Both TLR2 and TRIF contribute to interferon-β production during Listeria infection

Abstract

Synthesis of interferon-β (IFN-β) is an innate response to cytoplasmic infection with bacterial pathogens. Our recent studies showed that Listeria monocytogenes limits immune detection and IFN-β synthesis via deacetylation of its peptidoglycan, which renders the bacterium resistant to lysozyme degradation. Here, we examined signaling requirements for the massive IFN-β production resulting from the infection of murine macrophages with a mutant strain of L. monocytogenes, ΔpgdA, which is unable to modify its peptidoglycan. We report the identification of unconventional signaling pathways to the IFN-β gene, requiring TLR2 and bacterial internalization. Induction of IFN-β was independent of the Mal/TIRAP adaptor protein but required TRIF and the transcription factors IRF3 and IRF7. These pathways were stimulated to a lesser degree by wild-type L. monocytogenes. They operated in both resident and inflammatory macrophages derived from the peritoneal cavity, but not in bone marrow-derived macrophages. The novelty of our findings thus lies in the first description of TLR2 and TRIF as two critical components leading to the induction of the IFN-β gene and in uncovering that individual macrophage populations adopt different strategies to link pathogen recognition signals to IFN-β gene expression.

Figure 1. PgdA-dependent IFN-β response to Listeria in peritoneal macrophages.

PEM from WT C57BL/6J mice were infected with the parental EGDe strain (black bars), the ΔpgdA mutant (grey bars) or the complemented ΔpgdA strain (white bars). After 7 h of infection, IFN-β levels were measured in supernatants by ELISA. Data are mean ± SD (***, p<0.0001, n = 5).

Figure 2. TLR2 is required for PgdA-mediated IFN-β response to Listeria in peritoneal but not bone-marrow macrophages.

(A) PEM from C57BL/6J or tlr2^−/− mice were infected with the parental EGDe strain. After 4 h of infection, IFN-β induction was measured by qRT-PCR. (B) BMM from C57BL/6J or tlr2^−/− mice were infected with the parental EGDe strain. After 4 h of infection, IFN-β induction was measured by qRT-PCR. Data are mean ± SD (NS, non significant; ***, p<0.0001, n = 3). PEM from WT C57BL/6J or tlr2 ^−/− mice were infected with the ΔpgdA mutant (C) or the complemented ΔpgdA strain (D). After 7 h of infection, IFN-β levels were measured in supernatants by ELISA. Data are mean ± SD (**, p<0.01, n = 5).

Figure 3. TRIF, but not Mal/TIRAP, is necessary for IFN-β response to Listeria in peritoneal macrophages.

(A) PEM from WT C57BL/6J or mal/tirap^−/− mice were infected with the parental EGDe strain (black bars), the ΔpgdA mutant (grey bars). After 7 h of infection, IFN-β levels were measured in supernatants by ELISA. (B) PEM from C57BL/6J or trif^−/− mice were infected with the parental EGDe strain (black bars) or the ΔpgdA mutant (grey bars). After 4 h of infection, IFN-β induction was measured by qRT-PCR. (C) Resident peritoneal macrophages from WT C57BL/6J, trif ^−/− or tlr2^−/− mice were infected with the parental EGDe strain (black bars), the ΔpgdA mutant (grey bars). After 4 h of infection, IFN-β induction was measured by qRT-PCR. Data are mean ± SD (NS, non significant; *, p<0.05; ***, p<0.0001; n = 3–4).

Figure 4. TLR3, but not TLR4, contributes to IFN-β response to Listeria in peritoneal macrophages.

(A) PEM from WT C57BL/6J or tlr3 ^−/− mice were infected with the parental EGDe strain (black bars), the ΔpgdA mutant (grey bars). After 7 h of infection, IFN-β levels were measured in supernatants by ELISA. Data are mean ± SD (n = 3). (B) PEM from WT C57BL/6J or tlr4 ^−/− mice were infected with the parental EGDe strain (black bars), or the ΔpgdA mutant (grey bars). After 7 h of infection, IFN-β levels were measured in supernatants by ELISA. Data are mean ± SD (***, p<0.0001; n = 3).

Figure 5. Internalization of bacteria is required for IFN-β response by peritoneal macrophages.

(A) PEM from WT C57BL/6J mice were pretreated with 50 µM of cytochalasin D, and left uninfected (hatched bars) or infected with the parental EGDe strain (black bars) or the ΔpgdA mutant (grey bars). 7 h post-infection, IFN-β levels were measured in cells supernatants by ELISA. (B) PEM from WT C57BL/6J were treated with 80 µM dynasore. After 4 h of infection with the parental EGDe strain (black bars) or the ΔpgdA mutant (grey bars), IFN-β induction was measured by qRT-PCR. (C) PEM from WT C57BL/6J mice were treated with 100 µM chloroquine, and left uninfected (hatched bars) or infected with the parental EGDe strain (black bars) or the ΔpgdA mutant (grey bars). 7 h post-infection, IFN-β concentrations were measured in cells supernatants by ELISA. Data are mean ± SD (***, p<0.0001, n = 3–4).

Figure 6. IFN-β response to Listeria is mediated by IRF3 and IRF7 in peritoneal macrophages.

PEM from WT C57BL/6J, irf3 ^−/− and irf7 ^−/− mice were infected with the parental EGDe strain (black bars) or the ΔpgdA mutant (grey bars). 4 h post-infection, mRNA was isolated and the IFN-β induction was measured by qRT-PCR. Data are mean ± SD (***, p<0.0001, n = 3).

Figure 7. Bacterial internalization and NF-κB are required for TLR2 and TRIF-dependent IFN-β response in peritoneal macrophages.

(A) PEM from WT C57BL/6J mice and pretreated with dynasore were infected with the parental EGDe strain (black bars) or the ΔpgdA mutant (grey bars). 4 h post-infection, IκB induction was measured by qRT-PCR. (B) PEM from WT C57BL/6J, tlr2 ^−/− and trif ^−/− mice were infected with the parental EGDe strain (black bars) or the ΔpgdA mutant (grey bars). 4 h post-infection, mRNA was isolated and IκB induction was measured by qRT-PCR. Data are mean ± SD (***, p<0.0001, n = 3).

Figure 8. Listeria nucleic acids trigger IFN-β production.

(A) The parental EGDe (black bars) ΔpgdA (grey bars) and complemented ΔpgdA strain (hatched bars) were incubated with lysozyme. The amount of DNA and RNA released after treatment was quantified by spectrophotometry. (B) THP-1 macrophages were transfected with DNA from the ΔpgdA mutant, pretreated or not with DNase, and IFN-β induction was determined using the HEK-blue assay. (C) The parental EGDe strain (black bars) or the ΔpgdA mutant (grey bars) were incubated with lysozyme. PEM were transfected with bacterial lysates, pretreated with DNase or not treated, and IFN-β production was quantified in cells supernatants 24 h after transfection by ELISA. Data are mean ± SD (**, p<0.01, n = 3; ***, p<0.0001, n = 3).