Während der letzten Jahrzehnte ist eine steigende Inzidenz von Infektionen durch Pilze der Ordnung Mucorales zu beobachten. Rhizopus arrhizus ist der häufigste Erreger dieser lebensbedrohlichen Infektionen, die vor allem immunsupprimierte Patienten betreffen. Aufgrund der oft schwierigen Diagnosestellung und limitierter therapeutischer Optionen liegt derzeit die Letalität von Mucormykosen zwischen 50 bis 100 %. Eine Voraussetzung für die Etablierung neuer Biomarker oder immuntherapeutischer Strategien ist ein verbessertes Verständnis der immunpathologischen Prozesse bei der Abwehr von Mucorales. In dieser Arbeit wurden daher verschiedene Immunzellpopulationen durch ruhende und ausgekeimte Stadien von R. arrhizus stimuliert und anschließend deren proinflammatorische Immunantwort gemessen. Als Vergleich diente die proinflammatorische Immunantwort der untersuchten Immunzellen nach Stimulation mit Aspergillus fumigatus. Darüber hinaus war es Gegenstand dieser Arbeit, zu charakterisieren welche Pattern Recognition Receptors (PRRs) an der Erkennung von Mucorales durch verschiedene innate Immunzellen beteiligt sind. Zugleich wurde untersucht, ob unterschiedliche Morphotypen der Pilzspezies Auswirkungen auf die Stimulation der jeweiligen PRRs haben. Hierfür wurden Koinkubations-Experimente mit neutrophilen Granulozyten sowie Peripheral Blood Mononuclear Cells (PBMCs), Monozyten und monocyte derived dendritic cells (moDCs) mit verschiedenen Morphotypen von R. arrhizus durchgeführt. Die Rezeptoren TLR2, TLR4 und/oder Dectin-1 wurden dabei durch neutralisierende Antikörper oder RNA-Interferenz blockiert. Ausgekeimte Stadien von A. fumigatus sowie R. arrhizus induzierten eine erhöhte ROS-Freisetzung in Neutrophilen, die durch isolierte oder kombinierte Blockade von TLR2, TLR4 und Dectin-1 abgeschwächt wurde. Ebenso wurde die Phagozytoseaktivität neutrophiler Granulozyten gegenüber R. arrhizus-Konidien durch Blockade von TLR4 und Dectin-1 deutlich reduziert. Im Gegensatz zu A. fumigatus induzierten sowohl ruhende Konidien als auch ausgekeimte Stadien (Keimschläuche und Hyphen) von R. arrhizus eine robuste pro-inflammatorische Zytokinantwort durch moDCs. Nach Inhibition der Dectin-1 Expression durch RNA-Interferenz zeigte sich die Transkription und Sekretion von Interleukin-1β in Gegenwart aller drei untersuchten Morphotypen von R. arrhizus deutlich vermindert (Transkription um 46 bis 68 % und Sekretion um 75 bis 79 % vermindert). Diese Ergebnisse legen nahe, dass Dectin-1 ein wichtiger Mediator bei der Einleitung der innaten Immunantwort verschiedener Zelltypen gegen R. arrhizus ist. Diese Beobachtung sollte in weiteren Studien eingehender untersucht werden, z. B. um die Eignung von Dectin-1 als Rezeptor für zelltherapeutische Ansätze wie T-Zell-Konstrukte mit chimären Antigen-Rezeptoren zu evaluieren. / While Aspergillus species remain the most common cause of opportunistic mold infections, emerging pathogens such as Mucorales account for an increasing share of invasive mycoses in immunocompromised patients. Unspecific clinical presentation, lack of reliable biomarkers, and limited therapeutic options contribute to poor outcomes of this devastating infection. Depending on the site of infection, mortality rates of mucormycoses reach up to 100%, highlighting an unmet need for advances in diagnostic and therapeutic strategies. To facilitate improvements in immune biomarker development and immunotherapy, better understanding of host defense and host-pathogen interplay is warranted. Therefore, this project sought to assess the activation of proinflammatory immune responses by resting and germinated stages of Rhizopus arrhizus, the most common pathogenic Mucorales species. Furthermore, we aimed to characterize the pattern recognition receptors (PRRs) which are involved in the detection of R. arrhizus. To that end, we co-incubated polymorphonuclear neutrophils (PMNs), peripheral blood mononuclear cells (PBMCs), monocytes, and monocyte derived dendritic cells (moDCs) from healthy donors with different morphotypes of R. arrhizus and compared immune responses to A. fumigatus stimulation. Oxidative burst of PMNs was only stimulated by germinated morphotypes of both fungi but not by resting conidia. Inhibition of TLR2, TLR4 and Dectin-1 by specific blocking antibodies significantly reduced the secretion of reactive oxygen species in response to R. arrhizus germlings and hyphae. Similarly, phagocytosis of R. arrhizus spores by PMNs was significantly reduced by blockade of TLR4 and Dectin-1, further corroborating a functional role of these receptors in the recognition of Mucorales. Co-culture studies with PBMCs, monocytes, and moDCs revealed that both resting and germinated stages of R. arrhizus induce a proinflammatory cytokine response of mononuclear phagocytes, whereas conidia of A. fumigatus were largely inert. To determine a functional role of Dectin-1 in the recognition of R. arrhizus morphotypes by mononuclear cells, moDCs were transfected with CLEC7A-siRNA, resulting in a knockdown of the Dectin-1 gene. The transfected moDCs were stimulated with different A. fumigatus and R. arrhizus morphotypes and mRNA expression and secretion of IL-1β were determined by qPCR and ELISA. Significantly weaker induction of IL-1β by germinated stages of both fungi and R. arrhizus spores was seen in siCLEC7A-transfected moDCs. Collectively, these results indicate that major pattern recognition pathways with known roles in the detection of A. fumigatus are also pivotal to mount a proinflammatory immune response to R. arrhizus.

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The innate immune system houses cellular signaling proteins called pattern recognition receptors (PRRs) that are responsible for recognizing highly-conserved molecular patterns associated with pathogens or damage to elicit an immune response. However, NLRX1 is a unique PRR in the NOD-like receptor (NLR) family that instead functions to attenuate pro-inflammatory pathways that are activated by other PRRs, such as NF-κB and type-1 interferon signaling which both have implications in cancer. NLRX1 can regulate additional cancer-associated pathways, such as MAPK and AKT, and cancer-associated functions like metabolism and reactive oxygen species (ROS) production. Interestingly, depending on the type and subtype of cancer, NLRX1 can either be tumor promoting or tumor suppressing. Here, we investigate the role of NLRX1 in two deadly cancers: triple-negative breast cancer (TNBC) and pancreatic cancer. In a murine mammary tumor model that highly mimics TNBC, we discovered that NLRX1 is protective against disease burden in vivo when NLRX1 is expressed in healthy host cells. NLRX1 exerts its protection through limiting the recruitment of eosinophils to the tumor, suppressing epithelial-mesenchymal transition (EMT), and attenuating the formation of the metastatic niche. Conversely, when NLRX1 is instead expressed by the mammary tumor cells, NLRX1 promotes cancer-associated characteristics in vitro and disease burden in vivo by promoting EMT. This indicates that the role of NLRX1 in TNBC is highly dependent on cellular context. Conversely, in murine pancreatic cancer cells, we found that NLRX1 expression by the tumor cells is protective against cancer-associated characteristics in vitro, and that this is likely driven by NF-κB, MAPK, AKT, and inflammasome signaling with a potential to also limit immune evasion. Together, this research indicates that the role of NLRX1 can be highly variable based on the cell and tumor type and identifies the underlying mechanisms through which NLRX1 functions in these two cancer models. This is critical information for drug development initiatives so therapies can be developed that target NLRX1 in the appropriate cell type and in the appropriate disease. / Doctor of Philosophy / Inflammation, which is characterized by redness, heat, pain, swelling, and sometimes loss of function, is a critical way in which our bodies fight infections and repair tissue damage. However, chronic inflammation occurs when our bodies are unable to turn inflammation off and can result in cancerous mutations. Therefore, the successful resolution of inflammation is critical to maintaining inflammatory balance and has previously been dubbed the "Goldilocks Conundrum". The immune system houses a class of cellular signaling proteins called pattern recognition receptors (PRRs), which often function to turn inflammation on. However, a unique PRR in the NOD-like receptor (NLR) family called "NLRX1" functions to turn inflammation off and therefore plays an important role in preventing damaging chronic inflammation. NLRX1 has historically been studied in the context of infectious diseases, but because NLRX1 is involved in inflammation and because inflammation is a critical factor of cancer, its role as a tumor suppressor or tumor promoter has recently become an area of interest. NLRX1 has also been found to regulate biological pathways beyond inflammation that are also important for cancer initiation and progression. Interestingly, depending on the type and subtype of cancer, NLRX1 can either be tumor promoting or tumor suppressing. Here, we investigate the role of NLRX1 in two deadly cancers: triple-negative breast cancer (TNBC) and pancreatic cancer. In a mouse mammary tumor model that highly mimics TNBC, we discovered that NLRX1 is protective against disease burden when NLRX1 is expressed in healthy, non-tumor cells. NLRX1 exerts its protection through impacting the immune cells recruited to the tumor, limiting the ability of the tumor cells to leave the original tumor and spread throughout the body in the process known as metastasis, and suppressing the formation of a favorable tumor metastasis environment in the lung. Conversely, when NLRX1 is instead expressed by the mammary tumor cells, NLRX1 promotes disease burden by helping tumor cells leave the original tumor and spread throughout the body. This indicates that the role of NLRX1 in TNBC is highly dependent on cellular context, including if the cell is healthy or cancerous. Conversely, in mouse pancreatic cancer cells, we found that NLRX1 expression by the tumor cells is protective against cancer-associated characteristics. Together, this research indicates that the role of NLRX1 can be highly variable based on the cell and tumor type. This is critical information for drug development initiatives so therapies can be developed that turn NLRX1 on or off in the appropriate cell type and in the appropriate disease.

AIM: The aim of this study was to determine the expression of microbial nucleic acid receptors including Toll like receptor 9 “TLR-9”, DNA-dependent activator of interferon-regulatory factors “DAI” and absent in melanoma “AIM-2” in chronic periodontitis (P) versus healthy (H) tissues. METHODS: 33 chronic periodontitis (P) and 27 periodontally-healthy (H) gingival biopsies were included. The gene and protein expression for each receptor was determined using real-time quantitative PCR and immunohistochemistry. RESULTS: Our results revealed statistically significant up-regulation of TLR-9 (p<0.006) and DAI (p<0.001) gene expression in P tissues compared to H sites. We were also able to demonstrate significant correlation among three DNA receptors (p<0.05). Immunohistochemistry further confirmed the expression of DNA sensors in gingival tissues. CONCLUSION: This study highlights a possible role for nucleic acid sensing in periodontal inflammation. Further investigations will determine whether cytoplasmic receptors and their ligands can be targeted to improve clinical outcomes in periodontitis.

OBJECTIVES: Bacterial infections in the normally sterile environment of the middle ear cavity usually trigger host immune response, whereby the innate immune system plays a dominant role as the host’s first line of defense. In this study we evaluated the expression levels of Toll-like receptors (TLRs) -2, -4, -5, -9, and nucleotidebinding oligomerization domain-containing proteins (NODs) -1 and -2, all of which are related to bacterial infection in pediatric patients with otitis media with effusion (OME). METHODS: The study sample consisted of 46 pediatric patients with OME, all of whom had ventilation tubes inserted. The expression levels of TLR-2, -4, -5, -9, NOD-1 and -2 mRNA in middle ear effusion were assessed by polymerase chain reaction. RESULTS: All effusion fluid samples collected from patients with OME showed expression of TLR-2, -4, -5, -9, NOD-1, and -2 mRNA. However, we found no correlations among expression levels of pattern recognition receptors (PRRs) in relation to characteristics of exudates, presence of bacteria, or frequencies of ventilation tube insertion (p>0.05). CONCLUSION: Our findings suggest that exudates of OME patients show PRR expressions that are related to the innate immune response regardless of the characteristics of effusion fluid, presence of bacteria in exudates, or frequency of ventilation tube insertion.

The adenosine deaminase acting on RNA (ADAR) enzymes catalyse the hydrolytic deamination of adenosine (A) to inosine (I) in double stranded (ds) RNA. Mutations in ADAR1 underlie the autoimmune disorder Aicardi Goutiѐres syndrome (AGS). Patients with AGS display heightened levels of type I interferon (IFN) and IFN stimulated genes (ISGs). The first aim of my thesis was to determine whether the mutations found in the human ADAR1 gene affected RNA editing. I found that the ADAR1 mutants identified in the AGS patients have reduced editing activity. Interestingly, the mutations have a greater effect on the IFN-inducible cytoplasmic isoform, ADAR1p150 than on the constitutive ADAR1p110 isoform. These results imply that A-to-I editing plays a role in regulating the type I IFN response. The Adar1 null mouse dies by E12.5 with a type I IFN signature similar to that observed in the AGS patients. The second aim of my thesis was to characterize the immune signalling pathway aberrantly activated in the absence of Adar1. A colleague in our research group rescued the Adar1 null mouse to birth by blocking the cellular response to cytoplasmic dsRNA by generating a double mutant with the mitochondrial antiviral signalling adaptor, Mavs. In the Adar1-/-; Mavs-/- mutant I found that the aberrant immune response is rescued at E11.5. This indicates that MAVS is the downstream adaptor in the aberrant immune response that underlies the embryonic lethality in the Adar1-/- mouse. The third aim of my thesis was to determine if the lack of inosine modification within cellular RNA was triggering the aberrant immune response in the Adar1-/- mouse. To study this, Adar1-/-; p53 -/- mouse embryonic fibroblasts (MEFs) were generated. By reintroducing various ADAR isoforms into the Adar1-/-; p53 -/- MEFs I found that to rescue the aberrant immune response requires both catalytic activity and the location of an ADAR protein within the cytoplasm. Moreover, I demonstrated that transfecting inosine-containing dsRNA oligonucleotides into Adar1-/-; p53 -/- MEFs suppresses the aberrant immune response. Overall my results suggest that A-to-I editing by ADAR1 is an essential RNA modification that is required by the cell to distinguish between ‘self’ and ‘non-self’ RNA. Editing of cellular RNAs prevents an autoimmune response whereas editing of viral RNA may act to suppress a heightened antiviral immune response and prevent long-term damage to the cell.

Orientador: James Venturini / Resumo: Dermatofitoses são infecções fúngicas causadas por fungos dermatofíticos que geralmente afetam a camada córnea da pele, cabelos e unhas, mas sob certas condições podem apresentar envolvimento invasivo. A espécie Trichophyton rubrum é o dermatófito mais frequentemente isolado em espécimes clínicos. Além disso, pacientes com diabetes mellitus tipo II (DM-II) são mais suscetíveis a esta infecção fúngica e a relação dermatófito-hospedeiro é pouco compreendida. Contudo, o perfil pró-inflamatório observado em pacientes com hiperglicemia é uma condição complicadora nessa relação. Os receptores de reconhecimento padrão (PRRs) presentes nos fagócitos, como os receptores de manose (MR), dectina-1, toll-like (TLR) -2 e -4, reconhecem estruturas fúngicas e são responsáveis por desencadear mecanismos de ativação e regulação celular durante o processo inflamatório. Assim, o presente estudo tem como objetivo determinar o papel desses receptores na ativação de monócitos de pacientes com DM-II e dermatofitoses por T. rubrum. Exoantígenos de T. rubrum foram obtidos de pacientes com diabetes e indivíduos normoglicêmicos. Os PRRs foram bloqueados em monócitos e monócitos THP-1 de pacientes com DM-II e indivíduos normoglicêmicos desafiados com Exo_Tr_NG e Exo_Tr_DM. Posteriormente, o TNF e MIP-1α e IL-1β foram dosados. A expressão desses receptores foi avaliada por citometria de fluxo. Nenhuma diferença estatística foi observada no ensaio de inibição de monócitos de pacientes diabéticos e indivíd... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Dermatophytoses are fungal infections caused by dermatophytic fungi that usually affect the cornea layer of the skin, hair and nails, but under certain conditions may present invasive involvement. The species Trichophyton rubrum is the dermatophyte most often isolated in clinical specimens. In addition patients with type II diabetes mellitus (DM-II) are more susceptible to this fungal infection. The dermatophyte-host relationship is poorly understood; However, the pro-inflammatory profile observed in patients with hyperglycemia is a complicating condition in this relationship. The pattern recognition receptors (PRRs) present in phagocytes, such as the mannose (MR), dectin-1, toll-like (TLR) -2 and -4 receptor, recognize fungal structures and are responsible for triggering mechanisms of cellular activation and regulation during the inflammatory process, as well as influence on the polarization of the adaptive immune response. Thus, any interference in this process may impair the host to effectively and pathogenically eliminate pathogens. Thus, the present study aims to determine the role of these receptors in the activation of monocytes of patients with DM-II. Exoantigens of T. rubrum were obtained from patients with diabetes and normoglycemic individuals. PRRs were blocked in THP-1 monocytes and monocytes from DM-II patients and normoglycemic individuals challenged with Exo_Tr_NG and Exo_Tr_DM. Subsequently, TNF and MIP-1α and IL-1β were dosed. The expression of these recepto... (Complete abstract click electronic access below) / Mestre

Σκοπός της μελέτης ήταν η αποσαφήνιση των μηχανισμών με τους οποίους η P.aeruginosa διαντιδρά με τους PRRs των μακροφάγων. Από τα δεδομένα μας οι υποδοχείς TLR2 και Mannose receptor συνεργάζονται προς μέγιστη ενεργοποίηση των μακροφάγων σε απάντηση στη λοίμωξη από P.aeruginosa / The aim of the study was to delineate the mechanisms of P.aeruginosa interaction with specific PRRs on macrophages. Our data suggest that TLR2 and Mannose receptor synergize for maximum activation of human macrophages during Pseudomonas infection.

Nos travaux ont permis d’étudier différents aspects des interactions hôte-pathogènes. L’étude de différents Pattern Recognition Receptors (PRR) autres que les TLR, ainsi que leurs associations a mis en évidence une redondance partielle entre les récepteurs des familles des Scavenger Receptors, lectines de type C et EMR1 in vitro et in vivo dans l’induction de la réponse immunitaire à Mycobacterium tuberculosis. Cette compensation entre les récepteurs contraste avec les rôles indépendants et non redondants des cytokines et de leurs voies associées comme le TNF, l’IL-1R1, L’IFNγR et MyD88, indispensables pour le contrôle de l’infection. Grâce à l’utilisation de nouvelles souris génétiquement modifiées, nous avons pu montrer un rôle minime de la lymphotoxine α dans le contrôle de l’infection par Mycobacterium tuberculosis contrairement au rôle primordial du TNF. Enfin, notre étude s’est poursuivie avec l’analyse de la modulation de la réponse immunitaire de l’hôte par des composants de la paroi des mycobactéries, les PIM. L’élaboration de PIM synthétiques a permis de montrer que ces molécules de faibles poids moléculaires inhibent l’induction des voies TLR4 et TLR2 et possèdent ainsi un potentiel anti-inflammatoire thérapeutique. / In these study, we aimed to investigate different aspects of the host-pathogen interactions. We investigated the involvement of various Pattern Recognition Receptors (PRR) other than TLR, and their associations for the control of M. tuberculosis infection. We highlighted a partial redundancy between members of the Scavenger Receptors family, C-type lectins and EMR1 in response to mycobacteria in vitro and in vivo. This is in sharp contrast with the cytokine pathways like TNF, IL-1R1, IFNγR and MyD88, essentials to control M. tuberculosis infection and which cannot compensate with each other. By using new genetically deficient mice, we showed a limited role for the lymphotoxin α in the control of the infection by Mycobacterium tuberculosis in contrast with the vital role for TNF. Finally, we analysed the modulation of the immune response by mycobacterial cell wall components, PIM. Use of synthetic PIM demonstrated that these small molecules exert an inhibitory activity on TLR4- and TLR2-signaling pathways and may have a therapeutic anti-inflammatory potential.

Zymosan is a particulate yeast preparation that elicits high levels of IL-2 and IL-10 from dendritic cells (DC) and engages multiple innate receptors, including the Syk-coupled receptor dectin-1 and the MyD88-coupled receptor TLR2. Here, we show that induction of IL-2 and IL-10 by zymosan requires activation of ERK MAP kinase in murine DC. Surprisingly, ERK activation in response to zymosan is completely blocked in Syk-deficient DC and unaffected by MyD88 deficiency. Conversely, ERK activation in response to the TLR2 agonist Pam3Cys is completely MyD88 dependent and unaffected by Syk deficiency. The inability of TLR2 ligands in zymosan to couple to ERK may explain the Syk dependence of the IL-2 and IL-10 response in DC and emphasises the importance of Syk-coupled pattern recognition receptors such as dectin-1 in the detection of yeasts. Furthermore, the lack of receptor compensation observed here suggests that responses induced by complex innate stimuli cannot always be predicted by the signalling pathways downstream of individual receptors.