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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 up-regulates the expression of RIG-I, MDA-5 and IPS-1 mRNAs and proteins.RIG-I, MDA-5 and IPS-1 mRNA and protein expression by RT–PCR (A) and Western blot (B). THP-1 cells were infected with MVA (MOI 5) for the indicated time. Results are expressed as the ratio of RIG-I, MDA-5 or IPS-1 mRNA levels to that of HPRT. Data are means±SD of triplicate samples from one experiment and are representative of three independent experiments. AU: arbitrary units. *p<0.05.
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ppat-1000480-g006: MVA up-regulates the expression of RIG-I, MDA-5 and IPS-1 mRNAs and proteins.RIG-I, MDA-5 and IPS-1 mRNA and protein expression by RT–PCR (A) and Western blot (B). THP-1 cells were infected with MVA (MOI 5) for the indicated time. Results are expressed as the ratio of RIG-I, MDA-5 or IPS-1 mRNA levels to that of HPRT. Data are means±SD of triplicate samples from one experiment and are representative of three independent experiments. AU: arbitrary units. *p<0.05.

Mentions: The RLR family of cytosolic pattern recognition receptors has been implicated in the sensing of RNA viruses [35], but very little is known about their role in host response to DNA viruses. Extending the observations by Guerra et al. who noted an increased expression of RIG-I and MDA-5 mRNA in human dendritic cells infected with MVA [24], we observed that MVA caused a time-dependent increase in RIG-I, MDA-5 and IPS-1 mRNA and protein expression in THP-1 cells (Figure 6A and B). RIG-I and MDA-5 mRNAs rose within 3 h of infection and remained elevated for up to 24 h (Figure 6A). In vivo, MVA up-regulated RIG-I and MDA-5 mRNA levels in peritoneal cells and splenocytes (Figure S4). When compared to MVA, NYVAC induced lower levels of MDA-5 and, to a lesser extent, RIG-I and IPS-1 mRNA and protein expressions (Figure S1 and data not shown). Using shRIG-I, shMDA-5 and shIPS-1 THP-1 cells (Figure S5), we then examined whether RIG-I and MDA-5 were involved in MVA-induced IFNβ production. IFNβ and IP-10 mRNA and protein levels were markedly reduced in shMDA-5 and shIPS-1 cells, but not in shRIG-I cells. By contrast, the time-course and magnitude of the IL-8 and IL-1β production was similar in shMDA-5, shIPS-1, shRIG-I and control THP-1 cells (Figure 7A and B). Sensing of MVA by the MDA-5/IPS-1 pathway is therefore critical for the production of IFNβ and IFNβ-dependent chemokines in macrophages. In line with these data, the production of IFNβ, but not of IL-8, was also dependent on the MDA-5/IPS-1 pathway in cells infected with NYVAC and the Western Reserve strain 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)

MVA up-regulates the expression of RIG-I, MDA-5 and IPS-1 mRNAs and proteins.RIG-I, MDA-5 and IPS-1 mRNA and protein expression by RT–PCR (A) and Western blot (B). THP-1 cells were infected with MVA (MOI 5) for the indicated time. Results are expressed as the ratio of RIG-I, MDA-5 or IPS-1 mRNA levels to that of HPRT. Data are means±SD of triplicate samples from one experiment and are representative of three independent experiments. AU: arbitrary units. *p<0.05.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1000480-g006: MVA up-regulates the expression of RIG-I, MDA-5 and IPS-1 mRNAs and proteins.RIG-I, MDA-5 and IPS-1 mRNA and protein expression by RT–PCR (A) and Western blot (B). THP-1 cells were infected with MVA (MOI 5) for the indicated time. Results are expressed as the ratio of RIG-I, MDA-5 or IPS-1 mRNA levels to that of HPRT. Data are means±SD of triplicate samples from one experiment and are representative of three independent experiments. AU: arbitrary units. *p<0.05.
Mentions: The RLR family of cytosolic pattern recognition receptors has been implicated in the sensing of RNA viruses [35], but very little is known about their role in host response to DNA viruses. Extending the observations by Guerra et al. who noted an increased expression of RIG-I and MDA-5 mRNA in human dendritic cells infected with MVA [24], we observed that MVA caused a time-dependent increase in RIG-I, MDA-5 and IPS-1 mRNA and protein expression in THP-1 cells (Figure 6A and B). RIG-I and MDA-5 mRNAs rose within 3 h of infection and remained elevated for up to 24 h (Figure 6A). In vivo, MVA up-regulated RIG-I and MDA-5 mRNA levels in peritoneal cells and splenocytes (Figure S4). When compared to MVA, NYVAC induced lower levels of MDA-5 and, to a lesser extent, RIG-I and IPS-1 mRNA and protein expressions (Figure S1 and data not shown). Using shRIG-I, shMDA-5 and shIPS-1 THP-1 cells (Figure S5), we then examined whether RIG-I and MDA-5 were involved in MVA-induced IFNβ production. IFNβ and IP-10 mRNA and protein levels were markedly reduced in shMDA-5 and shIPS-1 cells, but not in shRIG-I cells. By contrast, the time-course and magnitude of the IL-8 and IL-1β production was similar in shMDA-5, shIPS-1, shRIG-I and control THP-1 cells (Figure 7A and B). Sensing of MVA by the MDA-5/IPS-1 pathway is therefore critical for the production of IFNβ and IFNβ-dependent chemokines in macrophages. In line with these data, the production of IFNβ, but not of IL-8, was also dependent on the MDA-5/IPS-1 pathway in cells infected with NYVAC and the Western Reserve strain 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