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BackgroundInterleukin-6 (IL-6), a pleiotropic cytokine with both degenerative and regenerative properties, is necessary and sufficient to provoke perioperative neurocognitive disorders after aseptic trauma in mice. IL-6 initiates its actions after binding to either membrane-bound IL-6 receptor α (mIL-6Rα) through classical signalling, or soluble IL-6 receptor (IL-6R) through trans-signalling; both signalling pathways require the transducer gp130. We investigated the site and type of IL-6 signalling that pertains in a tibial fracture aseptic trauma model of perioperative neurocognitive disorder.MethodsWild-type or genetically altered adult mice that lacked molecules unique to either classical or trans-IL-6 signalling underwent tibial fracture under isoflurane anaesthesia. In separate cohorts, we assessed postoperative memory using a trace fear conditioning paradigm (72 h postoperatively), and post-receptor IL-6 signalling (24 h postoperatively) using phosphorylation of signal transducer and activator of transcription 3 (pSTAT3) in CA1 hippocampal neurones. Fracture healing was assessed at postoperative day 15 after inhibiting either both forms of IL-6 signalling with BE0047 or only trans-signalling with sgp130Fc.ResultsThe surgical phenotype of memory decline (decrease in freezing in trace fear conditioning) and upregulated IL-6 signalling (pSTAT3) did not occur after pretreatment before surgery with either BE0047 or sgp130Fc, or after depleting gp130 from CA1 neurones. The surgical phenotype still occurred when IL-6Rα was depleted in either CA1 hippocampal neurones (freezing time, 38.9% [11.5%] vs 58.4% [12.3%]; pSTAT+ CA1 neurones, 31.7 [4.9] vs 7.0 [3.1]) or microglia (freezing time, 40.1% [13.9%] vs 65.2% [12.6%]; pSTAT+ CA1 neurones, 30.1 [5.5] vs 7.9 [3.2]). In global IL-6Rα-/- mice, hyper-IL-6, the trans-signalling agonist, produced the surgical phenotype when administered i.c.v. (freezing time, 42.4% [8.8%] vs 59.7% [10.4%]; pSTAT+ cells, 29.3 [4.3] vs 10.0 [4.4]). Bone-fracture healing (% of fracture callus comprised of new collagen) was significantly greater with sgp130Fc than with BE0047 (52.2% [8.3%] vs 39.7% [7.9%]).ConclusionsAfter orthopaedic trauma, IL-6 produces perioperative neurocognitive disorders through IL-6 trans-signalling in mouse CA1 neurones. Druggable targets of the trans-signalling pathway should be sought to reduce perioperative neurocognitive disorders while allowing the healing properties of classical IL-6 signalling.

The research work was supported by the Spanish Institute of Health Carlos III (COV-20/00792) and by the European Commission - NextgenerationEU (Regulation EU 2020/2094), through CSIC's Global Health Platform (PTI Salud Global). MAR-H acknowledges support from the Spanish Institute of Health Carlos III and the European Commission - NextgenerationEU (Regulation EU 2020/2094), through CSIC's Global Health Platform (PTI Salud Global). DC is supported by a predoctoral iPFIS (IFI 19/00048) funded by Spanish Institute of Health Carlos III. MN-N is supported by the Rio Hortega contract (CM20/00074)., IL-6 is one of the major mediators of the hyper-inflammatory responses with complex biological functions as it can signal via different modes of action. IL-6 by classical signalling has anti-inflammatory and antibacterial activities, while trans-signalling mediates proinflammatory effects. The net biological effect of IL-6 is established by multiple factors beyond its absolute concentration. Here, we assess the relationship between IL-6 signalling variables [IL-6, soluble IL-6R (sIL-6R) and soluble gp130 (sgp130)] and outcomes in a cohort of 366 COVID-19 patients. The potential trans-signalling was evaluated by a ratio between the pro-inflammatory binary IL-6:sIL-6R complex and the inactive ternary IL-6:sIL-6R:sgp130 complex (binary/ternary complex) and the fold molar excess of sgp130 over sIL-6R (FME). Our data provide new evidence that high levels of IL- 6, sIL-6R, sgp130, binary/ternary complex ratio, and low FME are independent predictors of COVID-19 severity in survivor patients (without death), and the combination of IL-6 + sIL-6R + sgp130 exhibited the most robust classification capacity. Conversely, in a subgroup of patients with a very poor prognosis, we found that high levels of IL-6 and low levels of sIL-6R, sgp130, and binary/ternary complex ratio were predictors of death. In this context, the highest predictive capacity corresponded to the combined analysis of IL-6 + FME + lymphopenia + creatinine. Herein, we present IL-6 signalling variables as a helpful tool for the early identification and stratification of patients with clear implications for treatment and clinical decision-making., Spanish Institute of Health Carlos III (COV-20/00792), European Commission - NextgenerationEU (Regulation EU 2020/2094), CSIC's Global Health Platform (PTI Salud Global), Spanish Institute of Health Carlos III and the European Commission - NextgenerationEU (Regulation EU 2020/2094), iPFIS (IFI 19/00048) funded by Spanish Institute of Health Carlos III, Rio Hortega contract (CM20/00074)

The cytokine Interleukin 6 (IL-6) is involved in many biological functions of the immune system, metabolism and the central nervous system, and in the bidirectional communication between these systems. In the brain, IL-6 is considered to affect the modulation of sleep-wake behaviour and synaptic plasticity. IL-6 is further well characterised for its dual role to maintain homeostasis as well as to contribute to inflammatory and autoimmune processes, both in brain and periphery. Signal transduction of IL-6 is induced by binding of IL-6 to the membrane-bound IL-6 receptor (IL-6R; classic signalling) or alternatively to the soluble form of the IL-6R (sIL-6R; trans-signalling). The membrane-bound glycoprotein 130 (gp130) is the signal-transducing receptor subunit in both signalling modes. Gp130 is ubiquitously expressed throughout the body, whereas IL-6R expression is restricted to distinct cell populations. Within the brain parenchyma the IL-6R is sparsely expressed, and thus the brain is primarily reliant on trans-signalling in its response to IL-6. As a previous study revealed an upregulation of the peripheral sIL-6R in humans during sleep (Dimitrov et al., 2006), the influence of IL-6 trans-signalling on sleep regulation/modulation was scrutinised in this work initially. The effect of the IL-6 trans-signalling mimetic designer cytokine Hyper-IL-6 (sIL-6R coupled to IL-6) was investigated on sleep architecture of rats by EEG/EMG sleep recordings. Hyper-IL-6 can activate almost all cells of the brain in contrast to IL-6 itself. Results of this present work demonstrated that an intracerebroventricular injection of Hyper-IL-6 (500 ng) before the dark phase into rats led to increased amounts of REM sleep accompanied by a reduced EEG power density. Non-REM sleep was not affected. These data define a new function of IL-6 trans-signalling within the complex network of REM sleep modulation. That might further be of importance for altered sleep behaviour under pathophysiological conditions in which sIL-6R and IL-6 are elevated (e.g. bacterial/viral infections, major depression). A further approach to explore the impact of IL-6 trans-signalling on brain-regulated functions included the central “knock-out” of this signal transduction mode. For that purpose transgenic mice were generated in this work expressing the competitive antagonist of IL-6 trans-signalling sgp130-Fc under transcriptional control of the astrocyte-specific promoter gfa2 within the brain. The protein sgp130-Fc is a dimer of the human extracellular part of gp130 fused to the Fc-portion of a human IgG antibody. Expression and secretion of sgp130-Fc in the brain of transgenic mice on the protein level could be verified. The biological activity of the transgenic sgp130-Fc protein was confirmed by Hyper-IL-6 precipitation tests. Likewise, the protein amounts of sgp130-Fc appeared to be sufficient for the blockade of sIL-6R-mediated cellular responses in the brain. The presence of sgp130-Fc mRNA in transgenic animals was detected predominantly in the brain. However, sgp130-Fc was also found in sera of the three different established mouse lines at varying protein levels, which did not correlate with the amounts observed within the brain. The gfa2–sgp130-Fc transgenic mice generated in this work offer an useful “in vivo tool” to further study the relevance of IL-6 trans-signalling for sleep-wake behaviour, memory consolidation, and models of peripheral and neuropathological inflammation. Das Zytokin Interleukin 6 (IL-6) ist an vielfältigen Funktionen des Immunsystems, des Stoffwechsels und des Zentralnervensystems beteiligt, sowie an der bidirektionalen Kommunikation zwischen diesen Systemen. Im Gehirn beeinflusst IL-6 die Modulation von Schlaf-Wach-Verhalten und die synaptische Plastizität. Charakteristisch für IL-6 ist weiterhin dessen duale Rolle bei der Aufrechterhaltung der Homeostasis einerseits aber auch bei der Förderung von entzündlichen und autoimmunen Prozessen andererseits, sowohl im Gehirn als auch in der Peripherie. Die Signaltransduktion von IL-6 wird durch Bindung des IL-6 an den membranständigen IL-6-Rezeptor (IL-6R; klassisches Signalling) oder alternativ an die lösliche Form des IL-6R (sIL-6R; IL-6-Trans-Signalling) induziert. Das membrangebundene Glykoprotein 130 (gp130) ist die signalweiterleitende Rezeptor-Untereinheit beider Signalling-Arten. Gp130 wird ubiquitär im Körper exprimiert, wohingegen die Expression des IL-6R auf bestimmte Zellpopulationen begrenzt ist. Im Gehirnparenchym ist der IL-6R wenig exprimiert, deshalb ist das Gehirn in seiner Reaktion auf IL-6 vornehmlich vom Trans-Signalling abhängig. Eine vorherige Studie beschrieb die Erhöhung des peripheren sIL-6R im Menschen während des Schlafes (Dimitrov et al., 2006). Deshalb wurde in der hier vorliegenden Arbeit erstmals der Einfluss des IL-6-Trans-Signalling auf die Schlaf-Modulation erforscht. Die Wirkung des IL-6-Trans-Signalling imitierenden Designer-Zytokins Hyper-IL-6 (sIL-6R gekoppelt an IL-6) auf das Schlaf-Profil von Ratten wurde in EEG/EMG-Schlaf-Messungen untersucht. Hyper-IL-6 kann fast alle Zellen des Gehirns aktivieren, im Gegensatz zu IL-6 allein. Ergebnisse dieser Arbeit zeigten, dass eine intracerebroventrikuläre Injektion von Hyper-IL-6 (500 ng) kurz vor der Dunkelphase in Ratten den Anteil an REM-Schlaf erhöhte, begleitet von einer reduzierten EEG-Power-Spektrum-Dichte. Dabei wurde der Non-REM-Schlaf nicht beeinflusst. Diese Daten definieren eine neue Funktion der IL-6-Trans-Signaltransduktion innerhalb des komplexen Netzwerks der REM-Schlaf-Modulation. Das könnte ebenso bei pathophysiologischen Zuständen, in denen der sIL-6R sowie IL-6 erhöht sind (z.B. bakterielle/virale Infektionen, Depression), von Bedeutung sein. Ein weiterer Ansatz, um die Auswirkung der IL-6-Trans-Signaltransduktion auf Gehirn-gesteuerte Prozesse zu untersuchen, beinhaltete der zentrale „Knock-out“ dieses Signaltransduktionsweges. Dafür wurden im Rahmen dieser Arbeit transgene Mäuse generiert, die den kompetitiven Antagonisten des IL-6-Trans-Signalling sgp130-Fc im Gehirn unter transkriptioneller Kontrolle des astrozytenspezifischen Promotors gfa2 exprimieren. Das Protein sgp130-Fc ist ein Dimer des humanen extrazellulären Teiles von gp130, das über den Fc-Teil eines humanen IgG-Antikörpers verbunden ist. Die Expression und Sekretion von sgp130-Fc konnnte im Gehirn transgener Mäusen auf Proteinebene nachgewiesen werden. Die biologische Aktivität des transgenen sgp130-Fc-Proteins wurde in Hyper-IL-6-Präzipitationsversuchen bestätigt. Zudem erschienen die Proteingehalte an sgp130-Fc für die Hemmung sIL-6R-vermittelter zellulärer Reaktionen im Gehirn ausreichend zu sein. Das Vorhandensein von sgp130-Fc-mRNA transgener Tiere wurde vorwiegend im Gehirn detektiert. Dennoch wurde sgp130-Fc auch in Seren der drei etablierten Mauslinien mit unterschiedlichen Proteingehalten gefunden, die nicht mit den Mengen im Gehirn korrelierten. Die in dieser Arbeit erzeugten gfa2–sgp130-Fc-transgenen Mäuse stellen ein geeignetes „in vivo-Werkzeug“ dar, um die Bedeutung des IL-6-Trans-Signalling für das Schlaf-Wach-Verhalten, die Gedächtniskonsolidierung sowie Modelle peripherer und neuropathologischer Entzündungen eingehend zu studieren.