Listeria monocytogenes induces IFNβ expression through an IFI16-, cGAS- and STING-dependent pathway

Abstract

Listeria monocytogenes is a gram-positive facultative intracellular bacterium, which replicates in the cytoplasm of myeloid cells. Interferon β (IFNβ) has been reported to play an important role in the mechanisms underlying Listeria disease. Although studies in murine cells have proposed the bacteria-derived cyclic-di-AMP to be the key bacterial immunostimulatory molecule, the mechanism for IFNβ expression during L. monocytogenes infection in human myeloid cells remains unknown. Here we report that in human macrophages, Listeria DNA rather than cyclic-di-AMP is stimulating the IFN response via a pathway dependent on the DNA sensors IFI16 and cGAS as well as the signalling adaptor molecule STING. Thus, Listeria DNA is a major trigger of IFNβ expression in human myeloid cells and is sensed to activate a pathway dependent on IFI16, cGAS and STING.

Keywords: Listeria monocytogenes; innate immunity; interferon beta.

Figure 1. IFNβ induction by Listeria monocytogenes, DNA and cyclic dinucleotides (CDNs) in human macrophages

A–E Human primary macrophages (A) and PMA-differentiated THP1 macrophages (B–E) were infected with 25 multiplicity of infection (MOI) of WT L. monocytogenes or listeriolysin O (LLO)-deficient (ΔLLO) L. monocytogenes (strain LO28). (A, B, D) The cells were lysed 6 h post-treatment, and total RNA was isolated or (C, E) supernatants were isolated 6 and 18 h post-infection. IFNβ and TNF-α mRNA, and levels was determined by RT-qPCR, IL-1β protein levels were determined by ELISA (6 h) and type I IFN bioactivity was determined by the HEK-BLUE IFN assay (18 h). F Lysates from PMA-differentiated THP1 cells treated as indicated for 6 h were subjected to Western blotting with antibodies specific for phosphor and total TBK1. G, H PMA-differentiated THP1 macrophages were transfected with increasing concentration of (G) CDNs or (H) dsDNA. Total RNA was isolated 6 h post-treatment and IFNβ mRNA levels were determined by RT-qPCR. I Supernatants from PMA-differentiated THP1 cells stimulated with dsDNA (2 μg) or cyclic-di-AMP (12.5 μM) for 6 h were analysed for IFN bioactivity. Data information: Data represent mean ± SD of duplicates, representative of at least three independent experiments.

Figure 2. IFNβ induction by Listeria monocytogenes in human macrophages does not correlate with expression of the multidrug efflux pump MdrT

A–D Mouse bone marrow-derived macrophages (BMMs) (A), Human primary monocyte-derived macrophages (B), PMA-differentiated THP1 cells (C) or PMA-differentiated U937 cells (D) were infected with the L. monocytogenes strains LO28 and 10403s or LO28 mutants with altered expression of the multidrug efflux pump MdrT (MOI 25). E PMA-differentiated THP1 cells were infected with L. monocytogenes strain LO28 for 2 h and treated with ampicillin in the indicated concentrations. F PMA-differentiated THP1 cells were infected with L. monocytogenes strain 10403s or the derived mutant Δlmo2473. Data information: Total RNA was isolated 6 h p.i. and RT-qPCR analysis for IFNβ was performed. Data represent mean ± SD of duplicates, representative of at least three independent experiments.

Figure 3. Induction of IFNβ by Listeria monocytogenes infection correlates with expression of IFI16

A THP1 cells either undifferentiated or PMA differentiated were infected with L. monocytogenes (strain LO28, multiplicity of infection, MOI 25). Total RNA was isolated 6 h p.i. and RT-qPCR analysis for IFNβ was performed. B Whole lysates from THP1 cells and hMDMs were subjected to Western blotting using antibodies against IFI16, cGAS, DDX41, STING and β-actin. C, D THP1-shCtrl, THP1-shIFI16 and THP1-shcGAS were seeded and either differentiated with PMA or left untreated prior to stimulation with dsDNA (2 μg/ml) for 6 h. Total RNA was isolated and IFNβ mRNA induction was determined by RT-qPCR. Data information: Data in (A), (C) and (D) represent mean ± SD of duplicates, representative of 2–3 independent experiments.

Figure 4. IFNβ induction by Listeria monocytogenes extracts is dependent on IFI16, cGAS and STING

A–L PMA-differentiated THP1 macrophages transduced with lentivirus-encoding control shRNA or a shRNA sequence targeted to cGAS, IFI16, DDX41 or STING were transfected with dsDNA (2 μg/ml) (A–D), L. monocytogenes (strain LO28, multiplicity of infection, MOI 25) total extracts or extracts pre-treated with either RNase or DNase (E-H), or infected with L. monocytogenes (I-L). The cells were lysed 6 h post-treatment, and IFNβ mRNA induction was determined by qPCR. M, N The Ctrl and gene-targeted shRNA cell lines were treated with DNA and L. monocytogenes as above. Total RNA was harvested at the indicated time points, and IFNβ mRNA induction was determined by qPCR. Data information: Data represent mean ± SD of duplicates, representative of 2–3 independent experiments.

Figure 5. L. monocytogenes infection in human myeloid cells stimulates phosphorylation of TBK1 in a manner dependent on IFI16, cGAS and STING

PMA-differentiated THP1 macrophages transduced with lentivirus-encoding control shRNA or a shRNA sequence targeted to cGAS, IFI16, DDX41 or STING were infected with L. monocytogenes (strain LO28, MOI 25). Whole-cell lysates were generated 3 h post-infection, and levels of phospho- and total TBK1 were determined by Western blotting.

Figure 6. IFI16 and STING co-localize with DNA in the cytoplasm after Listeria monocytogenes infection

A–C PMA-differentiated THP1 macrophages were (A) transfected with 2 μg/ml of FITC-labelled dsDNA and (B) 3.6 μM of cyclic-di-AMP, or (C) infected with L. monocytogenes (strain LO28, MOI25). Four hours post-treatment, the cells were fixed and stained with anti-IFI16 and anti-STING specific antibodies. DNA was visualized with DAPI (blue).