Type I Interferon Production Induced by Streptococcus pyogenes-Derived Nucleic Acids Is Required for Host Protection

Streptococcus pyogenes is a Gram-positive human pathogen that is recognized by yet unknown pattern recognition receptors (PRRs). Engagement of these receptor molecules during infection with S. pyogenes, a largely extracellular bacterium with limited capacity for intracellular survival, causes innate immune cells to produce inflammatory mediators such as TNF, but also type I interferon (IFN). Here we show that signaling elicited by type I IFNs is required for successful defense of mice against lethal subcutaneous cellulitis caused by S. pyogenes. Type I IFN signaling was accompanied with reduced neutrophil recruitment to the site of infection. Mechanistic analysis revealed that macrophages and conventional dendritic cells (cDCs) employ different signaling pathways leading to IFN-beta production. Macrophages required IRF3, STING, TBK1 and partially MyD88, whereas in cDCs the IFN-beta production was fully dependent on IRF5 and MyD88. Furthermore, IFN-beta production by macrophages was dependent on the endosomal delivery of streptococcal DNA, while in cDCs streptococcal RNA was identified as the IFN-beta inducer. Despite a role of MyD88 in both cell types, the known IFN-inducing TLRs were individually not required for generation of the IFN-beta response. These results demonstrate that the innate immune system employs several strategies to efficiently recognize S. pyogenes, a pathogenic bacterium that succeeded in avoiding recognition by the standard arsenal of TLRs

Dendritic cells in cancer immunology and immunotherapy

Dendritic cells (DCs) are a diverse group of specialized antigen-presenting cells with key roles in the initiation and regulation of innate and adaptive immune responses. As such, there is currently much interest in modulating DC function to improve cancer immunotherapy. Many strategies have been developed to target DCs in cancer, such as the administration of antigens with immunomodulators that mobilize and activate endogenous DCs, as well as the generation of DC-based vaccines. A better understanding of the diversity and functions of DC subsets and of how these are shaped by the tumour microenvironment could lead to improved therapies for cancer. Here we will outline how different DC subsets influence immunity and tolerance in cancer settings and discuss the implications for both established cancer treatments and novel immunotherapy strategies.S.K.W. is supported by a European Molecular Biology Organization Long- Term Fellowship (grant ALTF 438– 2016) and a CNIC–International Postdoctoral Program Fellowship (grant 17230–2016). F.J.C. is the recipient of a PhD ‘La Caixa’ fellowship. Work in the D.S. laboratory is funded by the CNIC, by the European Research Council (ERC Consolidator Grant 2016 725091), by the European Commission (635122-PROCROP H2020), by the Ministerio de Ciencia, Innovación e Universidades (MCNU), Agencia Estatal de Investigación and Fondo Europeo de Desarrollo Regional (FEDER) (SAF2016-79040-R), by the Comunidad de Madrid (B2017/BMD-3733 Immunothercan- CM), by FIS- Instituto de Salud Carlos III, MCNU and FEDER (RD16/0015/0018-REEM), by Acteria Foundation, by Atresmedia (Constantes y Vitales prize) and by Fundació La Marató de TV3 (201723). The CNIC is supported by the Instituto de Salud Carlos III, the MCNU and the Pro CNIC Foundation, and is a Severo Ochoa Centre of Excellence (SEV-2015-0505).S

Role of toll-like receptor signaling in innate immune cell function

Die Dendritische Zelle (DC) fungiert als eine der potentesten Antigen-Präsentierenden Zellen (APC) und ist eine wichtige Schaltstelle zwischen dem angeborenen und erworbenen Immunsystem. DC erkennen fremde Mikroorganismen, wie Viren und Bakterien, durch Toll-like Rezeptoren (TLR). Ligation von TLR führt zur Aktivierung einer Signalkaskade an deren Ende DC-Reifung und dadurch eine Stimulation der Immunantwort steht. Während die Rolle des Transkriptionsfaktors Nukleärer Faktor kappa B (NFkB) in TLR-Induzierter Reifung und Differenzierung von DC bereits eingehend untersucht wurde, ist über die Funktion der Aktivierungsproteine 1 (AP-1) nur wenig bekannt. AP-1 Moleküle spielen eine wichtige Rolle bei der Expression von Entzündungsmediatoren und Wachstumsfaktoren, sowie in chronisch-entzündlichen Erkrankungen wie Psoriasis. In diesem Projekt wurde mit Hilfe von transgenen Mäusen untersucht, ob und wie AP-1 Transkriptionsfaktoren eine Rolle in TLR-aktivierten DC spielen. Überraschendenderweise führte die Inaktivierung von c-Jun/JunB in konditionellen Mäusen mit Hilfe einer transgenen Mx-cre Linie, welche unter anderem in Knochenmarkszellen von Mäusen aktiv ist (c-jun/jun-mx), innerhalb von 14 Tagen zu einer schweren Entzündung der Haut, einer Rückbildung des Thymus, drastischem Gewichtsverlust, und schlussendlich zum Tod der Mäuse. Nähere Analysen zeigten, dass es in der Haut und der Milz, zu einer Verschiebung innerhalb der Immunzellpopulationen von lymphoiden zu myeloischen Zellen kam. Weiteres entwickelten Knochenmarks-Kulturen von c-jun/junB-mx signifikant weniger DC. Interessanterweise zeigten c-jun/junB-mx-DC auch Beeinträchtigungen in der Reifung und bei der Aktivierung von Co-Stimulationsmoleküle auf ihrer Zelloberfläche. In Mäusen mit nur einem fehlenden AP-1 Transkriptionsfaktor wurde kein vergleichbarer Phänotyp beobachtet, jedoch zeigten die in vitro generierte DC Funktionsstörungen. DC in denen c-Jun, JunB, c-Fos oder c-Jun/JunB fehlten zeigten Beeinträchtigungen in der Produktion von Zytokinen, wie zum Bespiel des monozyten-chemoattraktiven Protein 1 (MCP-1) und des Interleukin 6 (IL6), sowohl in nicht aktiviertem Zustand als auch nach TLR-Stimulation. Weiteres konnte ich zeigen, daß AP-1 Proteine die Antigenpräsentation in TLR-aktivierten DC regulieren. Diese Studie zeigt klar auf, das AP-1 Transkriptionsfaktoren eine wichtige Rolle in der Differenzierung und in diversen Funktionen von DC spielen, welche auch in Entzündungs- und Krebserkrankungen Einfluß haben könnten. Die auf DC-basierende Immuntherapie bekommt einen immer größeren Stellenwert. Im Blut findet man zwei natürliche DC Subtypen, nämlich die myleoiden DC und die plasmazytoiden DC (pDC). Einer der auffälligsten Unterschiede dieser beiden Zellen ist sicherlich das TLR Repertoire, welches auf deren unterschiedliche Aufgaben im Immunsystem hinweist. Um mehr über den Einfluss von TLR auf eine Immunantwort zur Krebsabwehr herauszufinden, wurde in einem weiteren Teil dieser Arbeit der Wirkmechanismus des Immunmodulators Imiquimod näher analysiert. Das Imidazoquinolin Imiquimod ist ein Ligand des TLR7 und wird in der Klinik erfolgreich zur topischen Behandlung von Basaliomen und bei bestimmten Melanomformen eingesetzt. Wir konnten in einem Melanom-Mausmodell zeigen, dass die Präsenz von pDC, im Tumor mit dem Therapieeffekt korreliert. PDC sind jene DC der Maus, welche TLR7 exprimieren. Der genaue Wirkmechanismus von Imiquimod konnte jedoch bislang nicht geklärt werden. Ich konnte zeigen, daß Hautzellen, wie Keratinozyten oder dermale Fibroblasten durch die Imiquimod-Behandlung direkt aktiviert werden in dem sie einerseits sterben und andererseits Zytokine sezernieren. Interessanterweise wirkt Imiquimod auf Zellen der Epidermis unabhängig von TLR7, während dermale Zellen TLR7 brauchen um stimuliert zu werden. Mit Tumorwachstumsstudien in verschiedenen Knock-out Mausmodellen konnte aber gezeigt werden, dass der therapeutische Effekt von Imiquimod von der TLR7-Expression in Immunzellen abhängig ist und Hautzellen kaum eine Rolle spielen. Mit Hilfe von Mäusen, in welchen verschiedene Immunzellpopulationen fehlten, konnte weiteres gezeigt werden, dass im Speziellen pDC für den Mechanismus von Imiquimod verantwortlich sind. pDC werden durch die topische Behandlung von Imiquimod in den Tumor rekrutiert und durch TLR7 aktiviert. Durch diese Stimulierung produzieren pDC Typ I Interferon, welches einerseits direkt auf die Tumorzellen wirken kann oder andererseits pDC selbst stimuliert um zelltoxische Moleküle hoch zu regulieren. Diese zelltoxischen Moleküle können dann den Zelltod von Tumorzellen induzieren. In dieser Studie konnte erstmals die genaue Wirkungsweise von Imiquimod gezeigt werden sowie dass pDC nicht nur indirekt eine Rolle bei der Aktivierung einer Immunantwort spielen, sondern abhängig von der Stimulierung auch direkt Tumorzellen angreifen können.Dendritic Cells (DCs) are the most potent antigen presenting cells and have long been recognized as key regulators of the immune system. DCs can be activated by Toll-like receptors (TLR) which recognize different types of invading microbes thereby stimulating immune responses. However, little is known about the intracellular mechanisms regulating DC function. Whereas the role of NFkB signaling is well studied not much is known about the role of activation protein 1 (AP-1) transcription factors in the activation, maturation and differentiation processes of DCs. The integrity of these pathways has been shown to be essential for the function of DCs. By employing genetically modified mice, the role of the AP-1 transcription factors JunB, c-Jun and c-Fos was determined during activation, maturation and function of DCs following TLR triggering in vivo and in vitro. Mice lacking c-jun and junB simultaneously developed a severe skin phenotype with ulcerative lesions in the face region and belly skin accompanied with massive infiltration of myeloid cells, in the skin and spleen. Additionally, bone-marrow dendritic cell-cultures of those mice showed a significant impairment in DC differentiation. In vitro generated DCs were impaired in upregulating co-stimulatory molecules after TLR triggering. Interestingly, these effects could not be observed in cultures lacking single AP-1 proteins. Moreover, AP-1 deficient DCs showed severe impairments in cytokine production, mainly in MCP-1 and IL6, under steady state conditions and upon TLR activation. In addition, the absence of Jun-proteins in DCs influenced the quality of cross-presentation. Whereas the absence of c-Jun and c-Jun/JunB in DCs led to reduced antigen-presentation; JunB deficient DCs induced increased T cell proliferation. So far these data suggested that AP-1 proteins regulate DCs differentiation and function, which further control immune responses under physiologic or pathologic inflammatory conditions. Several studies revealed that DCs are playing a major role in tumor immunity. To date, DC-based immunotherapy is explored worldwide. Two major types of naturally occurring DCs circulate in peripheral blood, myeloid DCs (mDCs) and plasmacytoid DCs (pDCs). These DC subsets express different surface molecules and are suggested to have distinct functions in evoking specific immune responses. The immunological functions of DCs, like sensing for pathogens, presenting antigens or secreting cytokines, are also related to TLRs expressed by DCs. To further analyze the role of TLR ligands in anti-tumor responses I analyzed the mechanisms underlying the function of the immune modulator Imiquimod, a synthetic TLR7/8 ligand, in anti-tumor immune responses. Topical treatment of melanomas with Imiquimod has been shown to induce tumor regression accompanied by an increased infiltration of pDCs, a DCs subset expressing TLR7. However, the mechanism by which Imiquimod mediated the anti-tumor activity was poorly understood. In this study, I investigated if pDCs or other cell types e.g. skin cells, play an active role in anti-tumor immune responses mediated by Imiquimod. Moreover, Imiquimod affected skin cells by either inducing apoptosis in keratinocytes or by increasing MCP-1 levels in the dermis. However, tumor growth studies in Tlr7-/-, Myd88-/- mice or chimeric mice demonstrated that, TLR7 and MyD88 expression is required for the anti-tumor response of Imiquimod and critically depends on their presence on bone marrow derived but not parenchymal cells. Surprisingly, Imiquimod still exerted its therapeutic effects in mice lacking either CD4+ cells, all T cells or NK cells, all of which are believed to be key players in anti-tumor immune responses. Strikingly, depletion of pDCs from tumor bearing mice completely abolished the Imiquimod induced tumor regression. Similar results were obtained in CD8+ cell-depleted mice suggesting that CD8+ pDCs were essential for the anti-tumor response of Imiquimod. When investigating the mechanism of how pDCs were involved in the tumoricidal effect of Imiquimod, I could show that pDCs stimulated with Imiquimod induced specific killing of melanoma cells by upregulating cytolytic molecules like TRAIL and GranzymeB in an IFNAR1 dependent manner. Taken together, this study demonstrated the first time the indispensible role of TLR7 activated pDCs for the anti-tumor effect induced by Imiquimod treatment and underlines the therapeutic potential of TLR ligands for anti-tumor therapy. Furthermore, I could show that Jun-proteins are regulating important features of dendritic cells when activated with TLR ligands. Moreover, the results of these studies will contribute to a better understanding of the role of DCs and TLR signaling in inflammatory pathologies such as skin diseases and cancer.submitted by Barbara DrobitsAbweichender Titel laut Übersetzung der Verfasserin/des VerfassersZsfassung in dt. SpracheWien, Med. Univ., Diss., 2011OeBB(VLID)171558

Imiquimod clears tumors in mice independent of adaptive immunity by converting pDCs into tumor-killing effector cells

Imiquimod is a synthetic compound with antitumor properties; a 5% cream formulation is successfully used to treat skin tumors. The antitumor effect of imiquimod is multifactorial, although its ability to modulate immune responses by triggering TLR7/8 is thought to be key. Among the immune cells suggested to be involved are plasmacytoid DCs (pDCs). However, a direct contribution of pDCs to tumor killing in vivo and the mechanism of their recruitment to imiquimod-treated sites have never been demonstrated. Using a mouse model of melanoma, we have now demonstrated that pDCs can directly clear tumors without the need for the adaptive immune system. Topical imiquimod treatment led to TLR7-dependent and IFN-α/β receptor 1–dependent (IFNAR1-dependent) upregulation of expression of the chemokine CCL2 in mast cells. This was essential to induce skin inflammation and for the recruitment of pDCs to the skin. The recruited pDCs were CD8α+ and induced tumor regression in a TLR7/MyD88- and IFNAR1-dependent manner. Lack of TLR7 and IFNAR1 or depletion of pDCs or CD8α+ cells from tumor-bearing mice completely abolished the effect of imiquimod. TLR7 was essential for imiquimod-stimulated pDCs to produce IFN-α/β, which led to TRAIL and granzyme B secretion by pDCs via IFNAR1 signaling. Blocking these cytolytic molecules impaired pDC-mediated tumor killing. Our results demonstrate that imiquimod treatment leads to CCL2-dependent recruitment of pDCs and their transformation into a subset of killer DCs able to directly eliminate tumor cells