study, we analyze two variants of the IL-11R described in craniosynostosis patients: p.T306_S308dup, which results in a duplication of three amino-acid residues within the membrane-proximal fibronectin type III domain, and p.E364_V368del, which results in a deletion of five amino-acid residues in the so-called stalk region adjacent to the plasma membrane. The stalk region connects the three extracellular domains to the transmembrane and intracellular region of the IL-11R and contains cleavage sites for different proteases that generate sIL-11R variants. Using a combination of bioinformatics and different biochemical, molecular, and cell biology methods, we show that the IL-11R-T306_S308dup variant does not mature correctly, is intracellularly retained, and does not reach the cell surface. In contrast, the IL-11R-E364_V368del variant is fully biologically active and processed normally by proteases, thus allowing classic and trans-signaling of IL-11. Our results provide evidence that mutations within the IL11RA gene may not be causative for craniosynostosis and suggest that other regulatory mechanism(s) are involved but remain to be identified.

The soluble IL-2 receptor α/CD25 as a modulator of IL-2 function.

The pleiotropic cytokine interleukin-2 (IL-2) is an integral regulator of healthy and pathological immune responses, with the most important role in regulating the homeostasis of regulatory T cells. IL-2 signalling involves three distinct receptors: The IL-2 receptor α (IL-2Rα/CD25), IL-2Rß, and IL-2Rγ/γc . While IL-2Rß and γc are essential for signal transduction, IL-2Rα regulates the affinity of the receptor complex towards IL-2. A soluble form of the IL-2Rα (sIL-2Rα) is present in the blood of healthy individuals and increased under various pathological conditions. Although it is known that the sIL-2Rα retains its ability to bind IL-2, it is not fully understood how this molecule affects IL-2 function and thus immune responses. Here, we summarize the current knowledge on the generation and function of the sIL-2Rα. We describe the molecular mechanisms leading to sIL-2Rα generation and discuss the different IL-2 modulating functions that have been attributed to the sIL-2Rα. Finally, we describe attempts to utilize the sIL-2Rα as a therapeutic tool.

The interleukin-11 receptor variant p.W307R results in craniosynostosis in humans.

Craniosynostosis is characterized by the premature fusion and ossification of one or more of the sutures of the calvaria, often resulting in abnormal features of the face and the skull. In cases in which growth of the brain supersedes available space within the skull, developmental delay or cognitive impairment can occur. A complex interplay of different cell types and multiple signaling pathways are required for correct craniofacial development. In this study, we report on two siblings with craniosynostosis and a homozygous missense pathogenic variant within the IL11RA gene (c.919 T > C; p.W307R). The patients present with craniosynostosis, exophthalmos, delayed tooth eruption, mild platybasia, and a basilar invagination. The p.W307R variant is located within the arginine-tryptophan-zipper within the D3 domain of the IL-11R, a structural element known to be important for the stability of the cytokine receptor. Expression of IL-11R-W307R in cells shows impaired maturation of the IL-11R, no transport to the cell surface and intracellular retention. Accordingly, cells stably expressing IL-11R-W307R do not respond when stimulated with IL-11, arguing for a loss-of-function mutation. In summary, the IL-11R-W307R variant, reported here for the first time to our knowledge, is most likely the causative variant underlying craniosynostosis in these patients.

Case Report: Arterial Wall Inflammation in Atherosclerotic Cardiovascular Disease is Reduced by Olamkicept (sgp130Fc).

Inflammation is a strong driver of atherosclerotic cardiovascular disease (ASCVD). There is a large unmet need for therapies that prevent or reduce excessive inflammation while avoiding systemic immunosuppression. We showed previously that selective inhibition of pro-inflammatory interleukin-6 (IL-6) trans-signalling by the fusion protein olamkicept (sgp130Fc) prevented and reduced experimental murine atherosclerosis in low-density lipoprotein receptor-deficient (Ldlr -/-) mice on a high-fat, high-cholesterol diet independently of low-density lipoprotein (LDL) cholesterol metabolism. Therefore, we allowed compassionate use of olamkicept (600 mg intravenously biweekly for 10 weeks) in a patient with very-high-risk ASCVD. Despite optimal LDL cholesterol under maximum tolerated lipid-lowering treatment, the patient had a remaining very high risk for future cardiovascular events related to significant arterial wall inflammation with lipoprotein (a) [Lp(a)]-cholesterol as the main contributor. 18Fluorodeoxyglucose positron emission tomography/computed tomography (18FDG PET/CT) measurements were performed before and after the treatment period. Olamkicept reduced arterial wall inflammation in this patient without interfering with lipoprotein metabolism. No clinical or laboratory side effects were observed during or after treatment with olamkicept. Our findings in this patient matched the results from our mechanistic study in Ldlr -/- mice, which were extended by additional analyses on vascular inflammation. Olamkicept may be a promising option for treating ASCVD independently of LDL cholesterol metabolism. A Phase II trial of olamkicept in ASCVD is currently being prepared.

The metalloprotease ADAM10 generates soluble interleukin-2 receptor alpha (sCD25) in vivo.

The cytokine interleukin-2 (IL-2) plays a critical role in controlling the immune homeostasis by regulating the proliferation and differentiation of immune cells, especially T cells. IL-2 signaling is mediated via the IL-2 receptor (IL-2R) complex, which consists of the IL-2Rα (CD25), the IL-2Rß, and the IL-2Rγ. While the latter are required for signal transduction, IL-2Rα controls the ligand-binding affinity of the receptor complex. A soluble form of the IL-2Rα (sIL-2Rα) is found constitutively in human serum, though its levels are increased under various pathophysiological conditions. The sIL-2Rα originates partly from activated T cells through proteolytic cleavage, but neither the responsible proteases nor stimuli that lead to IL-2Rα cleavage are known. Here, we show that the metalloproteases ADAM10 and ADAM17 can cleave the IL-2Rα and generate a soluble ectodomain, which functions as a decoy receptor that inhibits IL-2 signaling in T cells. We demonstrate that ADAM10 is mainly responsible for constitutive shedding of the IL-2Rα, while ADAM17 is involved in IL-2Rα cleavage upon T cell activation. In vivo, we found that mice with a CD4-specific deletion of ADAM10, but not ADAM17, show reduced steady-state sIL-2Rα serum levels. We propose that the identification of proteases involved in sIL-2Rα generation will allow for manipulation of IL-2Rα cleavage, especially as constitutive and induced cleavage of IL-2Rα are executed by different proteases, and thus offer a novel opportunity to alter IL-2 function.

The role of proteolysis in interleukin-11 signaling.

Although interleukin-11 (IL-11) was discovered more than 30 years ago, it remains an understudied member of the IL-6 family of cytokines. While it was originally discovered as a secreted factor that could foster megakaryocyte maturation and was therefore used as a recombinant protein to increase platelet production in patients with thrombocytopenia, recent research has established important roles for IL-11 in inflammation, fibrosis and cancer. In order to initiate signal transduction, IL-11 binds first to a non-signaling membrane-bound IL-11 receptor (IL-11R, classic signaling), which subsequently induces the formation of a heterodimer of the signal-transducing receptor gp130 that is shared with the other family members. Complex formation initiates several intracellular signaling cascades, most notably the Janus kinase/Signal Transducer and Activator of Transcription (Jak/STAT) pathway. We have recently identified a trans-signaling mechanism, in which IL-11 binds to soluble forms of the IL-11R (sIL-11R) and the agonistic IL-11/sIL-11R complex can activate cells that do not express the IL-11R and would usually not respond to IL-11. The generation of sIL-11R and thus the initiation of IL-11 trans-signaling is mediated by proteolytic cleavage. In this review, we summarize the current state of knowledge regarding IL-11R cleavage, highlight recent developments in IL-11 biology and discuss therapeutic opportunities and challenges in the light of IL-11 classic and trans-signaling.

Function and proteolytic generation of the soluble interleukin-6 receptor in health and disease.

The pleiotropic cytokine interleukin-6 (IL-6) is involved in numerous physiological and pathophysiological functions that include development, immune cell differentiation, inflammation and cancer. IL-6 can signal via the membrane-bound IL-6 receptor (IL-6R, classic signaling) or via soluble forms of the IL-6R (sIL-6R, trans-signaling). Both modes of signaling induce the formation of a homodimer of the signal transducing ß-receptor glycoprotein 130 (gp130) and the activation of several intracellular signaling cascades, e.g. the Jak/STAT pathway. Intriguingly, only IL-6 trans-signaling is required for the pro-inflammatory properties of IL-6, while regenerative and anti-inflammatory functions are mediated via classic signaling. The sIL-6R is generated by different molecular mechanisms, including alternative mRNA splicing, proteolysis of the membrane-bound IL-6R and the release of extracellular vesicles. In this review, we give an in-depth overview on these molecular mechanisms with a special emphasize on IL-6R cleavage by the metalloprotease ADAM17 and other proteases. We discuss the biological functions of the sIL-6R and highlight attempts to selectively block IL-6 trans-signaling in pre-clinical animal models as well as in clinical studies in patients with inflammatory bowel disease.

Anti-IL-6 cytokine treatment has no impact on elevated hematocrit or splenomegaly in a polycythemia vera mouse model.

Somatic mutations in JAK2, MPL and Calreticulin and inflammation play a key role in pathophysiology of chronic myeloproliferative neoplasia (CMN). One of the most prominent cytokines elevated in serum of Polycythemia vera patients is interleukin-6 (IL-6). Currently, it is being discussed whether suppression of inflammation by anti-cytokine approaches as anti-IL-6 treatment may be therapeutically useful in CMN. We here sought to investigate the efficacy of anti-IL-6 treatment on inflammatory cytokines, hematocrit and splenomegaly in CMN like disease. JAK2-V617F knock-in mice (JAK2+/V617F) were treated for three weeks with anti-IL-6 antibody (Ab) or IgG-control. Upon anti-IL-6 Ab treatment, serum levels of CXCL2 and CXCL10 were significantly reduced. In addition, CXCL1, CCL11, M-CSF, G-CSF, IL-17, IL-12p40 and CCL2 were reduced by a factor of 0.3 -- 0.8. Partly, this was also achieved by applying high-dose IgG. Hematocrit, erythrocyte and leukocyte counts were elevated in JAK2+/V617F mice but were not reduced by anti-IL6 Ab treatment. In addition, there was no apparent amelioration of splenomegaly and spleen histopathology. In conclusion, anti-IL-6 Ab treatment did not result in improvement of hematological disease parameters but was shown to modulate the serum cytokine signature.

Interleukin-6-interleukin-11 receptor chimeras reveal ionomycin-induced proteolysis beyond ADAM10.

Interleukin-6 (IL-6) and interleukin-11 (IL-11) are two important pleiotropic cytokines, both of which signal through a homodimer of the ß-receptor gp130. Specificity is gained through the unique, nonsignaling α-receptors IL-6R and IL-11R. Soluble variants of IL-6R and IL-11R also exist. Both membrane-bound receptors can be cleaved by the metalloprotease ADAM10. Here, we use ten different chimeric receptors consisting of different parts of IL-6R and IL-11R and analyze their susceptibility toward cleavage by ADAM10. As expected, all chimeras are substrates of ADAM10. However, we observed that cleavage of chimeric receptors containing the stalk region of the IL-11R could be blocked by the protease inhibitor GI (selective for ADAM10), but not by the protease inhibitor GW (selective for both ADAM10 and ADAM17), suggesting that another protease besides ADAM10 is involved in cleavage of these chimeras.

A single aromatic residue in sgp130Fc/olamkicept allows the discrimination between interleukin-6 and interleukin-11 trans-signaling.

Blocking the activity of cytokines is an efficient strategy to combat inflammatory diseases. Interleukin-6 (IL-6) fulfills its pro-inflammatory properties via its soluble receptor (IL-6 trans-signaling). The selective trans-signaling inhibitor olamkicept (sgp130Fc) is currently in clinical development. We have previously shown that sgp130Fc can also efficiently block trans-signaling of the closely related cytokine IL-11, which elicits the question how selectivity for one of the two cytokines can be achieved. Using structural information, we show that the interfaces between IL-6R-gp130 and IL-11R-gp130, respectively, within the so-called site III are different between the two cytokines. Modification of an aromatic cluster around Q113 of gp130 within these interfaces allows the discrimination between IL-6 and IL-11 trans-signaling. Using recombinant sgp130Fc variants, we demonstrate that these differences can indeed be exploited to generate a truly selective IL-6 trans-signaling inhibitor. Our data highlight how the selectivity of a clinically relevant designer protein can be further improved.

Characterization of the Cancer-Associated Meprin Βeta Variants G45R and G89R.

Meprin ß is a metalloprotease associated with neurodegeneration, inflammation, extracellular matrix homeostasis, transendothelial cell migration, and cancer. In this study, we investigated two melanoma-associated variants of meprin ß, both exhibiting a single amino acid exchange, namely, meprin ß G45R and G89R. Based on the structural data of meprin ß and with regard to the position of the amino acid exchanges, we hypothesized an increase in proteolytic activity in the case of the G45R variant due to the induction of a potential new activation site and a decrease in proteolytic activity from the G89R variant due to structural instability. Indeed, the G89R variant showed, overall, a reduced expression level compared to wild-type meprin ß, accompanied by decreased activity and lower cell surface expression but strong accumulation in the endoplasmic reticulum. This was further supported by the analysis of the shedding of the interleukin-6 receptor (IL-6R) by meprin ß and its variants. In transfected HEK cells, the G89R variant was found to generate less soluble IL-6R, whereas the expression of meprin ß G45R resulted in increased shedding of the IL-6R compared to wild-type meprin ß and the G89R variant. A similar tendency of the induced shedding capacity of G45R was seen for the well-described meprin ß substrate CD99. Furthermore, employing an assay for cell migration in a collagen IV matrix, we observed that the transfection of wild-type meprin ß and the G45R variant resulted in increased migration of HeLa cells, while the G89R variant led to diminished mobility.

Interleukin-11 receptor expression on monocytes is dispensable for their recruitment and pathogen uptake during Leishmania major infection.

Interleukin-11 (IL-11) is an important member of the IL-6 family of cytokines. IL-11 activates its target cells via binding to a non-signaling α-receptor (IL-11R), which results in recruitment and activation of a gp130 homodimer. The cytokine was initially described as an anti-inflammatory protein, but has recently gained attention as a potent driver in certain types of cancer and different fibrotic conditions. Leishmania spp. are a group of eukaryotic parasites that cause the disease leishmaniasis. They infect phagocytes of their hosts, especially monocytes recruited to the site of infection, and are able to replicate within this rather harsh environment, often resulting in chronic infections of the patient. However, the molecular mechanisms underlying parasite and host cell interactions and factors of the immune cells that are crucial for Leishmania uptake are so far largely unspecified. Recently, increased IL-11 expression in the lesions of patients with cutaneous leishmaniasis has been reported, but the functional relevance is unknown. In this study, we show that monocytes express IL-11R on their cell surface. Furthermore, using an adoptive transfer model of IL-11R-/- monocytes, we analyze the contribution of IL-11 signaling on monocyte recruitment and monocyte infection in a mouse model of cutaneous leishmaniasis and find that IL-11 signaling is dispensable for monocyte recruitment and pathogen uptake during Leishmania major infection.

The iRhom homology domain is indispensable for ADAM17-mediated TNFα and EGF receptor ligand release.

Membrane-tethered signalling proteins such as TNFα and many EGF receptor ligands undergo shedding by the metalloproteinase ADAM17 to get released. The pseudoproteases iRhom1 and iRhom2 are important for the transport, maturation and activity of ADAM17. Yet, the structural and functional requirements to promote the transport of the iRhom-ADAM17 complex have not yet been thoroughly investigated. Utilising in silico and in vitro methods, we here map the conserved iRhom homology domain (IRHD) and provide first insights into its structure and function. By focusing on iRhom2, we identified different structural and functional factors within the IRHD. We found that the structural integrity of the IRHD is a key factor for ADAM17 binding. In addition, we identified a highly conserved motif within an unstructured region of the IRHD, that, when mutated, restricts the transport of the iRhom-ADAM17 complex through the secretory pathway in in vitro, ex vivo and in vivo systems and also increases the half-life of iRhom2 and ADAM17. Furthermore, the disruption of this IRHD motif was also reflected by changes in the yet undescribed interaction profile of iRhom2 with proteins involved in intracellular vesicle transport. Overall, we provide the first insights into the forward trafficking of iRhoms which is critical for TNFα and EGF receptor signalling.

The cytokine interleukin-11 crucially links bone formation, remodeling and resorption.

Bone development is a complex process that requires the activity of several different signaling pathways and cell types. It involves the coordinated action of osteoclasts (cells that are capable of resorbing bone), osteoblasts (cells that are able to form bone), osteocytes (cells that form a syncytial network within the bone), skeletal muscle cells and the bone marrow. In recent years, the cytokine interleukin-11 (IL-11), a member of the IL-6 family of cytokines, has emerged as an important regulatory protein for bone formation, remodeling and resorption. Furthermore, coding missense mutations in the IL11RA gene, which encodes the IL-11 receptor (IL-11R), have recently been linked to craniosynostosis, a human disease in which the sutures that line the head bones close prematurely. This review summarizes current knowledge about IL-11 and highlights its role in bone development and homeostasis. It further discusses the specificity and redundancy provided by the other members of the IL-6 cytokine family and how they facilitate signaling and cross-talk between skeletal muscle cells, bone cells and the bone marrow. We describe their actions in physiological and in pathological states and discuss how this knowledge could be translated into therapy.

Natural Glycoforms of Human Interleukin 6 Show Atypical Plasma Clearance.

A library of glycoforms of human interleukin 6 (IL-6) comprising complex and mannosidic N-glycans was generated by semisynthesis. The three segments were connected by sequential native chemical ligation followed by two-step refolding. The central glycopeptide segments were assembled by pseudoproline-assisted Lansbury aspartylation and subsequent enzymatic elongation of complex N-glycans. Nine IL-6 glycoforms were synthesized, seven of which were evaluated for in vivo plasma clearance in rats and compared to non-glycosylated recombinant IL-6 from E. coli. Each IL-6 glycoform was tested in three animals and reproducibly showed individual serum clearances depending on the structure of the N-glycan. The clearance rates were atypical, since the 2,6-sialylated glycoforms of IL-6 cleared faster than the corresponding asialo IL-6 with terminal galactoses. Compared to non-glycosylated IL-6 the plasma clearance of IL-6 glycoforms was delayed in the presence of larger and multibranched N-glycans in most cases.

Therapeutic Interleukin-6 Trans-signaling Inhibition by Olamkicept (sgp130Fc) in Patients With Active Inflammatory Bowel Disease.

BACKGROUND & AIMS: A large unmet therapeutic need exists in inflammatory bowel disease (IBD). Inhibition of interleukin (IL)-6 appears to be effective, but the therapeutic benefit of a complete IL6/IL6 receptor (IL6R) blockade is limited by profound immunosuppression. Evidence has emerged that chronic proinflammatory activity of IL6 is mainly mediated by trans-signaling via a complex of IL6 bound to soluble IL6R engaging the gp130 co-receptor without the need for membrane-bound IL6R. We have developed a decoy protein, sgp130Fc, that exclusively blocks IL6 proinflammatory trans-signaling and has shown efficacy in preclinical models of IBD, without signs of immunosuppression. METHODS: We present a 12-week, open-label, prospective phase 2a trial (FUTURE) in 16 patients with active IBD treated with the trans-signaling inhibitor olamkicept (sgp130Fc) to assess the molecular mechanisms, safety, and effectiveness of IL6 trans-signaling blockade in vivo. We performed in-depth molecular profiling at various timepoints before and after therapy induction to identify the mechanism of action of olamkicept. RESULTS: Olamkicept was well tolerated and induced clinical response in 44% and clinical remission in 19% of patients. Clinical effectiveness coincided with target inhibition (reduction of phosphorylated STAT3) and marked transcriptional changes in the inflamed mucosa. An olamkicept-specific transcriptional signature, distinguishable from remission signatures of anti-tumor necrosis factor (infliximab) or anti-integrin (vedolizumab) therapies was identified. CONCLUSIONS: Our data suggest that blockade of IL6 trans-signaling holds great promise for the therapy of IBD and should undergo full clinical development as a new immunoregulatory therapy for IBD. (EudraCT no., Nu 2016-000205-36).

Interleukin-6 controls recycling and degradation, but not internalization of its receptors.

Interleukin-6 (IL-6) is a cytokine implicated in proinflammatory as well as regenerative processes and acts via receptor complexes consisting of the ubiquitously expressed, signal-transducing receptor gp130 and the IL-6 receptor (IL-6R). The IL-6R is expressed only on hepatocytes and subsets of leukocytes, where it mediates specificity of the receptor complex to IL-6 as the subunit gp130 is shared with all other members of the IL-6 cytokine family such as IL-11 or IL-27. The amount of IL-6R at the cell surface thus determines the responsiveness of the cell to the cytokine and might therefore be decisive in the development of inflammatory disorders. However, how the expression levels of IL-6R and gp130 at the cell surface are controlled is largely unknown. Here, we show that IL-6R and gp130 are constitutively internalized independent of IL-6. This process depends on dynamin and clathrin and is temporally controlled by motifs within the intracellular region of gp130 and IL-6R. IL-6 binding and internalization of the receptors is a prerequisite for activation of the Jak/STAT signaling cascade. Targeting of gp130, but not of the IL-6R, to the lysosome for degradation depends on stimulation with IL-6. Furthermore, we show that after internalization and activation of signaling, both the IL-6R and gp130 are recycled back to the cell surface, a process that is enhanced by IL-6. These data reveal an important function of IL-6 beyond the pure activation of signaling.

Interleukin-11 (IL-11) receptor cleavage by the rhomboid protease RHBDL2 induces IL-11 trans-signaling.

Interleukin-11 (IL-11) is a pleiotropic cytokine with both pro- and anti-inflammatory properties. It activates its target cells via binding to the membrane-bound IL-11 receptor (IL-11R), which then recruits a homodimer of the ubiquitously expressed, signal-transducing receptor gp130. Besides this classic signaling pathway, IL-11 can also bind to soluble forms of the IL-11R (sIL-11R), and IL-11/sIL-11R complexes activate cells via the induction of gp130 homodimerization (trans-signaling). We have previously reported that the metalloprotease ADAM10 cleaves the membrane-bound IL-11R and thereby generates sIL-11R. In this study, we identify the rhomboid intramembrane protease RHBDL2 as a so far unrecognized alternative sheddase that can efficiently trigger IL-11R secretion. We determine the cleavage site used by RHBDL2, which is located in the extracellular part of the receptor in close proximity to the plasma membrane, between Ala-370 and Ser-371. Furthermore, we identify critical amino acid residues within the transmembrane helix that are required for IL-11R proteolysis. We also show that ectopically expressed RHBDL2 is able to cleave the IL-11R within the early secretory pathway and not only at the plasma membrane, indicating that its subcellular localization plays a central role in controlling its activity. Moreover, RHBDL2-derived sIL-11R is biologically active and able to perform IL-11 trans-signaling. Finally, we show that the human mutation IL-11R-A370V does not impede IL-11 classic signaling, but prevents RHBDL2-mediated IL-11R cleavage.