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Innate immune sensing of modified vaccinia virus Ankara (MVA) is mediated by TLR2-TLR6, MDA-5 and the NALP3 inflammasome.

Delaloye J, Roger T, Steiner-Tardivel QG, Le Roy D, Knaup Reymond M, Akira S, Petrilli V, Gomez CE, Perdiguero B, Tschopp J, Pantaleo G, Esteban M, Calandra T - PLoS Pathog. (2009)

Bottom Line: Transcription of the Il1b gene was markedly impaired in TLR2(-/-) and MyD88(-/-) BMDM, whereas mature and secreted IL-1beta was massively reduced in NALP3(-/-) BMDMs or in human THP-1 macrophages with reduced expression of NALP3, ASC or caspase-1 by shRNAs.Innate immune sensing of MVA and production of chemokines, IFNbeta and IL-1beta by macrophages is mediated by the TLR2-TLR6-MyD88, MDA-5-IPS-1 and NALP3 inflammasome pathways.Delineation of the host response induced by MVA is critical for improving our understanding of poxvirus antiviral escape mechanisms and for designing new MVA vaccine vectors with improved immunogenicity.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Infectious Diseases Service, Centre Hospitalier Universitaire Vaudois and University of Lausanne, Lausanne, Switzerland.

ABSTRACT
Modified vaccinia virus Ankara (MVA) is an attenuated double-stranded DNA poxvirus currently developed as a vaccine vector against HIV/AIDS. Profiling of the innate immune responses induced by MVA is essential for the design of vaccine vectors and for anticipating potential adverse interactions between naturally acquired and vaccine-induced immune responses. Here we report on innate immune sensing of MVA and cytokine responses in human THP-1 cells, primary human macrophages and mouse bone marrow-derived macrophages (BMDMs). The innate immune responses elicited by MVA in human macrophages were characterized by a robust chemokine production and a fairly weak pro-inflammatory cytokine response. Analyses of the cytokine production profile of macrophages isolated from knockout mice deficient in Toll-like receptors (TLRs) or in the adapter molecules MyD88 and TRIF revealed a critical role for TLR2, TLR6 and MyD88 in the production of IFNbeta-independent chemokines. MVA induced a marked up-regulation of the expression of RIG-I like receptors (RLR) and the IPS-1 adapter (also known as Cardif, MAVS or VISA). Reduced expression of RIG-I, MDA-5 and IPS-1 by shRNAs indicated that sensing of MVA by RLR and production of IFNbeta and IFNbeta-dependent chemokines was controlled by the MDA-5 and IPS-1 pathway in the macrophage. Crosstalk between TLR2-MyD88 and the NALP3 inflammasome was essential for expression and processing of IL-1beta. Transcription of the Il1b gene was markedly impaired in TLR2(-/-) and MyD88(-/-) BMDM, whereas mature and secreted IL-1beta was massively reduced in NALP3(-/-) BMDMs or in human THP-1 macrophages with reduced expression of NALP3, ASC or caspase-1 by shRNAs. Innate immune sensing of MVA and production of chemokines, IFNbeta and IL-1beta by macrophages is mediated by the TLR2-TLR6-MyD88, MDA-5-IPS-1 and NALP3 inflammasome pathways. Delineation of the host response induced by MVA is critical for improving our understanding of poxvirus antiviral escape mechanisms and for designing new MVA vaccine vectors with improved immunogenicity.

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MVA induces the production of cytokines, chemokines and IFNβ by human macrophages.Human THP-1 cells (A–E) and primary human macrophages (F) were infected with GFP-positive (A, B) or wild-type (C–F) MVA (MOI 5). Expression of viral-derived GFP protein by THP-1 cells analyzed by flow cytometry (A, B). Cytokines, chemokines and IFNβ production by THP-1 cells stimulated for 24 h with MVA as assessed by the Luminex technology (C) or by ELISA (D). IL-8 (CXCL8), MIP-1α (CCL3), RANTES (CCL5), IP-10 (CXCL10) and IFNβ mRNA levels were analyzed by RT-PCR and results expressed as the ratio of chemokines or IFNβ to HPRT mRNA levels. AU: arbitrary units (E, F). Data are means±SD of duplicate (C) or triplicate (D to F) samples from one experiment and are representative of one (C) to three (D to F) independent experiments. p<0.05 for all conditions (D to F).
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ppat-1000480-g003: MVA induces the production of cytokines, chemokines and IFNβ by human macrophages.Human THP-1 cells (A–E) and primary human macrophages (F) were infected with GFP-positive (A, B) or wild-type (C–F) MVA (MOI 5). Expression of viral-derived GFP protein by THP-1 cells analyzed by flow cytometry (A, B). Cytokines, chemokines and IFNβ production by THP-1 cells stimulated for 24 h with MVA as assessed by the Luminex technology (C) or by ELISA (D). IL-8 (CXCL8), MIP-1α (CCL3), RANTES (CCL5), IP-10 (CXCL10) and IFNβ mRNA levels were analyzed by RT-PCR and results expressed as the ratio of chemokines or IFNβ to HPRT mRNA levels. AU: arbitrary units (E, F). Data are means±SD of duplicate (C) or triplicate (D to F) samples from one experiment and are representative of one (C) to three (D to F) independent experiments. p<0.05 for all conditions (D to F).

Mentions: Dissection of the molecular mechanisms of MVA-induced innate immune responses was preformed in PMA-differentiated human THP-1 macrophages and primary human macrophages. Flow cytometry analyses performed with GFP-expressing MVA (MOI 5) indicated that MVA rapidly infected THP-1 cells (Figure 3A and B). More than 60% of cells became GFP positive within 2 h followed by a progressive decline of GFP fluorescence thereafter, which could result either from MVA-induced apoptosis as observed in human HeLa and monocyte-derived dendritic cells [24],[25] or from the shutting down of protein synthesis through activation of the PKR pathway by MVA [26]. Indeed, the number of apoptotic cells increased from 5% at 6 h to 35% at 24 h post-infection as assessed by annexin V and propidium iodine staining (data not shown).


Innate immune sensing of modified vaccinia virus Ankara (MVA) is mediated by TLR2-TLR6, MDA-5 and the NALP3 inflammasome.

Delaloye J, Roger T, Steiner-Tardivel QG, Le Roy D, Knaup Reymond M, Akira S, Petrilli V, Gomez CE, Perdiguero B, Tschopp J, Pantaleo G, Esteban M, Calandra T - PLoS Pathog. (2009)

MVA induces the production of cytokines, chemokines and IFNβ by human macrophages.Human THP-1 cells (A–E) and primary human macrophages (F) were infected with GFP-positive (A, B) or wild-type (C–F) MVA (MOI 5). Expression of viral-derived GFP protein by THP-1 cells analyzed by flow cytometry (A, B). Cytokines, chemokines and IFNβ production by THP-1 cells stimulated for 24 h with MVA as assessed by the Luminex technology (C) or by ELISA (D). IL-8 (CXCL8), MIP-1α (CCL3), RANTES (CCL5), IP-10 (CXCL10) and IFNβ mRNA levels were analyzed by RT-PCR and results expressed as the ratio of chemokines or IFNβ to HPRT mRNA levels. AU: arbitrary units (E, F). Data are means±SD of duplicate (C) or triplicate (D to F) samples from one experiment and are representative of one (C) to three (D to F) independent experiments. p<0.05 for all conditions (D to F).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2691956&req=5

ppat-1000480-g003: MVA induces the production of cytokines, chemokines and IFNβ by human macrophages.Human THP-1 cells (A–E) and primary human macrophages (F) were infected with GFP-positive (A, B) or wild-type (C–F) MVA (MOI 5). Expression of viral-derived GFP protein by THP-1 cells analyzed by flow cytometry (A, B). Cytokines, chemokines and IFNβ production by THP-1 cells stimulated for 24 h with MVA as assessed by the Luminex technology (C) or by ELISA (D). IL-8 (CXCL8), MIP-1α (CCL3), RANTES (CCL5), IP-10 (CXCL10) and IFNβ mRNA levels were analyzed by RT-PCR and results expressed as the ratio of chemokines or IFNβ to HPRT mRNA levels. AU: arbitrary units (E, F). Data are means±SD of duplicate (C) or triplicate (D to F) samples from one experiment and are representative of one (C) to three (D to F) independent experiments. p<0.05 for all conditions (D to F).
Mentions: Dissection of the molecular mechanisms of MVA-induced innate immune responses was preformed in PMA-differentiated human THP-1 macrophages and primary human macrophages. Flow cytometry analyses performed with GFP-expressing MVA (MOI 5) indicated that MVA rapidly infected THP-1 cells (Figure 3A and B). More than 60% of cells became GFP positive within 2 h followed by a progressive decline of GFP fluorescence thereafter, which could result either from MVA-induced apoptosis as observed in human HeLa and monocyte-derived dendritic cells [24],[25] or from the shutting down of protein synthesis through activation of the PKR pathway by MVA [26]. Indeed, the number of apoptotic cells increased from 5% at 6 h to 35% at 24 h post-infection as assessed by annexin V and propidium iodine staining (data not shown).

Bottom Line: Transcription of the Il1b gene was markedly impaired in TLR2(-/-) and MyD88(-/-) BMDM, whereas mature and secreted IL-1beta was massively reduced in NALP3(-/-) BMDMs or in human THP-1 macrophages with reduced expression of NALP3, ASC or caspase-1 by shRNAs.Innate immune sensing of MVA and production of chemokines, IFNbeta and IL-1beta by macrophages is mediated by the TLR2-TLR6-MyD88, MDA-5-IPS-1 and NALP3 inflammasome pathways.Delineation of the host response induced by MVA is critical for improving our understanding of poxvirus antiviral escape mechanisms and for designing new MVA vaccine vectors with improved immunogenicity.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Infectious Diseases Service, Centre Hospitalier Universitaire Vaudois and University of Lausanne, Lausanne, Switzerland.

ABSTRACT
Modified vaccinia virus Ankara (MVA) is an attenuated double-stranded DNA poxvirus currently developed as a vaccine vector against HIV/AIDS. Profiling of the innate immune responses induced by MVA is essential for the design of vaccine vectors and for anticipating potential adverse interactions between naturally acquired and vaccine-induced immune responses. Here we report on innate immune sensing of MVA and cytokine responses in human THP-1 cells, primary human macrophages and mouse bone marrow-derived macrophages (BMDMs). The innate immune responses elicited by MVA in human macrophages were characterized by a robust chemokine production and a fairly weak pro-inflammatory cytokine response. Analyses of the cytokine production profile of macrophages isolated from knockout mice deficient in Toll-like receptors (TLRs) or in the adapter molecules MyD88 and TRIF revealed a critical role for TLR2, TLR6 and MyD88 in the production of IFNbeta-independent chemokines. MVA induced a marked up-regulation of the expression of RIG-I like receptors (RLR) and the IPS-1 adapter (also known as Cardif, MAVS or VISA). Reduced expression of RIG-I, MDA-5 and IPS-1 by shRNAs indicated that sensing of MVA by RLR and production of IFNbeta and IFNbeta-dependent chemokines was controlled by the MDA-5 and IPS-1 pathway in the macrophage. Crosstalk between TLR2-MyD88 and the NALP3 inflammasome was essential for expression and processing of IL-1beta. Transcription of the Il1b gene was markedly impaired in TLR2(-/-) and MyD88(-/-) BMDM, whereas mature and secreted IL-1beta was massively reduced in NALP3(-/-) BMDMs or in human THP-1 macrophages with reduced expression of NALP3, ASC or caspase-1 by shRNAs. Innate immune sensing of MVA and production of chemokines, IFNbeta and IL-1beta by macrophages is mediated by the TLR2-TLR6-MyD88, MDA-5-IPS-1 and NALP3 inflammasome pathways. Delineation of the host response induced by MVA is critical for improving our understanding of poxvirus antiviral escape mechanisms and for designing new MVA vaccine vectors with improved immunogenicity.

Show MeSH
Related in: MedlinePlus