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Innate antiviral defence depends on virus recognition and cytokines like interferon (IFN) that upregulate many interferon-stimulated genes (ISGs). Virus recognition normally relies on pattern recognition receptors binding to virus-associated nucleic acid motifs, but virus activity may provide an additional means for the cell to recognize infection. Enveloped viruses fuse with a cell membrane during entry and membrane fusion by virus-like particles or the purified protein p14 are sufficient to upregulate ISGs in the absence of viral nucleic acid. This thesis examines the mechanism by which cells recognize membrane fusion and how this affects downstream signalling and upregulation of ISGs. We found that membrane perturbation by enveloped virus particles or p14 triggered cytosolic Ca2+ oscillations important for antiviral defence. Surprisingly, Ca2+ signalling seemed to act upstream of nucleic acid sensing pathways during enveloped virus infection. In the absence of viral nucleic acid, p14 triggered a Ca2+-dependent antiviral response to dsRNA. It is still unclear how p14 might trigger recognition of endogenous dsRNA. We found that enveloped virus particles trigger IRF3-mediated upregulation of interferon as well as direct IFN-independent upregulation of ISGs. Furthermore, while some viruses like HCMV trigger widespread IRF3 activation, other viruses like SeV upregulate IRF3 and IFN in a minority of infected cells. This disparate response to infection can lead to different biological outcomes when measured at the population level. Our work highlights the complexity of the response to enveloped virus particles, despite the absence of replication. Further work is necessary to understand how membrane perturbation is recognized and how this interfaces with nucleic acid sensing. While nucleic acid sensing is sufficient to upregulate antiviral ISGs, other signals like membrane perturbation may provide important contextual cues during infection. This will be important to understand moving forward as virus-like particles are used more and more for research and clinical applications. / Thesis / Doctor of Philosophy (PhD) / Cells rely on pattern recognition receptors for innate antiviral defence. While the study of pattern recognition has focused on virus-associated nucleic acid motifs, disruptions of the cellular environment during virus infection could similarly warn the cell. Membrane perturbation during enveloped virus entry is associated with upregulation of antiviral interferon-stimulated genes. This thesis examines the mechanism of membrane perturbation sensing and different antiviral signalling pathways activated by non-replicating enveloped virus particles. We found evidence that membrane perturbation triggers cytosolic Ca2+ signalling which may act as a co-stimulatory signal for recognition of incoming viral nucleic acid. We initially thought enveloped virus particles were recognized through a common pathway, but have since learned that recognition is more complex. Further work is necessary to understand how membrane perturbation and nucleic acid sensing interface during enveloped virus infection and what role this plays in antiviral defence.

Circulating monocytes have the capacity to mature into either macrophages or dendritic cells in tissue, both of which play important roles in the induction and effector phase of immune response. In TB, the macrophages are the central player in the host-bacteria interaction as the main mycobacterial reservoir. In HIV disease, monocyte lineage cells are one of the two main cell types (along with CD4+ T-cells) in sustaining intracellular HIV infection. Monocytes are heterogeneous population with three functionally distinct subsets namely classical, intermediate and non-classical monocytes. The three subsets exist in a continuum, and have a certain plasticity or flexibility to develop into multiple roles depending on the local and tissue environment. In the current study we sought to evaluate the frequencies of these three subsets in participants with TB, HIV and TB/HIV co-infection. While previous studies had shown that the intermediate and non-classical monocyte subsets were elevated relative to classical monocytes, very little had been done in these disease groups regarding more comprehensive characterization of these subsets. In particular, we wished to quantitate the expression of multiple sets of cell surface and intracellular molecules of high relevance using multi-parameter flow cytometry from a functional point of view. In publication I, we evaluated Toll-like receptors (TLRs) expression in each of the study cohorts. TLRs are vital pattern recognition receptors by monocyte lineage cells and signal the induction of crucial functions. We focused on three such TLRs (TLR2, TLR4 and TLR9) which have been shown to be involved in many monocyte lineage cell interactions with mycobacterial and HIV infections. We observed enhanced expression of TLR2 and TLR4, but not TLR9 in TB and HIV. TLR4 was particularly high in patients with TB, but also in HIV. We observed comparable increase of TLR4 irrespective of monocyte subset. However, TLR2 expression exhibited a different pattern. Levels among the most prominent classical monocyte subsets were identical in all four cohorts, healthy controls (HC), HIV, TB, and TB/HIV co-infection. In contrast, TLR2 expression was significantly elevated in both participants with HIV and TB, but not with participants with TB/HIV co-infection in the intermediate monocyte subset. We also observed correlations between TLRs and plasma cytokines that were disease and TLR specific. In publication II, we observed elevated chemokine receptors (CRs) expression which above healthy controls and exhibit a pattern of disease preference. Thus, CCR2 and CX3CR1 were the highest in participants with TB, followed by HIV and TB/HIV co-infection, whereas CCR4 and CCR5 were highest in participants with HIV, and less elevated in TB. CCR2 and CX3CR1 are critical for migration of monocytes to sites of TB infection, as determined by murine models. CCR4 and especially CCR5 have been implicated in migration of cells to distant organs but more as co-receptors for HIV infection. Thus, the observed pattern of CRs expression in these monocytes in different disease states would predict greater availability of these cells or their receptors for interaction with either TB or HIV organisms. From the perspective of the pathogen this would lead to enhanced “substrate”, whereas from the perspective of the host, this could lead to greater immune potential. As a final point, we also observed that the pattern of disease association of CRs was independent of the monocyte subset. In publication III, we explored the expression of Programmed cell death-ligand 1 (PDL1) on the three monocyte subsets. Like many of the other molecules we have addressed in this thesis, PDL1 expression was enhanced in participants with HIV, TB, and TB/HIV co-infection. Among participants with HIV, PDL1 was correlated with HIV-1 viral load. The enhanced expression was apparent in all three subsets, but it was particularly prominent in the intermediate monocyte subset. Moreover, PDL1 expression was the highest in participants with TB/HIV co-infection. The implications behind these observations is that the subset thought to have the greatest potential for T cell antigen presentation had the highest levels of the T cell down-regulatory PDL1 molecule in the cohort of patients particularly participants with TB/HIV co-infection. Participants with TB/HIV co-infection have the greatest potential to be immuno-compromised and as a result the very need for enhanced not depressed APCs function. In addition, we also observed the PDL1 levels were correlated with multiple plasma, mostly pro-inflammatory,cytokines. We analyzed cytokine mRNA levels of total monocytes to address the source of the cytokines but mRNA levels did correlate with neither plasma cytokine nor PDL1 levels. Considering all the phenotype analysis in each of the three studies together we could see two patterns emerging. In one scenario,surface molecules expression patterns were disease specific but independent of monocyte subset expression. In other words, whatever the underlying mechanism(s) involved in their regulation, those mechanisms apparently acted similarly in all three subsets. In another scenario, expression of surface molecules showed disease specific patterns, but molecules were particularly enhanced in the intermediate monocyte subsets. These findings imply that there exist mechanisms to modulate surface phenotypes and functions that are unique to a given subset. In conclusion, we have comprehensively defined the density of multiple molecules expressed by different subsets of monocytes and explored their differences in participants with TB, HIV and TB/HIV co-infection, as well as their correlations with microbial indices and plasma cytokines. Many molecules levels were elevated to some extent in all disease cohorts, but we observed patterns of expression which were particularly elevated in TB (CCR2, CX3CR1, and TLR2), those in HIV (CCR4, CCR5) and those on both (TLR4, PDL1). Molecule-disease associations were either independent of monocyte subset, or most readily revealed in a single monocyte subset. TB/HIV co-infection did not follow a consistent pattern in association with monocytes markers, in some cases more resembling TB, in others HIV, in others neither. Finally, to proof one possible mechanism of association between disease and monocyte phenotype, we explored correlations between monocyte markers and plasma cytokines. We observed significant positive and negative associations, frequently unique to a single monocyte subset or disease cohort, such as TB/HIV co-infected cohort. Collectively, the results imply that there are likely multiple mechanisms involved at many levels regulating the phenotype and function of monocytes, and these differ in different disease states.:Abbreviations ............................................................................................................ 3 Abstract ..................................................................................................................... 4 1. Introduction ........................................................................................................... 6 1.1 Epidemiology of Tuberculosis and Human Immunodeficiency virus ................... 6 1.2 The immunological response to TB and HIV ....................................................... 7 1.2.1 Innate immunity of TB ...................................................................................... 7 1.2.2 Innate immunity of HIV .................................................................................... 11 1.2.3 Immune checkpoint regulation in TB and HIV.................................................. 13 1.3 The role of monocytes in TB, HIV and TB/HIV ................................................... 14 1.3.1 Monocytes ....................................................................................................... 14 1.3.2 Abnormalities of monocytes in TB ................................................................... 15 1.3.3 Abnormalities of monocytes in HIV ................................................................. 16 1.3.4 Abnormalities on monocytes in TB/HIV co-infection ....................................... 17 1.4 The rationale for the thesis ................................................................................ 17 2. Objectives ............................................................................................................ 19 3. Publications .......................................................................................................... 20 4. Summary .............................................................................................................. 65 References ............................................................................................................... 69 Annex I: Author contribution ..................................................................................... 76 Annex II: Declaration of independent writing of the work ......................................... 77 Annex III: Curriculum Vitea ....................................................................................... 78 Annex V: Acknowledgment........................................................................................ 82 Annex VI ................................................................................................................... 84

Les adénovirus humains (HAdV) provoquent un large spectre de maladies cliniques chez les patients immunodéprimés et immunocompétents et sont également des outils polyvalents pour le transfert de gènes et la vaccination. L’immunité humorale acquise peut être en partie responsable des réactions indésirables envers les vecteurs AdV révélées dans plusieurs essais cliniques de vaccination. De plus, plusieurs protéines de l'hôte comme le facteur X de coagulation de la souris (FX) ou les immunoglobulines de type G se lient aux HAdV et exacerbent la réponse pro-inflammatoire. L’évaluation des risques précliniques se fait souvent chez la souris, même s’il existe plusieurs différences entre les humains et les souris dans l'interaction avec les HAdV. La liaison de FX aux HAdV active une réponse pro-inflammatoire chez la souris par l'intermédiaire des récepteurs Toll-like 4. Dans un autre scénario clinique pertinent, le complexe immun HAdV (IC-HAdV-C5) induit une activation plus forte de l’inflammasome dans les phagocytes humains que l’HAdV-C5 non complexé, mais par un mécanisme inconnu. Dans ce contexte, j’ai participé à deux études. Premièrement, nous avons étudié le rôle potentiel de FX et de TLR4 dans la réponse innée à l ‘HAdV-C5 en utilisant uniquement des cellules et des protéines d’origine humaine. Nous avons constaté qu'il n'y a pas d’activation de la voie de signalisation via TLR4 chez l’homme en présence de FX-HAdV. De plus, le FX n'a pas affecté la forte réponse immunitaire innée induite par IC-HAdV-C5 dans les phagocytes humains. Deuxièmement, nous avons abordé le mécanisme sous-jacent de l'inflammation induite par IC-HAdV-C5. Nous avons démontré que l‘IC-HAdV-C5 induit la formation de l’inflammasome dans les cellules dendritiques dérivées de monocytes et cela dépend de l’échappement endosomal dépendant de la protéine VI et de l'activation des récepteurs cytosoliques de l’inflammasome. Nos résultats nous aident à mieux comprendre les différences entre les tests précliniques réalisés chez la souris et les essais cliniques réalisés chez l'homme. De plus, cela nous permet de mieux comprendre comment l’immunité préexistante façonne la réponse immunitaire innée face aux HAdV, afin d’améliorer le traitement des maladies liées aux HAdV ainsi que l’efficacité des vecteurs HAdV. / Human adenoviruses (HAdV) cause a broad spectrum of clinical diseases in immunocompromised and –competent patients and are also versatile tools for gene transfer and vaccination. Pre-existing humoral immunity may be in part responsible for the adverse responses towards AdV vectors seen in several clinical vaccine trials. Furthermore, a variety of host proteins like mouse coagulation factor X (FX) or immunoglobulin G bind HAdV exacerbate the pro-inflammatory response. Pre-clinical risk assessment is often done in mice, albeit there are multiple differences between human and mice in the interaction with HAdV. The binding of FX to HAdV activates a pro-inflammatory response in mouse via Toll-like receptor 4. In another clinical relevant scenario, immune complexed-HAdV (IC-HAdV-C5) induces more inflammasome activation in human phagocytes than HAdV-C5 alone but by unknown mechanism. In this regard, I participated in two studies. First, we investigated a potential role of FX and TLR4 in the innate response to HAdV-C5 by using only human components. We found that there is no detectable FX-HAdV-TLR4 axis in human and FX did not affect the innate immune response elevated by IC-HAdV-C5 in human phagocytes.Second, we addressed the underlying mechanism of IC-HAdV-C5-induced inflammation. We found that IC-HAdV-C5 induces inflammasome formation in monocyte-derived dendritic cells and this is dependent on pVI-mediated endosomal escape and activation of cytosolic inflammasome sensors. Our findings help us to better understand the differences in preclinical testing in mice and clinical use in humans and how pre-existing immunity shapes the innate immune response to HAdV to improve treatment for HAdV diseases and HAdV vector effectiveness.

Malarial infection in naïve individuals induces a robust innate immune response, but our understanding of the mechanisms by which the innate immune system recognizes malaria and regulates its response remain incomplete. Our group previously showed that stimulation of macrophages with Plasmodium falciparum genomic DNA (gDNA) and AT-rich oligodeoxynucleotides (ODNs) derived from this gDNA induces the production of type I interferons (IFN-I) through a STING/TBK1/IRF3-dependent pathway; however, the identity of the upstream cytosolic DNA receptor remained elusive. Here, we demonstrate that this IFN-I response is dependent on cyclic GMP-AMP synthase (cGAS). cGAS produced the cyclic dinucleotide 2’3’-cGAMP in response to P. falciparum gDNA and AT-rich ODNs, inducing IRF3 phosphorylation and IFNB transcription. In the recently described model of innate immune memory, an initial stimulus primes the innate immune system to either hyperrespond (termed “training”) or hyporespond (“tolerance”) to subsequent immune challenge. Previous work in mice and humans demonstrated that infection with malaria can both serve as a priming stimulus and promote tolerance to subsequent infection. In this study, we demonstrate that initial stimulation with P. falciparum-infected red blood cells (iRBCs) or the malaria crystal hemozoin (Hz) induced human adherent peripheral blood mononuclear cells (PBMCs) to hyperrespond to subsequent Toll-like receptor (TLR) challenge. This hyperresponsiveness correlated with increased H3K4me3 at important immunometabolic promoters, and these epigenetic modifications were also seen in monocytes from Kenyan children naturally infected with malaria. However, the use of epigenetic and metabolic inhibitors indicated that malaria-induced trained immunity may occur via previously unrecognized mechanism(s).

The cells and molecules that comprise the immune system are essential for mounting an effective response against microbes. A successful immune response limits pathology within the host while simultaneously eliminating the pathogen. The key to this delicate balance is the correct recognition of the pathogen and the appropriate response of immune cells. Cellular activation originates through receptors that relay information about the state of the microenvironment to different compartments within the cell. The rapid relay of information is called signal transduction and employs a network of signaling mediators such as kinases, phosphatases, adaptor molecules, and transcription factors. IL-2 inducible T cell kinase (Itk) is a non-receptor tyrosine kinase that is an integral component of signal transduction downstream of many immunoreceptors. This dissertation describes two distinct pathways that utilize Itk in both phases of the immune response. T cells use the TCR to sense a multitude of peptide-based ligands and to transmit signals inside the cell to activate cellular function. In this regard, the diversity of ligands the T cells encounter can be portrayed as analog inputs. Once a critical threshold is met, signaling events transpire in close proximity to the plasma membrane to activate major downstream pathways in the cell. The majority of these pathways are digital in nature resulting in the on or off activation of T cells. We find, however, that altering the TCR signal strength that a T cell receives can result in an analog-based response. Here, the graded expression of a transcription factor, IRF4, is modulated through the activity of Itk. We link this graded response to an NFAT-mediated pathway in which the digital vs. analog nature has been previously uncharacterized. Finally, we demonstrate that the repercussions of an analog signaling pathway is the altered expression of a second transcription factor, Eomes, which is important in the differentiation and function of T cells. These results suggest that Itk is crucial in the modulation of TCR signal strength. Mast cells primarily rely on the IgE-bound FcεR1 for pathogen recognition. Crosslinking this receptor activates mast cells and results in degranulation and cytokine production via an expansive signaling cascade. Upon stimulation, Itk is recruited to the plasma membrane and phosphorylated. Little else is known about how Itk operates inside of mast cells. We find that mast cells lacking Itk are hyperresponsive to FcεR1-mediated activation. This is most apparent in the amount of IL-4 and IL-13 produced in comparison to wild-type mast cells. Increased cytokine production was accompanied by elevated and sustained signaling downstream of the FcεR1. Finally, biochemical evidence demonstrates that Itk is part of an inhibitory complex containing the phosphatase SHIP-1. These results indicate a novel function for Itk as a negative regulator in FcεR1- mediated mast cell activation.