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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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Pathways activated by MVA in the macrophage.Infection of macrophages with MVA stimulates the TLR2-TLR6-MyD88, MDA-5/IPS-1 and NALP3 inflammasome pathways leading to the activation of NF-κB, ERK-1/2, JNK, IRF3, IRF7 and STAT-1 that are involved in the transcriptional activation of genes encoding for cytokines, chemokines and type I IFN. At the cell surface, MVA is sensed by the TLR2-TLR6 heterodimer that induces the production of IFNβ-independent chemokines (IL-8, MIP-1 and MIP-2) (1) and pro-IL-1β (2). Upon virus entry into the cell, cytosolic MVA or MVA-derived viral components (possibly envelope or core proteins, early mRNA or DNA) activate the MDA-5-IPS-1 pathway to release IFNβ (3) and subsequent induction of IFNβ-dependent chemokines (such as RANTES, IP-10) following activation of the type I IFN receptor (4). Finally, MVA infection leads to the activation of the NALP3 inflammasome (composed of NALP3, ASC and pro-caspase 1) enabling caspase-1 processing, pro-IL-1β maturation and IL-1β secretion (5). For simplicity, the same diagram for MVA is shown outside and inside the cell.
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ppat-1000480-g010: Pathways activated by MVA in the macrophage.Infection of macrophages with MVA stimulates the TLR2-TLR6-MyD88, MDA-5/IPS-1 and NALP3 inflammasome pathways leading to the activation of NF-κB, ERK-1/2, JNK, IRF3, IRF7 and STAT-1 that are involved in the transcriptional activation of genes encoding for cytokines, chemokines and type I IFN. At the cell surface, MVA is sensed by the TLR2-TLR6 heterodimer that induces the production of IFNβ-independent chemokines (IL-8, MIP-1 and MIP-2) (1) and pro-IL-1β (2). Upon virus entry into the cell, cytosolic MVA or MVA-derived viral components (possibly envelope or core proteins, early mRNA or DNA) activate the MDA-5-IPS-1 pathway to release IFNβ (3) and subsequent induction of IFNβ-dependent chemokines (such as RANTES, IP-10) following activation of the type I IFN receptor (4). Finally, MVA infection leads to the activation of the NALP3 inflammasome (composed of NALP3, ASC and pro-caspase 1) enabling caspase-1 processing, pro-IL-1β maturation and IL-1β secretion (5). For simplicity, the same diagram for MVA is shown outside and inside the cell.

Mentions: Up to now the retinoic acid-inducible gene-I-like receptors (RLR) RIG-I and MDA-5 had been viewed as master cytosolic sensors of RNA viruses [29]. However, recent observations suggested a role for the RLR pathway in the recognition of DNA viruses. Mouse embryo fibroblasts deficient in IPS-1 displayed reduced induction of IFNβ in response to MVA lacking the E3 protein [44]. Adenovirus and HSV1 have also been shown to replicate at much higher titers in RIG-I mutant than in RIG-I wild-type human hepatoma cell lines [45]. Moreover, microarray analyses revealed that RIG-I and MDA-5 expression was upregulated in human monocyte-derived dendritic cells infected with MVA [24]. Here we also showed that MVA caused a strong up-regulation of RIG-I, MDA-5 and IPS-1, yet only MDA-5 and IPS-1 were found to mediate MVA-induced IFNβ and IFNβ-dependent chemokine production by macrophages (Figure 10). As anticipated, transcriptional activation of IFNb and IFNb-dependent chemokine genes was associated with the activation of IRF3 and IRF7 and STAT-1. To the best of our knowledge this is the first demonstration of a direct role for MDA-5 in innate sensing of a DNA virus. Moreover, the MDA-5/IPS-1 pathway was also implicated in the production of IFNβ by macrophages infected with the NYVAC and the Western Reserve strains of vaccinia virus (Figure S1 and S2).


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)

Pathways activated by MVA in the macrophage.Infection of macrophages with MVA stimulates the TLR2-TLR6-MyD88, MDA-5/IPS-1 and NALP3 inflammasome pathways leading to the activation of NF-κB, ERK-1/2, JNK, IRF3, IRF7 and STAT-1 that are involved in the transcriptional activation of genes encoding for cytokines, chemokines and type I IFN. At the cell surface, MVA is sensed by the TLR2-TLR6 heterodimer that induces the production of IFNβ-independent chemokines (IL-8, MIP-1 and MIP-2) (1) and pro-IL-1β (2). Upon virus entry into the cell, cytosolic MVA or MVA-derived viral components (possibly envelope or core proteins, early mRNA or DNA) activate the MDA-5-IPS-1 pathway to release IFNβ (3) and subsequent induction of IFNβ-dependent chemokines (such as RANTES, IP-10) following activation of the type I IFN receptor (4). Finally, MVA infection leads to the activation of the NALP3 inflammasome (composed of NALP3, ASC and pro-caspase 1) enabling caspase-1 processing, pro-IL-1β maturation and IL-1β secretion (5). For simplicity, the same diagram for MVA is shown outside and inside the cell.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2691956&req=5

ppat-1000480-g010: Pathways activated by MVA in the macrophage.Infection of macrophages with MVA stimulates the TLR2-TLR6-MyD88, MDA-5/IPS-1 and NALP3 inflammasome pathways leading to the activation of NF-κB, ERK-1/2, JNK, IRF3, IRF7 and STAT-1 that are involved in the transcriptional activation of genes encoding for cytokines, chemokines and type I IFN. At the cell surface, MVA is sensed by the TLR2-TLR6 heterodimer that induces the production of IFNβ-independent chemokines (IL-8, MIP-1 and MIP-2) (1) and pro-IL-1β (2). Upon virus entry into the cell, cytosolic MVA or MVA-derived viral components (possibly envelope or core proteins, early mRNA or DNA) activate the MDA-5-IPS-1 pathway to release IFNβ (3) and subsequent induction of IFNβ-dependent chemokines (such as RANTES, IP-10) following activation of the type I IFN receptor (4). Finally, MVA infection leads to the activation of the NALP3 inflammasome (composed of NALP3, ASC and pro-caspase 1) enabling caspase-1 processing, pro-IL-1β maturation and IL-1β secretion (5). For simplicity, the same diagram for MVA is shown outside and inside the cell.
Mentions: Up to now the retinoic acid-inducible gene-I-like receptors (RLR) RIG-I and MDA-5 had been viewed as master cytosolic sensors of RNA viruses [29]. However, recent observations suggested a role for the RLR pathway in the recognition of DNA viruses. Mouse embryo fibroblasts deficient in IPS-1 displayed reduced induction of IFNβ in response to MVA lacking the E3 protein [44]. Adenovirus and HSV1 have also been shown to replicate at much higher titers in RIG-I mutant than in RIG-I wild-type human hepatoma cell lines [45]. Moreover, microarray analyses revealed that RIG-I and MDA-5 expression was upregulated in human monocyte-derived dendritic cells infected with MVA [24]. Here we also showed that MVA caused a strong up-regulation of RIG-I, MDA-5 and IPS-1, yet only MDA-5 and IPS-1 were found to mediate MVA-induced IFNβ and IFNβ-dependent chemokine production by macrophages (Figure 10). As anticipated, transcriptional activation of IFNb and IFNb-dependent chemokine genes was associated with the activation of IRF3 and IRF7 and STAT-1. To the best of our knowledge this is the first demonstration of a direct role for MDA-5 in innate sensing of a DNA virus. Moreover, the MDA-5/IPS-1 pathway was also implicated in the production of IFNβ by macrophages infected with the NYVAC and the Western Reserve strains of vaccinia virus (Figure S1 and S2).

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