Apoptose induziert die proteolytische Prozessierung des IL-6 Rezeptors und trägt zum proinflammatorischen Trans-Signalling von Neutrophilen bei

Interleukin-6 (IL-6) trans-signalling has emerged as a prominent regulator of immune responses during both innate and acquired immunity. Regulation of IL-6 trans-signalling is reliant upon the release of soluble IL-6R (sIL-6R), which binds IL-6 to create an agonistic IL-6/sIL-6R complex capable of activating cell types that would normally not respond to IL-6 alone. Soluble IL-6R is generated by proteolytic cleavage of the membrane-anchored precursor (shedding) or from alternative spliced mRNA. Several inducers of IL-6R shedding such as bacterial pore forming toxins, bacterial metalloproteases, the phorbol ester PMA, cholesterol depleting agents and C-reactive protein have been identified in previous studies. IL-6R shedding induced by PMA and cholesterol depletion is dependent on the metalloprotease ADAM17 (A Disintegrin And Metalloprotease), and to a lesser extent on the related sheddase ADAM10. In this study apoptosis was identified as a natural stimulus of IL-6R shedding. Apoptosis, or programmed cell death, plays an important role in regulating growth, tissue homeostasis, development and immune responses. The induction of apoptosis is mediated by extrinsic and intrinsic pathways which require the cooperation of a series of molecules including signal molecules and receptors, enzymes and gene regulating proteins. Among them, the caspase-cascade signalling system is crucial for induction, transduction and amplification of intracellular apoptotic signals. This study demonstrates that intrinsic and extrinsic apoptotic stimulation of cells by DNA-damage, cytokine deprivation, UV irradiation and Fas ligation promotes shedding of the IL-6R from the cell surface thereby generating sIL-6R. Apoptosis-induced shedding of the IL-6R was caspase dependent but PKC, MAPK and ROS independent. Inhibition of ADAM17 during apoptosis by small pharmacological inhibitors or a dominant-negative ADAM17 variant resulted in prevention of IL-6R shedding. The sIL-6R generated during apoptosis was biological active and formed complexes with IL-6 thereby facilitating IL-6 trans-signalling in nonapoptotic cells. Neutrophils have a short life span and die rapidly via apoptosis in vivo and in vitro. In this study neutrophils were used as a cellular model. Induction of apoptosis by Fas-ligation or UV irradiation of primary human neutrophils promoted IL-6R shedding thereby resulting in a two-fold increase of sIL-6R release. Neutrophils represent the first cell population infiltrating inflamed areas and play a crucial role during inflammatory responses due to their extraordinary microbial capacity. Following phagocytosis of invaded microorganisms neutrophils undergo apoptosis as a mechanism to promote resolution of inflammation. The second cell population which migrates into the inflamed area consists mostly of mononuclear phagocytes (monocytes) which are responsible for the clearance of apoptotic neutrophils thus leading to resolution of the inflammatory reaction. Findings in this study indicate that apoptosis-induced shedding of the IL-6R from the neutrophils surface facilitates formation of an [IL-6/sIL-6R] complex which directs IL-6 trans-signalling presumably on endothelial cells to promote recruitment of mononuclear phagocytic cells involved in the nonphlogisic removal of apoptotic neutrophils. Using a mouse model of acute inflammation, it was demonstrated in this study that blocking IL-6R-mediated signal transduction by neutralizing antibodies or soluble gp130-Fc during acute inflammation resulted in a normal influx of neutrophils but a severely impaired influx of monocytes into the inflamed area. Therefore, IL-6R shedding and subsequent IL-6 trans-signalling represent important steps for the resolution of inflammatory responses by controlling the recruitment of mononuclear phagocytes. Thus, shedding of the IL-6R during neutrophil apoptosis may have profound effects on the outcome of the inflammatory response.Interleukin-6 (IL-6) vermitteltes Trans-signalling ist ein wichtiger regulatorischer Signalweg für die Steuerung der angeborenen und erworbenen Immunität. IL-6 Trans-signalling wird durch die Freisetzung vom löslichen Interleukin-6-Rezeptor (sIL-6R) moduliert; dieser bindet an IL-6, um einen IL-6/sIL-6R Komplex zu bilden. Der Komplex aus IL-6 und sIL-6R kann dann Zellen stimulieren, die lediglich gp130 exprimieren und normallerweise nicht in der Lage sind auf IL-6 zu reagieren, da ihnen die ligandenbindende Untereinheit (IL-6R) fehlt. Die lösliche Form des IL-6Rs kann entweder durch limitierte Proteolyse des membranständigen Rezeptors oder durch alternatives Spleißen der IL-6R mRNA generiert werden. In vorherigen Arbeiten konnte bereits gezeigt werden, dass die limitierte Proteolyse des IL-6Rs (Shedding) durch bakterielle porenformende Toxine, bakterielle Metalloproteasen, den Phorbolester PMA, Cholesterinentzug und C-reaktivem Protein induziert werden kann. Shedding des IL-6Rs durch Phorbolester und Cholesterinentzug wird hauptsächlich durch die Metalloprotease ADAM17 (A Disintegrin And Metalloprotease 17) und zum geringen Teil von der verwandten Protease ADAM10 vermittelt. Im Rahmen dieser Arbeit konnte gezeigt werden, dass apoptotische Zellen vermehrt den IL-6R spalten; demnach stellen apoptotische Prozesse einen physiologischen Stimulus für induzierte IL-6R Proteolyse dar. Apoptose wird auch als programmierter Zelltod bezeichnet und spielt in der Regulation von Zellwachstum, Gewebehomöostase und Immunreaktionen eine entscheidende Rolle. Apoptose kann abhängig von der Art des auslösenden Reizes auf verschiedene Wege eingeleitet werden; zum einen durch Aktivierung von Todesrezeptoren auf der Zellmembran und zum anderen durch mitochondrial vermittelte Signalwege. Hauptmerkmal beider Apoptosewege sind Caspasen (Cysteinproteasen), welche die morphologischen und biochemischen Merkmale der Apoptose durch Spaltung von zellulären Bestandteilen hervorrufen. Im Rahmen dieser Arbeit konnte gezeigt werden, dass sowohl todesrezeptoren- als auch mitochondrialvermittelte Apoptose zu vermehrter Proteolyse des IL-6Rs führt. Dabei wurde zelluläre Apoptose durch verschiedene Wege induziert, wie zum Beispiel durch toxische Substanzen, die zur Schädigung des genetischen Materials führen, UV-Bestrahlung, Aktivierung des Todesrezeptors CD95 (Fas) oder Entzug von Überlebensfaktoren. Das durch Apoptose induzierte IL-6R Shedding war Caspase-abhängig jedoch PKC-, MAPK- und ROS- unabhängig. Des Weiteren führte eine Inhibition von ADAM17 durch kleine pharmakologische Inhibitoren oder durch eine dominant-negative ADAM17 Variante während des programmierten Zelltodes zur kompletten Unterbindung von apoptosevermitteltem IL-6R Shedding. Der lösliche IL-6R, welcher während der Apoptose vermehrt gebildet wurde, war biologisch aktiv und konnte im Komplex mit IL-6 in nicht-apoptotischen gp130 exprimierenden Zellen Trans-signalling auslösen. Polymorphkernige Neutrophile sind kurzlebige Zellen mit einer Lebensdauer von 6-8 Stunden; danach sterben Neutrophile durch Apoptose. Im Rahmen dieser Arbeit wurden Neutrophile als zelluläres System verwendet. CD95 (Fas) Aktivierung und UV-Bestrahlung lösten in primären humanen Neutrophilen Apoptose aus, welche mit erhöhtem Shedding des IL-6Rs einherging. Bei Entzündungsprozessen stellen Neutrophile die erste Verteidigungslinie gegen Mikroorganismen dar. Neutrophile phagozytieren Mikroorganismen und töten diese durch vermehrte Produktion von Sauerstoffradikalen ab. Um die inflammatorische Reaktion zu beenden, müssen die aktivierten Neutrophilen eliminiert werden. Dies passiert durch die Einleitung von Apoptose in aktivierten Neutrophilen und anschließender Phagocytose durch mononukleäre Immunzellen (Monocyten), welche als zweite Zellpopulation in den Entzündungsort einwandern. Ergebnisse in dieser Arbeit zeigen, dass apoptotische Neutrophile verstärkt den IL-6R spalten. Dieser bildet einen Komplex mit IL-6. Der Komplex aus sIL-6R/IL-6 stimuliert daraufhin endotheliale Zellen, Mediatoren auszuschütten, die an der Rekrutierung von Monocyten beteiligt sind. Mit Hilfe eines murinen Krankheitsmodelles (Air-pouch-Modell) konnte gezeigt werden, dass Blockierung von sIL-6R-vermittelten Signalwegen durch neutralisierende Antikörper oder rekombinantem löslichen gp130-Fc zu einer stark verminderten Akkumulation von Monocyten in den Entzündungsherd führt. Demnach wird der sIL-6R von apoptotischen Neutrophilen freigesetzt und beeinflusst bei entzündlichen Prozessen die Einwanderung von Monocyten, welche für das Beenden der inflammatorischen Reaktion verantwortlich sind

Structural basis for inflammation-driven shedding of CD163 ectodomain and tumor necrosis factor-α in macrophages

The haptoglobin-hemoglobin receptor CD163 and proTNF-α are transmembrane macrophage proteins subjected to cleavage by the inflammation-responsive protease ADAM17. This leads to release of soluble CD163 (sCD163) and bioactive TNF-α. Sequence comparison of the juxtamembrane region identified similar palindromic sequences in human CD163 ((1044)Arg-Ser-Ser-Arg) and proTNF-α ((78)Arg-Ser-Ser-Ser-Arg). In proTNF-α the Arg-Ser-Ser-Ser-Arg sequence is situated next to the previously established ADAM17 cleavage site. Site-directed mutagenesis revealed that the sequences harbor essential information for efficient cleavage of the two proteins upon ADAM17 stimulation. This was further evidenced by analysis of mouse CD163 that, like CD163 in other non-primates, does not contain the palindromic CD163 sequence in the juxtamembrane region. Mouse CD163 resisted endotoxin- and phorbol ester-induced shedding, and ex vivo analysis of knock-in of the Arg-Ser-Ser-Arg sequence in mouse CD163 revealed a receptor shedding comparable with that of human CD163. In conclusion, we have identified an essential substrate motif for ADAM17-mediated CD163 and proTNF-α cleavage in macrophages. In addition, the present data indicate that CD163, by incorporation of this motif in late evolution, underwent a modification that allows for an instant down-regulation of surface CD163 expression and inhibition of hemoglobin uptake. This regulatory modality seems to have coincided with the evolution of an enhanced hemoglobin-protecting role of the haptoglobin-CD163 system in primate

ADAM17 substrate release in proximal tubule drives kidney fibrosis

Kidney fibrosis following kidney injury is an unresolved health problem and causes significant morbidity and mortality worldwide. In a study into its molecular mechanism, we identified essential causative features. Acute or chronic kidney injury causes sustained elevation of a disintegrin and metalloprotease 17 (ADAM17); of its cleavage-activated proligand substrates, in particular of pro-TNFα and the EGFR ligand amphiregulin (pro-AREG); and of the substrates\u27 receptors. As a consequence, EGFR is persistently activated and triggers the synthesis and release of proinflammatory and profibrotic factors, resulting in macrophage/neutrophil ingress and fibrosis. ADAM17 hypomorphic mice, specific ADAM17 inhibitor-treated WT mice, or mice with inducible KO of ADAM17 in proximal tubule (Slc34a1-Cre) were significantly protected against these effects. In vitro, in proximal tubule cells, we show that AREG has unique profibrotic actions that are potentiated by TNFα-induced AREG cleavage. In vivo, in acute kidney injury (AKI) and chronic kidney disease (CKD, fibrosis) patients, soluble AREG is indeed highly upregulated in human urine, and both ADAM17 and AREG expression show strong positive correlation with fibrosis markers in related kidney biopsies. Our results indicate that targeting of the ADAM17 pathway represents a therapeutic target for human kidney fibrosis

Critical role of the disintegrin metalloprotease ADAM17 for intestinal inflammation and regeneration in mice

The protease a disintegrin and metalloprotease (ADAM) 17 cleaves tumor necrosis factor (TNF), L-selectin, and epidermal growth factor receptor (EGF-R) ligands from the plasma membrane. ADAM17 is expressed in most tissues and is up-regulated during inflammation and cancer. ADAM17-deficient mice are not viable. Conditional ADAM17 knockout models demonstrated proinflammatory activities of ADAM17 in septic shock via shedding of TNF. We used a novel gene targeting strategy to generate mice with dramatically reduced ADAM17 levels in all tissues. The resulting mice called ADAM17ex/ex were viable, showed compromised shedding of ADAM17 substrates from the cell surface, and developed eye, heart, and skin defects as a consequence of impaired EGF-R signaling caused by failure of shedding of EGF-R ligands. Unexpectedly, although the intestine of unchallenged homozygous ADAM17ex/ex mice was normal, ADAM17ex/ex mice showed substantially increased susceptibility to inflammation in dextran sulfate sodium colitis. This was a result of impaired shedding of EGF-R ligands resulting in failure to phosphorylate STAT3 via the EGF-R and, consequently, in defective regeneration of epithelial cells and breakdown of the intestinal barrier. Besides regulating the systemic availability of the proinflammatory cytokine TNF, our results demonstrate that ADAM17 is needed for vital regenerative activities during the immune response. Thus, our mouse model will help investigate ADAM17 as a potential drug target

STAT3 regulated ARF expression suppresses prostate cancer metastasis.

Prostate cancer (PCa) is the most prevalent cancer in men. Hyperactive STAT3 is thought to be oncogenic in PCa. However, targeting of the IL-6/STAT3 axis in PCa patients has failed to provide therapeutic benefit. Here we show that genetic inactivation of Stat3 or IL-6 signalling in a Pten-deficient PCa mouse model accelerates cancer progression leading to metastasis. Mechanistically, we identify p19(ARF) as a direct Stat3 target. Loss of Stat3 signalling disrupts the ARF-Mdm2-p53 tumour suppressor axis bypassing senescence. Strikingly, we also identify STAT3 and CDKN2A mutations in primary human PCa. STAT3 and CDKN2A deletions co-occurred with high frequency in PCa metastases. In accordance, loss of STAT3 and p14(ARF) expression in patient tumours correlates with increased risk of disease recurrence and metastatic PCa. Thus, STAT3 and ARF may be prognostic markers to stratify high from low risk PCa patients. Our findings challenge the current discussion on therapeutic benefit or risk of IL-6/STAT3 inhibition.Lukas Kenner and Jan Pencik are supported by FWF, P26011 and the Genome Research-Austria project “Inflammobiota” grants. Helmut Dolznig is supported by the Herzfelder Family Foundation and the Niederösterr. Forschungs-und Bildungsges.m.b.H (nfb). Richard Moriggl is supported by grant SFB-F2807 and SFB-F4707 from the Austrian Science Fund (FWF), Ali Moazzami is supported by Infrastructure for biosciences-Strategic fund, SciLifeLab and Formas, Zoran Culig is supported by FWF, P24428, Athena Chalaris and Stefan Rose-John are supported by the Deutsche Forschungsgemeinschaft (Grant SFB 877, Project A1and the Cluster of Excellence --“Inflammation at Interfaces”). Work of the Aberger lab was supported by the Austrian Science Fund FWF (Projects P25629 and W1213), the European FP7 Marie-Curie Initial Training Network HEALING and the priority program Biosciences and Health of the Paris-Lodron University of Salzburg. Valeria Poli is supported by the Italian Association for Cancer Research (AIRC, No IG13009). Richard Kennedy and Steven Walker are supported by the McClay Foundation and the Movember Centre of Excellence (PC-UK and Movember). Gerda Egger is supported by FWF, P27616. Tim Malcolm and Suzanne Turner are supported by Leukaemia and Lymphoma Research.This is the final version of the article. It first appeared from Nature Publishing Group via http://dx.doi.org/10.1038/ncomms873

Epithelial RNase H2 Maintains Genome Integrity and Prevents Intestinal Tumorigenesis in Mice

BACKGROUND & AIMS: RNase H2 is a holoenzyme, composed of 3 subunits (ribonuclease H2 subunits A, B, and C), that cleaves RNA:DNA hybrids and removes mis-incorporated ribonucleotides from genomic DNA through ribonucleotide excision repair. Ribonucleotide incorporation by eukaryotic DNA polymerases occurs during every round of genome duplication and produces the most frequent type of naturally occurring DNA lesion. We investigated whether intestinal epithelial proliferation requires RNase H2 function and whether RNase H2 activity is disrupted during intestinal carcinogenesis. METHODS: We generated mice with epithelial-specific deletion of ribonuclease H2 subunit B (H2bΔIEC) and mice that also had deletion of tumor-suppressor protein p53 (H2b/p53ΔIEC); we compared phenotypes with those of littermate H2bfl/fl or H2b/p53fl/fl (control) mice at young and old ages. Intestinal tissues were collected and analyzed by histology. We isolated epithelial cells, generated intestinal organoids, and performed RNA sequence analyses. Mutation signatures of spontaneous tumors from H2b/p53ΔIEC mice were characterized by exome sequencing. We collected colorectal tumor specimens from 467 patients, measured levels of ribonuclease H2 subunit B, and associated these with patient survival times and transcriptome data. RESULTS: The H2bΔIEC mice had DNA damage to intestinal epithelial cells and proliferative exhaustion of the intestinal stem cell compartment compared with controls and H2b/p53ΔIEC mice. However, H2b/p53ΔIEC mice spontaneously developed small intestine and colon carcinomas. DNA from these tumors contained T>G base substitutions at GTG trinucleotides. Analyses of transcriptomes of human colorectal tumors associated lower levels of RNase H2 with shorter survival times. CONCLUSIONS: In analyses of mice with disruption of the ribonuclease H2 subunit B gene and colorectal tumors from patients, we provide evidence that RNase H2 functions as a colorectal tumor suppressor. H2b/p53ΔIEC mice can be used to study the roles of RNase H2 in tissue-specific carcinogenesis

The pro- and anti-inflammatory properties of the cytokine interleukin-6

AbstractInterleukin-6 is a cytokine not only involved in inflammation and infection responses but also in the regulation of metabolic, regenerative, and neural processes. In classic signaling, interleukin-6 stimulates target cells via a membrane bound interleukin-6 receptor, which upon ligand binding associates with the signaling receptor protein gp130. Gp130 dimerizes, leading to the activation of Janus kinases and subsequent phosphorylation of tyrosine residues within the cytoplasmic portion of gp130. This leads to the engagement of phosphatase Src homology domains containing tyrosin phosphatase-2 (SHP-2) and activation of the ras/raf/Mitogen-activated protein (MAP) kinase (MAPK) pathway. In addition, signal transducer and activator of transcription factors are recruited, which are phosphorylated, and consequently dimerize whereupon they translocate into the nucleus and activate target genes. Interestingly, only few cells express membrane bound interleukin-6 receptor whereas all cells display gp130 on the cell surface. While cells, which only express gp130, are not responsive to interleukin-6 alone, they can respond to a complex of interleukin-6 bound to a naturally occurring soluble form of the interleukin-6 receptor. Therefore, the generation of soluble form of the interleukin-6 receptor dramatically enlarges the spectrum of interleukin-6 target cells. This process has been named trans-signaling. Here, we review the involvement of both signaling modes in the biology of interleukin-6. It turns out that regenerative or anti-inflammatory activities of interleukin-6 are mediated by classic signaling whereas pro-inflammatory responses of interleukin-6 are rather mediated by trans-signaling. This is important since therapeutic blockade of interleukin-6 by the neutralizing anti-interleukin-6 receptor monoclonal antibody tocilizumab has recently been approved for the treatment of inflammatory diseases

Mouse neutrophils express the decoy type 2 interleukin-1 receptor (IL-1R2) constitutively and in acute inflammatory conditions

The proinflammatory activities of IL-1 are tightly controlled at different levels. IL-1R2 acts as a decoy receptor and has been shown to regulate the biological effects of IL-1 in vitro and in vivo. However, little is known about its natural expression in the mouse in physiologic and pathologic conditions. In this study, we examined IL-1R2 mRNA and protein expression in isolated cells and tissues in response to different stimulatory conditions. Data obtained using ex vivo CD11b(+)Ly6G(+) peripheral blood cells and in vitro-differentiated CD11b(+)Ly6G(+) BMG indicated that neutrophils are the major source of constitutively expressed IL-1R2 in the mouse. The expression of IL-1R2 on BMG and ex vivo Ly6G(+) peripheral blood cells was highly up-regulated by HC. IL-1R2 pull-down experiments showed that mouse rIL-1β binds to BMG IL-1R2, whereas binding of IL-1Ra could not be detected. Furthermore, LPS treatment induced shedding of IL-1R2 from the neutrophil membrane in vitro and in vivo, executed mainly by ADAM17. Finally, in in vivo models of inflammation, including thioglycolate-induced acute peritonitis and acute lung injury, infiltrating Ly6G(+) neutrophils, expressed IL-1R2. Our data show that in the mouse, neutrophils mainly express the decoy receptor IL-1R2 under naïve and inflammatory conditions. These data suggest that neutrophils may contribute to the resolution of acute inflammation