A host type I interferon response is induced by cytosolic sensing of the bacterial second messenger cyclic-di-GMP

Abstract

The innate immune system responds to unique molecular signatures that are widely conserved among microbes but that are not normally present in host cells. Compounds that stimulate innate immune pathways may be valuable in the design of novel adjuvants, vaccines, and other immunotherapeutics. The cyclic dinucleotide cyclic-di-guanosine monophosphate (c-di-GMP) is a recently appreciated second messenger that plays critical regulatory roles in many species of bacteria but is not produced by eukaryotic cells. In vivo and in vitro studies have previously suggested that c-di-GMP is a potent immunostimulatory compound recognized by mouse and human cells. We provide evidence that c-di-GMP is sensed in the cytosol of mammalian cells via a novel immunosurveillance pathway. The potency of cytosolic signaling induced by c-di-GMP is comparable to that induced by cytosolic delivery of DNA, and both nucleic acids induce a similar transcriptional profile, including triggering of type I interferons and coregulated genes via induction of TBK1, IRF3, nuclear factor kappaB, and MAP kinases. However, the cytosolic pathway that senses c-di-GMP appears to be distinct from all known nucleic acid-sensing pathways. Our results suggest a novel mechanism by which host cells can induce an inflammatory response to a widely produced bacterial ligand.

Figure 1.

c-di-GMP is a potent cytosolic inducer of type I IFNs. (A) Bone marrow macrophages were overlaid or transfected with the indicated amounts of synthetic, purified c-di-GMP. The amount of type I IFN in the supernatant was assessed after 6 h by use of an L929-ISRE-luc bioassay (Jiang et al., 2005) using recombinant IFN-β as a standard. *, P < 0.01 as compared with transfected c-di-GMP (Student's t test). (B) Bone marrow macrophages were transfected with 3.3 μg/ml of the indicated molecules (or overlaid with 100 ng/ml LPS), and type I IFN was assessed after 6 h by bioassay as in A. *, P < 0.001 as compared with unstimulated cells (Student's t test). (C) Bone marrow macrophages were transfected with 3.3 μg/ml of the indicated molecules (or overlaid with 100 ng/ml LPS), and transcription of the IFN-β gene (Ifnb) was assessed by quantitative RT-PCR, with normalization to ribosomal protein rps17 message. n.d., not detectably induced above background (lipofectamine alone). *, P < 0.001 as compared with unstimulated cells (Student's t test). (D) As in C, but transcription of the IFN-α5 gene was assessed. n.d., not detectably induced above background (lipofectamine alone). *, P < 0.01 as compared with unstimulated cells (Student's t test). (E) c-di-GMP, treated or untreated with snake venom phosphodiesterase, was transfected at 3.3 μg/ml into B6 bone marrow macrophages and analyzed after 6 h for IFN-β production by bioassay, as in A. *, P < 0.02 as compared with c-di-GMP treatment alone (Student's t test). Results in A–E are representative of at least three independent experiments. Data are means ± SD (n = 3). (F) Bone marrow macrophages were transfected with poly dA:dT (DNA) or with c-di-GMP. Total RNA was isolated after 6 h of stimulation. Probes were amplified and hybridized to whole-transcriptome spotted MEEBO microarrays. Each dot represents a single gene, and its position is determined by its induction in response to DNA (x axis) or c-di-GMP (y axis). Most genes lie on the diagonal, indicative of similar induction ratios by both treatments. Selected highly induced genes are labeled. Two independent microarray experiments gave similar results.

Figure 2.

Induction of type I IFN by c-di-GMP is independent of TLRs and requires TBK1 and IRF3. (A) Bone marrow macrophages from Myd88^−/−Trif^−/− mice were transfected with 3.3 μg/ml c-di-GMP or pdA:dT (DNA), or overlayed with 100 ng/ml LPS as indicated. After 6 h of stimulation, induction of IFN-β mRNA was analyzed by quantitative RT-PCR, with normalization to ribosomal protein rps17 message. n.d., not detectable. (B) As in A, except Tnfr1^−/− or Tnfr1^−/−Tbk1^−/− macrophages were analyzed. Tbk1 deficiency is embryonic lethal, but viability can be rescued on the TNF receptor 1 (Tnfr1)–deficient background. (C) As in A except Ikbke (IKKϵ/IKK-i)-deficient mice were analyzed. (D) as in A, except Irf3^−/− and Irf7^−/− macrophages were analyzed. (E) as in A, except macrophages deficient in the type I IFN receptor (Ifnar^−/−) were analyzed. (F) as in A, except Irf5^−/− and Irf1^−/− macrophages were analyzed. Results are representative of at least three independent experiments. Data are means ± SD (n = 3). *, P < 0.05; and **, P < 0.001 as compared with wild-type bone marrow macrophages (Student's t test).

Figure 3.

The host transcription factors IRF3 and NF-κB are induced by c-di-GMP, as are the p38, JNK, and ERK1/2 MAP kinases. (A) Nuclear extracts from Myd88/Trif^−/− macrophages stimulated for the indicated times with c-di-GMP, poly dA:dT (pdA:dT, DNA), or poly I:C (pI:C, RNA) were probed with the indicated antibodies. (B) Nuclear extracts from Myd88/Trif^−/− macrophages stimulated with c-di-GMP, poly dA:dT (pdA:dT, DNA), or poly I:C (pI:C, RNA) for the indicated times were used in a gel shift assay with an NF-κB consensus binding sequence probe. (C) RAW264.7 macrophages stably expressing an NF-κB luciferase reporter were stimulated with c-di-GMP, poly dA:dT (pdA:dT, DNA), or poly I:C (pI:C, RNA) for 6 h and analyzed for luciferase activity. (D) Whole-cell extracts from Myd88/Trif^−/− macrophages stimulated for the indicated times with c-di-GMP, poly dA:dT (pdA:dT, DNA), or poly I:C (pI:C, RNA) were probed with the indicated antibodies. Results are representative of at least three independent experiments. Data are means ± SD (n = 3). *, P < 0.05 compared with unstimulated cells (Student's t test).

Figure 4.

Responsiveness to cytosolic c-di-GMP varies among cell types and does not correlate with responsiveness to cytosolic DNA or RNA. (A) Thioglycollate-elicited peritoneal macrophages were stimulated with 3.3 μg/ml c-di-GMP, poly dA:dT (pdA:dT, DNA), or poly I:C (pI:C, RNA), and type I IFN was measured by bioassay as in Fig. 1 A. (B) GM-CSF–differentiated dendritic cells were stimulated as in A. SeV, Sendai virus. (C) L929 cells were stimulated as in A. (D) RAW264.7 macrophage-like cells were stimulated as in A. (E) MEFs were stimulated as in A. (F) 293T cells were stimulated as in A. In B, D, and F, induction of IFN-β transcripts was assessed by quantitative RT-PCR. Results are representative of at least three independent experiments. Data are means ± SD (n = 3). n.d., not detectable. *, P < 0.01; and **, P < 0.001 as compared with unstimulated cells (Student's t test).

Figure 5.

c-di-GMP does not stimulate known cytosolic pathways for sensing nucleic acids. (A) Mavs^−/− and littermate wild-type macrophages were transfected with 3.3 μg/ml c-di-GMP, poly dA:dT (pdA:dT, DNA), or poly I:C (pI:C, RNA), and transcription of the IFN-β gene (Ifnb) was assessed by quantitative RT-PCR, with normalization to ribosomal protein rps17 message. n.d., not detectable. (B) Mda5^−/− and wild-type control macrophages were stimulated and analyzed as in A. (C) Zbp1^−/− (DAI knockout) and wild-type control macrophages were stimulated and analyzed as in A. SeV, Sendai virus. Results are representative of at least three independent experiments. Data are means ± SD (n = 3). *, P < 0.01; and **, P < 0.001 as compared with wild-type bone marrow macrophages (Student's t test).

Figure 6.

c-di-GMP activates IRF3/7-dependent responses in vivo. (A and B) B6 and Irf3/7^−/− mice were injected intraperitoneally with 200 nmol c-di-GMP, and (A) serum IFN was measured by bioassay 18h later, or (B) 36 h after injection, splenic NK responsiveness to YAC-1 target cells (or PBS control) was measured ex vivo by intracellular staining for IFN-γ. (C) B6 and Irf3/7^−/− mice were injected intraperitoneally with HSA ± 200 nmol c-di-GMP, and 2 wk later serum IgG1 specific for HSA was determined by ELISA. The experiments in A and B were repeated twice, and the experiment in C was performed once. Horizontal bars represent means. **, P < 0.01; and *, P < 0.05 (Student's t test).