Publications by authors named "Stefan Rose-John"

381 Publications

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

Biochim Biophys Acta Mol Cell Res 2021 Oct 6;1869(1):119143. Epub 2021 Oct 6.

Department of Pathology, Otto-von-Guericke-University Magdeburg, Medical Faculty, Magdeburg, Germany. Electronic address:

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 β-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.
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http://dx.doi.org/10.1016/j.bbamcr.2021.119143DOI Listing
October 2021

ADAM17 orchestrates Interleukin-6, TNFα and EGF-R signaling in inflammation and cancer.

Biochim Biophys Acta Mol Cell Res 2021 Oct 2;1869(1):119141. Epub 2021 Oct 2.

Biochemical Institute, University of Kiel, Germany. Electronic address:

It was realized in the 1990s that some membrane proteins such as TNFα, both TNF receptors, ligands of the EGF-R and the Interleukin-6 receptor are proteolytically cleaved and are shed from the cell membrane as soluble proteins. The major responsible protease is a metalloprotease named ADAM17. So far, close to 100 substrates, including cytokines, cytokine receptors, chemokines and adhesion molecules of ADAM17 are known. Therefore, ADAM17 orchestrates many different signaling pathways and is a central signaling hub in inflammation and carcinogenesis. ADAM17 plays an important role in the biology of Interleukin-6 (IL-6) since the generation of the soluble Interleukin-6 receptor (sIL-6R) is needed for trans-signaling, which has been identified as the pro-inflammatory activity of this cytokine. In contrast, Interleukin-6 signaling via the membrane-bound Interleukin-6 receptor is mostly regenerative and protective. Probably due to its broad substrate spectrum, ADAM17 is essential for life and most of the few human individuals identified with ADAM17 gene defects died at young age. Although the potential of ADAM17 as a therapeutic target has been recognized, specific blockade of ADAM17 is not trivial since the metalloprotease domain of ADAM17 shares high structural homology with other proteases, in particular matrix metalloproteases. Here, the critical functions of ADAM17 in IL-6, TNFα and EGF-R pathways and strategies of therapeutic interventions are discussed.
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http://dx.doi.org/10.1016/j.bbamcr.2021.119141DOI Listing
October 2021

Identification of the factor XII contact activation site enables sensitive coagulation diagnostics.

Nat Commun 2021 09 22;12(1):5596. Epub 2021 Sep 22.

Institute of Clinical Chemistry and Laboratory Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.

Contact activation refers to the process of surface-induced activation of factor XII (FXII), which initiates blood coagulation and is captured by the activated partial thromboplastin time (aPTT) assay. Here, we show the mechanism and diagnostic implications of FXII contact activation. Screening of recombinant FXII mutants identified a continuous stretch of residues Gln317-Ser339 that was essential for FXII surface binding and activation, thrombin generation and coagulation. Peptides spanning these 23 residues competed with surface-induced FXII activation. Although FXII mutants lacking residues Gln317-Ser339 were susceptible to activation by plasmin and plasma kallikrein, they were ineffective in supporting arterial and venous thrombus formation in mice. Antibodies raised against the Gln317-Ser339 region induced FXII activation and triggered controllable contact activation in solution leading to thrombin generation by the intrinsic pathway of coagulation. The antibody-activated aPTT allows for standardization of particulate aPTT reagents and for sensitive monitoring of coagulation factors VIII, IX, XI.
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http://dx.doi.org/10.1038/s41467-021-25888-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8458485PMC
September 2021

Blocking only the bad side of IL-6 in inflammation and cancer.

Authors:
Stefan Rose-John

Cytokine 2021 Dec 30;148:155690. Epub 2021 Aug 30.

Biochemical Institute, University of Kiel, Germany. Electronic address:

Interleukin-6 (IL-6) is considered an inflammatory cytokine, which is involved not only in most inflammatory states but it also plays a prominent role in inflammation associated cancers. The response of cells to the cytokine strictly depends on the presence of the IL-6 receptor (IL-6R),which presents IL-6 to the signal transducing receptor subunit gp130, which is expressed on all cells of the body. The expression of IL-6R is limited to some cells, which are therefore IL-6 target cells. The IL-6R can be cleaved by proteases and the thus generated soluble IL-6R (sIL-6R) still binds the ligand IL-6. The complex of IL-6 and sIL-6R can bind to gp130 on any cell, induce dimerization of gp130 and intracellular signaling. This process has been named IL-6 trans-signaling. A fusion protein of soluble gp130 with the constant portion of human IgG1 (sgp130Fc) turned out to be a potent and specific inhibitor of IL-6 trans-signaling. In many animal models of human diseases the significance of IL-6 trans-signaling has been analyzed. It turned out that the activities of IL-6 mediated by the sIL-6R are the pro-inflammatory activities of the cytokine whereas activities of IL-6 mediated by the membrane-bound IL-6R are rather protective and regenerative. The sgp130Fc protein has recently been developed into a biologic. The possible consequences of a specific IL-6 trans-signaling blockade is discussed in the light of the recent successfully concluded phase II clinical trials in patients with inflammatory bowel disease.
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http://dx.doi.org/10.1016/j.cyto.2021.155690DOI Listing
December 2021

Macrophage-derived IL-6 trans-signaling as a novel target in the pathogenesis of bronchopulmonary dysplasia.

Eur Respir J 2021 Aug 26. Epub 2021 Aug 26.

Department of Pediatric and Adolescent Medicine, University of Cologne, Faculty of Medicine and University Hospital Cologne, Translational Experimental Pediatrics - Experimental Pulmonology, Koln, Germany

Rationale: Premature infants exposed to oxygen are at risk for bronchopulmonary dysplasia (BPD), which is characterised by lung growth arrest. Inflammation is important, but the mechanisms remain elusive. Here, we investigated inflammatory pathways and therapeutic targets in severe clinical and experimental BPD.

Methods And Results: First, transcriptomic analysis with cellular deconvolution identified a lung-intrinsic M1-like-driven cytokine pattern in newborn mice after hyperoxia. These findings were confirmed by gene expression of macrophage-regulating chemokines (, , ) and markers (, , ). Second, hyperoxia-activated IL-6/STAT3 signaling was measured and related to loss of alveolar epithelial type II cells (ATII) as well as increased mesenchymal marker. null mice exhibited preserved ATII survival, reduced myofibroblasts and improved elastic fiber assembly, thus enabling lung growth and protecting lung function. Pharmacological inhibition of global IL-6 signaling and IL-6 trans-signaling promoted alveolarisation and ATII survival after hyperoxia. Third, hyperoxia triggered M1-like polarisation, possibly Klf4; hyperoxia-conditioned medium of macrophages and IL-6 impaired ATII proliferation. Finally, clinical data demonstrate elevated macrophage-related plasma cytokines as potential biomarkers that identify infants receiving oxygen at increased risk of developing BPD. Moreover, macrophage-derived and active STAT3 were related to loss of epithelial cells in BPD lungs.

Conclusion: We present a novel IL-6-mediated mechanism by which hyperoxia activates macrophages in immature lungs, impairs ATII homeostasis, and disrupts elastic fiber formation, thereby inhibiting lung growth. The data provide evidence that IL-6 trans-signaling could offer an innovative pharmacological target to enable lung growth in severe neonatal chronic lung disease.
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http://dx.doi.org/10.1183/13993003.02248-2020DOI Listing
August 2021

Corrigendum: Cathepsin D Variants Associated With Neurodegenerative Diseases Show Dysregulated Functionality and Modified α-Synuclein Degradation Properties.

Front Cell Dev Biol 2021 23;9:671532. Epub 2021 Jun 23.

Institute of Biochemistry, Christian-Albrechts-Universität zu Kiel, Kiel, Germany.

[This corrects the article DOI: 10.3389/fcell.2021.581805.].
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http://dx.doi.org/10.3389/fcell.2021.671532DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8262674PMC
June 2021

New insights into IL-6 family cytokines in metabolism, hepatology and gastroenterology.

Nat Rev Gastroenterol Hepatol 2021 Nov 1;18(11):787-803. Epub 2021 Jul 1.

Institute of Biomedicine of Seville (IBIS), Spanish National Research Council (CSIC), University of Seville, Virgen del Rocio University Hospital, Seville, Spain.

IL-6 family cytokines are defined by the common use of the signal-transducing receptor chain glycoprotein 130 (gp130). Increasing evidence indicates that these cytokines are essential in the regulation of metabolic homeostasis as well as in the pathophysiology of multiple gastrointestinal and liver disorders, thus making them attractive therapeutic targets. Over the past few years, therapies modulating gp130 signalling have grown exponentially in several clinical settings including obesity, cancer and inflammatory bowel disease. A newly engineered gp130 cytokine, IC7Fc, has shown promising preclinical results for the treatment of type 2 diabetes, obesity and liver steatosis. Moreover, drugs that modulate gp130 signalling have shown promise in refractory inflammatory bowel disease in clinical trials. A deeper understanding of the main roles of the IL-6 family of cytokines during homeostatic and pathological conditions, their signalling pathways, sources of production and target cells will be crucial to the development of improved treatments. Here, we review the current state of the role of these cytokines in hepatology and gastroenterology and discuss the progress achieved in translating therapeutics targeting gp130 signalling into clinical practice.
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http://dx.doi.org/10.1038/s41575-021-00473-xDOI Listing
November 2021

Endosomes as Signaling Platforms for IL-6 Family Cytokine Receptors.

Front Cell Dev Biol 2021 1;9:688314. Epub 2021 Jun 1.

Institute of Biochemistry, Christian-Albrechts-University Kiel, Kiel, Germany.

Interleukin-6 (IL-6) is the name-giving cytokine of a family of eleven members, including IL-6, CNTF, LIF, and IL-27. IL-6 was first recognized as a B-cell stimulating factor but we now know that the cytokine plays a pivotal role in the orchestration of inflammatory processes as well as in inflammation associated cancer. Moreover, IL-6 is involved in metabolic regulation and it has been shown to be involved in major neural activities such as neuroprotection, which can help to repair and to reduce brain damage. Receptor complexes of all members formed at the plasma membrane contain one or two molecules of the signaling receptor subunit GP130 and the mechanisms of signal transduction are well understood. IL-6 type cytokines can also signal from endomembranes, in particular the endosome, and situations have been reported in which endocytosis of receptor complexes are a prerequisite of intracellular signaling. Moreover, pathogenic GP130 variants were shown to interfere with spatial activation of downstream signals. We here summarize the molecular mechanisms underlying spatial regulation of IL-6 family cytokine signaling and discuss its relevance for pathogenic processes.
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http://dx.doi.org/10.3389/fcell.2021.688314DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8204807PMC
June 2021

Multiple Roles of IL6 in Hepatic Injury, Steatosis, and Senescence Aggregate to Suppress Tumorigenesis.

Cancer Res 2021 Sep 11;81(18):4766-4777. Epub 2021 Jun 11.

Goldyne Savad Institute of Gene Therapy, Hadassah Hebrew University Hospital, Jerusalem, Israel.

Hepatocellular carcinoma (HCC) typically develops on a background of chronic hepatitis for which the proinflammatory cytokine IL6 is conventionally considered a crucial driving factor. Paradoxically, IL6 also acts as a hepatoprotective factor in chronic liver injury. Here we used the multidrug-resistant gene 2 knockout (Mdr2) mouse model to elucidate potential roles of IL6 in chronic hepatitis-associated liver cancer. Long-term analysis of three separate IL6/Stat3 signaling-deficient Mdr2 strains revealed aggravated liver injury with increased dysplastic nodule formation and significantly accelerated tumorigenesis in all strains. Tumorigenesis in the IL6/Stat3-perturbed models was strongly associated with enhanced macrophage accumulation and hepatosteatosis, phenotypes of nonalcoholic steatohepatitis (NASH), as well as with significant reductions in senescence and the senescence-associated secretory phenotype (SASP) accompanied by increased hepatocyte proliferation. These findings reveal a crucial suppressive role for IL6/Stat3 signaling in chronic hepatitis-associated hepatocarcinogenesis by impeding protumorigenic NASH-associated phenotypes and by reinforcing the antitumorigenic effects of the SASP. SIGNIFICANCE: These findings describe a context-dependent role of IL6 signaling in hepatocarcinogenesis and predict that increased IL6-neutralizing sgp130 levels in some patients with NASH may herald early HCC development..
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http://dx.doi.org/10.1158/0008-5472.CAN-21-0321DOI Listing
September 2021

A variant in IL6ST with a selective IL-11 signaling defect in human and mouse.

Bone Res 2020 Jun 11;8(1):24. Epub 2020 Jun 11.

Translational Gastroenterology Unit, John Radcliffe Hospital, University of Oxford, Oxford, UK.

The GP130 cytokine receptor subunit encoded by IL6ST is the shared receptor for ten cytokines of the IL-6 family. We describe a homozygous non-synonymous variant in IL6ST (p.R281Q) in a patient with craniosynostosis and retained deciduous teeth. We characterize the impact of the variant on cytokine signaling in vitro using transfected cell lines as well as primary patient-derived cells and support these findings using a mouse model with the corresponding genome-edited variant Il6st p.R279Q. We show that human GP130 p.R281Q is associated with selective loss of IL-11 signaling without affecting IL-6, IL-27, OSM, LIF, CT1, CLC, and CNTF signaling. In mice Il6st p.R279Q lowers litter size and causes facial synostosis and teeth abnormalities. The effect on IL-11 signaling caused by the GP130 variant shows incomplete penetrance but phenocopies aspects of IL11RA deficiency in humans and mice. Our data show that a genetic variant in a pleiotropic cytokine receptor can have remarkably selective defects.
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http://dx.doi.org/10.1038/s41413-020-0098-zDOI Listing
June 2020

Initiation of Pancreatic Cancer: The Interplay of Hyperglycemia and Macrophages Promotes the Acquisition of Malignancy-Associated Properties in Pancreatic Ductal Epithelial Cells.

Int J Mol Sci 2021 May 11;22(10). Epub 2021 May 11.

Institute for Experimental Cancer Research, Kiel University (CAU) and University Medical Center Schleswig-Holstein (UKSH), Campus Kiel, 24105 Kiel, Germany.

Pancreatic ductal adenocarcinoma (PDAC) is still one of the most aggressive solid malignancies with a poor prognosis. Obesity and type 2 diabetes mellitus (T2DM) are two major risk factors linked to the development and progression of PDAC, both often characterized by high blood glucose levels. Macrophages represent the main immune cell population in PDAC contributing to PDAC development. It has already been shown that pancreatic ductal epithelial cells (PDEC) undergo epithelial-mesenchymal transition (EMT) when exposed to hyperglycemia or macrophages. Thus, this study aimed to investigate whether concomitant exposure to hyperglycemia and macrophages aggravates EMT-associated alterations in PDEC. Exposure to macrophages and elevated glucose levels (25 mM glucose) impacted gene expression of EMT inducers such as IL-6 and TNF-α as well as EMT transcription factors in benign (H6c7-) and premalignant (H6c7-) PDEC. Most strikingly, exposure to hyperglycemic coculture with macrophages promoted downregulation of the epithelial marker E-cadherin, which was associated with an elevated migratory potential of PDEC. While blocking IL-6 activity by tocilizumab only partially reverted the EMT phenotype in H6c7- cells, neutralization of TNF-α by etanercept was able to clearly impair EMT-associated properties in premalignant PDEC. Altogether, the current study attributes a role to a T2DM-related hyperglycemic, inflammatory micromilieu in the acquisition of malignancy-associated alterations in premalignant PDEC, thus providing new insights on how metabolic diseases might promote PDAC initiation.
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http://dx.doi.org/10.3390/ijms22105086DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8151031PMC
May 2021

The two facets of gp130 signalling in liver tumorigenesis.

Semin Immunopathol 2021 08 28;43(4):609-624. Epub 2021 May 28.

Institute of Biochemistry, Christian-Albrechts-University Kiel, Kiel, Germany.

The liver is a vital organ with multiple functions and a large regenerative capacity. Tumours of the liver are the second most frequently cause of cancer-related death and develop in chronically inflamed livers. IL-6-type cytokines are mediators of inflammation and almost all members signal via the receptor subunit gp130 and the downstream signalling molecule STAT3. We here summarize current knowledge on how gp130 signalling and STAT3 in tumour cells and cells of the tumour micro-environment drives hepatic tumorigenesis. We furthermore discuss very recent findings describing also anti-tumorigenic roles of gp130/STAT3 and important considerations for therapeutic interventions.
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http://dx.doi.org/10.1007/s00281-021-00861-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8443519PMC
August 2021

Therapeutic targeting of IL-6 trans-signaling.

Authors:
Stefan Rose-John

Cytokine 2021 08 19;144:155577. Epub 2021 May 19.

Biochemical Institute, University of Kiel, Germany. Electronic address:

Interleukin-6 (IL-6) is a cytokine, which is involved in innate and acquired immunity, in neural cell maintenance and in metabolism. IL-6 can be synthesized by many different cells including myeloid cells, fibroblasts, endothelial cells and lymphocytes. The synthesis of IL-6 is strongly stimulated by Toll like receptors and by IL-1. Therefore, IL-6 levels in the body are high during infection and inflammatory processes. Moreover, IL-6 is a prominent growth factor of tumor cells and plays a major role in inflammation associated cancer. On target cells, IL-6 binds to an IL-6 receptor, which is not signaling competent. The complex of IL-6 and IL-6 receptor associate with a second receptor subunit, glycoprotein gp130, which dimerizes and initiates intracellular signaling. Cells, which do not express the IL-6 receptor are not responsive to IL-6. They can, however, be stimulated by the complex of IL-6 and a soluble form of the IL-6 receptor, which is generated by limited proteolysis and to a lesser extent by translation from an alternatively spliced mRNA. This process has been named IL-6 trans-signaling. This review article will explain the biology of IL-6 trans-signaling and the specific inhibition of this mode of signaling, which has been recognized to be fundamental in inflammation and cancer.
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http://dx.doi.org/10.1016/j.cyto.2021.155577DOI Listing
August 2021

Interleukin-6: obstacles to targeting a complex cytokine in critical illness.

Lancet Respir Med 2021 06 16;9(6):643-654. Epub 2021 Apr 16.

Department of Medicine, Royal College of Surgeons in Ireland, Dublin, Ireland; Beaumont Hospital, Dublin, Ireland. Electronic address:

Circulating concentrations of the pleiotropic cytokine interleukin-6 (IL-6) are known to be increased in pro-inflammatory critical care syndromes, such as sepsis and acute respiratory distress syndrome. Elevations in serum IL-6 concentrations in patients with severe COVID-19 have led to renewed interest in the cytokine as a therapeutic target. However, although the pro-inflammatory properties of IL-6 are widely known, the cytokine also has a series of important physiological and anti-inflammatory functions. An adequate understanding of the complex processes by which IL-6 signalling occurs is crucial for the correct interpretation of IL-6 concentrations in the blood or lung, the use of IL-6 as a critical care biomarker, or the design of effective anti-IL-6 strategies. Here, we outline the role of IL-6 in health and disease, explain the different types of IL-6 signalling and their contribution to the net biological effect of the cytokine, describe the approaches to IL-6 inhibition that are currently available, and discuss implications for the future use of treatments such as tocilizumab in the critical care setting.
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http://dx.doi.org/10.1016/S2213-2600(21)00103-XDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8051931PMC
June 2021

Natural Glycoforms of Human Interleukin 6 Show Atypical Plasma Clearance.

Angew Chem Int Ed Engl 2021 06 6;60(24):13380-13387. Epub 2021 May 6.

Bioorganic Chemistry, University of Bayreuth, Universitätsstraße 30, 95447, Bayreuth, Germany.

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.
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http://dx.doi.org/10.1002/anie.202101496DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8251587PMC
June 2021

Cathepsin D Variants Associated With Neurodegenerative Diseases Show Dysregulated Functionality and Modified α-Synuclein Degradation Properties.

Front Cell Dev Biol 2021 11;9:581805. Epub 2021 Feb 11.

Institute of Biochemistry, Christian-Albrechts-Universität zu Kiel, Kiel, Germany.

Cathepsin D (CTSD) is a lysosomal protease important for the degradation of various substrates, including disease-associated proteins like α-synuclein (a-syn), amyloid precursor protein (APP) and tau, all of which tend to aggregate if not efficiently degraded. Hence, it is not surprising that genetic variants within the gene have been linked to neurodegenerative diseases, like Parkinson's and Alzheimer's disease (PD, AD), as well as the lysosomal storage disorder neuronal ceroid lipofuscinosis type-10 (NCL10). Although recent studies have shown the molecular dependence of substrate degradation via CTSD within autophagic pathways, only little is known about the precise role of lysosomal CTSD function in disease development. We here performed biochemical, cellular and structural analyses of eleven disease-causing CTSD point mutations found in genomic sequencing data of patients to understand their role in neurodegeneration. These CTSD variants were analyzed for cellular localization, maturation and enzymatic activity in overexpression analyses. Moreover, for PD-associated mutants, intracellular degradation of a-syn was monitored. In summary, our results suggest that NCL10-associated CTSD variants are significantly impaired in lysosomal maturation and enzymatic activity, whereas the AD- and PD-associated variants seemed rather unaffected, indicating normal maturation, and lysosomal presence. Interestingly, a PD-associated CTSD variant (A239V) exhibited increased enzymatic activity accompanied by enhanced a-syn degradation. By structural analyses of this mutant utilizing molecular dynamics simulation (MDS), we identified a structural change within a loop adjacent to the catalytic center leading to a higher flexibility and potentially accelerated substrate exchange rates. Our data sheds light onto the role of CTSD in disease development and helps to understand the structural regulation of enzymatic function, which could be utilized for targeted CTSD activation. Because of the degradative function of CTSD, this enzyme is especially interesting for therapeutic strategies tackling protein aggregates in neurodegenerative disorders.
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http://dx.doi.org/10.3389/fcell.2021.581805DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7928348PMC
February 2021

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

Gastroenterology 2021 Jun 2;160(7):2354-2366.e11. Epub 2021 Mar 2.

Institute of Clinical Molecular Biology, Kiel University and University Medical Center Schleswig-Holstein, Campus Kiel, Kiel, Germany. Electronic address:

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).
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http://dx.doi.org/10.1053/j.gastro.2021.02.062DOI Listing
June 2021

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

J Biol Chem 2021 Jan-Jun;296:100434. Epub 2021 Feb 19.

Department of Pathology, Otto-von-Guericke-University Magdeburg, Medical Faculty, Magdeburg, Germany. Electronic address:

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.
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http://dx.doi.org/10.1016/j.jbc.2021.100434DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8010714PMC
August 2021

Transgenic inhibition of interleukin-6 trans-signaling does not prevent skeletal pathologies in mucolipidosis type II mice.

Sci Rep 2021 Feb 11;11(1):3556. Epub 2021 Feb 11.

Department of Osteology and Biomechanics, University Medical Center Hamburg-Eppendorf, 20246, Hamburg, Germany.

Severe skeletal alterations are common symptoms in patients with mucolipidosis type II (MLII), a rare lysosomal storage disorder of childhood. We have previously reported that progressive bone loss in a mouse model for MLII is caused by an increased number of bone-resorbing osteoclasts, which is accompanied by elevated expression of the cytokine interleukin-6 (IL-6) in the bone microenvironment. In the present study we addressed the question, if pharmacological blockade of IL-6 can prevent the low bone mass phenotype of MLII mice. Since the cellular IL-6 response can be mediated by either the membrane-bound (classic signaling) or the soluble IL-6 receptor (trans-signaling), we first performed cell culture assays and found that both pathways can increase osteoclastogenesis. We then crossed MLII mice with transgenic mice expressing the recombinant soluble fusion protein sgp130Fc, which represents a natural inhibitor of IL-6 trans-signaling. By undecalcified histology and bone-specific histomorphometry we found that high circulating sgp130Fc levels do not affect skeletal growth or remodeling in wild-type mice. Most importantly, blockade of IL-6 trans-signaling did neither reduce osteoclastogenesis, nor increase bone mass in MLII mice. Therefore, our data clearly demonstrate that the bone phenotype of MLII mice cannot be corrected by blocking the IL-6 trans-signaling.
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http://dx.doi.org/10.1038/s41598-021-82802-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7878873PMC
February 2021

NOTCH Activation via gp130/STAT3 Signaling Confers Resistance to Chemoradiotherapy.

Cancers (Basel) 2021 Jan 26;13(3). Epub 2021 Jan 26.

Institute of Cellular and Molecular Immunology, University Medical Center Goettingen, 37073 Goettingen, Germany.

Resistance of tumor cells to chemoradiotherapy represents a fundamental problem in clinical oncology. The underlying mechanisms are actively debated. Here we show that blocking inflammatory cytokine receptor signaling via STAT3 re-sensitized treatment-refractory cancer cells and abolished tumor growth in a xenograft mouse model when applied together with chemoradiotherapy. STAT3 executed treatment resistance by triggering the expression of RBPJ, the key transcriptional regulator of the NOTCH pathway. The mandatory RBPJ interaction partner, NOTCH intracellular domain, was provided by tumor cell-intrinsic expression of NOTCH ligands that caused tonic NOTCH proteolysis. In fact, NOTCH inhibition phenocopied the effect of blocking STAT3 signaling. Moreover, genetic profiling of rectal cancer patients revealed the importance of the STAT3/NOTCH axis as NOTCH expression correlated with clinical outcome. Our data uncovered an unprecedented signal alliance between inflammation and cellular development that orchestrated resistance to chemoradiotherapy. Clinically, our findings allow for biomarker-driven patient stratification and offer novel treatment options.
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http://dx.doi.org/10.3390/cancers13030455DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7865718PMC
January 2021

Genetic IL-6R variants and therapeutic inhibition of IL-6 receptor signalling in COVID-19.

Lancet Rheumatol 2021 Feb 15;3(2):e96-e97. Epub 2020 Dec 15.

Institute of Biochemistry, Kiel University, Kiel, Germany.

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http://dx.doi.org/10.1016/S2665-9913(20)30416-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7834724PMC
February 2021

Overlapping and distinct biological effects of IL-6 classic and trans-signaling in vascular endothelial cells.

Am J Physiol Cell Physiol 2021 04 20;320(4):C554-C565. Epub 2021 Jan 20.

Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia, Canada.

IL-6 affects tissue protective/reparative and inflammatory properties of vascular endothelial cells (ECs). This cytokine can signal to cells through classic and trans-signaling mechanisms, which are differentiated based on the expression of IL-6 receptor (IL-6R) on the surface of target cells. The biological effects of these IL-6-signaling mechanisms are distinct and have implications for vascular pathologies. We have directly compared IL-6 classic and trans-signaling in ECs. Human ECs expressed IL-6R in culture and in situ in coronary arteries from heart transplants. Stimulation of human ECs with IL-6, to model classic signaling, triggered the activation of phosphatidylinositol 3-kinase (PI3K)-Akt and ERK1/2 signaling pathways, whereas stimulation with IL-6 + sIL-6R, to model trans-signaling, triggered activation of STAT3, PI3K-Akt, and ERK1/2 pathways. IL-6 classic signaling reduced persistent injury of ECs in an allograft model of vascular rejection and inhibited cell death induced by growth factor withdrawal. When inflammatory effects were examined, IL-6 classic signaling did not induce ICAM or CCL2 expression but was sufficient to induce secretion of CXCL8 and support transmigration of neutrophil-like cells. IL-6 trans-signaling induced all inflammatory effects studied. Our findings show that IL-6 classic and trans-signaling have overlapping but distinct properties in controlling EC survival and inflammatory activation. This has implications for understanding the effects of IL-6 receptor-blocking therapies as well as for vascular responses in inflammatory and immune conditions.
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http://dx.doi.org/10.1152/ajpcell.00323.2020DOI Listing
April 2021

The role of interleukin-6 trans-signalling on cardiovascular dysfunction in inflammatory arthritis.

Rheumatology (Oxford) 2021 06;60(6):2852-2861

CREATE Centre, Division of Infection and Immunity, , Cardiff, UK.

Objectives: Cardiovascular (CV) mortality in RA patients is 50% higher than in the general population. There is increasing recognition that systemic inflammation is a major driver of this. IL-6 is implicated in cardiovascular disease (CVD) in the general population but its role in CVD in RA is undefined. Of the two modes of IL-6 signalling, trans-signalling is pro-inflammatory whereas classical signalling is linked with inflammation resolution. This study examines the role of IL-6 trans-signalling in CVD in a mouse model and patients with RA.

Methods: Myography determined the effect of IL-6 trans-signalling blockade, using sgp130Fc, on aortic constriction in murine collagen-induced arthritis. Serum CCL2 and sVCAM-1 as soluble biomarkers of sIL-6R trans-signalling were investigated in a human cross-sectional study. An observational longitudinal study investigated the association between these biomarkers and progression of subclinical atherosclerosis in early RA by measuring carotid intima-media thickness (CIMT).

Results: sgp130Fc reduced arthritis severity, serum CCL2 and sVCAM-1 and restored vascular function in collagen-induced arthritis (CIA). In established RA, sVCAM-1 correlated with the 28-joint DAS (DAS28) and CV risk. In early RA, baseline DAS28 was associated with CIMT change at 6 months. CIMT 'rapid progressors' at 12 months had higher baseline sVCAM-1, haemoglobin A1c, cholesterol:high-density lipoprotein cholesterol ratio and LDL cholesterol.

Conclusions: IL-6 trans-signalling plays a pivotal role in vascular dysfunction in CIA. In early RA, sVCAM-1 was associated with progression of subclinical atherosclerosis. Inflammation from RA onset in CVD-susceptible individuals may accelerate atherosclerosis. IL-6 trans-signalling blockade may be beneficial to RA patients and perhaps for atherosclerosis in the general population.
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http://dx.doi.org/10.1093/rheumatology/keaa725DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8213430PMC
June 2021

Cathepsin S provokes interleukin-6 (IL-6) trans-signaling through cleavage of the IL-6 receptor in vitro.

Sci Rep 2020 12 10;10(1):21612. Epub 2020 Dec 10.

Department of Pathology, Medical Faculty, Otto-Von-Guericke-University Magdeburg, Magdeburg, Germany.

The cytokine interleukin-6 (IL-6) fulfills its pleiotropic functions via different modes of signaling. Regenerative and anti-inflammatory activities are mediated via classic signaling, in which IL-6 binds to the membrane-bound IL-6 receptor (IL-6R). For IL-6 trans-signaling, which accounts for the pro-inflammatory properties of the cytokine, IL-6 activates its target cells via soluble forms of the IL-6R (sIL-6R). We have previously shown that the majority of sIL-6R in human serum originates from proteolytic cleavage and mapped the cleavage site of the IL-6R. The cleavage occurs between Pro-355 and Val-356, which is the same cleavage site that the metalloprotease ADAM17 uses in vitro. However, sIL-6R serum levels are unchanged in hypomorphic ADAM17 mice, making the involvement of ADAM17 questionable. In order to identify other proteases that could be relevant for sIL-6R generation in vivo, we perform a screening approach based on the known cleavage site. We identify several candidate proteases and characterize the cysteine protease cathepsin S (CTSS) in detail. We show that CTSS is able to cleave the IL-6R in vitro and that the released sIL-6R is biologically active and can induce IL-6 trans-signaling. However, CTSS does not use the Pro-355/Val-356 cleavage site, and sIL-6R serum levels are not altered in Ctss mice. In conclusion, we identify a novel protease of the IL-6R that can induce IL-6 trans-signaling, but does not contribute to steady-state sIL-6R serum levels.
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http://dx.doi.org/10.1038/s41598-020-77884-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7730449PMC
December 2020

ADAM17 Deficiency Protects against Pulmonary Emphysema.

Am J Respir Cell Mol Biol 2021 02;64(2):183-195

Centre for Innate Immunity and Infectious Diseases, Hudson Institute of Medical Research, Clayton, Victoria, Australia.

Pulmonary emphysema is the major debilitating component of chronic obstructive pulmonary disease (COPD), which is a leading cause of morbidity and mortality worldwide. The ADAM17 (A disintegrin and metalloproteinase 17) protease mediates inflammation via ectodomain shedding of numerous proinflammatory cytokines, cytokine receptors, and adhesion molecules; however, its role in the pathogenesis of emphysema and COPD is poorly understood. This study aims to define the role of the protease ADAM17 in the pathogenesis of pulmonary emphysema. ADAM17 protein expression and activation was investigated in lung biopsies from patients with emphysema, as well as lungs of the emphysematous mouse model and an acute (4 d) cigarette smoke (CS)-induced lung pathology model. The mice, which display significantly reduced global ADAM17 expression, were coupled with emphysema-prone mice to produce :. Both and wild-type mice were subjected to acute CS exposure. Histological, immunohistochemical, immunofluorescence, and molecular analyses as well as lung function tests were performed to assess pulmonary emphysema, inflammation, and alveolar cell apoptosis. ADAM17 was hyperphosphorylated in the lungs of patients with emphysema and also in emphysematous and CS-exposed mice. ADAM17 deficiency ameliorated the development of pulmonary emphysema in mice by suppressing elevated alveolar cell apoptosis. In addition, genetic blockade of ADAM17 protected mice from CS-induced pulmonary inflammation and alveolar cell apoptosis. Our study places the protease ADAM17 as a central molecular switch implicated in the development of pulmonary emphysema, which paves the way for using ADAM17 inhibitors as potential therapeutic agents to treat COPD and emphysema.
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http://dx.doi.org/10.1165/rcmb.2020-0214OCDOI Listing
February 2021

Functional Characterization of Colon-Cancer-Associated Variants in Affecting the Catalytic Domain.

Biomedicines 2020 Oct 30;8(11). Epub 2020 Oct 30.

Institute of Biochemistry Christian-Albrechts-Universität zu Kiel, 24118 Kiel, Germany.

Although extensively investigated, cancer is still one of the most devastating and lethal diseases in the modern world. Among different types, colorectal cancer (CRC) is most prevalent and mortal, making it an important subject of research. The metalloprotease ADAM17 has been implicated in the development of CRC due to its involvement in signaling pathways related to inflammation and cell proliferation. ADAM17 is capable of releasing membrane-bound proteins from the cell surface in a process called A deficiency of ADAM17 activity has been previously shown to have protective effects against CRC in mice, while an upregulation of ADAM17 activity is suspected to facilitate tumor development. In this study, we characterize ADAM17 variants found in tissue samples of cancer patients in overexpression studies. We here focus on point mutations identified within the catalytic domain of ADAM17 and could show a functional dysregulation of the CRC-associated variants. Since the catalytic domain of ADAM17 is the only region structurally determined by crystallography, we study the effect of each point mutation not only to learn more about the role of ADAM17 in cancer, but also to investigate the structure-function relationships of the metalloprotease.
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http://dx.doi.org/10.3390/biomedicines8110463DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7692748PMC
October 2020

Interleukin-6 trans-signaling is a candidate mechanism to drive progression of human DCCs during clinical latency.

Nat Commun 2020 10 5;11(1):4977. Epub 2020 Oct 5.

Department of Pathology, University of Regensburg, 93053, Regensburg, Germany.

Although thousands of breast cancer cells disseminate and home to bone marrow until primary surgery, usually less than a handful will succeed in establishing manifest metastases months to years later. To identify signals that support survival or outgrowth in patients, we profile rare bone marrow-derived disseminated cancer cells (DCCs) long before manifestation of metastasis and identify IL6/PI3K-signaling as candidate pathway for DCC activation. Surprisingly, and similar to mammary epithelial cells, DCCs lack membranous IL6 receptor expression and mechanistic dissection reveals IL6 trans-signaling to regulate a stem-like state of mammary epithelial cells via gp130. Responsiveness to IL6 trans-signals is found to be niche-dependent as bone marrow stromal and endosteal cells down-regulate gp130 in premalignant mammary epithelial cells as opposed to vascular niche cells. PIK3CA activation renders cells independent from IL6 trans-signaling. Consistent with a bottleneck function of microenvironmental DCC control, we find PIK3CA mutations highly associated with late-stage metastatic cells while being extremely rare in early DCCs. Our data suggest that the initial steps of metastasis formation are often not cancer cell-autonomous, but also depend on microenvironmental signals.
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http://dx.doi.org/10.1038/s41467-020-18701-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7536220PMC
October 2020

Cell-autonomous hepatocyte-specific GP130 signaling is sufficient to trigger a robust innate immune response in mice.

J Hepatol 2021 02 25;74(2):407-418. Epub 2020 Sep 25.

Institute of Biochemistry, Christian-Albrechts-University Kiel, Germany. Electronic address:

Background & Aims: Interleukin (IL)-6 cytokine family members contribute to inflammatory and regenerative processes. Engagement of the signaling receptor subunit gp130 is common to almost all members of the family. In the liver, all major cell types respond to IL-6-type cytokines, making it difficult to delineate cell type-specific effects. We therefore generated mouse models for liver cell type-specific analysis of IL-6 signaling.

Methods: We produced mice with a Cre-inducible expression cassette encoding a designed pre-dimerized constitutive active gp130 variant. We bred these mice to different Cre-drivers to induce transgenic gp130 signaling in distinct liver cell types: hepatic stellate cells, cholangiocytes/liver progenitor cells or hepatocytes. We phenotyped these mice using multi-omics approaches, immunophenotyping and a bacterial infection model.

Results: Hepatocyte-specific gp130 activation led to the upregulation of innate immune system components, including acute-phase proteins. Consequently, we observed peripheral mobilization and recruitment of myeloid cells to the liver. Hepatic myeloid cells, including liver-resident Kupffer cells were instructed to adopt a bactericidal phenotype which ultimately conferred enhanced resistance to bacterial infection in these mice. We demonstrate that persistent hepatocyte-specific gp130 activation resulted in amyloid A amyloidosis in aged mice. In contrast, we did not observe overt effects of hepatic stellate cell- or cholangiocyte/liver progenitor cell-specific transgenic gp130 signaling.

Conclusions: Hepatocyte-specific gp130 activation alone is sufficient to trigger a robust innate immune response in the absence of NF-κB activation. We therefore conclude that gp130 engagement, e.g. by IL-6 trans-signaling, represents a safe-guard mechanism in innate immunity.

Lay Summary: Members of the interleukin-6 cytokine family signal via the receptor subunit gp130 and are involved in multiple processes in the liver. However, as several liver cell types respond to interleukin-6 family cytokines, it is difficult to delineate cell type-specific effects. Using a novel mouse model, we provide evidence that hepatocyte-specific gp130 activation is sufficient to trigger a robust systemic innate immune response.
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http://dx.doi.org/10.1016/j.jhep.2020.09.021DOI Listing
February 2021
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