Inhibition de la réplication des orthopoxvirus par le phénomène d'ARN interférence (perspectives thérapeutiques)

A l heure actuelle, face à une menace de réémergence de la variole, les recherches se sont axées vers la sélection de molécules anti-poxvirus. Dans ce contexte, les travaux réalisés au cours de cette thèse décrivent la mise en place de la technologie de l ARN interférence comme stratégie inhibitrice de la réplication des orthopoxvirus. Nous avons évalué l activité antivirale de plusieurs petits ARN interférents (siRNA), ayant pour cible des transcrits de gènes codant des protéines essentielles à la réplication du virus de la vaccine (VACV). A la suite de cette sélection, trois siRNA ont été retenus : siD5R- 2, ciblant le gène codant la protéine nucléoside triphosphate D5 ; siB1R-2, dirigé contre le gène B1R codant la protéine kinase B1 ; et siG7L-1, dirigé contre le gène G7L codant une protéine clé, G7, de la morphogenèse virale. Une inhibition significative de plus de 90% de la réplication virale des virus VACV, du cowpox (CPXV) et du monkeypox (MPXV), dans différentes lignées cellulaires, a été observée avec chacun de ces siRNA. Nous avons démontré que cette action antivirale est spécifique : les siRNA n induisent pas l activation du système interféron, et ils diminuent uniquement l expression du transcrit ciblé (D5R, B1R ou G7L), comme démontré par RT-PCR en temps réel. De plus, le pouvoir antiviral de ces siRNA, administrés par différentes voies, a été évalué in vivo dans un modèle souris d infection intra-nasale avec le CPXV ou le VACV. Le cidofovir (Vistide®) reste la molécule de première intention recommandée en cas de réémergence de la variole. Nous avons ainsi démontré pour la première fois qu une association du cidofovir avec chacun des trois siRNA sélectionnés avait un effet synergique contre la propagation du VACV in vitro. De plus, l activité antivirale des trois siRNA a étéévaluée contre cinq souches du VACV connues pour être résistantes au cidofovir, et à l un de ses dérivés diaminopurique, HPMPDAP, du fait de la présence de mutations dans le gène codant l ADN polymérase virale (E9L). Enfin, nous avons développé un siRNA en tant qu outil expérimental afin d étudier le mécanisme d action de la molécule anti-orthopoxvirus, ST-246. Un siRNA, siF13L, dirigé contre la protéine virale F13, cible de ST-246, nous a permis de confirmer que l activité antivirale de la molécule ST-246 était dépendante du mode de propagation de l orthopoxvirus ciblé. Ces travaux démontrent l efficacité des siRNA contre la réplication virale des orthopoxvirus in vitro et suggèrent de poursuivre leur développement in vivo en tant qu outil thérapeutique ou prophylactique pour traiter les infections par poxvirus.The potential release of the etiological agent of smallpox, Variola virus, by bioterrorists, has prompted renewed interest in the development of new therapeutic molecules that inhibit poxvirus replication. Here we report the use of the RNA interference technology as a sequence-specific inhibitory approach against orthopoxvirus replication in vitro and in vivo. We have assessed the antiviral activities of several siRNAs targeting different genes that are essential for viral replication of vaccinia virus (VACV). Three siRNAs have been selected : siD5R-2, siB1R-2 and siG7L-1 designed to target, respectively, the D5R gene encoding the DNA-independant nucleoside triphosphatase, the essential viral gene B1R (i.e., protein kinase) and the G7L gene encoding the protein G7 involved in virus morphogenesis. Each siRNA led to a significant decrease of VACV, cowpox virus (CPXV) and monkeypox virus (MPXV) replication (i.e., up to 90%) in different cell lines. Our results have also demonstrated the specificity of the antiviral effect of each siRNA: they only knocked down the transcripts of the targeted genes, as shown by real time RT-PCR, and they did not induce any interferon response. Moreover, the antiviral potencies of these three siRNAs, following several routes of administration (i.e., intranasal, intratracheal, intravenous or topical), have been investigated in vivo in a mouse model of orthopoxvirus infection. To date, cidofovir (Vistide®) is permitted for use as an emergency treatment in the case of smallpox outbreak. Thus, for the first time, we demonstrated the synergistic effect of cidofovir combined with each siRNA against VACV growth in cell culture. Moreover, we evaluated the antiviral potencies of these siRNAs against five vaccinia virus strains bearing mutations in the viral DNA polymerase gene (E9L), which are know to confer resistance to cidofovir and to one of its derivative, HPMPDAP. We finally developed a siRNA as an experimental tool to investigate the mechanism of action of the novel anti-orthopoxvirus compound ST-246. A siRNA (siF13L) designed to silence the F13L gene encoding the viral F13 protein, target of ST-246, confirmed our previous hypothesis: orthopoxviruses exhibit different levels of sensitivity to ST-246 due to their way of propagation. Our findings demonstrate the anti-orthopoxvirus potency of siRNAs and suggest to pursue their development in vivo as therapeutics for the treatment of poxvirus infections.AIX-MARSEILLE2-BU Méd/Odontol. (130552103) / SudocSudocFranceF

The mouse interleukin (Il)33 gene is expressed in a cell type- and stimulus-dependent manner from two alternative promoters

GenBank entries for mouse Il33 reveal the existence of two transcripts, Il33a and Il33b, with different 5`UTRs but coding for the same protein. We investigated expression of these transcripts in different mouse organs and cell types in basal and inflammatory conditions. Il33a and Il33b mRNAs start with different noncoding first exons, transcribed from different promoter regions, which both contain a consensus TATA-like sequence. Constitutive Il33a mRNA expression was detected in mouse stomach, lung, spleen, and brain, whereas basal Il33b mRNA expression was observed only in the stomach. Expression of both transcripts increased after systemic LPS administration. In vitro, we observed high constitutive expression of Il33 transcripts in MEFs. Constitutive Il33a mRNA expression was observed also in BMDCs, where it was preferentially increased in response to poly(I:C), whereas LPS increased levels of Il33a and Il33b mRNA. In contrast, BMMs and Raw 264.7 cells did not express Il33 mRNA constitutively, and LPS stimulation selectively induced expression of Il33b mRNA in these cells. Our data indicate that the Il33 gene is expressed from two alternative promoters in the mouse and that the relative expression of Il33a and Il33b transcripts is cell type- and stimulus-dependent

Interleukin-27 drives oxysterol production that regulate the adaptive Immune response

Oxysterols, oxidized forms of cholesterol, have pleiotropic roles on the immune response aside from their involvement in lipid metabolism. The oxysterols 25-hydroxycholesterol (25-OHC) and 725-dihydroxycholesterol (725-OHC) regulate antiviral immunity and immune cell chemotaxis. However their physiological effects on adaptive immune response in particular on CD4+ T lymphocytes are largely unknown. Here we assessed oxysterol levels in subset of CD4+ T cells and demonstrated that 25-OHC and transcript levels of its synthesizing enzyme, cholesterol 25 hydroxylase (Ch25h), were specifically increased in IL-27-induced Type 1 regulatory T (TR1) cells. We further showed that 25-OHC acts as negative regulator of TR1 cells in particular of IL-10 secretion via LXR signaling. Not only do these findings unravel novel molecular mechanisms accounting for IL-27 signaling but they also highlight lipids as critical modulators of adaptive immunity

Severe Neutrophil-Dominated Inflammation and Enhanced Myelopoiesis in IL-33-Overexpressing CMV/IL33 Mice

IL-33 is a cytokine of the IL-1 family, which signals through the ST2 receptor. Previous studies emphasized a role for IL-33 in shaping innate and adaptive immune responses. IL-33 was also reported to modulate myelopoiesis and myeloid cell activity. In this article, we describe IL-33-overexpressing CMV/IL33 and LysM/IL33 mice, which display an inflammatory phenotype associated with growth retardation and paw swelling. The phenotype of CMV/IL33 mice is dependent on activation of the ST2 receptor and is characterized by extensive neutrophil infiltration into different organs, including the paws. Local or systemic levels of proinflammatory mediators such as IL-1β, Cxcl-1, G-CSF, and IL-6 are increased. CMV/IL-33 mice also suffer from anemia, thrombocytosis, and a marked dysregulation of myelopoiesis, leading to an important increase in myeloid cell production or accumulation in bone marrow (BM), spleen, and peripheral blood. Consistently, recombinant IL-33 induced proliferation of myeloid lineage cells in BM-derived granulocyte cultures, whereas IL-33 knockout mice exhibited minor deficiencies in spleen and BM myeloid cell populations. Our observations reveal a neutrophil-dominated inflammatory phenotype in IL-33-overexpressing CMV/IL33 and LysM/IL33 mice, and highlight important regulatory effects of IL-33 on myelopoiesis in vitro and in vivo, where excessive IL-33 signaling can translate into the occurrence of a myeloproliferative disorder

Mouse neutrophils express the decoy type 2 interleukin-1 receptor (IL-1R2) constitutively and in acute inflammatory conditions

The proinflammatory activities of IL-1 are tightly controlled at different levels. IL-1R2 acts as a decoy receptor and has been shown to regulate the biological effects of IL-1 in vitro and in vivo. However, little is known about its natural expression in the mouse in physiologic and pathologic conditions. In this study, we examined IL-1R2 mRNA and protein expression in isolated cells and tissues in response to different stimulatory conditions. Data obtained using ex vivo CD11b(+)Ly6G(+) peripheral blood cells and in vitro-differentiated CD11b(+)Ly6G(+) BMG indicated that neutrophils are the major source of constitutively expressed IL-1R2 in the mouse. The expression of IL-1R2 on BMG and ex vivo Ly6G(+) peripheral blood cells was highly up-regulated by HC. IL-1R2 pull-down experiments showed that mouse rIL-1β binds to BMG IL-1R2, whereas binding of IL-1Ra could not be detected. Furthermore, LPS treatment induced shedding of IL-1R2 from the neutrophil membrane in vitro and in vivo, executed mainly by ADAM17. Finally, in in vivo models of inflammation, including thioglycolate-induced acute peritonitis and acute lung injury, infiltrating Ly6G(+) neutrophils, expressed IL-1R2. Our data show that in the mouse, neutrophils mainly express the decoy receptor IL-1R2 under naïve and inflammatory conditions. These data suggest that neutrophils may contribute to the resolution of acute inflammation

Articular inflammation is controlled by myeloid cell-derived interleukin 1 receptor antagonist during the acute phase of arthritis in mice

To define the cell type (myeloid vs other cells) specific effect of interleukin 1 (IL-1) receptor antagonist (IL-1Ra) deficiency on the acute inflammatory phase of arthritis

IL-27-Induced Type 1 Regulatory T-Cells Produce Oxysterols that Constrain IL-10 Production

IF 6.429International audienceThe behaviors of lymphocytes, including CD4(+) T helper cells, are controlled on many levels by internal metabolic properties. Lipid metabolites have recently been ascribed a novel function as immune response modulators and perturbation of steroids pathways modulates inflammation and potentially promotes a variety of diseases. However, the impact of lipid metabolism on autoimmune disease development and lymphocyte biology is still largely unraveled. In this line, oxysterols, oxidized forms of cholesterol, have pleiotropic roles on the immune response aside from their involvements in lipid metabolism. The oxysterols 25-hydroxycholesterol (25-OHC) and 7α,25-dihydroxycholesterol (7α,25-OHC) regulate antiviral immunity and immune cell chemotaxis. However, their physiological effects on adaptive immune response in particular on various subset CD4(+) T lymphocytes are largely unknown. Here, we assessed oxysterol levels in subset of CD4(+) T cells and demonstrated that 25-OHC and transcript levels of its synthesizing enzyme, cholesterol 25-hydroxylase, were specifically increased in IL-27-induced type 1 regulatory T (TR1) cells. We further showed that 25-OHC acts as a negative regulator of TR1 cells in particular of IL-10 secretion via liver X receptor signaling. Not only do these findings unravel molecular mechanisms accounting for IL-27 signaling but also they highlight oxysterols as pro-inflammatory mediators that dampens regulatory T cell responses and thus unleash a pro-inflammatory response