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

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.

Figure 1. MVA stimulates cytokine, chemokine and IFNβ production in vivo.

BALB/c mice were injected i.p. with MVA (10⁷ PFU). Peritoneal cells (A) and peritoneal lavage fluid (B) were collected 12 h after infection as described in Materials and Methods . TNF, IL-1β, IL-12p40, IP-10, RANTES and IFNβ mRNA contents of peritoneal cells were analyzed by RT-PCR (A). Results are expressed as the ratio of cytokines, chemokines or IFNβ mRNA levels to that of HPRT. AU: arbitrary units. Cytokine concentrations in peritoneal lavage fluid were measured by ELISA (B). Data are means±SD of triplicate samples from one experiment comprising three mice per experimental condition and are representative of two independent experiments. p<0.05 for all conditions when comparing PBS versus MVA.

Figure 2. TNF, IL-1β, IL-6, IL-8, MIP-1α and IP-10 release by human whole blood exposed to MVA.

Whole blood from 3 healthy volunteers (#1, 2 and 3) was incubated for 24 h with (+) or without (−) MVA (MOI 1) in triplicates. Cell-free supernatants were collected to quantify the concentrations of TNF, IL-1β, IL-6, IL-8, MIP-1α and IP-10. Data are means±SD of triplicate samples from one experiment. MVA significantly increased cytokine production (p<0.05 for all conditions).

Figure 3. 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).

Figure 4. TLR2, TLR6 and MyD88 are critical for IFNβ-independent chemokine production after MVA infection.

MIP-2 (A) and IFNβ (B) produced by wild-type, TLR1^−/−, TLR2^−/−, TLR4^−/−, TLR6^−/−, MyD88^−/− and TRIF^−/− bone marrow-derived macrophages infected with MVA (MOI 5 and 20) or stimulated with lipopolysaccharide (LPS, 100 ng/ml), Pam₂CSK₄ (P₂CSK₄, 10 µg/ml), Pam₃CSK₄ (P₃CSK₄, 10 µg/ml) for 24 h. Data are means±SD of triplicate samples from one experiment and are representative of 2 to 4 experiments.

Figure 5. 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.

Figure 6. 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.

Figure 7. MVA is sensed by MDA-5 and not by RIG-I.

THP-1 cells stably transduced with control, MDA-5, RIG-I or IPS-1 shRNAs were infected with MVA (MOI 5 unless specified otherwise) for the indicated time. IFNβ, IP-10, IL-8 and IL-1β mRNA and protein expression by RT-PCR and ELISA (A–B). Results are expressed as the ratio of IFNβ, IP-10, IL-8 or IL-1β mRNA levels to that of HPRT. Data are means±SD of triplicate samples from one experiment and are representative of four independent experiments. AU: arbitrary units. Concentrations of IFNβ and IL-8 in cell-culture supernatants were measured 24 h after stimulation. shMDA-5 and shIPS-1 THP-1 cells produced significantly less IFNβ and IP-10 mRNA and protein than control cells as measured 24 h post-infection (A and B) (p<0.05).

Figure 8. 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).

Figure 9. MVA activates the NF-κB, ERK1/2, JNK, IRF3, IRF7 and STAT-1 signalling pathways.

Electrophoretic mobility shift assay of NF-κB DNA binding activity and Western blots of phosphorylated ERK1/2 (P-ERK1/2), JNK (P-JNK), IRF3 (P-IRF3) and STAT-1 (P-STAT-1) and total ERK1/2, JNK and IRF7 (A). Nuclear (NF-κB) and cytosolic (ERK1/2, JNK, IRF3, IRF7, STAT-1 and tubulin) extracts were prepared from THP-1 cells infected with MVA (MOI 5) for the indicated time. Results are representative of three independent experiments. The retarded complex detected by EMSA was dose-dependently inhibited by cold wild-type but not mutant NF-κB oligonucleotide, and supershifted using anti-p65 antibody (data not shown). NF-κB- (B) and IRF3- (C) mediated transcriptional activities measured in THP-1 cells transiently transfected with trimeric κB sites or IRF3-dependent IFNβ promoter luciferase reporter vectors and infected with MVA (MOI 5 and 20) for 18 h. Results are expressed as the ratio of luciferase activity to Renilla luciferase activity. Data are means±SD of triplicate samples from one experiment and are representative of four independent experiments. p = 0.05, 0.02, 0.04 and 0.02 for MVA-infected (MOI 5 and 20) vs. control cells.

Figure 10. 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.