RIG-I detects infection with live Listeria by sensing secreted bacterial nucleic acids

Abstract

Immunity against infection with Listeria monocytogenes is not achieved from innate immune stimulation by contact with killed but requires viable Listeria gaining access to the cytosol of infected cells. It has remained ill-defined how such immune sensing of live Listeria occurs. Here, we report that efficient cytosolic immune sensing requires access of nucleic acids derived from live Listeria to the cytoplasm of infected cells. We found that Listeria released nucleic acids and that such secreted bacterial RNA/DNA was recognized by the cytosolic sensors RIG-I, MDA5 and STING thereby triggering interferon β production. Secreted Listeria nucleic acids also caused RIG-I-dependent IL-1β-production and inflammasome activation. The signalling molecule CARD9 contributed to IL-1β production in response to secreted nucleic acids. In conclusion, cytosolic recognition of secreted bacterial nucleic acids by RIG-I provides a mechanistic explanation for efficient induction of immunity by live bacteria.

Figure 1

RIG-I and STING detect cytosolic infection with Listeria monocytogenes in macrophages. IFNβ in cell-culture supernatants was determined by ELISA 24 h after transfection or 18 h after infection; IFNβ mRNA was determined by qRT-PCR 6 h after transfection or infection. (A) IFNβ, TNF and IL-6 from cell-culture supernatant of bone marrow-derived macrophages (Mø) generated from TRIF^−/−, MyD88^−/−, TRIF/MyD88^−/− and wild-type (wt) littermate mice. In all, 2 × 10⁵ Mø after infection with 2 × 10⁶ CFU wt Listeria, LLO-deficient Listeria mutant (Δhly) (MOI of 10) or 2 × 10⁸ of HKLM or mock treatment. (B) IFNβ in cell-culture supernatant of RIG-I^−/−, STING^−/− or wt Mø infected with Listeria. LPS (500 ng/ml) as positive control. (C) Time kinetics of IFNβ mRNA from wt, STING^−/− or RIG-I^−/− Mø after infection. NS, not significant; **P=0.01, ***P=0.001 (unpaired Student’s t-test). Data are representative of at least three separate experiments (mean and s.d. of triplicates).

Figure 2

Listeria monocytogenes-secreted RNA triggers type I IFN induction through RIG-I in macrophages. (A) IFNβ in cell-culture supernatants of wt, TRIF^−/− or MyD88^−/− Mø after transfection with seRNA (10 ng/10⁵ cells) or lysRNA (1 μg/10⁵ cells). (B) Dose kinetic of IFNβ mRNA in Mø after transfection with seRNA or lysRNA. (C) IFNβ in cell-culture supernatant of wt, RIG-I^−/−, STING^−/− or MDA5^−/− Mø after transfection of seRNA (10 ng/10⁵ cells). (D) ATPase assay of purified recombinant human RIG-I protein after incubation with increasing concentration of Listeria RNAs (as described in Materials and methods). EC50 values for the different RNAs were determined by non-linear regression analysis. (E) ELISA of IFNβ in cell-culture supernatant of Mø 24 h after transfection with seRNA (10 ng/10⁵ cells) or lysRNA (1 μg/10⁵ cells) subjected to CIAP treatment. 3pRNA as control. NS, not significant; *P=0.05, ***P=0.001 (unpaired Student’s t-test). Data are representative of at least three separate experiments (mean and s.d. of triplicates).

Figure 3

Secreted Listeria DNA is recognized by RIG-I via the RNA-polymerase III pathway. (A–C) IFNβ in cell-culture supernatants of (A) wt, TRIF^−/− or MyD88^−/− Mø transfected with seDNA (20 ng/10⁵ cells), (B) wt, STING^−/−, RIG-I^−/− or MDA5^−/− Mø transfected with seDNA (20 ng/10⁵ cells) or (C) wt, MDA5^−/− or RIG-I^−/− Mø transfected with seDNA (20 ng/10⁵ cells) or lysDNA (2 μg/10⁵ cells). (D) IFNβ mRNA assessed by qRT-PCR in wt macrophages after transfection with seRNA (10 ng/10⁵ cells) or seDNA (20 ng/10⁵ cells), lysRNA (1 μg/10⁵ cells) or lysDNA (2 μg/10⁵ cells) after treatment with the RNA polymerase III inhibitor (ML-60218, 4 μM) for 10 h prior to transfection and during the incubation time. NS, not significant; *P=0.05, **P=0.01, ***P=0.001 (unpaired Student’s t-test). Data are representative of at least three separate experiments (mean and s.d. of triplicates).

Figure 4

SecA2 mutant Listeria (ΔsecA2) induce less IFNβ than wild-type Listeria. (A) IFNβ in cell-culture supernatants of Mø infected with wild-type Listeria, Δhly (MOI 10) or heat-killed Listeria monocytogenes (HKLM, MOI 100) in the absence or presence of the pore-forming protein LLO. (B) IFNβ in cell-culture supernatants of Mø infected with wild-type or Listeria mutants (MOI=10). (C) Secreted acids from wild-type Listeria or ΔsecA2 were determined in the supernatant from the same number of bacteria. (D) Confocal microscopy of actin polymerization in Mø after infection with wild-type Listeria, ΔsecA2 or Δhly mutants. Representative images from three independent experiments. (E) IFNβ in cell-culture supernatants of wt, STING^−/− or RIG-I^−/− Mø after infection with wild-type Listeria, ΔsecA2 or Δhly mutants (MOI 10). LPS (500 ng/ml) as positive control. NS, not significant; *P=0.05, **P=0.01, ***P=0.001 (unpaired Student’s t-test). Data are representative of at least three separate experiments.

Figure 5

Listeria infection triggers IL-1β release and inflammasome activation in an RIG-I-dependent manner. (A) IL-1β in cell-culture supernatants of wild-type or ASC^−/− Mø 18 h after infection with wt Listeria or Δhly. (B) Immunoblot of full-length (pro)caspase-1 in cell lysates or cleaved caspase 1 in the supernatant of wild-type or ASC^−/− Mø 6 h after infection with wt Listeria or Δhly. (C) IL-1β in cell-culture supernatants of wild-type Mø 18 h after infection with wt, or different Listeria mutants. (D) Immunoblot of procaspase-1 in cell lysates or cleaved caspase 1 in the supernatant of wild-type Mø 6 h after infection with wt or different Listeria mutants. (E) IL-1β in cell-culture supernatants of wild-type, MDA5^−/− or RIG-I^−/− Mø 18 h after infection with wt, Δhly or ΔsecA2 Listeria. (F) Immunoblot of procaspase-1 and pro-IL-1β in cell lysates or cleaved caspase 1 and IL-1β in the supernatant of wild-type, MDA5^−/− or RIG-I^−/− Mø 6 h after infection with wt, Δhly or ΔsecA2 Listeria. (G) Like in (E) but macrophages were pretreated with 500 U/ml of IFNα or IFNβ for 2 h prior to and during infection. (H) Immunoblot 6 h after infection of procaspase-1 in cell lysates or cleaved caspase 1 in the supernatant of wild-type or RIG-I^−/− Mø pretreated with IFNα or IFNβ as in (G). (I) IL-1β in cell-culture supernatants of wild-type and CARD9^−/− macrophages 18 h after infection with wt Listeria, Δhly and ΔsecA2. (J) Immunoblot of wild-type, CARD9^−/−, MDA5^−/− or RIG-I^−/− macrophages 6 h after infection with wt Listeria, Δhly and ΔsecA2. *P=0.05, **P=0.01, ***P=0.001. Figure source data can be found with the Supplementary data.

Figure 6

Secreted Listeria nucleic acids cause IL-1β release and inflammasome activation in an RIG-I-dependent manner. (A, B) IL-1β in cell-culture supernatants of wild-type or ASC^−/− Mø 18 h after transfection with (A) seRNA (10 ng/10⁵ cells) or lysRNA (1 μg/10⁵ cells) or (B) seDNA (20 ng/10⁵ cells) or lysDNA (2 μg/10⁵ cells). (C) IL-1β in cell-culture supernatants of wild-type or RIG-I^−/− Mø 18 h after transfection of seRNA and seDNA incubated before with RNAse and/or DNAse. (D) Immunoblot of supernatant and cell lysates of cells treated as in (C) 6 h after transfection. **P=0.01. Figure source data can be found with the Supplementary data.