Chlamydia muridarum and Chlamydia caviae have equivalent growth rates in mouse epithelial cells but only C. muridarum replicates inside mouse macrophages, while C. caviae does not. Macrophages infected with C. muridarum or C. caviae were used to address the hypothesis that the early signaling pathways initiated during infection depend on the fate of chlamydiae in the host cell. Transmission electron microscopy of C. muridarum-infected macrophages showed intact chlamydial elementary bodies and reticulate bodies 2 h postinfection in compact vacuoles. Conversely, in macrophages infected with C. caviae, chlamydiae were observed in large phagocytic vacuoles. Furthermore, C. caviae infections failed to develop into inclusions or produce viable bacteria. Expression of proinflammatory cytokines TNFα, IL-1β and MMP13 was similar in C. caviae- or C. muridarum-infected macrophages at 3 h postinfection, indicating that chlamydial survival is not required for initiation of these responses. IL-1β secretion, dependent on inflammasome activation, occurred in C. caviae-infected macrophages despite no chlamydial growth. Conversely, IFNβ mRNA was observed only in C. muridarum- but not in C. caviae-infected macrophages. These data demonstrate that differential signaling events are initiated during a productive versus nonproductive chlamydial infection in a macrophage.

Les cellules épithéliales pulmonaires constituent la première ligne de défense face aux virus respiratoires via la sécrétion de mucus, de peptides, de cytokines et chimiokines qui déterminent l'élimination ou la progression de l’infection. Les principales cytokines antivirales produites par les cellules épithéliales alvéolaires (AEC) sont les interférons (IFN) type I (α/β) et III (λ). La liaison d’IFNβ à son récepteur induit une voie antivirale bien caractérisée qui aboutit à l’activation du complexe ISGF3 (STAT1, STAT2 et IRF9) qui permet la transcription de multiples gènes codant pour des protéines à activité antivirale et immunorégulatrice. Il a récemment été démontré que la costimulation des cellules épithéliales pulmonaires par l’IFNβ et le Tumor Necrosis Factor α (TNFα), également produit lors d’une infection, synergisent pour induire un état antiviral tardif distinct. D’autre part, il a été montré que la synergie entre le TNFα et l'IFNβ induit une voie de signalisation impliquant STAT2 et IRF9, mais indépendante de STAT1 permettant l’expression du gène DUOX2. Notre but est de déterminer l’importance de cette nouvelle voie de signalisation induite par la costimulation du TNFα+IFNβ, impliquant STAT2 et IRF9 indépendamment de STAT1 dans la régulation d’un programme transcriptionnel antiviral et immunorégulateur tardif. Notre premier objectif est de déterminer si des gènes antiviraux et immunorégulateurs qui sont induits par la costimulation par TNFα+IFNβ sont dépendants de la voie STAT2/IRF9, indépendamment de STAT1. En utilisant la technique de qRT-PCR, nous avons identifié 3 gènes immunorégulateurs, CXCL10, IDO et APOBEC3G, induits de manière synergique en réponse à TNFα+IFNβ dans les cellules A549, un modèle de cellules épithéliales pulmonaires. Afin de confirmer que ces gènes sont induits indépendamment de STAT1, nous avons validé leur expression dans la lignée cellulaire U3A déficiente en STAT1. Par l'utilisation d'ARN interférants (ARNi) dirigés contre STAT2 et IRF9, nous avons confirmé que l’induction de ces gènes est dépendante de STAT2 et IRF9. Finalement, l’analyse de l’activité du promoteur de CXCL10 en réponse à TNFα+IFNβ par des essais rapporteurs luciférases a permis de montrer que la régulation se fait au niveau transcriptionnel. Notre deuxième objectif, est de déterminer si STAT6 pourrait remplacer STAT1 dans la voie de signalisation induite par TNFα+IFNβ. En effet, STAT6 est un inducteur connu de l’expression de DUOX2 en réponse à IL4+IL13. Contrairement à notre hypothèse, l’inhibition de STAT6 par ARNi augmente l’expression de DUOX2 en réponse à TNFα+IFNβ suggérant que STAT6 est un régulateur négatif. Nos résultats ont permis de comprendre de manière plus détaillée les mécanismes mis en place dans le développement d’une réponse antivirale. D’autre part, l’étude de l’effet de l’IFNβ et du TNFα est également pertinente pour les maladies chroniques inflammatoires et autoimmunes. De plus, nos résultats illustrent un nouveau paradigme concernant les mécanismes de signalisation cellulaire impliqués dans la synergie entre deux cytokines qui pourrait être applicable à des combinaisons de cytokines autres que TNFα+IFNβ. / Lung epithelial cells are the first line of defense against respiratory viruses via mucus secretion, peptides, cytokines and chemokines that determine the progression of the infection. The main antiviral cytokines produced by alveolar epithelial cells (AEC) are the interferons (IFN) type I (α / β) and III (λ). IFNβ binding to its receptor induces an antiviral pathway that is well characterized and leads to activation of the ISGF3 complex (STAT1, STAT2 and IRF9) which allows the transcription of multiple genes encoding proteins with antiviral and immunoregulatory activity. It has recently been shown that the costimulation of lung epithelial cells by IFNβ and Tumor Necrosis Factor α (TNFα), also produced during infection, induces a separate and late antiviral state, through synergy. On the other hand, it has been shown that the synergy between IFNβ+TNFα induces a signaling pathway involving STAT2 and IRF9 independently of STAT1 permitting the expression of the DUOX2 gene. Our goal is to determine the importance of this new signaling pathway induced by costimulation of TNFα+IFNβ involving STAT2 and IRF9 regardless of STAT1 in regulating the antiviral immunoregulatory and late transcriptional program. Our first objective is to determine whether antiviral and immunomodulatory genes that are induced by costimulation TNFα+IFNβ are dependent on the STAT2/IRF9 way, indenpant of STAT1. Using the technique of qRT-PCR, we identified 3 immunoregulatory genes, CXCL10, IDO and APOBEC3G, synergistically induced in response to TNFα+IFNβ in A549 cells, a model of pulmonary epithelial cells. To confirm that these genes are induced independantly of STAT1, we validated their expression in the STAT1 deficient cell line, U3A. By the use of interfering RNA (siRNA) directed against STAT2 and IRF9, we confirmed that the induction of these genes is dependent STAT2 and IRF9. Finally, the analysis of the activity of CXCL10 promoter in response to TNFα+IFNβ by luciferase reporter assays has shown that the regulation is at the transcriptional level. Our second objective is to determine whether STAT6 could replace the STAT1 in the signaling pathway induced by TNFα+IFNβ. Indeed, STAT6 is a known inducer of the expression of DUOX2 in response to IL4+IL13. Contrarily to our hypothesis, inhibition of STAT6 by RNAi increases the expression of DUOX2 in response to TNFα+IFNβ suggesting that STAT6 is a negative regulator. Our results allow the understanding of the mechanisms in the development of an antiviral response in more detail. On the other hand, the study of the effect of IFNβ and TNFα is also relevant for chronic inflammatory and autoimmune diseases. In addition, our results illustrate a new paradigm for cell signaling mechanisms involved in the synergy between two cytokines that may be applicable to combinations of cytokines other than TNFα+IFNβ.

<p dir="ltr">Introduction: Radiotherapy is a treatment modality that is prescribed for more than 50% of cancer patients around the globe. Through decades of clinical application, RT has witnessed considerable advancements achieving significant tumor control with minimal damage to healthy tissues. Recently, a paradigm shift has recognized RT's potential to induce anti-tumor immune responses, where patients receiving radiation to the primary tumor also resolved lesions outside the treatment field. This out-of-field response also referred to as an abscopal effect, is believed to promote immunogenic cell death (ICD) initiated by the radiation-induced DNA damage and subsequent activation of the cGAS-STING-IFNβ pathways. However, clinical realization of an abscopal effect remains rare. We <i><u>hypothesize</u></i> by selectively irradiating cancer cells with high metastatic potential within a solid tumor (intra-tumor radiotherapy treatment planning) with high metastatic potential, a more efficient anti-tumor response can be achieved while minimizing inflammatory responses from surrounding tumor and normal tissues, obfuscating a potential adaptive immune response, thus help in overcoming the rarity observed in the clinical practice. To achieve this <i><u>objective</u></i>, radiotherapy treatment plans targeting hypoxic regions (known to harbor a metastatic phenotype) within a solid tumor and optimally activating IFNβ will be investigated.</p><p dir="ltr">Methods: Hypoxic conditions within tumor microenvironments significantly reduce DNA damage, conferring a radioresistant phenotype that leads to RT failure. To address the inherent radioresistance and immunosuppression of hypoxic tumors, high linear energy transfer (LET) modalities are used. Our research aims to enhance the specificity and efficiency of ICD, particularly in highly metastatic (hypoxic) regions within the tumor, by employing heavy charged particle (HCP) beams to optimize DSB induction. Empirical mathematical models have been developed to predict the dose-response of IFNb based on in vitro data and Monte Carlo methods of DSB-induction. These methods are used in maximizing type I interferon (IFNβ) production and subsequent immune response while minimizing the inflammatory response and damage to surrounding tissue. Immunogenic treatment plans, iTPS, have been developed to integrate charged particle beam models for proton, helium, and carbon ions and the above-empirical models into FLUKA Monte Carlo simulations and subsequently evaluated in clinical case studies of brain and lung cancer. Next, new biophysical models accounting for tumor hypoxia were developed and integrated into the iTPS, and clinical case studies were reevaluated.</p><p dir="ltr">Results: SA(1): Developed and integrated charged particle beam models into FLUKA MC for both homogeneous and heterogeneous treatment planning. Empirical equations for RBE_{DSB, pO2}, LET, and IFNβ dose-response were incorporated into FLUKA for voxel-based simulations across oxygen levels. SA(2): RBE_DSB-weighted optimization yielded uniform IFNβ production. High LET enabled carbon ion beams to require the lowest doses, achieving superior peak-to-entrance ratios of 15.85 compared to 10.78 and 7.60 for helium and proton beams, respectively. Patient simulations demonstrated carbon ions' superiority, with D_95% values of 7.68 Gy for the brain and 7.60 Gy for lung tumors, excelling in IFNβ production. SA(3): An optimized treatment plan for uniform IFNβ in hypoxia utilizing empirical equations for RBE_DSB across hypoxia levels was created for different charged particles. MCC13 adjustments based on OER_DSB from MCDS were confirmed by measured data in U251 cell lines, showing an OER of 1.5 between normoxia and 1% hypoxia, closely matching MCDS predictions within a 7% discrepancy. Carbon ions achieved optimal IFNβ at 11.02 Gy for brain tumors under 0.1% hypoxia in FLUKA simulations.</p><p dir="ltr">Conclusions: Our results from both homogeneous target and patient cases demonstrate that charged particles have the potential to elicit higher levels of IFNβ at lower doses compared to photon irradiations in different pO₂ levels. High LET irradiation not only ensures a highly localized IFNβ response in the target but also effectively spares surrounding normal tissues, thereby minimizing treatment-related toxicity. This finding underscores the superiority of high LET irradiation in achieving targeted immunogenic effects while enhancing the therapeutic window by reducing damage to normal cells.</p>