TMEM203 is a putative co-receptor of innate immune adaptor protein STING

Acute inflammation is the innate immune defence against environmental disturbances. Macrophages are one of the central immune cells that react to infections and maintain tissue homeostasis, and they exhibit their functions via numerous inflammatory signalling regulators. In addition to previously identified immune mediators, novel proteins involved in inflammation continue to emerge. A previous cDNA functional screening in murine macrophages has identified a novel protein named transmembrane protein 203 (Tmem203) displaying pro-inflammatory characteristics. Tmem203-promoted inflammatory activities were found to be TLR independent but dependent on STING, a cytosolic innate immune adaptor for DNA detection. STING responds to upstream DNA sensors and microbial cyclic dinucleotides, and instigates type I interferon response via TBK1-IRF3 axis. The work in this thesis investigated the function of TMEM203 in STING-dependent type I interferon responses. TMEM203 has been found to colocalise, interact and migrate with STING. Further studies revealed a critical role for TMEM203 in STING-dependent type I interferon response in both human and mouse primary macrophages. We showed that TMEM203-STING association was highly dependent on STING’s N-terminal transmembrane domains. Finally, TMEM203 showed a distinct regulation of STING-interferon signalling between stimulation by natural and synthetic STING ligands, and this difference was also reflected in TMEM203-STING interaction. Thus, this novel mechanism of TMEM203-dependent STING regulation has brought new insights to better understand critical regulators of pathogen infections and interferon-associated autoimmune diseases. Additionally, a brief research was conducted to explore STING regulation in flavivirus infected primary macrophages. Flaviviruses Dengue virus and Zika virus infect humans to cause global pandemics. Dengue virus is known to specifically and potently interrupt STING-interferon pathway. The emerging flavivirus Zika virus is genetically-closely related to Dengue virus and thus it has been hypothesised to adopt similar strategies in STING antagonism. We have investigated Dengue and Zika virus-induced type I interferon stimulated ISG response in the M-CSF differentiated primary macrophage model, and tested the role of STING in such conditions. Contradictory to previous report, our experiments showed a potent and persistent ISG induction in virus-infected macrophages. Prior virus infections were unable to intercept ISG induction cause by STING ligands, whereas the downregulation of STING dampens virus-induced ISG response. Therefore, this primary macrophage model highlights alternative regulatory mechanisms via STING in response to Dengue and Zika virus

Development of poxvirus-derived vectors to analyze novel ISG15 functions

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Medicina Preventiva y Salud Pública y Microbiología. Fecha de Lectura: 28-04-2023The current coronavirus pandemic highlights how essential it is for humankind to develop effective antiviral strategies to protect individuals from infectious outbreaks. In this regard, a better knowledge of the immune system and the sensitivity of pathogens to these strategies is essential. Our research is focused on the use of poxviruses, viral vectors known for their safety and stability largely used in vaccination and cancer studies, in combination with a ubiquitin-like protein, ISG15, with a central role in the antiviral response of the host organism. ISG15 is an interferon-stimulated protein that exists in different forms, depending on its location, with different activities. The ISG15 intracellular form better characterized mechanism is the post-translational modification process, known as ISGylation, through which the ISG15 carboxy terminal motif LRLRGG, essential for this process, is covalently bound to lysine residues of new synthesized target proteins, causing an alteration in the localization and cellular function in the conjugated proteins. While, when ISG15 is released in extracellular space as free molecule act as a cytokine, interacting with LFA-1 receptor and inducing IFN-γ and IL-10 release form NK cells, this form is defined as neutrophil chemotactic factor. This work focuses on expanding the knowledge of ISG15 for its use in biotechnology. Two main objectives have been defined, both based on the use of poxviruses expressing ISG15GG, its wild-type form or ISG15AA, its mutant form unable to ISGylate target proteins. To investigate the immunostimulatory power of ISG15 as adjuvant, we analyze its possible function in prime-boost protocols against HIV-1, responsible of the Acquired Immune Deficiency Syndrome, a major global public health issue with no cure currently available. This study reveals the immunomodulatory properties of either ISG15GG or ISG15AA proteins when expressed by a DNA vector, conferring immune potency to the HIV-1 vaccine candidate MVA-B, with a possible role for ISGylation in the connection between innate and adaptive immunities. We investigated further by studying the expression of ISG15 from a poxvirus platform that reveals the properties of the free form of ISG15. The MVA-Δ3-ISG15AA virus results in increased transcription of Ifn-I both in vitro and in vivo system, its coadministration with MVA-B significantly enhances the magnitude of the HIV-1-specific CD8 T cells in mice acute immune response. Additionally, to highlight the importance of the ISG15 response pathway its highly advantageous to identify the ISGylation targets. The generation of a novel poxviral-based tool to capture ISGylated proteins allow the overexpression of the V5 tagged ISG15GG, or ISG15AA as control, using WR VACV as platform to coimmunoprecipitate interacting proteins. The use of this technique reveals several proteins downregulated in the WR-ISG15AA infected cells, highlighting the impact of ISGylation and allowing the identification of USP3 as target of ISGylation, involved in IFN-I downregulation deubiquitinating RIG-I. This work highlights that ISG15 should be taken into consideration as a novel component to be incorporated in prime boost immunization regimens against HIV-1 infection and advance the hypothesis of USP3 as possible ISGylation candidate involved in the downregulation of IFN-

Microbiota-induced tonic type I interferons instruct a transcriptional, epigenetic and metabolic program that defines the poised basal state of conventional dendritic cells

Border surfaces in the human body are colonized in high density by various microbes, known as microbiota. It has becoming increasingly clear that, depending on environmental factors and host genetics, the microbiota has profound effects on shaping host physiology, including the education of the host’s immune system. In my dissertation, I present evidence that microbiota-derived signals are required to program splenic conventional dendritic cells (cDCs) during steady-state, so that they can immediately respond to pathogen encounter by producing pro-inflammatory cytokines and activating T cells. The molecular mechanisms behind this microbiota-dependent instructive program of cDCs are not well understood though. Here, type I interferons (IFNs), which are constantly produced in steady-state by mainly plasmacytoid (p)DCs, are identified as signaling molecules required for the cDC instruction process. Transcriptome and epigenome analyses revealed that tonic type I IFN receptor signaling instructs a specific epigenetic and metabolic basal state in cDCs that is indispensable for their functionality in pathogen defense. Collectively, I provide new insights into how the indigenous microbiota affects immunological functions of cDCs residing in non-mucosal tissue on a transcriptional, epigenetic and metabolic level, contributing to a better understanding of the evolutionary trade-offs that come with the successful adaptation of vertebrates to their microbial environment.Zelloberflächen im menschlichen Körper sind von einer Vielzahl an Mikroben besiedelt, die als Mikrobiota bezeichnet wird. Diese hat, abhängig von Umwelt- und genetischen Faktoren, einen wesentlichen Einfluss auf zahlreiche physiologische Funktionen ihres Wirts, worunter sowohl das angeborene als auch adaptive Immunsystem fällt. Diese Arbeit zeigt, dass in der Milz lokalisierte konventionelle dendritische Zellen (cDCs) von der Mikrobiota kommende Signale im Steady-State benötigen, um sofort gegen eindringende Pathogene agieren zu können, indem sie pro-inflammatorische Zytokine ausschütten und T-Zellen des adaptiven Immunsystems aktivieren. Die molekularen Mechanismen, die diesem Mikrobiota-induzierten Instruktionsprogramm zugrunde liegen, sind allerdings kaum entschlüsselt. In dieser Studie werden Typ I Interferone (IFN), die permanent im Steady-State hauptsächlich von plasmazytoiden (p)DCs produziert werden, als Schlüsselmoleküle identifiziert, die essentiell für den Instruktionsprozess von cDCs sind. Analysen des Transkriptoms und Epigenoms zeigen, dass der durch IFN-I induzierte Bereitschaftszustand in cDCs mit epigenetischen und metabolischen Veränderungen assoziiert ist und für ihre biologische Funktion in der Immunabwehr essentiell ist. Zusammenfassend liefert die vorliegende Arbeit neue Erkenntnisse darüber, wie die Mikrobiota die immunologische Funktion von in nicht-mukosalen Geweben residierenden cDCs auf transkriptioneller, epigenetischer und metabolischer Ebene beeinflusst. Sie trägt zu einem besseren Verständnis über der evolutionär bedingten biologischen Konflikte bei, die mit der erfolgreichen Adaption von Vertebraten an ihr mikrobielles Umfeld einhergehen.I, 131 Seiten ; Illustrationen, Diagramm

Immunity to Emerging RNA Viruses

In the past decade, newly emerged and re-emerged pathogenic RNA viruses such as Ebola, H1N1, and Zika virus have become substantial threats to global health. Two notable examples include the explosive re-appearance of chikungunya virus in the 2000s in Asia and the Western Hemisphere and, more recently, the global COVID-19 pandemic. Therefore, an improved understanding of immunity to emerging RNA viruses is critical for defining host susceptibility and restriction factors to viral infection as well as appropriate vaccine and antiviral therapeutic design. Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that is maintained in an urban epidemic transmission cycle. While most CHIKV infections cause an acute febrile illness lasting weeks, a subset of individuals progresses to debilitating, persistent musculoskeletal pain and inflammation lasting months to years. The magnitude and duration of viremia in humans correlate with mosquito infection and transmission. Although alphavirus viremia is a key determinant of disease severity and vector transmission, little is known about the acquired and genetic factors that impact the levels of virus in the human host circulation. Herein, I describe one of these factors, the impact of the mammalian intestinal microbiome on alphavirus infection and dissemination. CHIKV infection of oral antibiotic-treated or germ-free mice resulted in increased viral burden in the blood and in tissues distant from the site of inoculation. Perturbation of the microbiome altered TLR7-MyD88 signaling in plasmacytoid dendritic cells (pDCs) and blunted systemic production of type I interferon (IFN). Consequently, circulating monocytes expressed fewer IFN-stimulated genes and become permissive for CHIKV infection. Reconstitution with a single bacterial species, Clostridium scindens, or its derived metabolite, the secondary bile acid deoxycholic acid, could restore pDC- and MyD88-dependent type I IFN responses to restrict systemic CHIKV infection and transmission back to vector mosquitoes. Thus, symbiotic intestinal bacteria modulate antiviral immunity and levels of circulating alphaviruses within hours of infection through a bile acid-pDC-IFN signaling axis, which affects viremia, dissemination, and potentially transmission. Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) is the recently emerged RNA virus responsible for the Coronavirus Disease 2019 (COVID-19) pandemic that has led to over 163 million infections and over 3 million deaths. The development of countermeasures that reduce COVID-19 morbidity and mortality has been a global priority, and animal models are essential for this effort. Although animal models have been evaluated for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, none have fully recapitulated the lung disease phenotypes seen in humans who have been hospitalized. Herein, I evaluated transgenic mice expressing the human angiotensin I-converting enzyme 2 (ACE2) receptor driven by the cytokeratin-18 (K18) gene promoter (K18-hACE2) as a model of SARS-CoV-2 infection and pathogenesis. Intranasal inoculation of SARS-CoV-2 in K18-hACE2 mice resulted in high levels of viral infection in lungs, with spread to other organs. A decline in pulmonary function occurred 4 days after peak viral titer and correlated with infiltration of monocytes, neutrophils and activated T cells. SARS-CoV-2-infected lung tissues showed a massively upregulated innate immune response with signatures of nuclear factor-κB-dependent, type I and II interferon signaling, and leukocyte activation pathways. Thus, the K18-hACE2 model of SARS-CoV-2 infection shares many features of severe COVID-19 infection and can be used to define the basis of lung disease and test immune and antiviral-based countermeasures. One approach for both prevention and treatment of COVID-19 has been the development of SARS-CoV-2-neutralizing monoclonal antibodies (mAbs) directed against the spike (S) glycoprotein of SARS-CoV-2. Although passively delivered neutralizing antibodies against SARS-CoV-2 show promise in clinical trials, mechanisms of protection in vivo can be due to multiple factors including direct virus neutralization and engagement of complement or Fc gamma receptors (FcRs) on leukocytes. Fc effector functions of antibodies can promote immune-mediated cellular clearance, enhance antigen presentation and CD8+ T cell responses, and reshape inflammation through engagement of FcRs on specific cells. In contrast, under certain circumstances, Fc-FcR interactions can promote antibody-dependent enhancement of virus infection (ADE) or cause pathological immune skewing, which is at least a theoretical concern of antibody-based therapies and vaccines against SARS-CoV-2. Thus, a more complete understanding of the contribution of Fc effector functions in the context of antibody-based therapies is needed. Herein, I define correlates of protection of neutralizing human monoclonal antibodies (mAbs) in SARS-CoV-2-infected animals. Whereas Fc effector functions are dispensable when representative neutralizing mAbs are administered as prophylaxis, they are required for optimal protection as therapy. When given after infection, intact mAbs reduce SARS-CoV-2 burden and lung disease in mice and hamsters better than loss-of-function Fc variant mAbs. Fc engagement of neutralizing antibodies mitigates inflammation and improves respiratory mechanics, and transcriptional profiling suggests these phenotypes are associated with diminished innate immune signaling and preserved tissue repair. Immune cell depletion experiments establish that neutralizing mAbs require monocytes and CD8+ T cells for optimal clinical and virological benefit. Thus, potently neutralizing mAbs utilize Fc effector functions during therapy to mitigate lung infection and disease

In the moonlight: non-catalytic functions of ubiquitin and ubiquitin-like proteases

Proteases that cleave ubiquitin or ubiquitin-like proteins (UBLs) are critical players in maintaining the homeostasis of the organism. Concordantly, their dysregulation has been directly linked to various diseases, including cancer, neurodegeneration, developmental aberrations, cardiac disorders and inflammation. Given their potential as novel therapeutic targets, it is essential to fully understand their mechanisms of action. Traditionally, observed effects resulting from deficiencies in deubiquitinases (DUBs) and UBL proteases have often been attributed to the misregulation of substrate modification by ubiquitin or UBLs. Therefore, much research has focused on understanding the catalytic activities of these proteins. However, this view has overlooked the possibility that DUBs and UBL proteases might also have significant non-catalytic functions, which are more prevalent than previously believed and urgently require further investigation. Moreover, multiple examples have shown that either selective loss of only the protease activity or complete absence of these proteins can have different functional and physiological consequences. Furthermore, DUBs and UBL proteases have been shown to often contain domains or binding motifs that not only modulate their catalytic activity but can also mediate entirely different functions. This review aims to shed light on the non-catalytic, moonlighting functions of DUBs and UBL proteases, which extend beyond the hydrolysis of ubiquitin and UBL chains and are just beginning to emerge

Immunology, Immunopathology, and Immunopharmacology of COVID-19

Respiratory viruses represent a continuous and growing threat to humanity. This is exemplified by the ongoing global COVID-19 pandemic, which is caused by a newly emerged coronavirus SARS-CoV-2. The immune system provides a formidable barrier to viral pathogens, while dysregulated immunity leads to deleterious infection outcomes. Understanding the involvement of immune responses in viral infections will provide crucial insights into disease pathogenesis and shed light on potential targets for therapeutic interventions. In this thesis, we interrogate the components of the immune response that control SARS-CoV-2 and contribute to its pathogenesis. Based on these insights, we develop distinct immunopharmacological strategies to strengthen protection against viral infection, disease, and transmission. In chapter 2, we investigated the prevalence, magnitude, and specificity of autoantibody (AAb) responses against the human exoproteome in SARS-CoV-2-infected individuals using a high-throughput AAb discovery technology called Rapid Extracellular Antigen Profiling (REAP). Infection with SARS-CoV-2 leads to diverse immunological and clinical outcomes. However, the impact of AAbs on these infection outcomes remains unknown. We found that patients with COVID-19 exhibit increases in autoreactivities against a broad array of immunological antigens, including cytokines, chemokines, and cell surface proteins. We uncovered mechanisms by which AAbs interfere with immunological functions, by perturbing immunoreceptor signaling, by depleting circulating leukocytes, and by dampening antiviral antibody responses. Murine AAb surrogates similarly hinder immune activation and exacerbate disease in a mouse model of SARS-CoV-2. Collectively, through the lens of an unbiased proteome-scale discovery campaign for AAbs, these findings implicate humoral immunopathology as an integral aspect of COVID-19 pathogenesis with diverse impacts on immune functionality and clinical outcomes. In chapter 3, we developed a therapeutic strategy that bolsters type I interferon (IFN-I)-dependent innate immunity against SARS-CoV-2 on the basis of a synthetic RNA ligand of RIG-I, stem-loop RNA 14 (SLR14). The IFN system presents a critical protective barrier against viral pathogens but is also capable of contributing to pathogenesis. Our discovery in chapter 2 that preexisting IFN-I-neutralizing AAbs are associated with poor disease prognosis in patients with COVID-19 suggests a protective role for IFN-I. We discovered that a single dose of SLR14 confers robust antiviral protection in mouse models of SARS-CoV-2 infection by eliciting systemic and mucosal IFN-I responses. SLR14 is the most protective when administered prophylactically or early after viral exposure. We further demonstrated that SLR14 elicits sterilizing immunity in immunodeficient mice chronically infected with SARS-CoV-2 independent of the adaptive immune system and confers broad-spectrum protection against emerging variants of concerns (VOCs). This study demonstrates the potential of leveraging host-directed nucleic acid therapeutics to induce protective antiviral immunity against SARS-CoV-2. In chapter 4, we developed a novel vaccine strategy, Prime and Spike, that elicits mucosal immunity against SARS-CoV-2. Parenteral vaccines induce robust systemic immunity, but poor immunity at the respiratory mucosa. We designed a vaccination regimen that converts existing circulating immunological effector mechanisms generated by primary vaccination (Prime) into mucosal immunity in the respiratory tract via unadjuvanted intranasal (IN) spike boosting (Spike). We established that Prime and Spike induces tissue-resident memory T cells and B cells, promotes mucosal IgA, boosts systemic immunity, protects mice with waning immunity from lethal disease, and reduces viral shedding in a hamster model of contact transmission. We demonstrated IN spike boosters can be delivered in the format of unadjuvanted recombinant spike proteins or spike-encoding mRNA encapsulated by immunosilent poly(amine-co-ester) (PACE) polymers. Using divergent spike proteins, we showed that Prime and Spike enables the induction of cross-reactive immunity against sarbecoviruses. These findings suggest that Prime and Spike can be exploited as a potent prophylactic vaccine platform to elicit pan-sarbecovirus mucosal immunity. In chapter 5, we employed a different, but just as devasting disease model to study factors governing the induction of protective immunity. In the context of melanoma, we identified type 1 conventional dendritic cells (cDC1s) as a crucial innate immunological determinant for effective anti-PD-1 (αPD-1) cancer immunotherapy. While cDC1s critically mediate the initiation of anti-tumor CD8+ T cell immunity, whether they contribute to effector CD8+ T cells during αPD-1 therapy remains unclear. We discovered that cDC1s enables αPD-1 therapy by unleashing the terminal differentiation and expansion of intratumoral CD8 T cells at the effector phase of αPD-1 therapy. We found that tumor cDC1 abundance predicts strong tumor infiltration by CD8+ T cells and associates with clinical responses to αPD-1 treatment in human cancer patients. Together, this study suggests that the development of efficacious cancer immunotherapeutics should incorporate strategies that engage cDC1 immunity. In conclusion, the collective work presented in this thesis addresses both discovery and application-oriented research questions, provides important insights into the role of immune system in the control of infection and tumorigenesis, and expands the therapeutic toolkit for tackling human viral diseases and malignancies

Charakterisierung von UNC93B-Toll-like Rezeptor Komplexen und deren Rolle für die angeborene Immunantwort

Toll-like receptors (TLRs) are key players of the innate immune system. Localized at the cell surface or intracellularly, they trigger immune responses upon recognition of pathogenic patterns. After their synthesis in the endoplasmic reticulum (ER), intracellular TLRs 3, 7, and 9 need to move to the endolysosome to meet their ligands. On their way they are escorted by the polytopic membrane protein UNC93B. UNC93B interacts with TLRs 3, 7, and 9 and delivers them from the ER to the endolysosome where they initiate the expression of proinflammatory cytokines and type I interferon (IFN) upon encounter of nucleic acids. In 3d mice, which express a missense mutant of UNC93B (H412R), binding of UNC93B to TLRs is disrupted and UNC93B H412R as well as intracellular TLRs fail to leave the ER. How function and trafficking of UNC93B-TLR complexes is regulated is not entirely understood. A comparison of innate immune cells derived from UNC93B knockout and 3d mice showed that the phenotype of cells from UNC93B knockout mice does not differ from the 3d phenotype. In a proteomics approach, cleft lip and palate transmembrane protein (Clptm), three prime repair exonuclease 1 (Trex1), and the protein tyrosine phosphatase 1B (PTP1B) have been identified as novel binding partners of UNC93B in macrophages. Co-immunoprecipitation experiments verified interactions between UNC93B, the previously uncharacterized protein Clptm, as well as the negative regulator of cytosolic DNA responses Trex1. PTP1B is known to be crucial for intracellular trafficking of receptor tyrosine kinases, but had not been linked to regulation of TLR signaling. Interaction of PTP1B with UNC93B, Clptm, TLR7, and TLR9 was verified by co-immunoprecipitation. The TLR-independent type I IFN response upon infection of bone marrow-derived macrophages with mouse cytomegalovirus, as well as the TLR7- and TLR9-dependent IFNalpha responses in plasmacytoid dendritic cells (pDC) were impaired in cells derived from PTP1B knockout mice compared to wild type cells. Furthermore, PTP1B knockout mice showed significantly reduced IFNalpha and interleukin (IL)-12p40 responses compared to wildtype mice upon stimulation of TLR9 by intravenous administration of CpG oligonucleotides in vivo. This suggests that PTP1B is a critical regulator of the TLR7- and TLR9-dependent IFNalpha response in pDC as well as the TLR-independent type I IFN response in macrophages.Toll-like Rezeptoren (TLRs) sind ein wichtiger Bestandteil des angeborenen Immunsystems. Sie werden an der Zelloberfläche oder intrazellulär exprimiert und lösen bei der Erkennung pathogener Strukturen Immunantworten aus. Nach ihrer Synthese im endoplasmatischen Retikulum müssen die intrazellulären TLRs 3, 7 und 9 ins Endolysosom gelangen, um auf ihre Liganden zu treffen. Das Membranprotein UNC93B interagiert mit den TLRs 3, 7 und 9 und eskortiert sie vom ER zum Endolysosom, wo sie die Expression von proinflammatorischen Zytokinen und Typ I Interferon (IFN) initiieren, wenn sie auf Nukleinsäuren treffen. In 3d Mäusen, die UNC93B mit einer missense-Mutation (H412R) exprimieren, kann UNC93B nicht an die TLRs binden, weshalb UNC93B H412R und intrazelluläre TLRs das ER nicht verlassen können. Wie die Funktion und der Transport von UNC93B-TLR-Komplexen reguliert werden, ist nicht vollständig geklärt. Ein Vergleich von Immunzellen, die aus UNC93B knockout oder 3d Mäusen generiert wurden, zeigte, dass der Phänotyp der UNC93B knockout Zellen sich nicht vom 3d Phänotyp unterscheidet. Cleft lip and palate transmembrane protein (Clptm), three prime repair exonuclease 1 (Trex1) und protein tyrosine phosphatase 1B (PTP1B) wurden mittels eines Proteomik-Ansatzes als neue Bindungspartner von UNC93B identifiziert. Die Interaktion zwischen UNC93B und dem uncharakterisierten Protein Clptm sowie Trex1, einem negativen Regulator der Immunantwort auf zytosolische DNA, wurde mittels Ko-Immunpräzipitation bestätigt. PTP1B ist wichtig für den intrazellulären Transport von Rezeptor-Tyrosinkinasen, wurde aber bisher nicht mit der Regulation von TLRs in Verbindung gebracht. Interaktionen zwischen PTP1B und UNC93B, Clptm, TLR7 und TLR9 wurden mittels Ko-Immunpräzipitation bestätigt. Die TLR-unabhängige Typ I IFN-Antwort nach der Infektion von Makrophagen, die aus PTP1B knockout Mäusen generiert wurden, mit Maus-Cytomegalievirus sowie die TLR7- und TLR9-abhängige IFNalpha-Antwort von plasmazytoiden dendritischen Zellen von PTP1B knockout Mäusen waren im Vergleich zu Wildtyp-Zellen vermindert. Außerdem waren die IFNalpha-Antwort und die Interleukin (IL)-12p40-Antwort nach intravenöser Verabreichung von CpG Oligonukleotiden in vivo in PTP1B knockout Mäusen im Vergleich zum Wildtyp reduziert. Diese Ergebnisse deuten darauf hin, dass PTP1B ein entscheidender Regulator der TLR7- und TLR9-abhängigen IFNalpha-Antwort in pDC sowie der TLR-unabhängigen Typ I IFN-Antwort in Makrophagen ist

Investigation of the roles and regulation of indoleamine 2,3-dioxygenase-1 (IDO1) in human antigen-presenting cells

Indoleamine 2,3-dioxygenase 1 (IDO1) is a critical immunoregulatory heme enzyme expressed in antigen-presenting cells (APCs). While IDO1’s immunoregulatory actions have been documented to alter a diverse range of diseases, less is understood about the biochemical pathways controlling the enzyme in APCs. Chapter 3 studies established that heme-iron metabolism regulates IDO1 in primary human APCs. Thus, iron chelators inhibited IDO1 expression independent of STAT1 or NF-κB, which represent important signals for IDO1 transcription. The heme precursor aminolevulinic acid (ALA) or heme biosynthesis inhibitor succinylacetone, stimulated or inhibited IDO1, respectively, at the posttranslational level. Prior upregulation of the heme catabolic enzyme heme oxygenase-1 (HO-1) inhibited IDO1 at the post-translational or transcriptional levels in IFNγ- or LPS-stimulated APCs, respectively. Mechanistic studies showed that HO-1 post-translationally controls IDO1 by reducing heme bioavailability for insertion into IDO1 and generating CO that directly inhibits IDO1 activity or suppressed IDO1 expression by inhibiting STAT1 and NF-κB. Chapter 4 studies showed that IFNγ and LPS differentially modulate glycolytic and mitochondrial parameters in human MDMs and that IDO1 is subject to transcriptional and/or post-translational control by different metabolic pathways including glycolysis, Krebs cycle metabolites itaconate and succinate, mitochondrial respiration and generation of reactive oxygen species, and mitochondrial fatty acid oxidation. These studies revealed complex regulatory crosstalk between cellular energy metabolism and IDO1. Chapter 5 examined the IDO1-dependent changes in the proteomic profile of MDMs stimulated with IFNγ/LPS or infected with Dengue or West Nile virus. Proteomic analysis indicated that IDO1 activity modulates various immune and metabolic pathways in MDMs, which have the potential to impact on their function. As cell immune responses play complex roles in the immunopathology accompanying virus infection, Chapter 6 established multiparametric flow cytometry workflows to comprehensively detail the changes in immune cell sub-set phenotypes in the lungs and lymph nodes of influenza-infected mice. These workflows are available to study the immunoregulatory actions of IDO1 in influenza. This work provides important new insights into the roles and regulation of IDO1 in APCs and established methods for studying IDO1’s immune function in vivo

Characterization of Atlantic salmon head kidney leukocyte culture

The Atlantic salmon (Salmo salar) is an economically important farmed and wild fish in several countries including Canada. Macrophages are white blood cells of the immune system of fish and other vertebrates, that are essential in fighting infection and disease. Elucidating how macrophages differentiate and function is necessary to fully understand how the fish immune system functions and to enable the development of methods to maintain healthy fish. Therefore, the objective of my Ph.D. thesis was to characterize the Atlantic salmon adherent head kidney leukocyte (HKL) culture, a macrophage-like model commonly used in fish immunological studies, using various genomic and complementary techniques. Using morphology (Giemsa stain) and functional (phagocytosis) assays, the results of this thesis showed that the Atlantic salmon adherent HKL population changes during culture time. At Day 1 of culture, the results suggest that adherent HKLs are a heterogeneous population of predominantly “monocyte-like”, cells but by Day 5 of culture, the cells become more homogenous selectively enriched with macrophages. RNA-sequencing identified a change in the microRNA (miRNA) profile of Day 1 and Day 5 adherent HKLs, as well as the extracellular vesciles (EVs) released from them. Many of the identified miRNAs are involved in macrophage function and/or differentiation in other species. Furthermore, using a 44K microarray, changes in the mRNA transciprtome were profiled. Macrophage-related transcripts, lipid-related transcripts, immune-related transcripts and transcription factors were identified as differentially expressed between the two cell populations. In addition, GO term enrichment and network analysis identified immune-related and immune-cell differentiation related terms. The results of this thesis provides evidence that the Atlantic salmon HKL culture changes to become predominantly “macrophage-like” by Day 5 of culture and this is something that should be kept in mind when using HKLs for in vitro fish immunology studies. This research provides novel insight into the genes, miRNAs and molecular pathways involved in the differentiation of Atlantic salmon adherent HKLs from monocyte-like cells to macrophage-like cells

HIV-Host Interactions

HIV remains the major global health threat, and neither vaccine nor cure is available. Increasing our knowledge on HIV infection will help overcome the challenge of HIV/AIDS. This book covers several aspects of HIV-host interactions in vitro and in vivo. The first section covers the interaction between cellular components and HIV proteins, Integrase, Tat, and Nef. It also discusses the clinical relevance of HIV superinfection. The next two chapters focus on the role of innate immunity including dendritic cells and defensins in HIV infection followed by the section on the impact of host factors on HIV pathogenesis. The section of co-infection includes the impact of Human herpesvirus 6 and Trichomonas vaginalis on HIV infection. The final section focuses on generation of HIV molecular clones that can be used in macaques and the potential use of cotton rats for HIV studies