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Mesenchymale Knochenmarksstammzellen (engl. Bone Marrow-Derived Mesenchymal Stem Cells (BMSCs)) sind hochproliferative multipotente Progenitorzellen mit einem hohen Regenerationspotential. Sie können aus dem Knochenmark in geschädigte Knorpelareale migrieren und dort zu Chondrozyten differenzieren. Somit können sie zur Reparatur traumatisch oder osteoarthrotisch bedingter Knorpelschäden beitragen. In verschiedenen Bereichen des Gelenks konnten zudem sympathische Nervenfasern sowie der sympathische Neurotransmitter Noradrenalin (NE) nachgewiesen werden. NE inhibiert die chondrogene Differenzierungskapazität von BMSCs und kann so zur Pathogenese der Osteoarthrose (OA) beitragen. Unbekannt ist zum derzeitigen Zeitpunkt, inwiefern NE die Proliferation von humanen BMSCs beeinflusst. Ziel unserer Studie war, den Einfluss von NE auf die Proliferationskapazität humaner BMSCs zu untersuchen und beteiligte intrazelluläre Signalwege zu identifizieren.
Zu diesem Zweck wurden BMSCs von Patienten nach stattgehabtem Gelenktrauma (Trauma BMSCs) und von Patienten mit diagnostizierter OA (OA BMSCs) untersucht. Zunächst erfolgte eine Analyse des Genexpressionsmusters der verschiedenen Adrenorezeptoren (ARs). Anschließend wurden sowohl Trauma als auch OA BMSCs mit NE in unterschiedlichen Konzentrationen sowie mit NE in Kombination mit verschiedenen AR-Antagonisten (Doxazosin (α1), Yohimbin (α2) oder Propranolol (β2)) behandelt. Die Aktivierung der AR-gekoppelten Signalwege wurde anhand der Phosphorylierung der beiden Hauptsignalwege der extrazellulären signalregulierten Kinasen 1/2 (ERK1/2) und der Proteinkinase A (PKA) via Western Blot untersucht.
Die Genexpression diverser AR-Subtypen konnte in Trauma (α2B-, α2C- und β2-AR) und OA BMSCs (α2A-, α2B- und β2-AR) nachgewiesen werden. Die Behandlung mit NE in hohen Konzentrationen führte zu einer statistisch signifikanten Inhibition der Proliferation von Trauma und OA BMSCs. Die Behandlung mit NE in niedrigen Konzentrationen hatte hingegen keinen Einfluss auf die Proliferation von Trauma und OA BMSCs. Sowohl ERK1/2 als auch PKA wurden in Trauma und OA BMSCs nach Behandlung mit NE aktiviert. Lediglich der β2-Antagonist Propranolol konnte sowohl die Effekte auf die Proliferation als auch auf die Aktivierung von ERK1/2 und PKA aufheben. Doxazosin und Yohimbin hatten hingegen keinen signifikanten Einfluss auf die Proliferation sowie die ERK1/2- und PKA-Phosphorylierung.
Unsere Untersuchungen zeigen, dass NE die Proliferation von Trauma und OA BMSCs konzentrationsabhängig inhibiert. Dieser Effekt wird vornehmlich über eine β2-AR-gekoppelte ERK1/2- und PKA-Aktivierung vermittelt. Über diesen Mechanismus kann NE das regenerative Potential von humanen BMSCs verringern und somit zur Pathogenese der OA beitragen. Über eine zielgerichtete Beeinflussung des β2-Signalweges könnten sich zukünftig neue therapeutische Optionen bei der Behandlung osteoarthrotisch oder traumatisch bedingter Knorpelschäden ergeben.
Molecular oxygen (O2) is essential for numerous metabolic processes. Not surprisingly, hypoxia and the resulting adaptations play a pivotal role in pathophysiology, e.g., in cancer or in inflammatory diseases. Of note, myeloid cells are known to accumulate in hypoxic regions such as tumor cores or rheumatoid arthritis joints and may contribute to disease progression. While most studies so far concentrated on transcriptional adaptation by the hypoxia-inducible factors (HIF) 1 and 2 under short term hypoxia, prolonged oxygen deprivation and alternative post-transcriptional regulation are rather poorly investigated.
Consequently, the aim of the study was to generate a comprehensive overview of mRNA de novo synthesis and degradation and its contribution to total mRNA changes in monocytic cells in the course of hypoxia.
To this end, I used thiol-linked alkylation for the metabolic sequencing of RNA (SLAM-Seq) to characterize RNA dynamics under hypoxia. Specifically, I labeled monocytic THP-1 cells under normoxia (N), acute hypoxia (AH; 8 h 1% O2), or chronic hypoxia (CH; 72 h 1% O2) with 4-thiouridine (4sU), which allows for transcriptome-wide identification of de novo synthesized mRNAs and estimation of their half-lives. Total mRNA expression analyses revealed that most changes occurred under CH. Considering that HIF accumulation and resulting transcriptional regulation was shown to decline again under CH, I further analyzed the impact of RNA stability on gene expression. I observed a global reduction in RNA half-lives under hypoxia, indicative for the attenuation of energy-consuming protein synthesis upon oxygen deprivation. Moreover, I observed a subgroup of hypoxic destabilized transcripts with resulting decreased mRNA expression under CH, which consisted of 59 nuclear-encoded mitochondrial mRNAs. This might prevent futile production of new mitochondria under conditions, where mitochondria are even actively degraded to prevent production of detrimental reactive oxygen species.
While stability-regulated transcripts were mainly destabilized under hypoxia, the vast majority of differentially de novo synthesized transcripts were upregulated.
Functional analyses revealed not only hypoxia, but also cholesterol homeostasis and inflammatory response as top enriched terms, corroborating findings on total mRNA level. Focusing on hypoxia-altered cholesterol metabolism, I observed an 9 accumulation of early and a decrease in late cholesterol precursors, which are separated by several oxygen-dependent enzymatic steps. Although total cholesterol levels were only slightly reduced, my data indicate locally lowered endoplasmic reticulum (ER) cholesterol levels under hypoxia, which cause feedback activation of the ER cholesterol-sensing transcription factor sterol regulatory element-binding protein 2 (SREBP2) and induction of cholesterol biosynthesis enzymes. Interestingly, a broad range of interferon-stimulated genes (ISGs), mainly known for their antiviral function, was also induced under hypoxia with similar kinetics as SREBP2 targets, suggesting an immunometabolic crosstalk. While the availability of certain cholesterol biosynthesis intermediates as well as a direct involvement of SREBP2 seemed rather unlikely to cause hypoxic ISG induction, changes in intracellular cholesterol distribution appeared crucial for the hypoxic induction of chemokine-ISGs. Mechanistically, I found that MyD88-dependent toll-like receptor 4 (TLR4) signaling contributes to enhanced hypoxic ISG induction, likely sensitized by changes in cholesterol dynamics. Importantly, hypoxia amplified induction of chemokine-ISGs in monocytes upon treatment with severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) spike protein via TLR4 similarly as after addition of infectious virus, which might contribute to systemic inflammation in hypoxemic patients with severe coronavirus disease-2019 (COVID-19).
Taken together, I comprehensively analyzed RNA dynamics in hypoxic monocytes. Specifically, I identified RNA stability as a modulating mechanism to limit production of mitochondria under oxygen-restricted conditions. Moreover, I characterized the immunometabolic crosstalk between disturbed cholesterol homeostasis and spontaneous induction of interferon (IFN)-signaling in hypoxic monocytes, which might contribute to systemic inflammation in severe cases of COVID-19.
Die Pathophysiologie der Bandscheibendegeneration (intervertebral disc degeneration, IVDD) und ihre molekularen Mechanismen sind noch in weiten Teilen unverstanden. Ihre Ursachen und Risikofaktoren sind vielfältig und schließen unter anderem Alter, Geschlecht, Umwelteinflüsse oder mechanische Belastungen mit ein.
Für das der Bandscheibe eng verwandte Knorpelgewebe wurde in aktuellen Studien der Einfluss des Sympathikus bzw. dessen Neurotransmitters Noradrenalin (NE) via adrenerger Rezeptoren (AR) auf die Zellproliferation, die Expression von Molekülen der extrazellulären Matrix und somit auch auf die Degeneration beschrieben. In Bandscheiben wurde bereits das Vorhandensein von sympathischen Nervenendigungen nachgewiesen, allerdings wurde die Expression der Adrenozeptoren hier noch nie untersucht. Das Ziel der vorliegenden Arbeit war also die Analyse der ARs im Gewebe der Bandscheibe und die Evaluation der Korrelation mit der Bandscheibendegeneration.
Das für die Analyse benötigte Gewebe stammt von Patienten, bei welchen eine Wirbelkörperverblockung (Spondylodese) durchgeführt wurde. Im Rahmen dieser Spondylodese wird das Bandscheibengewebe des betroffenen Segmentes entfernt. Der Degenerationsgrad der anonymisierten Proben wurde prä- und intraoperativ bestimmt und im entnommenen Gewebe sowie in isolierten Zellen die Expression aller bekannten ARs mittels reverse transcription polymerase chain reaction (RT-PCR) untersucht. Zum Nachweis der ARs auf Proteinebene wurden einzelne humane Proben auch immunhistochemisch analysiert. Des Weiteren wurde anhand von Wildtyp- und sogenannten SM/J-Mäusen, die eine spontane IVDD entwickeln, die Proteinexpression der ARs und der extrazellulären Matrix (ECM) von gesunden und geschädigten Bandscheiben an histologischen Schnitten verglichen. Schließlich wurde an isolierten und kultivierten humanen Zellen ein Stimulationsversuch mit Noradrenalin durchgeführt, um zu prüfen, ob es nach Aktivierung der ARs zu einer intrazellulären Signalweiterleitung kommt.
In Nativgewebe der humanen Bandscheibe konnte die messenger Ribonukleinsäure (mRNA) von α1a-, α1b-, α2a-, α2b-, α2c-, β1- und β2-ARs nachgewiesen werden. Nach siebentägiger Zellkultur im Monolayer präsentierte sich ein nur dezent abweichendes Genexpressionsmuster. Auf Proteinebene war das Signal des β2-AR nur im Bereich des Annulus fibrosus (AF) detektierbar jedoch nicht im Nucleus pulposus (NP). Selbiges war auch in murinen Schnitten festzustellen, wobei sich bei Wildtype (WT)-Mäusen hauptsächlich im inneren AF β2-positive Zellen fanden, während sich das Signal bei der SM/J-Maus weiter in Richtung des äußeren AF und des NP ausdehnte. α2a-AR und α2c-AR waren hingegen auf Proteinebene nicht nachweisbar. Bei der immunhistochemischen Untersuchung relevanter ECM-Moleküle zeigte sich für Kollagen II, Kollagen XII, cartilage oligomeric matrix protein (COMP) und Decorin (DCN) eine Verteilung, die mit der des β2-AR-Signals korreliert. Der Stimulationsversuch in humaner Zellkultur ergab eine Aktivierung der für die ARs relevanten Proteinkinase A (PKA)- und extracellular signal–regulated kinases (ERK1/2) -Signalwege.
In der vorliegenden Arbeit konnte zum ersten Mal die Existenz und Funktionalität von Adrenozeptoren im Bandscheibengewebe nachgewiesen werden. Unterschiede in der Expression der ARs, kombiniert mit Veränderungen der ECM-Zusammensetzung könnten ein Hinweis auf den Einfluss des Sympathikus bei IVDD sein. Die aktuelle demographische Entwicklung und die sich hieraus ergebende gesundheitsökonomische Belastung machen die Ergründung molekularer Mechanismen der IVDD und die daraus resultierende Entwicklung innovativer Behandlungsmethoden zu Kardinalfragen moderner orthopädischer Grundlagenforschung.
Tumor-associated macrophages (TAM) are a major supportive component within neoplasms and by their plasticity promote all phases of tumor development. Mechanisms of macrophage (M Phi) attraction and differentiation to a tumor-promoting phenotype, defined among others by distinct cytokine patterns such as pronounced immunosuppressive interleukin 10 (IL-10) production, are largely unknown. However, a high apoptosis index within tumors and strong M Phi infiltration correlate with poor prognosis. Thus, I aimed at identifying signaling pathways contributing to generation of TAM-like M Phi by using supernatant of apoptotic cancer cells (ACM) as stimulus.
To distinguish novel factors involved in generating TAM-like M Phi, I used an adenoviral RNAi-based approach. The primary read-out was production of IL-10. However, mediators modulating IL-10 were re-validated for their impact on regulation of the cytokines IL-6, IL-8 and IL-12. Following assay development, optimization and down-scaling to a 384-well format, primary human M Phi were transduced with 8495 constructs of the adenoviral shRNA SilenceSelect® library of Galapagos BV, followed by activation to a TAM-like phenotype using ACM. I identified 96 genes involved in IL-10 production in response to ACM and observed a pronounced cluster of 22 targets regulating IL-10 and IL-6. Principal validation of five targets of the IL-10/IL-6 cluster was performed using siRNA or pharmacological inhibitors. Among those, IL-4 receptor-alpha and cannabinoid receptor 2 were confirmed as regulators of IL-10 and IL-6 secretion.
One protein identified in the screen, the nerve growth factor (NGF) receptor TRKA was chosen for in-depth validation, based on its involvement in IL-10, IL-6 and IL-12 secretion from ACM-stimulated human M Phi. TRKA possesses a cardinal role in neuronal development, but compelling evidence emerges suggesting participation of TRKA in cancer development. First experiments using pharmacological inhibitors principally confirmed the involvement of TRKA in IL-10 secretion by ACM-stimulated M Phi and revealed PI3K/AKT and to a lesser extend MAPK p38 as important signaling molecules downstream of TRKA activation. Signaling through TRKA required the presence of its ligand NGF, as indicated by NGF neutralization experiments. NGF was not induced by or present in ACM, but was constitutively secreted by M Phi. Interestingly, M Phi responded to authentic NGF with neither AKT and p38 phosphorylation nor IL-10 production. TRKA is well known to be transactivated by other receptors and in neurons its cellular localization is decisive for its function. Inhibitors of common transactivation partners did not influence IL-10 production by human M Phi. Rather, ACM-treatment provoked pronounced translocation of TRKA to the plasma membrane within 10 minutes as observed by immunofluorescence staining. Consequently, I was intrigued to clarify mechanisms of TRKA trafficking in response to ACM.
The bioactive lipid sphingosine-1-phosphate (S1P) has been previously identified as important apoptotic cell-derived mediator involved in TAM-like M Phi polarization. Indeed, I observed S1P and src kinase involvement in ACM-mediated IL-10 induction. Furthermore, inhibition of S1P receptor (S1PR) signaling or src kinase activity prevented TRKA translocation, whereas a TRKA inhibitor or anti-NGF did not block TRKA trafficking to the plasma membrane in response to ACM. Thus, autocrine secreted NGF activated TRKA to promote IL-10 secretion, which required previous S1PR/src-dependent translocation of TRKA to the plasma membrane. Following the detailed analysis of IL-10 regulation, I was interested whether other TAM phenotype markers were influenced by ACM and whether their expression was regulated through TRKA-dependent signaling. Five of six markers were up-regulated on mRNA level by ACM, and secretion of IL-6, IL-8 and TNF-alpha was triggered. S1PR-signaling was essential for induction of all but one marker, whereas TRKA signaling was only required for cytokine secretion. Interestingly, none of the investigated TAM markers was regulated identically to IL-10, emphasizing a tight and exclusive regulation machinery of this potent immunosuppressive cytokine.
Finally, I aimed to validate the in vitro findings in human ACM-stimulated M Phi. Therefore, I isolated murine TAM as well as other major mononuclear phagocyte populations from primary oncogene-induced breast cancer tissue. Indeed, TRKA-dependent signaling was required for spontaneous cytokine production selectively by primary murine TAM. Besides IL-10, the TRKA pathway was decisive for secretion of IL-6, TNF-alpha and monocyte chemotactic protein-1, indicating its relevance in cancer-associated inflammation.
In summary, my findings highlight a fine-tuned regulatory system of S1P-dependent TRKA trafficking and autocrine NGF signaling in TAM biology. Both factors, S1P as well as NGF, might be interesting targets for future cancer therapy.
Prostaglandin D2 (PGD2) is involved in a variety of physiological and pathophysiological processes, but its role in fever is poorly understood and the data obtained so far are rather controversial. Here we investigated the effects of central PGD2 delivery and of systemic prostaglandin D synthase (PGDS) or cyclooxygenase (COX) inhibition on core body temperature (TC) and on prostaglandin levels in the cerebrospinal fluid (CSF) of rats. Both PGE2 and PGD2 were detectable in CSF samples from control rats (6.2 ± 1.1 and 17.3 ± 3.1 pg/ml, respectively). Lipopolysaccharide (LPS) injection (50 μg i.p.) induced fever during the 5-hour observation period. Five hours after LPS injection, the levels of PGE2 and PGD2 were increased in the CSF about 90-fold (541.0 ± 47.5 pg/ml) and 5-fold (95.4 ± 23.1 pg/ml), respectively. Administration of PGD2 (50 - 500 ng) into the cisterna magna (i.c.m) evoked a delayed fever response in a dose-dependent manner that was accompanied by increased levels of PGE2 in the CSF. RT-PCR analyses revealed that the increased levels of PGE2 after PGD2 administration were not caused by up-regulation of COX-2 or microsomal prostaglandin E synthase 1 (mPGES-1) in the hypothalamus. Interestingly, i.c.m. pretreatment of animals with PGD2 considerably sustained the pyrogenic effects of i.c.m. administered PGE2. Pretreatment with a novel PGDS inhibitor, EDJ300520 (10 – 40 mg/kg p.o.), 1 h prior to the LPS injection impaired the LPS-induced increase of both PGD2 and PGE2 in the CSF and inhibited the fever response. In contrast, administration of EDJ300520 3 h after LPS injection did not ameliorate the LPS-induced fever. Accordingly, the concentration of PGE2 in the CSF was not decreased after EDJ300520 treatment. However, the CSF levels of PGD2 were reduced after administration of a high dose of EDJ300520 (40 mg/kg). We also investigated the effects of antipyretic drugs on the CSF levels of PGE2 and PGD2 during LPS-induced fever. Four antipyretic drugs with different mechanisms of action were used, including ibuprofen (5 - 20 mg/kg), celecoxib (10 - 50 mg/kg), SC560 5 - 20 mg/kg), and paracetamol (50 - 150 mg/kg). Each drug was used in three different doses and was orally administered 3 h after the LPS injection. All drugs were capable to attenuate the LPS-induced fever. The decrease of TC paralleled the reduction of PGE2 levels in the CSF. Of note, there was a tendency to reduced PGD2 levels in the CSF after treatment with the antipyretic drugs. However, only SC560 and the high dose of celecoxib (50 mg/kg) reduced the PGD2 levels significantly. In summary, our experiments underscore the pivotal role of PGE2 as the principal downstream mediator of fever. Moreover, we demonstrate that PGD2 is also involved in the mechanisms underlying fever. Our data suggest that PGD2 exerts an indirect pyrogenic effect by modulating the availability of PGE2 in the CSF. Additional studies are needed to explore the exact mechanism by