Induction and properties of SOCS1 in myeloid cells by activation of Dectin-1

Die präzise Regulation der Sekretion pro- und anti-inflammatorischer Zytokine stellt einen der wichtigsten Mechanismen bei der Immunantwort gegen pathogene Pilzinfektionen dar. Suppressor of Cytokine Signalling (SOCS) Proteine stellen eine Klasse von induzierbaren feedback Inhibitoren für den JAK/STAT (janus kinase/signal transducer and activator of transcription) Signalweg dar. Diese intrazellulären Proteine werden durch Zytokine, aber auch durch Toll-like Rezeptor (TLR) Liganden induziert. Neben TLRs ist Dectin-1 als weiteres Mitglied der PRRs (pattern recognition receptors) beschrieben. Dectin-1 erkennt Zellwandbestandteile pathogener Pilze. In dieser Arbeit sollte untersucht werden, ob Dectin-1 Aktivierung mit der Induktion von SOCS Proteinen und damit der Modulation von Zytokinantworten einhergeht. Es kann gezeigt werden, dass SOCS1 (mRNA und Protein) nach Dectin-1 Stimulation muriner Makrophagen (BMMs) oder Dendritischer Zellen (BMDCs) induziert wird. Dabei erfolgte die SOCS1 Expression durch einen Dectin-1 Liganden, der keine TLR-Bindekapazität aufweist (modifiziertes Zymosan), direkt und TLR unabhängig. Die SOCS1 Induktion wurde über einen Signalweg vermittelt, der in Zusammenhang mit der SOCS1 Expression noch nicht beschrieben war. Dabei war der Transkriptionsfaktor NF-κB in BMMs, im Gegensatz zu BMDCs, nicht für die SOCS1 Induktion verantwortlich. Nach Dectin-1 Stimulation wurden Tyrosin Kinasen der Src und Syk Familie aktiviert; diese stimulierten wiederum die nachgeschaltete Tyrosin Kinase Pyk2 (Proline rich tyrosine kinase 2). In BMMs induzierte phosphoryliertes Pyk2 die Aktivierung der MAPK (Mitogen-activated kinase) ERK (extracellular signal-regulated kinase). Phosphoryliertes ERK induzierte die NF-κB unabhängige Expression von SOCS1. Eine Inhibition der Pyk2 Aktivierung führte zu einer spezifischen Hemmung der ERK Phosphorylierung sowie der Induktion des SOCS1 Proteins. Die Aktivität der MAP Kinasen JNKII und p38 blieb unbeeinflusst. SOCS1 wurde weiterhin auf seine Wirkung im Dectin-1 und TLR Signalweg untersucht. Es konnte eine inhibitorische Wirkung des SOCS1 Proteins auf den TLR Signalweg beobachtet werden, jedoch nicht auf den Dectin-1 Signalweg. Die Stimulation mit modifiziertem Zymosan führte zu einer verlängerten NF-κB Aktivierung in SOCS1 defizienten BMMs, die zusätzlich über TLR9 stimuliert wurden. Zudem war die TLR9 induzierte Sekretion der Zytokine IL12 und IL10 durch Dectin-1 induziertes SOCS1 Protein inhibiert. Zusätzlich konnte ein signifikanter Anstieg der IL17 Produktion in T-Helfer (Th) Zellen beobachtet werden, der abhängig von der Dectin-1 induzierten SOCS1 Expression in BMMs war. Dazu wurden BMMs aus SOCS1 knockout Mäusen und den entsprechenden Kontrollen via TLR9 und Dectin-1 kostimuliert und mit isolierten Wildtyp CD4+ Th-Zellen kokultiviert. Eine erhöhte IL17 Produktion konnte nur in Kokulturen mit BMMs gezeigt werden, die noch endogenes SOCS1 Protein exprimierten. SOCS1 ist somit assoziiert mit einem Th-Zell Phänotyp mit erhöhter IL17 Produktion. Die Ergebnisse zeigen, dass SOCS1 in Dectin-1 stimulierten myeloiden Zellen über einen neuartigen Signalweg induziert wird. In BMMs wird SOCS1 NF-κB unabhängig exprimiert. Darüber hinaus beeinflusst SOCS1 TLR-Signalwege und Th-Zell Antworten und wirkt somit an der Regulation von Immunantworten bei Pilzinfektionen mit

Hsa-miR-99b/let-7e/miR-125a Cluster Regulates Pathogen Recognition Receptor-Stimulated Suppressive Antigen-Presenting Cells

Antigen-presenting cells (APCs) regulate the balance of our immune response toward microbes. Whereas immunogenic APCs boost inflammation and activate lymphocytes, the highly plastic cells can switch into a tolerogenic/suppressive phenotype that dampens and resolves the response. Thereby the initially mediated inflammation seems to prime the switch of APCs while the strength of activation determines the grade of the suppressive phenotype. Recently, we showed that pathogen recognition receptor-mediated pro-inflammatory cytokines reprogram differentiating human blood monocytes in vitro toward an immunosuppressive phenotype through prolonged activation of signal transducer and activator of transcription (STAT) 3. The TLR7/8 ligand R848 (Resiquimod) triggers the high release of cytokines from GM-CSF/IL-4-treated monocytes. These cytokines subsequently upregulate T cell suppressive factors, such as programmed death-ligand 1 (PD-L1) and indolamin-2,3-dioxygenase (IDO) through cytokine receptor-mediated STAT3 activation. Here, we reveal an essential role for the microRNA (miR, miRNA) hsa-miR-99b/let-7e/miR-125a cluster in stabilizing the suppressive phenotype of R848-stimulated APCs on different levels. On the one hand, the miR cluster boosts R848-stimulated cytokine production through regulation of MAPkinase inhibitor Tribbles pseudokinase 2, thereby enhancing cytokine-stimulated activation of STAT3. One the other hand, the STAT3 inhibitor suppressor of cytokine signaling-1 is targeted by the miR cluster, stabilizing the STAT3-induced expression of immunosuppressive factors PD-L1 and IDO. Finally, hsa-miR-99b/let-7e/miR-125a cluster regulates generation of the suppressive tryptophan (Trp) metabolite kynurenine by targeting the tryptophanyl-tRNA synthetase WARS, the direct competitor of IDO in terms of availability of Trp. In summary, our results reveal the hsa-miR-99b/let-7e/miR-125a cluster as an important player in the concerted combination of mechanisms that stabilizes STAT3 activity and thus regulate R848-stimulated suppressive APCs

IL-1β As Mediator of Resolution That Reprograms Human Peripheral Monocytes toward a Suppressive Phenotype

During infection pathogen-associated molecular patterns activate immune cells to initiate a cascade of reactions leading to inflammation and the activation of the adaptive immune response culminating in the elimination of foreign pathogens. However, shortly after activation of the host defense machinery, a return to homeostasis is preferred to prevent inflammation-induced tissue damage. This switch from the initial immunogenic to the subsequent tolerogenic phase after clearance of the infection can be mediated through highly plastic peripheral monocytes. Our studies reveal that an early encounter with toll-like receptor 7/8-ligand R848 mediates a strong pro-inflammatory monocytic phenotype that primes its own reprogramming toward an immunosuppressive one. Previously, we showed that these R848-treated antigen-presenting cells (APCs) fail to activate allogeneic T cells and induce regulatory T cells (Tregs) through signal transducer and activator of transcription 3 (STAT3)-dependent PD-L1. Here, we further demonstrate that R848-treated APCs suppress CD3/CD28-mediated and dendritic cell-mediated T cell activation and that adenosine and indoleamine 2,3-dioxygenase/kynurenin pathways are involved in tolerance induction. Reprogramming of monocytes after R848 stimulation requires the pro-inflammatory cytokine IL-1β and a boosted IL-6 release. The subsequent autocrine prolonged activation of STAT3 induces direct upregulation of tolerogenic factors which finally downregulate proliferation of activated T cells and mediate Tregs. Thereby our study suggests that inflammatory cytokines, such as IL-1β and IL-6, should be considered as mediators of resolution of inflammation

table_1_Hsa-miR-99b/let-7e/miR-125a Cluster Regulates Pathogen Recognition Receptor-Stimulated Suppressive Antigen-Presenting Cells.PDF

<p>Antigen-presenting cells (APCs) regulate the balance of our immune response toward microbes. Whereas immunogenic APCs boost inflammation and activate lymphocytes, the highly plastic cells can switch into a tolerogenic/suppressive phenotype that dampens and resolves the response. Thereby the initially mediated inflammation seems to prime the switch of APCs while the strength of activation determines the grade of the suppressive phenotype. Recently, we showed that pathogen recognition receptor-mediated pro-inflammatory cytokines reprogram differentiating human blood monocytes in vitro toward an immunosuppressive phenotype through prolonged activation of signal transducer and activator of transcription (STAT) 3. The TLR7/8 ligand R848 (Resiquimod) triggers the high release of cytokines from GM-CSF/IL-4-treated monocytes. These cytokines subsequently upregulate T cell suppressive factors, such as programmed death-ligand 1 (PD-L1) and indolamin-2,3-dioxygenase (IDO) through cytokine receptor-mediated STAT3 activation. Here, we reveal an essential role for the microRNA (miR, miRNA) hsa-miR-99b/let-7e/miR-125a cluster in stabilizing the suppressive phenotype of R848-stimulated APCs on different levels. On the one hand, the miR cluster boosts R848-stimulated cytokine production through regulation of MAPkinase inhibitor Tribbles pseudokinase 2, thereby enhancing cytokine-stimulated activation of STAT3. One the other hand, the STAT3 inhibitor suppressor of cytokine signaling-1 is targeted by the miR cluster, stabilizing the STAT3-induced expression of immunosuppressive factors PD-L1 and IDO. Finally, hsa-miR-99b/let-7e/miR-125a cluster regulates generation of the suppressive tryptophan (Trp) metabolite kynurenine by targeting the tryptophanyl-tRNA synthetase WARS, the direct competitor of IDO in terms of availability of Trp. In summary, our results reveal the hsa-miR-99b/let-7e/miR-125a cluster as an important player in the concerted combination of mechanisms that stabilizes STAT3 activity and thus regulate R848-stimulated suppressive APCs.</p

data_sheet_1_Hsa-miR-99b/let-7e/miR-125a Cluster Regulates Pathogen Recognition Receptor-Stimulated Suppressive Antigen-Presenting Cells.PDF

<p>Antigen-presenting cells (APCs) regulate the balance of our immune response toward microbes. Whereas immunogenic APCs boost inflammation and activate lymphocytes, the highly plastic cells can switch into a tolerogenic/suppressive phenotype that dampens and resolves the response. Thereby the initially mediated inflammation seems to prime the switch of APCs while the strength of activation determines the grade of the suppressive phenotype. Recently, we showed that pathogen recognition receptor-mediated pro-inflammatory cytokines reprogram differentiating human blood monocytes in vitro toward an immunosuppressive phenotype through prolonged activation of signal transducer and activator of transcription (STAT) 3. The TLR7/8 ligand R848 (Resiquimod) triggers the high release of cytokines from GM-CSF/IL-4-treated monocytes. These cytokines subsequently upregulate T cell suppressive factors, such as programmed death-ligand 1 (PD-L1) and indolamin-2,3-dioxygenase (IDO) through cytokine receptor-mediated STAT3 activation. Here, we reveal an essential role for the microRNA (miR, miRNA) hsa-miR-99b/let-7e/miR-125a cluster in stabilizing the suppressive phenotype of R848-stimulated APCs on different levels. On the one hand, the miR cluster boosts R848-stimulated cytokine production through regulation of MAPkinase inhibitor Tribbles pseudokinase 2, thereby enhancing cytokine-stimulated activation of STAT3. One the other hand, the STAT3 inhibitor suppressor of cytokine signaling-1 is targeted by the miR cluster, stabilizing the STAT3-induced expression of immunosuppressive factors PD-L1 and IDO. Finally, hsa-miR-99b/let-7e/miR-125a cluster regulates generation of the suppressive tryptophan (Trp) metabolite kynurenine by targeting the tryptophanyl-tRNA synthetase WARS, the direct competitor of IDO in terms of availability of Trp. In summary, our results reveal the hsa-miR-99b/let-7e/miR-125a cluster as an important player in the concerted combination of mechanisms that stabilizes STAT3 activity and thus regulate R848-stimulated suppressive APCs.</p