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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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Crosstalk between TLR2-MyD88 and the NALP3 inflammasome for IL-1β expression and processing.(A) Wild-type, TLR2−/− and MyD88−/− BMDMs were primed overnight with ultra-pure LPS (100 ng/ml) and infected with MVA (MOI 5). IL-1β mRNA expression was quantified by RT-PCR (p<0.05 for TLR2−/− or MyD88−/− vs. wild-type BMDMs). THP-1 cells stably transduced with control, NALP3, ASC and caspase 1 (casp1) shRNAs were infected with MVA (MOI 5 unless specified otherwise) for the indicated time (B–C). (B) Western blots of intracellular pro-IL-1β and secreted IL-1β p17. (C) IL-1β concentrations measured by ELISA in cell-culture supernatants collected 24 h after infection (p<0.05 for cells transduced with NALP3, ASC and casp1 shRNAs vs. control shRNA). LPS-primed wild-type and NALP3−/− BMDMs were infected with MVA (MOI 5 in D) for 6 h (D–E). (D) Western blots of intracellular pro-IL-1β and secreted IL-1β p17. (E) IL-1β concentrations measured by ELISA in cell-culture supernatants collected 24 h after infection. Results are expressed as the ratio of IL-1β mRNA levels to that of HPRT. Data are means±SD of triplicate samples from one experiment and are representative of two independent experiments (p<0.05 for NALP3−/− vs. wild-type BMDMs).
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ppat-1000480-g008: Crosstalk between TLR2-MyD88 and the NALP3 inflammasome for IL-1β expression and processing.(A) Wild-type, TLR2−/− and MyD88−/− BMDMs were primed overnight with ultra-pure LPS (100 ng/ml) and infected with MVA (MOI 5). IL-1β mRNA expression was quantified by RT-PCR (p<0.05 for TLR2−/− or MyD88−/− vs. wild-type BMDMs). THP-1 cells stably transduced with control, NALP3, ASC and caspase 1 (casp1) shRNAs were infected with MVA (MOI 5 unless specified otherwise) for the indicated time (B–C). (B) Western blots of intracellular pro-IL-1β and secreted IL-1β p17. (C) IL-1β concentrations measured by ELISA in cell-culture supernatants collected 24 h after infection (p<0.05 for cells transduced with NALP3, ASC and casp1 shRNAs vs. control shRNA). LPS-primed wild-type and NALP3−/− BMDMs were infected with MVA (MOI 5 in D) for 6 h (D–E). (D) Western blots of intracellular pro-IL-1β and secreted IL-1β p17. (E) IL-1β concentrations measured by ELISA in cell-culture supernatants collected 24 h after infection. Results are expressed as the ratio of IL-1β mRNA levels to that of HPRT. Data are means±SD of triplicate samples from one experiment and are representative of two independent experiments (p<0.05 for NALP3−/− vs. wild-type BMDMs).

Mentions: IL-1β is a key cytokine of antimicrobial host defenses, whose expression is regulated at a transcriptional and post-transcriptional level [36]. IL-1β is likely to play an important role during poxvirus infection, as suggested by the fact that poxviruses encode for IL-1β decoy receptor and disrupt intracellular IL-1 receptor signalling [37],[38]. We therefore examined whether activation of the TLR2-MyD88 pathway was implicated in the activation of the IL1b gene. As shown in Figure 8A, up-regulation of IL-1β mRNA was markedly impaired in TLR2−/− and MyD88−/− BMDMs infected with MVA, indicating that activation of the TLR2-MyD88 signalling pathway is critical for transcription of the IL1b gene during MVA infection. Secretion of mature IL-1β p17 in response to endogenous and exogenous danger signals requires the cleavage of the inactive pro-IL-1β precursor by the cysteine protease caspase-1. Conversion of pro-caspase-1 into caspase-1 is tightly regulated by the NALP3 inflammasome composed of NALP3, ASC and pro-caspase-1 [22]. To examine the contribution of the NALP3 inflammasome in the production of IL-1β triggered by MVA, we analyzed the expression of pro-IL-1β and IL-1β p17 in THP-1 cells deficient in NALP3, ASC or caspase-1 [39]. Knocking down of either one of the three components of the NALP3 inflammasome (i.e. NALP3, ASC or caspase-1) was associated with a massive reduction of mature and secreted IL-1β (Figure 8B and C). Similar results were obtained in THP-1 cells infected with NYVAC (Figure S1) and in NALP3−/− BMDMs infected with MVA (Figure 8D and E). Of note, in THP-1 cells and in BMDMs the expression of pro-IL-1β was unaffected by the absence of either NALP3, ASC or caspase-1 clearly indicating that NALP3 inflammasome does not itself regulate the transcriptional and translation control of the IL-1β precursor. The NALP3 inflammasome was also dispensable for activation of the IRF3 transcription factor and IFNβ secretion (Figure S6). Altogether, these data demonstrate that IL-1β production after MVA infection requires a crosstalk between TLR2-MyD88 (initiation of the transcription and translational of IL-1β) and the NALP3 inflammasome (processing of pro-IL-1β into mature IL-1β).


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)

Crosstalk between TLR2-MyD88 and the NALP3 inflammasome for IL-1β expression and processing.(A) Wild-type, TLR2−/− and MyD88−/− BMDMs were primed overnight with ultra-pure LPS (100 ng/ml) and infected with MVA (MOI 5). IL-1β mRNA expression was quantified by RT-PCR (p<0.05 for TLR2−/− or MyD88−/− vs. wild-type BMDMs). THP-1 cells stably transduced with control, NALP3, ASC and caspase 1 (casp1) shRNAs were infected with MVA (MOI 5 unless specified otherwise) for the indicated time (B–C). (B) Western blots of intracellular pro-IL-1β and secreted IL-1β p17. (C) IL-1β concentrations measured by ELISA in cell-culture supernatants collected 24 h after infection (p<0.05 for cells transduced with NALP3, ASC and casp1 shRNAs vs. control shRNA). LPS-primed wild-type and NALP3−/− BMDMs were infected with MVA (MOI 5 in D) for 6 h (D–E). (D) Western blots of intracellular pro-IL-1β and secreted IL-1β p17. (E) IL-1β concentrations measured by ELISA in cell-culture supernatants collected 24 h after infection. Results are expressed as the ratio of IL-1β mRNA levels to that of HPRT. Data are means±SD of triplicate samples from one experiment and are representative of two independent experiments (p<0.05 for NALP3−/− vs. wild-type BMDMs).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2691956&req=5

ppat-1000480-g008: Crosstalk between TLR2-MyD88 and the NALP3 inflammasome for IL-1β expression and processing.(A) Wild-type, TLR2−/− and MyD88−/− BMDMs were primed overnight with ultra-pure LPS (100 ng/ml) and infected with MVA (MOI 5). IL-1β mRNA expression was quantified by RT-PCR (p<0.05 for TLR2−/− or MyD88−/− vs. wild-type BMDMs). THP-1 cells stably transduced with control, NALP3, ASC and caspase 1 (casp1) shRNAs were infected with MVA (MOI 5 unless specified otherwise) for the indicated time (B–C). (B) Western blots of intracellular pro-IL-1β and secreted IL-1β p17. (C) IL-1β concentrations measured by ELISA in cell-culture supernatants collected 24 h after infection (p<0.05 for cells transduced with NALP3, ASC and casp1 shRNAs vs. control shRNA). LPS-primed wild-type and NALP3−/− BMDMs were infected with MVA (MOI 5 in D) for 6 h (D–E). (D) Western blots of intracellular pro-IL-1β and secreted IL-1β p17. (E) IL-1β concentrations measured by ELISA in cell-culture supernatants collected 24 h after infection. Results are expressed as the ratio of IL-1β mRNA levels to that of HPRT. Data are means±SD of triplicate samples from one experiment and are representative of two independent experiments (p<0.05 for NALP3−/− vs. wild-type BMDMs).
Mentions: IL-1β is a key cytokine of antimicrobial host defenses, whose expression is regulated at a transcriptional and post-transcriptional level [36]. IL-1β is likely to play an important role during poxvirus infection, as suggested by the fact that poxviruses encode for IL-1β decoy receptor and disrupt intracellular IL-1 receptor signalling [37],[38]. We therefore examined whether activation of the TLR2-MyD88 pathway was implicated in the activation of the IL1b gene. As shown in Figure 8A, up-regulation of IL-1β mRNA was markedly impaired in TLR2−/− and MyD88−/− BMDMs infected with MVA, indicating that activation of the TLR2-MyD88 signalling pathway is critical for transcription of the IL1b gene during MVA infection. Secretion of mature IL-1β p17 in response to endogenous and exogenous danger signals requires the cleavage of the inactive pro-IL-1β precursor by the cysteine protease caspase-1. Conversion of pro-caspase-1 into caspase-1 is tightly regulated by the NALP3 inflammasome composed of NALP3, ASC and pro-caspase-1 [22]. To examine the contribution of the NALP3 inflammasome in the production of IL-1β triggered by MVA, we analyzed the expression of pro-IL-1β and IL-1β p17 in THP-1 cells deficient in NALP3, ASC or caspase-1 [39]. Knocking down of either one of the three components of the NALP3 inflammasome (i.e. NALP3, ASC or caspase-1) was associated with a massive reduction of mature and secreted IL-1β (Figure 8B and C). Similar results were obtained in THP-1 cells infected with NYVAC (Figure S1) and in NALP3−/− BMDMs infected with MVA (Figure 8D and E). Of note, in THP-1 cells and in BMDMs the expression of pro-IL-1β was unaffected by the absence of either NALP3, ASC or caspase-1 clearly indicating that NALP3 inflammasome does not itself regulate the transcriptional and translation control of the IL-1β precursor. The NALP3 inflammasome was also dispensable for activation of the IRF3 transcription factor and IFNβ secretion (Figure S6). Altogether, these data demonstrate that IL-1β production after MVA infection requires a crosstalk between TLR2-MyD88 (initiation of the transcription and translational of IL-1β) and the NALP3 inflammasome (processing of pro-IL-1β into mature IL-1β).

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