[Show abstract][Hide abstract] ABSTRACT: Anti-cytokine therapies have substantially improved the treatment of inflammatory and autoimmune diseases. Cytokine-targeting drugs are usually biologics such as antibodies or other engineered proteins. Production of biologics, however, is complex and intricate and therefore expensive which might limit therapeutic application. To overcome this limitation we developed a strategy that involves the design of an optimized, monogenic cytokine inhibitor and the protein producing capacity of the host. Here, we engineered and characterized a receptor fusion protein, mIL-6-RFP-Fc, for the inhibition of interleukin-6 (IL-6), a well-established target in anti-cytokine therapy. Upon application in mice mIL-6-RFP-Fc inhibited IL-6-induced activation of the transcription factor STAT3 and ERK1/2 kinases in liver and kidney. mIL-6-RFP-Fc is encoded by a single gene and therefore most relevant for gene transfer approaches. Gene transfer through hydrodynamic plasmid delivery in mice resulted in hepatic production and secretion of mIL-6-RFP-Fc into the blood in considerable amounts, blocked hepatic acute phase protein synthesis and improved kidney function in an ischemia and reperfusion injury model. Our study establishes receptor fusion proteins as promising agents in anti-cytokine therapies through gene therapeutic approaches for future targeted and cost-effective treatments. The strategy described here is applicable for many cytokines involved in inflammatory and other diseases. Anti-cytokine therapy is a significant step forward in the current treatment of immunologic and other inflammatory diseases and has therapeutic potential in cancer. Anti-inflammatory biologics in clinical use today are mainly engineered antibodies which block cytokines such as TNF, IL-6 and IL-1 or one of their receptors 1–5. Engineered soluble cytokine-binding receptors are an effective alternative to anti-bodies offering easier production, better suitability for gene therapeutic delivery and therefore add to the available choice of therapeutics. Surprisingly, far fewer substances of this class have been developed to clinical maturity 6. Most cytokines signal through heteromeric receptors and often two or more different receptor chains contribute to high-affinity binding of the ligand. In the case of IL-6, the α-receptor subunit in its soluble form (sIL-6Rα) is an agonistic molecule that in combination with IL-6 stimulates cells expressing the β-subunit gp130 7. A soluble form of gp130 (sgp130), in turn, potently antagonizes the complex of IL-6 and sIL-6Rα 8,9. Based on these observations, we and others developed a strategy to block IL-6 with a fusion protein consisting of the ligand-binding domains of IL-6Rα and gp130 10,11. These receptor fusion
[Show abstract][Hide abstract] ABSTRACT: The IL-6 signaling complex is described as a hexamer, formed by the association of two IL-6/IL-6R/gp130 trimers, with gp130 being the signal transducer inducing cis- and trans-mediated signaling via a membrane-bound (mb) or soluble (s) form of the IL-6R, respectively. 25F10 is an anti-mouse IL-6R mAb that binds to both mbIL-6R and sIL-6R with the unique property of specifically inhibiting trans-mediated signaling events. In this study, epitope mapping revealed that 25F10 interacts at site IIb of IL-6R yet allows the binding of IL-6 to the IL-6R and the recruitment of gp130 forming a trimer complex. Binding of 25F10 to IL-6R prevented the formation of the hexameric complex obligate for trans-mediated signaling suggesting that the cis- and trans- modes of IL-6 signaling adopt different mechanisms for receptor complex assembly. To study this phenomenon also in the human system, we developed NI-1201, a mAb that targets, in the human IL-6R sequence, the epitope recognized by 25F10 for mice. Interestingly, NI-1201, however, did not selectively inhibit human IL-6 trans-signaling although both mAbs produced beneficial outcomes in conditions of exacerbated IL-6 as compared to a site I-directed mAb. These findings shed light on the complexity of IL-6 signaling. First, triggering cis- versus trans-mediated IL-6 signaling occurs via distinctive mechanisms for receptor complex assembly in mice. Second, the formation of the receptor complex leading to cis- and trans-signaling biology in mice and humans is different which should be taken into account when developing strategies to inhibit IL-6 clinically.
Journal of Biological Chemistry 09/2015; DOI:10.1074/jbc.M115.682138 · 4.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: IL-23, composed of the cytokine subunit p19 and the soluble α receptor subunit p40, binds to a receptor complex consisting
of the IL-23 receptor (IL-23R) and the IL-12 receptor β1 (IL-12Rβ1). Complex formation was hypothesized to follow the “site
I-II-III” architectural paradigm, with site I of p19 being required for binding to p40, whereas sites II and III of p19 mediate
binding to IL-12Rβ1 and IL-23R, respectively. Here we show that the binding mode of p19 to p40 and of p19 to IL-23R follow
the canonical site I and III paradigm but that interaction of IL-23 to IL-12Rβ1 is independent of site II in p19. Instead,
binding of IL-23 to the cytokine binding module of IL-12Rβ1 is mediated by domains 1 and 2 of p40 via corresponding site II
amino acids of IL-12Rβ1. Moreover, domains 2 and 3 of p40 were sufficient for complex formation with p19 and to induce binding
of p19 to IL-23R. The Fc-tagged fusion protein of p40_D2D3/p19 did, however, not act as a competitive IL-23 antagonist but,
at higher concentrations, induced proliferation via IL-23R but independent of IL-12Rβ1. On the basis of our experimental validation,
we propose a non-canonical topology of the IL-23·IL-23R·IL-12Rβ1 complex. Furthermore, our data help to explain why p40 is
an antagonist of IL-23 and IL-12 signaling and show that site II of p19 is dispensable for IL-23 signaling.
[Show abstract][Hide abstract] ABSTRACT: Interleukin-6 (IL-6) is a multifunctional cytokine that orchestrates the immune response to a wide variety of pathophysiologic challenges but also contributes to tissue homeostasis. Furthermore, IL-6 is elevated in patients with acute myocardial infarction. Hyaluronan (HA) is an extracellular carbohydrate that has been implicated in wound healing and accumulates after acute myocardial infarction (AMI). Aim of this study was to investigate the involvement of IL-6 in the regulation of the HA-matrix in the early phase of infarct healing. In the present study, we show by the use of a blocking anti-IL-6 antibody, that endogenous IL-6 rapidly but transiently increased HA-synthase (HAS) 1 and 2 expression resulting in the formation of a HA-rich matrix acutely after AMI in mice. In vitro, IL-6 induced HAS1 and 2 via STAT3 phosphorylation in cardiac fibroblasts (CF) and supported a myofibroblastic phenotype in a HA-dependent manner. Furthermore, CCL5 and MCP1 expression were dependent on IL-6, HA-synthesis and the HA-receptor CD44 as shown in cultured CF derived from CD44 knockout mice. In vivo after AMI, blocking IL-6 decreased HA-matrix formation in the peri-infarct region and alpha-smooth muscle actin-positive myofibroblasts. Blocking IL-6 also reduced neutrophil infiltration in infarcted left ventricles. Moreover, treatment with the blocking IL-6 antibody reduced cardiac ejection fraction and increased infarct size 3 weeks after AMI. These findings support a functionally important role for IL-6 in CF by transiently inducing a HA-rich matrix that in turn promotes a myofibroblastic phenotype and inflammatory responses, and ultimately establishes a cardioprotective program after AMI.
Archiv für Kreislaufforschung 11/2014; 109(6):440. DOI:10.1007/s00395-014-0440-y · 5.41 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Objective:
Interleukin (IL)-17A is regarded as an important cytokine to drive psoriasis, an inflammatory skin disease marked by increased cardiovascular mortality. We aimed to test the hypothesis that overproduction of IL-17A in the skin leading to dermal inflammation may systemically cause vascular dysfunction in psoriasis-like skin disease.
Approach and results:
Conditional overexpression of IL-17A in keratinocytes caused severe psoriasis-like skin inflammation in mice (K14-IL-17A(ind/+) mice), associated with increased reactive oxygen species formation and circulating CD11b(+) inflammatory leukocytes in blood, with endothelial dysfunction, increased systolic blood pressure, left ventricular hypertrophy, and reduced survival compared with controls. In K14-IL-17A(ind/+) mice, immunohistochemistry and flow cytometry revealed increased vascular production of the nitric oxide/superoxide reaction product peroxynitrite and infiltration of the vasculature with myeloperoxidase(+)CD11b(+)GR1(+)F4/80(-) cells accompanied by increased expression of the inducible nitric oxide synthase and the nicotinamide dinucleotide phosphate (NADPH) oxidase, nox2. Neutrophil depletion by anti-GR-1 antibody injections reduced oxidative stress in blood and vessels. Neutralization of tumor necrosis factor-α and IL-6 (both downstream of IL-17A) reduced skin lesions, attenuated oxidative stress in heart and blood, and partially improved endothelial dysfunction in K14-IL-17A(ind/+) mice.
Dermal overexpression of IL-17A induces systemic endothelial dysfunction, vascular oxidative stress, arterial hypertension, and increases mortality mainly driven by myeloperoxidase(+)CD11b(+)GR1(+)F4/80(-) inflammatory cells. Depletion of the GR-1(+) immune cells or neutralization of IL-17A downstream cytokines by biologicals attenuates the vascular phenotype in K14-IL-17A(ind/+) mice.
[Show abstract][Hide abstract] ABSTRACT: The Interleukin (IL)-12 family contains several heterodimeric composite cytokines which share subunits among each other. IL-12 consists of the subunits p40 (shared with IL-23) and p35. p35 is shared with the composite cytokine IL-35 which comprises of the p35/EBI3 heterodimer (EBI3 shared with IL-27). IL-35 signals via homo- or heterodimers of IL-12Rβ2, gp130 and WSX-1, which are shared with IL-12 and IL-27 receptor complexes, respectively. p35 was efficiently secreted in complex with p40 as IL-12 but not with EBI3 as IL-35 in several transfected cell lines tested which complicates the analysis of IL-35 signal transduction. p35 and p40 but not p35 and EBI3 form an inter-chain disulfide bridge. Mutation of the responsible cysteine residue (p40C197A) reduced IL-12 formation and activity only slightly. Importantly, the p40C197A mutation prevented the formation of antagonistic p40 homodimers which enabled the in vitro reconstitution of biologically active IL-12 with p35 produced in bacteria (p35bac). Reconstitution of IL-35 with p35bac and EBI3 did, however, fail to induce signal transduction in Ba/F3 cells expressing IL-12Rβ2 and gp130. In summary, we describe the in vitro reconstitution of IL-12, but fail to produce recombinant IL-35 by this novel approach.
PLoS ONE 09/2014; 9(9):e107990. DOI:10.1371/journal.pone.0107990 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Myocardial inflammation is critical for ventricular remodeling after ischemia. Phospholipid mediators play an important role in inflammatory processes. In the plasma membrane they are degraded by phospholipase D1 (PLD1). PLD1 was shown to be critically involved in ischemic cardiovascular events. Moreover, PLD1 is coupled to tumor necrosis factor-α signaling and inflammatory processes. However, the impact of PLD1 in inflammatory cardiovascular disease remains elusive. Here, we analyzed the impact of PLD1 in tumor necrosis factor-α–mediated activation of monocytes after myocardial ischemia and reperfusion using a mouse model of myocardial infarction. PLD1 expression was highly up-regulated in the myocardium after ischemia/reperfusion. Genetic ablation of PLD1 led to defective cell adhesion and migration of inflammatory cells into the infarct border zone 24 hours after ischemia/reperfusion injury, likely owing to reduced tumor necrosis factor-α expression and release, followed by impaired nuclear factor-κB activation and interleukin-1 release. Moreover, PLD1 was found to be important for transforming growth factor-β secretion and smooth muscle α-actin expression of cardiac fibroblasts because myofibroblast differentiation and interstitial collagen deposition were altered in Pld1−/− mice. Consequently, infarct size was increased and left ventricular function was impaired 28 days after myocardial infarction in Pld1−/− mice. Our results indicate that PLD1 is crucial for tumor necrosis factor-α–mediated inflammation and transforming growth factor-β–mediated collagen scar formation, thereby augmenting cardiac left ventricular function after ischemia/reperfusion.
American Journal Of Pathology 09/2014; DOI:10.1016/j.ajpath.2014.06.005 · 4.59 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Immune cells regulate cell surface receptor expression during their maturation, activation, and motility. Although many of these receptors are regulated largely at the level of expression, protease-mediated ectodomain shedding represents an alternative means of refashioning the surface of immune cells. Shedding is largely attributed to a family of a disintegrin and metalloprotease domain (ADAM) metalloproteases, including ADAM17. Although ADAM17 is well known to contribute to the innate immune response, mainly by releasing TNF-α, much less is known about whether/how this metalloprotease regulates adaptive immunity. To determine whether ADAM17 contributes to regulating adaptive immune responses, we took advantage of ADAM17 hypomorphic (ADAM17(ex/ex)) mice, in which ADAM17 expression is reduced by 90-95% compared with wild-type littermates. In this study, we show that that ADAM17 deficiency results in spleen and lymph node enlargement, as well as increased levels of Ag-specific class-switched Ig production following immunization with OVA together with anti-CD40 mAbs and polyinosinic-polycytidylic acid. Moreover, we demonstrate that the costimulatory ligand ICOS ligand (ICOSL) is selectively downregulated on the surface of B cells in an ADAM17-specific manner, although it is not proteolitically processed by recombinant ADAM17 in vitro. Finally, we show that higher cell surface levels of ICOSL in ADAM17(ex/ex) mice may contribute to the development of excessive Ab responses. Therefore, our data suggest a functional link between ADAM17 and ICOSL in controlling adaptive immune responses.
The Journal of Immunology 08/2014; DOI:10.4049/jimmunol.1302893 · 4.92 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Interleukin (IL)-6-deficient, but not IL-6 receptor (IL-6R)‑deficient mice present with a delayed skin wound healing phenotype. Since IL-6 solely signals via the IL-6R and glycoprotein 130 (gp130), Il-6r-deficient mice are expected to exhibit a similar phenotype as Il-6-deficient mice. However, p28 (IL-30) and ciliary neurotrophic factor (CNTF) have been identified as additional low‑affinity ligands of the IL-6R/gp130/LIFR complex. IL-6 plays an inflammatory and regenerative role in inflammatory bowel disease (IBD). In the present study, we compared Il-6r-deficient mice with mice treated with neutralizing IL-6 monoclonal antibody (mAb) in a model of dextran sodium sulfate (DSS)-induced colitis. Our results, in agreement with those of previous reports, demonstrated that IL-6 mAbs slightly attenuated DSS-induced colitis during the regeneration phase. Il-6r-deficient mice and mice with tissue-specific deletion of the Il-6r in the myeloid cell lineage (LysMCre) with acute and chronic DSS-induced colitis were, however, indistinguishable from wild-type mice. Our data suggest that IL-6 and IL-6R have an additional role in colitis, apart from the IL-6/IL-6R classic and trans-signaling.
International Journal of Molecular Medicine 06/2014; 34(3). DOI:10.3892/ijmm.2014.1825 · 2.09 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Interleukin (IL)-6 signals via a receptor complex composed of the signal-transducing β-receptor gp130 and the non-signaling
membrane-bound or soluble IL-6 receptor α (IL-6R, sIL-6R), which is referred to as classic and trans-signaling, respectively.
IL-6 trans-signaling is functionally associated with the development of chronic inflammatory diseases and cancer. Soluble
gp130 (sgp130) variants are natural inhibitors of trans-signaling. Differential splicing yields sgp130 isoforms. Here, we
describe that alternative intronic polyadenylation in intron 10 of the gp130 transcript results in a novel mRNA coding for
an sgp130 protein isoform (sgp130-E10) of 70–80 kDa. The sgp130-E10 protein was expressed in vivo in human peripheral blood mononuclear cells. To assess the biological activity of sgp130-E10, we expressed this variant as
Fc-tagged fusion protein (sgp130-E10Fc). Recombinant sgp130-E10Fc binds to a complex of IL-6 and sIL-6R, but not to IL-6 alone,
and specifically inhibits IL-6 trans-signaling. Thus, it might play an important role in the regulation of trans-signaling
[Show abstract][Hide abstract] ABSTRACT: The pleiotropic activities of Interleukin (IL-)6 are controlled by membrane-bound and soluble forms of the IL-6 receptor (IL-6R) in processes called classic and trans-signaling, respectively. The coding single nucleotide polymorphism (SNP) rs2228145 of the Interleukin 6 receptor (IL-6R Asp358Ala variant) is associated with an 2fold increase in soluble IL-6R (sIL-6R) serum levels resulting in reduced IL-6-induced C-reactive protein (CRP) production and a reduced risk for coronary heart disease. It was suggested that the increased sIL-6R level lead to decreased IL-6 classic or increased IL-6 trans-signaling. Irrespective of the functional outcome of increased sIL-6R serum level, it is still under debate, whether the increased sIL-6R serum levels emerged from differential splicing or ectodomain shedding. Here we show that increased proteolytic ectodomain shedding mediated by the A Disintegrin and metalloproteinase domain (ADAM) proteases ADAM10 and ADAM17 caused increased sIL-6R serum level in vitro as well as in healthy volunteers homozygous for the IL-6R Asp358Ala allele. Differential splicing of the IL-6R appears to have only a minor effect on sIL-6R level. Increased ectodomain shedding resulted in reduced cell-surface expression of the IL-6R Asp358Ala variant compared to the common IL-6R variant. In conclusion, increased IL-6R ectodomain shedding is a mechanistic explanation for the increased serum IL-6R levels found in persons homozygous for the rs2228145 IL-6R Asp358Ala variant.
Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease 05/2014; 1842(9). DOI:10.1016/j.bbadis.2014.05.018 · 4.88 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Ciliary neurotrophic factor (CNTF) is a neurotrophic factor with therapeutic potential for neurodegenerative diseases. Moreover, therapeutic application of CNTF reduced body weight in mice and humans. CNTF binds to high or low affinity receptor complexes consisting of CNTFR/gp130/LIFR or IL-6R/gp130/LIFR, respectively. Clinical studies of the CNTF derivative Axokine revealed intolerance at higher concentrations, which may rely on the low-affinity binding of CNTF to the IL-6R. Here, we aimed to generate a CNTFR-selective CNTF variant (CV). CV-1 contained the single amino acid exchange R28E. R28 is in close proximity to the CNTFR binding site. Using molecular modelling we hypothesised that R28 might contribute to IL-6R/CNTFR plasticity of CNTF. CV-2 to CV-5 were generated by transferring parts of the CNTFR binding site from Cardiotrophin-like cytokine (CLC) to CNTF. CLC selectively signals via the CNTFR/gp130/LIFR complex, albeit with a much lower affinity compared to CNTF. As shown by immunoprecipitation, all CNTF variants retained the ability to bind to CNTFR. CV-1, CV-2 and CV-5, however, lost the ability to bind to IL-6R. Although all variants induced cytokine-dependent cellular proliferation and STAT3 phosphorylation via CNTFR/gp130/LIFR, only CV-3 induced STAT3 phosphorylation via IL-6R/gp130/LIFR. Quantification of CNTF-dependent proliferation of CNTFR-gp130-LIFR expressing cells indicated that only CV-1 was as biologically active as CNTF. Thus, the CNTFR-selective CV-1 will allow discriminating between CNTFR- and IL-6R-mediated effects in vivo.
Journal of Biological Chemistry 05/2014; DOI:10.1074/jbc.M114.568857 · 4.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A disintegrin and metalloprotease 17 (ADAM17) is a major sheddase involved in the regulation of a wide range of biological
processes. Key substrates of ADAM17 are the IL-6 receptor (IL-6R) and TNF-α. The extracellular region of ADAM17 consists of
a prodomain, a catalytic domain, a disintegrin domain, and a membrane-proximal domain as well as a small stalk region. This
study demonstrates that this juxtamembrane segment is highly conserved, α-helical, and involved in IL-6R binding. This process
is regulated by the structure of the preceding membrane-proximal domain, which acts as molecular switch of ADAM17 activity
operated by a protein-disulfide isomerase. Hence, we have termed the conserved stalk region “Conserved ADAM seventeen dynamic interaction sequence” (CANDIS). Finally, we identified the region in IL-6R that binds to CANDIS. In contrast to the type I transmembrane
proteins, the IL-6R, and IL-1RII, CANDIS does not bind the type II transmembrane protein TNF-α, demonstrating fundamental
differences in the respective shedding by ADAM17.
[Show abstract][Hide abstract] ABSTRACT: Tumor necrosis factor alpha (TNF) plays a pivotal role in chronic inflammatory diseases such as rheumatoid arthritis and Crohn's disease. Although anti-TNF antibody therapy is now commonly used to treat patients suffering from these inflammatory conditions, the cost of treatment continues to be a concern. Here, we developed a rice transgenic system for the production of a llama variable domain of a heavy-chain antibody fragment (VHH) specific for mouse TNF in rice seeds (MucoRice-mTNF-VHH). MucoRice-mTNF-VHH was produced at high levels in the rice seeds when we used our most recent transgene-overexpression system with RNA interference technology that suppresses the production of major rice endogenous storage proteins while enhancing the expression of the transgene-derived protein. Production levels of mTNF-VHH in rice seeds reached an average of 1.45% (w/w). Further, approximately 91% of mTNF-VHH was released easily when the powder form of MucoRice-mTNF-VHH was mixed with PBS. mTNF-VHH purified by means of single-step gel filtration from rice PBS extract showed high neutralizing activity in an in vitro mTNF cytotoxicity assay using WEHI164 cells. In addition, purified mTNF-VHH suppressed progression of collagen-induced arthritis in mice. These results show that this rice-expression system is useful for the production of neutralizing VHH antibody specific for mTNF.
Journal of Biotechnology 04/2014; 175(1). DOI:10.1016/j.jbiotec.2014.02.005 · 2.87 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The Janus kinase / signal transducer and activator of transcription (Jak/STAT) pathway can be activated by many different cytokines, among them all members of the Interleukin (IL-)6 family. Dysregulation of this pathway, resulting in its constitutive activation, is associated with chronic inflammation and cancer development. In the present study, we show that activity of protein kinase II (CK2), a ubiquitously expressed serine/threonine kinase, is needed for induced activation of STAT1 and STAT3 by IL-6 classic and trans-signaling, IL-11, IL-27, oncostatin M (OSM), leukemia inhibitory factor (LIF) and cardiotrophin-1 (CT-1). Inhibition of CK2 efficiently prevented STAT phosphorylation and inhibited cytokine-dependent cell proliferation in a Jak1-dependent manner. Conversely, forced activation of CK2 alone was not sufficient to induce activation of the Jak/STAT signaling pathway. Inhibition of CK2 in turn inhibited Jak1-dependent STAT activation by oncogenic gp130 mutations. Furthermore, CK2 inhibition diminished the Jak1- and Src kinase-dependent phosphorylation of a constitutively active STAT3 mutant recently described in human large granular lymphocytic leukemia. In conclusion, we characterize CK2 as an essential component of the Jak/STAT pathway. Pharmacologic inhibition of this kinase is therefore a promising strategy to treat human inflammatory diseases and malignancies associated with constitutive activation of the Jak/STAT pathway.