abstract: Vaccination remains one of the most effective means for preventing infectious diseases. During viral infection, activated CD8 T cells differentiate into cytotoxic effector cells that directly kill infected cells and produce anti-viral cytokines. Further T cell differentiation results in a population of memory CD8 T cells that have the ability to self-renew and rapidly proliferate into effector cells during secondary infections. However during persistent viral infection, T cell differentiation is disrupted due to sustained antigen stimulation resulting in a loss of T cell effector function. Despite the development of vaccines for a wide range of viral diseases, efficacious vaccines for persistent viral infections have been challenging to design. Immunization against virus T cell epitopes has been proposed as an alternative vaccination strategy for persistent viral infections, such as HIV. However, vaccines that selectively engage T cell responses can result in inappropriate immune responses that increase, rather than prevent, disease. Quantitative models of virus infection and immune response were used to investigate how virus and immune system variables influence pathogenic versus protective T cell responses generated during persistent viral infection. It was determined that an intermediate precursor frequency of virus-specific memory CD8 T cells prior to LCMV infection resulted in maximum T cell mediated pathology. Increased pathology was independent of antigen sensitivity or the diversity of TCR in the CD8 T cell response, but was dependent on CD8 T cell production of TNF and the magnitude of initial virus exposure. The threshold for exhaustion of responding CD8 T cells ultimately influences the precursor frequency that causes enhanced disease.In addition, viral infection can occur in the context of co-infection by heterologous pathogens that modulate immune responses and/or disease. Co-infection of two unrelated viruses in their natural host, Ectromelia virus (ECTV) and Lymphocytic Choriomeningitis virus (LCMV) infection in mice, were studied. ECTV infection can be a lethal infection in mice due in part to the blockade of antiviral cytokines, including Type I Interferons (IFN-I). It was determined that ECTV/LCMV co-infection results in decreased ECTV viral load and amelioration of ECTV-induced disease, presumably due to IFN-I induction by LCMV. However, immune responses to LCMV in ECTV co-infected mice were also lower compared to mice infected with LCMV alone and biased toward effector-memory cell generation. Thus, providing evidence for bi-directional effects of viral co-infection that modulate disease and immunity. Together the results suggest heterogeneity in T cell responses during vaccination with viral vectors may be in part due to heterologous virus infection or vaccine usage and that TNF-blockade may be useful for minimizing pathology while maintaining protection during virus infection. Lastly, quantitative mathematical models of virus and T cell immunity can be useful to generate predictions regarding which molecular and cellular pathways mediate T cell protection versus pathology. / Dissertation/Thesis / Doctoral Dissertation Molecular and Cellular Biology 2015

Cell death is an imperative mechanism for the development, homeostasis and survival of an organism. Various forms of cell death have been documented and recent reports indicate that the mode of cell death elicited can have a profound influence on the development and perpetuation of inflammation. Apoptosis is the predominant, programmed pathway of cell death, which ensures physiological elimination of unwanted cells. On the other hand, another cell death pathway described as programmed necrosis (necroptosis), has recently been revealed. The induction of necroptosis and its impact in host biology is not clear. Herein I have evaluated the mechanisms of necroptosis in macrophages, an important cell type of the immune system. My experiments indicate that type I interferon (IFN-I) signaling through transcription factors STAT1, STAT2 and IRF9, collectively described as the ISGF3 complex, is indispensable for necroptosis of macrophages. Furthermore, my results indicate that IFN-I signaling promotes the sustained phosphorylation of receptor interacting protein kinase 3 (Rip3), a key protein required for the execution of necroptosis. My findings also reveal that dynamin-dependent endocytosis following IFNβ stimulation and caspase inhibition is necessary for the induction of necroptosis. The results presented in this thesis provide new insights into the molecular mechanisms of necroptosis and therefore contribute to a deeper understanding of multiple inflammatory pathologies.

Conventional Dendritic cells (cDCs), a specialized group of immunological sentinels with tree-like or dendritic shapes, are critical for recognition of danger signals, presentation of antigens and control of a spectrum of innate and adaptive immune responses. Type I interferons (IFNs), as important danger signals, activate cDCs through the canonical type I IFN receptor signaling. Type I IFNs are the first line of host defense against viral infection by up-regulating IFN-stimulated genes (ISGs). However, there are circumstances in which the silencing of excessive type I IFNs could be beneficial to the host, such as IFN-dependent autoimmune diseases, gene therapy that uses viral vectors and transplantation. The role of type I IFNs in DC development, activation and antigen presentation function remains to be completely investigated. In this dissertation, we studied the regulation of Type I IFNs in murine DCs, both cDCs and plasmacytoid DCs (pDCs), and specifically we investigated the role of two molecules, Signal Transducer and Activator of Transcription 2 (STAT2) and Three prime Repair EXonuclease 1 (Trex1), in DC biology. Our research furthers our understanding of DC development, activation and function, and provides important data for the therapeutic application of modified DCs to induce immunological tolerance in gene therapy, IFN-dependent autoimmune diseases and transplantation. STAT2 is a nuclear transcription factor downstream of type I IFN receptor-mediated signaling, the role of which has been mostly explored in antiviral responses mediated by type I IFNs. However, the involvement of STAT2 in cDC activation and function such as cross-presentation remains hitherto unclear. We report that STAT2 is essential for murine cDC activation upon TLR3, -4, -7 and -9 stimulation. In the absence of STAT2, cDCs displayed impaired up-regulation of type I IFN response (costimulatory molecules and type I IFN-stimulated genes), and reduced inflammatory cytokine production when stimulated with TLR ligands. STAT2 was required in all of the DC responses to exogenous IFNα, suggesting that the canonical STAT1-STAT2 heterodimers are the major signaling transducers downstream of type I IFNs in DCs. Of interest, LPS-induced TNFα and IL6 production were reduced in STAT2-/- DCs but not in IFNAR1-/- DCs, suggesting a novel STAT2-dependent pathway mediated by LPS, bypassing type I IFN-receptor signaling. STAT2-deficient cDCs showed impaired cross-presentation leading to decreased CD8+ T cell response both in vitro and CTL killing in vivo, indicating that STAT2 is essential for TLR-induced cross-presentation. These results demonstrate that STAT2 is critical in the regulation of TLR-induced DC activation and cross-presentation, suggesting an essential role for STAT2 in anti-viral and anti-tumor immune responses. We also propose a novel regulatory function of STAT2 in the LPS response independent of type I IFN receptor signaling. Trex1 mutations are associated with a spectrum of type I IFN-dependent autoimmune diseases such as Aicardi-Goutières syndrome and systemic lupus erythematosus. Trex1 plays an essential role in preventing accumulation of excessive cytoplasmic DNA, avoiding cell-intrinsic innate DNA sensor activation and suppressing activation of both type I IFN-stimulated and IFN-independent antiviral genes. Trex1 also helps HIV escape cytoplasmic detection by DNA sensors. However, regulation of Trex1 in DC biology is lacking. We report that murine cDCs have high constitutive expression of Trex1 in vitro and in vivo in the spleen. In resting bone marrow-derived cDCs, type I IFNs up-regulate Trex1 expression via the canonical IFN receptor signaling pathway (STAT1-, STAT2-dependent). DC activation induced by TLR3, -4, -7 and -9 ligands also augments Trex1 expression through autocrine IFNß production and triggering of the IFN signaling pathway, while TLR4 ligand LPS also stimulates an early expression of Trex1 through an IFN-independent NFΚB-dependent signaling pathway. Furthermore, retroviral infection also induces Trex1 up-regulation in cDCs, as we found that a gene therapy HIV-1-based lentiviral vector induces significant Trex1 expression, suggesting that Trex1 may affect local and systemic administration of gene therapy vehicles. Our data indicate that Trex1 is induced in cDCs during activation upon IFN- and TLR- stimulation through the canonical IFN signaling pathway, and suggest that Trex1 may play a role in cDC activation during infection and autoimmunity. Finally, these results suggest that HIV-like viruses may up-regulate Trex1 to increase their ability to escape immunosurveillance. In order to dissect the role of Trex1 in DC functions, we compared DCs from Trex1-/- and wild-type mice. We report that Trex1 deficiency reduces absolute number of pDCs in BM but not in spleen of male over female mice. Furthermore, Trex1 deficiency preferentially represses Flt3L-induced DC development both in vitro and in vivo but not GM-CSF-dependent DC development, suggesting that Trex1 plays an indispensable role in Flt3L-induced DC development and GM-CSF may compensate the effect of Trex1 deficiency. This defect is only limited to male Trex1-/- DCs, and mimics the effect of mTOR inhibition. Furthermore, although Flt3L-induced Trex1-/- DCs expressed a type I IFN signature, they also exhibited decreased pDC development markers, indicating Trex1 regulates pDC development at the transcriptional level. Thus, we propose a novel and essential role of Trex1 in Flt3L-induced DC development, and the effect of Trex1 regulation is gender-dependent. Together, these findings enhance our understanding of regulatory roles of Type I IFNs in DC development, activation and function, supporting the beneficial role of STAT2/type I IFN axis in TLR-induced DC activation and cross-presentation. Our study in Trex1 reveals Trex1 induction by viral infection, type I IFNs and TLRs in DCs, and a new role of Trex1 in early development of Flt3L-induced DCs in a gender-dependent manner, whereby a balance between type I IFNs and Trex1 is important for DC activation and hemostasis. / Microbiology and Immunology

Intestinal homeostasis is a delicate balance between suppression of immune responses against innocuous antigens and stimulation of immune responses against pathogens. Type I interferon (IFN-1) cytokines have both immunostimulatory and immunomodulatory effects. Colon mononuclear phagocytes (MP) constitutively produced IFN-1 in a TRIFdependent manner. We explored the function of endogenous IFN-1 in the colon using the T cell adoptive transfer model of colitis. Transfer of CD4⁺CD45RB^hi naïve T cells from wild type (WT) or IFNAR subunit 1 knockout (IFNAR1^-/-) mice into RAG^-/- hosts resulted in similar onset and severity of colitis. In contrast, RAG^-/- x IFNAR1^-/- double knockout (DKO) mice developed accelerated severe colitis compared to RAG^-/- hosts when transferred WT CD4⁺CD45RB^hi T cells. Although WT or IFNAR1^-/- regulatory T (Treg) cells equally prevented disease caused by CD45RB^hi naïve T cells, WT Treg cells co-transferred with naïve CD4⁺ T cells into DKO recipients failed to expand or maintain Foxp3 expression and gained effector functions in the colon. IFNAR signaling on host hematopoietic cells inhibited T cell-mediated colitis, but not innate colitis. MPs isolated from the colon lamina propria (cLP) required IFNAR signaling for the production of the anti-inflammatory cytokines, IL-10, IL-27, and IL-1RA, but not for the production of classic pro-inflammatory cytokines. IFN-1-dependent secretion of IL-1RA was particularly important in inhibiting the migration of inflammatory DCs with potent T cell proliferative capacity from the cLP to the mesenteric lymph nodes. Finally, preliminary results suggested that IFN-1 may shape the commensal microbiota, but is not essential for controlling specific colitis-inducing bacteria.

RSV is the most common cause of infant bronchiolitis, leading to morbidity and mortality in both infants and the elderly. The relationship between RSV and asthma development further highlights the need to fully understand the immune responses involved in order to develop effective vaccines and therapeutics to aid prevention and treatment of RSV infection respectively. Helminths have long been studied both as a major pathogen of humans, infecting approximately 3 billion people worldwide, and also their ability to modulate the host immune response to allow survival and chronic infection to ensue. Specifically, helminth infections are thought to modulate the host immune response through regulatory mechanisms which are not fully understood. This not only confers protection and survival of the parasites themselves, but also modulates the immune response to unrelated antigens and pathogens. In this thesis, the potential role of a strictly enteric helminth infection, with Heligmosomoides polygyrus, in the modulation of respiratory syncytial virus (RSV) infection was investigated and the associated immune mechanisms were investigated. Firstly, the effects of prior H. polygyrus infection on RSV infection and immune responses in the lung were analysed. H. polygyrus significantly reduced the number of natural killer cells, CD8+ T cells, B cells and conventional dendritic cells in the lung following RSV infection. Co-infection also reduced the production of pro-inflammatory cytokines IL-6 and TNF-α in the lungs. All of these reductions were associated with significantly lower viral titres on day 4 of RSV infection. Interestingly, this attenuation of immune responses and viral titres, correlated with reduced severity of clinical disease, as assessed by weight loss and lung function. H. polygyrus excretory secretory product (HES) was not found to be the immune-modulatory factor in this system, as HES failed to suppress viral titres and reduce immune cell responses to RSV infection. However, irradiated larvae with stunted maturation to adult worms, revealed that larval stages were sufficient to suppress viral titres. Next, the role of type 2 signalling for H. polygyrus effects on RSV infection were examined, using IL-4Rα-/- mice. H. polygyrus infection maintained the ability to attenuate RSV infection and subsequent immune responses in IL-4Rα-/- mice. Furthermore, the presence of the adaptive immune response was not required for H. polygyrus-induced attenuation of RSV infection, as demonstrated in recombinase-activating gene (RAG-/-) deficient mice. H. polygyrus induces innate type 2 immune responses indicating the release of the innate alarmin, IL-33, in the lung and consequently an accumulation of group 2 innate lymphoid cells (ILC2). Their contribution to H. polygyrus effects remain to be fully elucidated. Finally, the role of antiviral responses was explored in H. polygyrus and RSV co-infection. H. polygyrus infection alone induced expression of antiviral genes, IFN-β, OAS1A, Viperin and the antimicrobial peptide CRAMP, in both the duodenum and the lung. Expression of these genes was still higher in the lung 1 hour after RSV in H. polygyrus co-infected mice compared to controls without co-infection. The importance of type I IFN signalling pathway was demonstrated using mice deficient in the type I IFN receptor in H. polygyrus co-infection, which failed to suppress RSV titres and subsequent lung immune cell infiltration. These data highlight the ability of the strictly enteric helminth H. polygyrus to attenuate RSV infection and subsequent immune responses in the lung through the potentiation of type I IFN signalling and consequent upregulation of antiviral immune responses in the lung.

<p>When bacterial pathogens infect their hosts, they illicit responses intended on containing and eliminating these invaders. This happens not only on the organismal level, but also on the cellular level. When a cell detects that it has been infected by an intracellular pathogen, it triggers a set of internal signaling events intended to contain the intruder. These events may allow the cell to produce antimicrobial agents or may help recruit members of the immune system to help fight the infection. In the case of closely evolved pathogens, such cell signaling events can be co-opted by the invading bacteria to its advantage. One example of this is infection with the gram-negative bacteria Chlamydia trachomatis. Infection with the obligate bacterial intracellular pathogen Chlamydia trachomatis leads to the sustained activation of the small GTPase Ras and many of its downstream signaling components. In particular, the mitogen-activated protein kinase ERK and the calcium-dependent phospholipase cPLA2 are activated and are important for the onset of inflammatory responses during chlamydial infection. In this study we tested if activation of ERK and cPLA2 occurred as a result of Ras signaling during infection and determined the relative contribution of these signaling components to chlamydial replication and survival. we provide genetic and pharmacological evidence that Ras, ERK and, to a lesser extent, cPLA2 activation are uncoupled during infection, suggesting that Chlamydia activates individual components of this signaling pathway in a non-canonical manner. In human cell lines, inhibition of ERK or cPLA2 signaling did not adversely impact C. trachomatis replication. In contrast, in murine cells cPLA2, and to a lesser extent ERK, signaling played a significant protective role against C. trachomatis. we determined that cPLA2-deficient murine cells are permissive for C. trachomatis replication because of their impaired expression of &#946; interferon and the induction of immunity-related GTPases (IRG) important for the containment of intracellular pathogens. Overall, these findings define a previously unrecognized role for cPLA2 in the induction of autonomous innate immune responses to Chlamydia infections.</p> / Dissertation

No description available.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23804号 / 医博第4850号 / 新制||医||1058(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 井上 治久, 教授 林 康紀, 教授 竹内 理 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Type I interferons (IFN) are a potent antiviral cytokine group that are key regulators of the immune response against virus infection. Not only does this group activate antiviral states within target cells, it can modulate the innate immune response. In the studies presented, we investigate the effects of type I IFN on the innate immune system during a mucosal vaginal virus infection, herpes simplex virus type 2 (HSV-2), a prominent sexually transmitted infection that causes genital herpes and increases risk of human immunodeficiency virus acquisition. It is well known that type I IFN is critical for natural killer (NK) cell activation. These cells contribute to the antiviral response by suppressing virus replication and aiding in the initiation of the adaptive immune response, particularly through the release of IFN-γ. In the work presented, we demonstrate that type I IFN does not act on NK cells directly for their activation, but instead activates NK cell IFN-γ production by inducing inflammatory monocytes to release IL-18, which in turn, signals NK cells to release IFN-γ during a mucosal HSV-2 infection. Rather, direct action of type I IFN on NK cells serves to negatively regulate their IFN-γ response. We also found that type I IFN was critical for suppressing virus-induced innate immunopathology during HSV-2 infection. Overall, our studies further our understanding of type I IFN and the many roles it plays during virus infection, which has become more relevant as specific therapies altering type I IFN are being used in the clinic. Further, we provide a fundamental understanding of type I IFN and its ability to shape the innate immune response to virus infection by suppressing dysregulated and immunopathological functions while promoting beneficial innate immune responses that can help fight the infection. / Thesis / Doctor of Philosophy (PhD) / Type I interferons (IFN) are a group of proteins that are rapidly produced early during infection and is important for combatting virus infections. We show that type I IFN is not just an antiviral molecule, but can modulate the initial immune response to virus infection. As part of the initial immune response, Natural killer (NK) cells are immune cells that respond rapidly to infection and are a key element in controlling the early stages of infection. We found that type I IFN is critical for activating NK cell function by signaling through an intermediary cell, but can also suppress that same function by directly acting on NK cells. We also found that type I IFN is critical for suppressing a dysregulated immune response that causes severe virus-induced vaginal pathology. Overall, our data suggests that type I IFN is a key antiviral molecule that shapes the immune response to virus infection.

L’activation des lymphocytes T nécessite un double signal. Le premier est antigénique et permet la reconnaissance d’un peptide spécifique présenté à la surface de cellules présentatrices d’antigène (APC). Le second signal est co-stimulateur et implique l’interaction avec des molécules activatrices exprimées par les APC et la présence de cytokines proinflammatoires. Les cellules dendritiques (DC) sont les uniques APC capables de délivrer ce double signal et d'activer les lymphocytes T naïfs, initiant ainsi les réponses immunes primaires. L’immaturité du système immunitaire du nouveau-né est responsable d’une plus grande susceptibilité aux maladies infectieuses ainsi qu’une faible réponse vaccinale. Des déficiences tant au niveau de l’immunité innée que de l’immunité acquise participe à une faible défense face aux agressions. A la naissance, les DC expriment des niveaux faibles de molécules co stimulatrices et présentent un défaut majeur de synthèse d'IL 12, cytokine cruciale pour l’établissement de réponses de type Th1. Le but de ce travail est d’évaluer la capacité des DC du nouveau-né humain à activer les lymphocytes T CD8+. Dans une première approche, nous avons utilisé un modèle unique d’induction de réponse primaire in vitro qui permet d'étudier l'activation de lymphocytes T CD8+ spécifiques de l’antigène Melan-A, une protéine du soi exprimée par les mélanocytes. Ces lymphocytes existent à des fréquences particulièrement élevées chez les individus sains HLA-A2 et présentent les caractéristiques de lymphocytes T naïfs. Dans ce modèle, nous avons d’abord analysé les capacités immunostimulatrices de différentes populations de DC différenciées in vitro. Nous avons observé que les DC différenciées par la culture de monocytes purifiés en présence d'IL-3 et d’IFN-beta sont capables d’initier une réponse fonctionnelle des lymphocytes T CD8+, analogue à celle induite par les DC différenciées en présence de GM-CSF et d’IL-4. Ce même modèle nous a permis de démontrer que, en dépit de leur défaut de production d’IL 12, les DC du nouveau-né sont capables d'induire efficacement une réponse lymphocytaire T CD8+ cytotoxique. Afin dévaluer la relevance in vivo de nos observations, nous avons étudié le phénotype et la fonction des DC circulantes chez des nouveau-nés infectés par le cytomégalovirus (CMV). L’infection par le CMV au cours de la vie fœtale représente une situation clinique où le nouveau-né développe une réponse mature et fonctionnelle des lymphocytes T CD8+, alors que celle des lymphocytes T CD4+ est déficiente. Ces expériences ont montré que le phénotype, la fonction et la réponse à différents stimuli des APC présentes en périphérie ne sont pas affectés par l’infection congénitale par le CMV. Ces résultats suggèrent que l’observation des DC circulantes des nouveau-nés infectés par le CMV ne permet pas d’analyser l’influence du virus sur la fonction des DC néonatales. Dans ce but, nous avons reproduit un modèle d’infection in vitro de DC par une souche primaire du CMV. L’utilisation de micropuces à ADN nous a permis de comparer l’expression de gènes différentiellement induits par l’infection des DC d’adultes et de nouveau-nés. Nous avons ainsi révélé une proportion importante de gènes différentiellement induits, parmi lesquels celui de l’IFN-beta. Nous avons confirmé ce défaut au niveau protéique et mis en évidence une production d’IL 12 déficiente en réponse à l’infection par CMV. L’ensemble de nos résultats indique que malgré leur immaturité, les DC du nouveau-né sont capables, dans certaines circonstances, d’induire une réponse lymphocytaire T CD8+ cytotoxique. Cependant, le défaut de production de certaines cytokines co-stimulatrices pourrait être impliqué dans la faible réponse des lymphocytes T CD4+ à l’infection par CMV. Ces observations ont d’importantes implications pour la compréhension de l’induction de réponses cytotoxiques au cours d’infections virales et pour l’élaboration de stratégies vaccinales en début de vie.