TRK-Fused Gene (TFG), une protéine impliquée dans le système de sécrétion de protéines, est une composante essentielle de la réponse antivirale innée

La production d’interférons de type I (IFN) représente un événement signalétique nécessaire à la formation d’une réponse antivirale. La détermination du rôle de nouveaux régulateurs de l’immunité innée pourrait permettre le développement d’un arsenal thérapeutique conçu de manière à renforcer les réponses antivirales de l’hôte. La production des IFNs et des IFN-stimulated genes (ISG) est soutenue par la reconnaissance de composantes virale par les retinoic acid-inducible gene-I (RIG-I)-like receptors (RLR). Suivant leur activation, tumor necrosis factor receptor associated factor 3 (TRAF3) est recruté auprès de son substrat, TRAF family member-associated nuclear factor (NF)-kappa-B activator (TANK)-binding kinase 1 (TBK1), au niveau des compartiments cellulaires contenant la mitochondrial antiviral signaling adaptor (MAVS). Toutefois, la régulation de tels événements demeure largement indéterminée. Ici, nous identifions tropomyosin-receptor kinase (TRK)-fused gene (TFG), une protéine impliquée dans la biogenèse des vésicules coat protein complex II (COPII), en tant que nouveau partenaire de TRAF3 facilitant son recrutement à MAVS et à TBK1. La diminution de l’expression de TFG compromet en fait la formation d’un état antiviral en affectant la phosphorylation de TBK1, l’activation d’IFN regulatory factor 3 (IRF3) et la production d’ISGs et d’IFN-bêta. Certains résultats préliminaires révèlent aussi un nouveau lien entre les voies de signalisation des RLR et de la mammalian target of Rapamycin (mTOR) jusqu’alors présumées indépendantes. En effet, suivant une infection virale, la phosphorylation de mTOR sur sa Ser2159 par TBK1, un événement nécessitant l’expression de TFG, permet l’activation de mTOR et la traduction subséquente d’ISGs. Notre étude identifie ainsi TFG en tant que composante essentielle de la réponse antivirale aux IFNs de type I.The type I interferons (IFN) response represents key signaling event leading to potent antiviral state. As such, deciphering roles of new regulators of innate immunity could transform the antiviral treatment paradigm by introducing novel panviral therapeutics designed to reinforce antiviral host responses. The IFNs and IFN-stimulated genes (ISGs) production is supported by the recognition of RNA viruses by patternrecognition receptors, including retinoic acid-inducible gene-I (RIG-I) like receptors (RLR). Upon sensing of viral RNA, the E3 ubiquitine ligase tumor necrosis factor receptor associated factor 3 (TRAF3) is recruited along with its substrate TRAF family member-associated nuclear factor (NF)-kappa-B activator (TANK)-binding kinase 1 (TBK1) to mitochondrial antiviral signaling adaptor (MAVS)-containing subcellular compartments. However, regulation of such events remains largely unresolved. Here, we identify tropomyosin-receptor kinase (TRK)-fused gene (TFG), a protein involved in coat protein complex II (COPII)-coated vesicle biogenesis, as a new TRAF3- interacting protein allowing the efficient recruitment of TRAF3 to MAVS and TBK1 upon RLR activation following viral infection. Silencing its expression also compromised virus-induced phosphorylation of TBK1 on Ser172, activation of IFN regulatory factor 3 (IRF3), and production of ISGs and IFN-beta globally resulting in a decreased antiviral state. Preliminary results also identify a new link between the previously unrelated RLR and mammalian target of Rapamycin (mTOR) signaling pathways. Indeed, following viral infection, TFG-dependant phosphorylation of mTOR on Ser2159 by TBK1 seems to allow the activation of mTOR and the following translation of ISGs. Our study therefore identifies TFG as an essential component of type I IFN antiviral response by stimulating both the transcription and translation of antiviral agents

BTN3A2 Expression in Epithelial Ovarian Cancer Is Associated with Higher Tumor Infiltrating T Cells and a Better Prognosis

BTN3A2/BT3.2 butyrophilin mRNA expression by tumoral cells was previously identified as a prognostic factor in a small cohort of high grade serous epithelial ovarian cancer (HG-EOC). Here, we evaluated the prognostic value of BT3.2 at the protein level in specimen from 199 HG-EOC patients. As the only known role of butyrophilin proteins is in immune regulation, we evaluated the association between BT3.2 expression and intratumoral infiltration of immune cells by immunohistochemistry with specific antibodies against BT3.2, CD3, CD4, CD8, CD20, CD68 and CD206. Epithelial BT3.2 expression was significantly associated with longer overall survival and lower risk of disease progression (HR = 0.651, p = 0.006 and HR = 0.642, p = 0.002, respectively) and significantly associated with a higher density of infiltrating T cells, particularly CD4+ cells (0.272, p<0.001). We also observed a strong association between the relative density of CD206+ cells, as evaluated by the ratio of intratumoral CD206+/CD68+ expression, and risk of disease progression (HR = 1.355 p = 0.044, respectively). In conclusion, BT3.2 protein is a potential prognostic biomarker for the identification of HG-EOC patients with better outcome. In contrast, high CD206+/CD68+ expression is associated with high risk of disease progression. While the role of BT3.2 is still unknown, our result suggest that BT3.2 expression by epithelial cells may modulates the intratumoral infiltration of immune cells

Roles of GSK-3 and β-Catenin in Antiviral Innate Immune Sensing of Nucleic Acids

The rapid activation of the type I interferon (IFN) antiviral innate immune response relies on ubiquitously expressed RNA and DNA sensors. Once engaged, these nucleotide-sensing receptors use distinct signaling modules for the rapid and robust activation of mitogen-activated protein kinases (MAPKs), the I&kappa;B kinase (IKK) complex, and the IKK-related kinases IKK&epsilon; and TANK-binding kinase 1 (TBK1), leading to the subsequent activation of the activator protein 1 (AP1), nuclear factor-kappa B (NF-&kappa;B), and IFN regulatory factor 3 (IRF3) transcription factors, respectively. They, in turn, induce immunomodulatory genes, allowing for a rapid antiviral cellular response. Unlike the MAPKs, the IKK complex and the IKK-related kinases, ubiquitously expressed glycogen synthase kinase 3 (GSK-3) &alpha; and &beta; isoforms are active in unstimulated resting cells and are involved in the constitutive turnover of &beta;-catenin, a transcriptional coactivator involved in cell proliferation, differentiation, and lineage commitment. Interestingly, studies have demonstrated the regulatory roles of both GSK-3 and &beta;-catenin in type I IFN antiviral innate immune response, particularly affecting the activation of IRF3. In this review, we summarize current knowledge on the mechanisms by which GSK-3 and &beta;-catenin control the antiviral innate immune response to RNA and DNA virus infections

Pearson correlation test (two-tailed) between intra-epithelial immune infiltrate and clinical parameters in EOC patients.

<p>Table showing the coefficient of correlation with a Pearson correlation test (Pearson Correlation) between clinical parameters and immune infiltrate density in the intra-epithelial ovarian tumor tissues. Grade and stage were evaluated according to the FIGO classification. Res. Dis. =  amount of residual disease at primary resection of the tumor as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038541#pone-0038541-t001" target="_blank">Table 1</a>. Recurrence is the first event of ovarian cancer recurrence after the primary resection of the tumor. Death is the event of death due to ovarian cancer. CA125 =  blood serum level CA125 at the date of primary resection of the ovarian tumor. P is the p value from Pearson correlation test. N is the number of cases included in the statistical analysis. C =  Pearson correlation coefficient. P = p value. N =  number of cases.</p

Pearson correlation test (two-tailed) between BT3.2 expression and markers of intra-epithelial immune infiltrate in EOC tissues.

<p>Table showing the Pearson coefficient of correlation C between staining observed for each marker in the intra-epithelial ovarian tumor tissues. Grade and stage were evaluated according to the FIGO classification. P is the p value from Pearson correlation test. N is the number of cases included in the statistical analysis for which the observed staining was reliable. CD206/CD68 =  ratio of CD206+/CD68+ staining in the intra-epithelial area of tumor. C =  Pearson correlation coefficient. P = p value.</p

BT3.2 expression in ovarian cancer cells.

<p><b>A</b>. Western-blot analysis of total protein extracts from TOV112D cell line infected with PLenti (vector control), BT3.1, BT3.2 or BT3.3 viral constructs. Extracts were loaded in triplicate on 10% SDS/PAGE gel and membranes were hybridized with anti-CD277 (eBioscience), anti-BT3.3 (Atlas Antibodies) and anti-BT3.2 (SDIX Inc.) as indicated at the bottom of the Figure. <b>B</b>. Immunohistochemistry analysis of paraffin-embedded cell pellets from TOV112D cell line infected with either PLenti (vector control), BT3.1, BT3.2 or the BT3.3 viral constructs. Only cells transfected with the BT3.2 construct stained positively with the anti-BT3.2 (SDIX Inc.). <b>C</b>. Immunohistochemistry of xenograft tumors from TOV112D transfected with either empty vector or BT3.2 construct. Only cells infected with the BT3.2 construct stained positively with the anti-BT3.2 (SDIX Inc.). <b>D</b>. Representative staining for immunohistochemistry of BT3.2 on a high-grade serous EOC TMA. From left to right: negative, low, moderate and high intensity.</p

Representative staining for immunohistochemistry of infiltrating macrophages.

<p>High and low density of CD68+ (tumor-associated macrophages, TAM) (A) and CD206+ (M2 subtype of TAM) cells (B). Magnification in the high infiltration area is shown on the right panel for each staining (top). C and D. Kaplan-Meier analysis of the ratio of infiltrating intraepithelial CD206+/CD68+ cells representing the relative density of CD206+ M2 TAM over the total density of CD68+ intraepithelial infiltrating macrophages. Kaplan-Meier curves of overall survival (C) and disease-free survival (D) in 180 and 174 patients, respectively. Significance (p) is indicated by log rank.</p

Pearson correlation test (two-tailed) between BT3.2 expression in EOC tissues and clinical parameters of patients.

<p>Table showing the coefficient of correlation with a Pearson correlation test (Correlation) between clinical parameters and BT3.2 staining intensity observed in the intra-epithelial area of ovarian tumor tissues. Grade and stage were evaluated according to the FIGO classification as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038541#s2" target="_blank">Materials and Methods</a>. Res. Disease is the amount of residual disease at primary resection of the tumor as classified in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038541#pone-0038541-t001" target="_blank">Table 1</a>. Recurrence is the first event of ovarian cancer recurrence after the primary resection of the tumor. CA125 =  blood serum level CA125 at the date of primary resection of the ovarian tumor. P is the p value from Pearson correlation test. N is the number of cases included in the statistical analysis.</p

Univariate and multivariate Cox regression analysis showing the statistical association between BT3.2 expression and outcome of EOC patients.

<p>OS =  overall survival is the time of survival from the date of primary resection until event of death due to ovarian cancer. Res. Dis. =  amount of residual disease at time of primary resection of the ovarian tumor. Age =  age of patient at time of the first resection of the ovarian tumor. DFS =  disease free survival is the time from the first resection of the primary tumor until the first event of recurrence. HR =  hazard ratio. CI =  confidence interval. P =  p value.</p

Representative staining for immunohistochemistry of tumor infiltrating lymphocytes.

<p>High and low density of CD3+ (T cells), CD4+ (T cells), CD8+ (T cells) and CD20+ (B cells) shown in left and middle panels. A magnification in the high infiltration area is shown on the right panel for each staining.</p