Viral evasion of intracellular innate immune sensing pathways
1. Hiding the viral genome 2. Inhibiting interactions with key host inducers of the IFN response 3. Regulating phosphorylation events 4. Regulating ubiquitinylation & related pathways 5. Cleavage & degradation 6.Transcriptional shut-off 7 . RNA processing & trafficking regulation 8. Translational shut-off 9. Decoys 10. Everything counts
Ten Strategies: Evasion of Induction of Interferons (1)
Ten Strategies: Inhibition of Interferon Signaling (2)
Ten Strategies: Inhibition of Host Gene Expression (3)
Dengue virus immunopathogenesis � � Viral evasion strategies � � Metabolic stress & innate immunity
Epidemiology and pathogenesis of dengue infection Ø Most common arthropod-borne viral pathogen - transmitted to humans by Aedes aegypti and Aedes albopictus Ø 2.5 billion people at risk in tropical regions Ø Estimated 300 million infections per year, with 25,000-50,000 deaths annually Ø Primary infection: often a self limiting acute infection (‘breakbone fever’) Ø Secondary heterologous infection generally lead to more severe immunopathogenic disease with risk of dengue hemorrhagic fever or shock syndrome (DHF/DSS) Ø No effective antiviral agents and no effective vaccine to treat or prevent dengue infection
Dendritic Cells and Dengue Virus Ø Skin-resident DC and Langerhans cells are the initial targets of infection Ø DC are able to mount a rapid inflammatory and antiviral response to DENV Ø DENV-infected DC: induce an innate immune response to infection, undergo maturation and are subject to viral evasion mechanisms (interference with STAT signaling; viral protease cleavage of STING) Ø Interaction between DENV and DC is crucial for both control of infection and the evolution of disease severity Understanding the mechanisms involved in DENV sensing and the innate immune response to infection is critical for the comprehension of DENV disease evolution and control of infection
Differentiated myeloid cells are the main targets for DENV infection Mo-DC Day 7 60 % of CD14- CD1a+ cells mRNA relative expression + DENV2 - DENV2 r 2 = 0.9829 79.6% 40 p <0.0001 n=15 *** SSC-A *** 20 *** Monocyte *** Day 0 DENV2 ** 0 0 20 40 60 80 100 CD1a % of DENV2+ cells . CD14
Differential gene expression appears early following DENV2 infection of dendritic cel ls 0 6 12 18 24 MT1A MT2A Antioxidant MT1E genes MT1G Up-regulated MT1H 6000 Down-regulated 6000 Source of ERO1L 4000 ROS 4000 Number of DEGs DDIT4 DNA-damaged 2000 induced gene 2000 ISG15 0 OAS2 0 HERC5 IFIT1 2000 IFIT2 Antiviral -2000 IFIT3 response OASL 4000 HES4 -4000 CCL5 6000 -6000 Time (h) 0 6 18 12 24
Stress-Associated Pathways Are Enriched Early After DENV2 Infection Antiviral Response Myeloid Signalling 6H Inflammatory Response Stress Response Apoptosis 24H Diverse Signalling 0 6 12 18 24
Is there a link between DENV2-induced oxidative stress response and DENV2-induced antiviral/ inflammatory responses?
NADPH oxidase-dependent ROS formation restricts DENV infection and replication IL-1 β IDO1 IFN- β CCL5 mRNA relative 70 expression 60 * * 50 40 * * * * * * * * * 30 * * * * * 20 * * * * * * * * * * * * * * * * * * 10 * * * * * * * * * * * * 0 DENV2 - + + + + + + + - + + + + + + + - + + + + + + + - + + + + + + + DPI Apocynin Tempol Ebselen PDTC Trolox uninfected % of DENV E+ cells p= 0.036 1.54±0.3 p= 0.044 Viral Titer DENV 38.7±3.9 p= 0.004 52.1±4.0 DENV2 - + + + - + + + DENV2 FSC-A DPI - - 0.1 1 - - 1 0.1 DPI DENV E
DENV infection stimulates an Nrf2-driven antioxidant response 0 6 12 18 24 Time (h) MT2A FTH1P11 TXNRD1 mRNA relative expression NQO1 EIF2AK3 MT1G FTH1P16 GCLM MT1E NQO2 SOD2 UGCG GCLC FTH1P2 SQSTM1 MT1A FTH1P3 Time (h) MT1X HMOX1 AKR1C3 * relative expression ** relative expression *** ** HMOX -1 mRNA SOD2 mRNA - 0.1 1 10 - 0.1 - - 0.1 1 DENV2 1 10 DENV2 10 0.1 1 10 + DPI - DPI - DPI + DPI
Nrf2 is a master regulator of the oxidative stress response Ø Nuclear factor-erythroid 2 related factor 2 - bZIP transcription factor - involved in the redox homeostasis and in the protection to oxidative stress - regulates the expression of cytoprotective and antioxidant genes Mitsuishi Y, Front Oncol, 2012
Nrf2 knockdown deregulates ROS production and antiviral/apoptotic responses in DENV infection Si Ctrl Nrf2 mRNA relative p= 0.031 expression 596 Events 1088 Si Ctrl+DV2 92.0% 1088 1996 Si Nrf2+DV2 88.8% - - DENV2 + + ROS Si-control Si-Nrf2 Si Ctrl+DV2 Si Ctrl Si Nrf2 Si Nrf2+DV2 DENV-E RSAD2 IFIT1 DDX58 CXCL10 IFNb NOXA BCLX RIPK1 160 80 mRNA relative 50 15 25 25 15 6 mRNA relative ** * expression ** 2.10 4 ** * ** expression 40 120 20 60 20 10 10 4 30 2.10 3 15 80 15 40 10 10 20 10 10 5 5 2 1.10 3 6 6 10 5 5 0 0 0 0 0 0 0 0 0 - - - + - - - - - - - - - - - - - DENV2 + + DENV2 + + + + + + + + + + + + + Si-control Si-Nrf2
Itaconate – suppresses inflammation via induction of the transcription factor Nrf2 through direct binding of the Nrf2 repressor Keap1
Modulation of Nrf2 & KEAP1 inversely control the antiviral response ITAC & SFN stimulate Nrf2 & anti-oxidant response element
Conclusions Ø DENV2 infection generates an NOX-dependent ROS production in mature DC Ø DENV2-induced ROS formation is essential for the activation of the innate immune response and the activation of IRF3/STAT1 and NF- κ B signaling in DC Ø DENV2-infected DC undergo NOX-mediated mitochondrial-dependent apoptosis Ø Nrf2 protects cells from stress-associated damages including over-activation of the immune and apoptotic responses
DENV induces NOX- dependent ROS production required for antiviral & apoptotic responses Olagnier et al Mol Therapy 2017
Metabolic re-programming inhibits antiviral responses via Nrf2 IRG1 Citrate ITACONIC ACID Antibacterial Activity Malate HBV AlphaKG HCV Fumarate HIV NRF2 Influenza HMOX-1 Succinate EV71 SDH RSV EBOV (CII) Glutamate Arginosuccin DENV ate Antiviral Activity mROS shunt Interferon Inflammation pathway HIF-1α IL1-β
Acknowledgements McGill University VGTI-FL MarieLine Goulet David Olagnier Julien van Grevenynghe Cindy Chiang Samar Bel Hadj Vladimir Beljanski Stephanie Oliere Nadine VanMontfoort Suzanne Paz Kevin Yin Rongtuan Lin Carmen Nichols Zhong He Fox Chase Cancer Center Elias Haddad Sid Balachandran Lydie Trautmann Suraj Peri Genomics/Bioinformatics VGTI-FL VGTI-Oregon Mark Cameron Peter Wilkinson Vic Defilippis Courtney Steel Dan Streblow Stephanie Richards Washington University St. Louis Andrew Smith Michael S. Diamond Leiden University Medical Center Martijn Van Hemert Mount Sinai School of Medicine Irina Albulescu Ana Fernandez-Sesma Florine Scholte Adolfo Garcia-Sastre Marjolein Kikkert Eric Snijder
Acknowledgements Luciano Castiello Michela Muscolini Alessandra Zevini Enrico Palermo Matteo Ferrari Dominga Lovecchio Fani Souvalidou Shirley Mann Thank you
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