36. Genome-Wide Insight Into the Transcriptional Modulations Triggered By Lentiviral Transduction in Human Hematopoietic Stem Cells

Recent studies suggest that hematopoietic stem cells (HSC) can sense foreign nucleic acids and pathogen-associated molecular patterns (PAMPs). Exposure to lentiviral vectors (LV) upon gene transfer may thus trigger acute host responses in HSC that could potentially impact on their biological properties, although no comprehensive studies are available to date. We have performed a high throughput RNA-Seq analysis on human cord-blood (CB)-derived CD34+ hematopoietic stem and progenitor cells (HSPC) exposed to research- or clinical-grade VSV-g pseudotyped (SIN) LV at a high multiplicity of infection, matching current clinical vector dose requirements. As controls, cells were exposed to non-transducing Env-less, genome-less or heat inactivated control vectors or kept in culture untreated. RNA was extracted at different times early after transduction, processed and ran in Illumina HiSeq2000. Analysis of Differential Expression in Time Course was performed using LIMMA R/BioConductor library. Key pathways were assessed by Term Enrichment Analysis considering KEGG pathways and Gene Ontology Biological processes. Transduction with both research-and clinical-grade LV significantly triggered DNA damage and apoptosis-related responses. In particular, p53 signaling was among the most significantly altered pathways (p<3.47×10−14) and induction of several key players, including a 8-fold increase in p21 mRNA, was further confirmed by Taqman. This signaling occurred also in bone-marrow-derived CD34+ cells and was integration-independent as Integrase-Defective LV (IDLV) induced p21 to a similar extent as LV. Furthermore, equal induction was observed in all CD34+ subpopulations, including in the most primitive CD38-CD133+ fraction. Finally, LV/IDLV exposure lead to a slight but significant increase in the percentage of apoptotic HSPC in culture (p<0.001) as compared to control vector exposed cells and untreated controls. Experiments are ongoing to further investigate the potential short and long-term consequences of this signaling on the biological properties of HSPC in vitro and in vivo. Overall, our results suggest for the first time that LV transduction triggers transcriptional changes in HSPC involving pathways pivotal for their biology. Better understanding of the potential functional consequences this may have will be important for the development of improved gene therapy protocols

HIV-1 transcriptional silencing caused by TRIM22 inhibition of Sp1 binding to the viral promoter

Background: Intracellular defense proteins, also referred to as restriction factors, are capable of interfering with different steps of the viral life cycle. Among these, we have shown that Tripartite motif 22 (TRIM22) suppresses basal as well as phorbol ester-induced HIV-1 long terminal repeat (LTR)-mediated transcription, independently of its E3 ubiquitin ligase activity, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) binding to the U3 region and Tat interaction with the TAR region of the HIV-1 LTR. As basal HIV-1 transcription is driven by the transcription factor specificity protein 1 (Sp1), we have investigated whether TRIM22 could interfere with Sp1-driven transcriptional activation of the HIV-1 LTR. Findings: 293T cells, devoid of endogenous TRIM22 expression, were transfected with a TRIM22-expressing plasmid together with reporter plasmids driven by the HIV-1 LTR promoter either containing or lacking Sp1 binding sites or with reporter plasmids driven by non-viral promoter sequences either containing or lacking the three Sp1 binding sites from the HIV-1 LTR. These reporter assays showed that TRIM22 efficiently inhibited Sp1-driven transcription. Knocking down TRIM22 expression in the CD4+ SupT1 T cell line increased the replication of Sp1-dependent HIV-1 variants. TRIM22 did not interact with Sp1, but prevented binding of Sp1 to the HIV-1 promoter, as demonstrated in protein-DNA pull down and chromatin immunoprecipitation assays. Conclusion: TRIM22 acts as a suppressor of basal HIV-1 LTR-driven transcription by preventing Sp1 binding to the HIV-1 promoter

D-mannose suppresses macrophage IL-1β production

D-mannose is a monosaccharide approximately a hundred times less abundant than glucose in human blood. Previous studies demonstrated that supraphysiological levels of D-mannose inhibit tumour growth and stimulate regulatory T cell differentiation. It is not known whether D-mannose metabolism affects the function of non-proliferative cells, such as inflammatory macrophages. Here, we show that D-mannose suppresses LPS-induced macrophage activation by impairing IL-1β production. In vivo, mannose administration improves survival in a mouse model of LPS-induced endotoxemia as well as decreases progression in a mouse model of DSS-induced colitis. Phosphomannose isomerase controls response of LPS-activated macrophages to D-mannose, which impairs glucose metabolism by raising intracellular mannose-6-phosphate levels. Such alterations result in the suppression of succinate-mediated HIF-1α activation, imposing a consequent reduction of LPS-induced Il1b expression. Disclosing an unrecognized metabolic hijack of macrophage activation, our study points towards safe D-mannose utilization as an effective intervention against inflammatory conditions

DNA damage contributes to neurotoxic inflammation in Aicardi-Goutières Syndrome astrocytes

Aberrant induction of type I IFN is a hallmark of the inherited encephalopathy Aicardi-Goutières syndrome (AGS), but the mechanisms triggering disease in the human central nervous system (CNS) remain elusive. Here, we generated human models of AGS using genetically modified and patient-derived pluripotent stem cells harboring TREX1 or RNASEH2B loss-of-function alleles. Genome-wide transcriptomic analysis reveals that spontaneous proinflammatory activation in AGS astrocytes initiates signaling cascades impacting multiple CNS cell subsets analyzed at the single-cell level. We identify accumulating DNA damage, with elevated R-loop and micronuclei formation, as a driver of STING- and NLRP3-related inflammatory responses leading to the secretion of neurotoxic mediators. Importantly, pharmacological inhibition of proapoptotic or inflammatory cascades in AGS astrocytes prevents neurotoxicity without apparent impact on their increased type I IFN responses. Together, our work identifies DNA damage as a major driver of neurotoxic inflammation in AGS astrocytes, suggests a role for AGS gene products in R-loop homeostasis, and identifies common denominators of disease that can be targeted to prevent astrocyte-mediated neurotoxicity in AGS

Impact du polymorphisme des gènes CD209 (DC-SIGN) et 2'-5 oligo-adénylate synthétase 1b (2' -5 Oas1b) sur leurs activités fonctionnelles en relation avec les flaviviroses dengue et fièvre du Nil occidental (West Nil)

PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Impact du polymorphisme des gènes CD209 (DC-SIGN) et 2'-5 oligo-adénylate synthétase 1b (2' -5 Oas1b) sur leurs activités fonctionnelles en relation avec les flaviviroses dengue et fièvre du Nil occidental (West Nil)

PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

HI and SN Titres in Human Sera against Human and Avian Viruses of the H1, H2, and H3 Subtype.

*<p>A/Wisconsin/67/2005/H3N2.</p>**<p>A/New Caledonia/20/99/H1N1.</p>***<p>A/Singapore/57/H2N2.</p><p>- = ≤1∶16 for HI, <1∶20 for SN.</p><p>∧ = Titres expressed as the highest dilution at which full haemagglutination inhibition is observed.</p><p>° = 0.5% chicken RBC solution.</p><p>°° = 1% horse RBC solution.</p><p>• = Not done.</p