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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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Endocytosis is required for IL-1β and IFNβ production after MVA infection.THP-1 cells were preincubated for 1 h with or without cytochalasin (2 µM) or chloroquine (100 µM) prior to exposure to MVA or UV-treated MVA (MOI 20). Cell-culture supernatants were harvested after 6 h (IL-1β) or 24 h (IFNβ and IL-8) and cytokine concentrations were measured by ELISA. Data are means±SD of triplicate samples from one experiment and are representative of two independent experiments.
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ppat-1000480-g005: Endocytosis is required for IL-1β and IFNβ production after MVA infection.THP-1 cells were preincubated for 1 h with or without cytochalasin (2 µM) or chloroquine (100 µM) prior to exposure to MVA or UV-treated MVA (MOI 20). Cell-culture supernatants were harvested after 6 h (IL-1β) or 24 h (IFNβ and IL-8) and cytokine concentrations were measured by ELISA. Data are means±SD of triplicate samples from one experiment and are representative of two independent experiments.

Mentions: Vaccinia virus penetrates into target cells either by endocytosis or by membrane fusion in a low pH-independent manner [31]. To determine the contribution of endocytosis to MVA-induced intracellular signalling and cytokine production, THP-1 cells were treated with cytochalasine D, an actin-depolymerizing drug that blocks the endocytotic trafficking, or with chloroquine, a lysosomotropic weak base to neutralize the acidic environment of endocytic vesicles. IL-1β and to a lesser extend IFNβ production were inhibited by cytochalasine D and chloroquine treatment. The inhibition was not related to drug toxicity because chloroquine did not affect IL-8 production and cell viability (Figure 5 and data not shown). The reason why the inhibition of cytokine production (particularly IFNβ) was only partial after treatment with the inhibitors remains uncertain. The data suggest that additional non-endocytic pathways may play a role in the production of IFNβ. In agreement with a key role for membrane-bound TLR2 for IL-8 induction, the production of IL-8 was not reduced after cytochalasine D or chloroquine treatment (Figure 5). UV treatment of MVA causing a nearly complete (i.e. 90%) inhibition of the expression of the early C6L gene (data not shown) did not affect IL-1β, IL-8 and IFNβ production (Figure 5). Although one cannot completely rule out a contribution of residual viral protein synthesis, these observations support the view that induction of cytokines by MVA is most likely independent of viral gene synthesis [32]–[34]. Overall, endocytosis of MVA was required for IL-1β and IFNβ release suggesting a role for intracellular pattern recognition receptors in the production of these cytokines.


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)

Endocytosis is required for IL-1β and IFNβ production after MVA infection.THP-1 cells were preincubated for 1 h with or without cytochalasin (2 µM) or chloroquine (100 µM) prior to exposure to MVA or UV-treated MVA (MOI 20). Cell-culture supernatants were harvested after 6 h (IL-1β) or 24 h (IFNβ and IL-8) and cytokine concentrations were measured by ELISA. Data are means±SD of triplicate samples from one experiment and are representative of two independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1000480-g005: Endocytosis is required for IL-1β and IFNβ production after MVA infection.THP-1 cells were preincubated for 1 h with or without cytochalasin (2 µM) or chloroquine (100 µM) prior to exposure to MVA or UV-treated MVA (MOI 20). Cell-culture supernatants were harvested after 6 h (IL-1β) or 24 h (IFNβ and IL-8) and cytokine concentrations were measured by ELISA. Data are means±SD of triplicate samples from one experiment and are representative of two independent experiments.
Mentions: Vaccinia virus penetrates into target cells either by endocytosis or by membrane fusion in a low pH-independent manner [31]. To determine the contribution of endocytosis to MVA-induced intracellular signalling and cytokine production, THP-1 cells were treated with cytochalasine D, an actin-depolymerizing drug that blocks the endocytotic trafficking, or with chloroquine, a lysosomotropic weak base to neutralize the acidic environment of endocytic vesicles. IL-1β and to a lesser extend IFNβ production were inhibited by cytochalasine D and chloroquine treatment. The inhibition was not related to drug toxicity because chloroquine did not affect IL-8 production and cell viability (Figure 5 and data not shown). The reason why the inhibition of cytokine production (particularly IFNβ) was only partial after treatment with the inhibitors remains uncertain. The data suggest that additional non-endocytic pathways may play a role in the production of IFNβ. In agreement with a key role for membrane-bound TLR2 for IL-8 induction, the production of IL-8 was not reduced after cytochalasine D or chloroquine treatment (Figure 5). UV treatment of MVA causing a nearly complete (i.e. 90%) inhibition of the expression of the early C6L gene (data not shown) did not affect IL-1β, IL-8 and IFNβ production (Figure 5). Although one cannot completely rule out a contribution of residual viral protein synthesis, these observations support the view that induction of cytokines by MVA is most likely independent of viral gene synthesis [32]–[34]. Overall, endocytosis of MVA was required for IL-1β and IFNβ release suggesting a role for intracellular pattern recognition receptors in the production of these cytokines.

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