Primary immunodeficiencies (PID) are genetic disorders characterized by increased susceptibility to infections and various degrees of immune dysregulation. With the expansion of massive parallel sequencing, an increasing number of defects in immune-related genes is being identified in PID. However, the biological impact of the found mutations is often unknown. It is necessary to devise methods to clarify their causality for disease development, which may also aid therapeutic decisions. One of the novel discoveries are gain-of-function mutations in STAT1 gene, resulting in chronic mucocutaneous candidiasis. Candidiasis may be ameliorated with antimycotics or with targeted JAK-STAT inhibitor, ruxolitinib. For our patient with a novel mutation in STAT1, we developed a simple test for the detection of phospho-STAT molecules in peripheral blood lymphocytes. The test confirmed the gain-of-function character of the identified mutation and was used to monitor ruxolitinib treatment efficacy. In the second patient, who presented with lymphadenopathy and immunodeficiency, the as yet undescribed mutation in CASP8 was found. We proved its loss-of-function property expressed as reduced caspase-8 and caspase-3 cleavage, impaired cellular apoptosis, and decreased NFB-related signaling. The third patient who...

Apoptosis, a form of genetically programmed cell death, plays a key role in regulation of cellularity of the arterial wall. During atherogenesis, improper apoptosis may cause abnormalities of arterial morphogenesis, wall structural stability, and metabolisms. It has been well established that vascular cells undergo apoptosis after uptake of oxidized low-density lipoprotein (oxLDL). Thus, an analysis of the signaling pathway of apoptotic induction by oxLDL is of value in understanding the development of atherosclerotic plaque. In order to elucidate the signaling pathway of apoptosis induced by oxLDL, we have used Chinese hamster ovary (CHO)-K1 cells treated with a potent oxysterol, 25-hydroxycholesterol (25-OHC). In the present study, we find that oxLDL can induce apoptosis in any cell types if cells present the specific receptors on their surface to take up oxLDL, and that apoptosis-inducing activity is associated with oxysterol components in oxLDL. Oxysterol-induced apoptosis does not involve regulation of sterol regulatory element-binding protein proteolysis pathway. 25-OHC stimulates calcium uptake by CHO-K1 cells within 2 min after addition. Treatment of CHO-K1 cells with the calcium channel blocker nifedipine prevents 25-OHC induction of apoptosis. One possible signal transduction pathway initiated by calcium ion fluxes is the activation of cytosolic phospholipase A2 (cPLA2). We demonstrate that activation of cPLA2 does occur in CHO-K1 treated with 25-OHC. Activation is evidenced by 25-OHC-induced relocalization of cPLA2 to the nuclear envelope and arachidonic acid (AA) release. Loss of cPLA2 activity by treatment with a cPLA2 inhibitor results in an attenuation of AA release as well as of the apoptotic response to 25-OHC in CHO-K1 cells. CPLA2ûmediated liberation of AA leads to the formation of a cyclooxygenase product, probably a prostaglandin, which activates the transcription factor PPARγ and induces apoptosis. We also examined the execution phase of the apoptotic pathway in CHO-K1 cell death induced by 25-OHC. Oxysterol-induced apoptosis in CHO-K1 is accompanied by caspase activation and is preceded by mitochondrial cytochrome C release. Furthermore, treatment with a cPLA2 inhibitor results in an inhibition of caspase-3 activation in CHO-K1 cells. These data provide strong evidence indicating that 25-OHC induces caspase-3-mediated apoptosis via an activation of calcium-dependent cPLA2.

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L’élimination des cellules infectées par apoptose constitue un mécanisme de défense antivirale. Les virus de l’herpès simplex (HSV) de type 1 et 2 encodent des facteurs qui inhibent l’apoptose induite par la réponse antivirale. La sous-unité R1 de la ribonucléotide réductase d’HSV-2 (ICP10) possède une fonction anti-apoptotique qui protège les cellules épithéliales de l’apoptose induite par les récepteurs de mort en agissant en amont ou au niveau de l’activation de la procaspase-8. Puisqu’une infection avec un mutant HSV-1 déficient pour la R1 diminue la résistance des cellules infectées vis à vis du TNFα, il a été suggéré que la R1 d’HSV-1 (ICP6) pourrait posséder une fonction anti-apoptotique. Le but principal de cette thèse est d’étudier le mécanisme et le potentiel de la fonction anti-apoptotique de la R1 d’HSV-1 et de la R1 d'HSV-2. Dans une première étude, nous avons investigué le mécanisme de la fonction anti-apoptotique de la R1 d’HSV en utilisant le TNFα et le FasL, deux inducteurs des récepteurs de mort impliqués dans la réponse immune anti-HSV. Cette étude a permis d’obtenir trois principaux résultats concernant la fonction anti-apoptotique de la R1 d’HSV. Premièrement, la R1 d’HSV-1 inhibe l’apoptose induite par le TNFα et par le FasL aussi efficacement que la R1 d’HSV-2. Deuxièmement, la R1 d’HSV-1 est essentielle à l’inhibition de l’apoptose induite par le FasL. Troisièmement, la R1 d’HSV interagit constitutivement avec la procaspase-8 d’une manière qui inhibe la dimérisation et donc l’activation de la caspase-8. Ces résultats suggèrent qu’en plus d’inhiber l’apoptose induite par les récepteurs de mort la R1 d’HSV peut prévenir l’activation de la caspase-8 induite par d’autres stimuli pro-apoptotiques. Les ARN double-brins (ARNdb) constituant un intermédiaire de la transcription du génome des HSV et activant l’apoptose par une voie dépendante de la caspase-8, nous avons testé dans une seconde étude l’impact de la R1 d’HSV sur l’apoptose induite par l’acide polyriboinosinique : polyribocytidylique (poly(I:C)), un analogue synthétique des ARNdb. Ces travaux ont montré qu’une infection avec les HSV protège les cellules épithéliales de l’apoptose induite par le poly(I:C). La R1 d’HSV-1 joue un rôle majeur dans l’inhibition de l’activation de la caspase-8 induite par le poly(I:C). La R1 d’HSV interagit non seulement avec la procaspase-8 mais aussi avec RIP1 (receptor interacting protein 1). En interagissant avec RIP1, la R1 d’HSV-2 inhibe l’interaction entre RIP1 et TRIF (Toll/interleukine-1 receptor-domain-containing adapter-inducing interferon β), l’adaptateur du Toll-like receptor 3 qui est un détecteur d’ARNdb , laquelle est essentielle pour signaler l’apoptose induite par le poly(I:C) extracellulaire et la surexpression de TRIF. Ces travaux démontrent la capacité de la R1 d’HSV à inhiber l’apoptose induite par divers stimuli et ils ont permis de déterminer le mécanisme de l’activité anti-apoptotique de la R1 d’HSV. Très tôt durant l’infection, cFLIP, un inhibiteur cellulaire de la caspase-8, est dégradé alors que la R1 d’HSV s’accumule de manière concomitante. En interagissant avec la procapsase-8 et RIP1, la R1 d’HSV se comporte comme un inhibiteur viral de l’activation de la procaspase-8 inhibant l’apoptose induite par les récepteurs de mort et les détecteurs aux ARNdb. / Elimination of infected cells by apoptosis constitutes an ancestral mechanism of host defense against viral infection. Herpes simplex viruses (HSVs) encode several viral factors to counteract the apoptotic antiviral response. Among them, the R1 subunit of HSV type-2 ribonucleotide reductase (HSV-2 R1, also named ICP10), protects cells by interrupting death receptor-mediated signaling at, or upstream of, caspase-8 activation. Since protection against tumor necrosis factor alpha (TNFα)-induced apoptosis is decreased un cells infected with an HSV type-1 R1 null mutant, it has been proposed that HSV-1 R1 (ICP6) could also possess an antiapoptotic activity. The fundamental goal of this thesis is to better understand the mechanism and the potential of the HSV R1s antiapoptotic activity. In a first study, we investigated the mechanism of the antiapoptotic activity of HSV R1s by using TNFα and Fas ligand (FasL), two death-receptor inducers involved in anti-HSVs immune response. From this work, we report three main findings on the antiapoptotic activity of HSV R1s. First, HSV-1 R1 like HSV-2 R1 has the ability to protect cells against TNFα- and FasL-induced apoptosis. Second, HSV-1 R1 contributes in protecting infected cells against FasL. Third, HSV R1s and procaspase-8 interact in a way that inhibits the dimerization/activation of caspase-8. These results suggest that in addition to counteracting death receptor-induced apoptosis, HSV R1s could inhibit apoptosis induced by other signals that trigger caspase-8 activation during HSV infection. Double-stranded RNA (dsRNA) are viral intermediates notably produced by HSVs and have been shown to induce apoptosis via caspase-8 activation. We tested in a second study whether HSV R1s have the ability to counteract apoptosis triggered by polyriboinosinic : polyribocytidylic acid (poly(I:C)), a synthetic analog of dsRNA that triggers caspase-8 activation. We showed that HSVs infection protect epithelial cells from apoptosis induced by poly(I:C). We established that HSV-1 R1 is essential for the protection of HSV-1-infected cells against poly(I:C)-induced caspase-8 activation. HSV R1s interact not only with procaspase-8 but also with the receptor interacting protein 1 (RIP1). The interaction between RIP1 and HSV-2 R1 inhibits the binding of RIP1 to the Toll/interleukine-1 receptor-domain-containing adapter-inducing interferon β (TRIF), the adaptor of Toll-like receptor 3 that is an extracellular dsRNA sensor, which is required to activate caspase-8 following extracellular poly(I:C) stimulation and TRIF overexpression. Thus, HSV R1s have the ability to inhibit poly(I:C)-induced apoptosis at several levels by preventing caspase-8 dimerization/activation and TRIF RIP1 interaction. This work sheds light on the ability of HSV R1s to manipulate apoptosis. Early during the lytic cycle, protein levels of the cellular inhibitors of caspase-8 as cFLIP drop but HSV R1s accumulate concomitantly and act as a viral inhibitor of apoptosis by binding to procaspase-8 and RIP1 in a way that impairs caspase-8 activation by death-receptors and dsRNA detectors stimulation.