Cell intrinsic immunity spreads to bystander cells via the intercellular transfer of cGAMP - PubMed (original) (raw)
. 2013 Nov 28;503(7477):530-4.
doi: 10.1038/nature12640. Epub 2013 Sep 29.
Affiliations
- PMID: 24077100
- PMCID: PMC4142317
- DOI: 10.1038/nature12640
Cell intrinsic immunity spreads to bystander cells via the intercellular transfer of cGAMP
Andrea Ablasser et al. Nature. 2013.
Abstract
The innate immune defence of multicellular organisms against microbial pathogens requires cellular collaboration. Information exchange allowing immune cells to collaborate is generally attributed to soluble protein factors secreted by pathogen-sensing cells. Cytokines, such as type I interferons (IFNs), serve to alert non-infected cells to the possibility of pathogen challenge. Moreover, in conjunction with chemokines they can instruct specialized immune cells to contain and eradicate microbial infection. Several receptors and signalling pathways exist that couple pathogen sensing to the induction of cytokines, whereas cytosolic recognition of nucleic acids seems to be exquisitely important for the activation of type I IFNs, master regulators of antiviral immunity. Cytosolic DNA is sensed by the receptor cyclic GMP-AMP (cGAMP) synthase (cGAS), which catalyses the synthesis of the second messenger cGAMP(2'-5'). This molecule in turn activates the endoplasmic reticulum (ER)-resident receptor STING, thereby inducing an antiviral state and the secretion of type I IFNs. Here we find in murine and human cells that cGAS-synthesized cGAMP(2'-5') is transferred from producing cells to neighbouring cells through gap junctions, where it promotes STING activation and thus antiviral immunity independently of type I IFN signalling. In line with the limited cargo specificity of connexins, the proteins that assemble gap junction channels, most connexins tested were able to confer this bystander immunity, thus indicating a broad physiological relevance of this local immune collaboration. Collectively, these observations identify cGAS-triggered cGAMP(2'-5') transfer as a novel host strategy that serves to rapidly convey antiviral immunity in a transcription-independent, horizontal manner.
Figures
Figure 1. cGAS overexpression activates STING in adjacent cells
a, Confocal microscopy of HEK STING cells 20 h after transfection with GFP (left) or a cGAS–GFP (right). Asterisks and arrows highlight STING complexes in GFP-positive cells and bystander cells. b, c, HEK STING cells were transfected with varying amounts of cGAS–GFP as indicated. The number of GFP-positive cells is plotted against the number of activated HEK STING cells (y = 0.27_x, R_2 = 0.84) (b) and the respective ratio of STING-activated cells over cGAS-expressing cells is depicted. Data are depicted as box plots with whiskers indicating minimum and maximum (c). One representative experiment out of two independent experiments is shown. *P < 0.05, **P < 0.01.
Figure 2. Cytosolic DNA sensing via cGAS propagates STING activation in trans
a, HEK or HEK cGASlow co-incubated with HEK STING cells were stimulated (4 h) as indicated and IRF3 phosphorylation was assessed. b, Confocal microscopy of HEK cGASlow co-incubated with HEK STING cells unstimulated or transfected with ISD (6 h). c, d, HEK cells and HEK STING cells were co-cultured with HEK cGASlow cells (c) or primary MEFs (d) (ratios ranging from 1:0.25 to 1:0.0156 HEK/HEK STING:cGASlow/MEFs) and transfected with pIFN-β-GLuc, whereas transactivation of the reporter was assessed after 20 h. Representative experiments of n = 2 (a and b) or mean and s.e.m. (biological duplicates) of one representative experiments out of six (c) or eight (d) are depicted. RLU, relative light unit.
Figure 3. cGAS-produced cGAMP(2′-5′) passes through gap junctions to trigger STING activation in bystander cells
a, Confocal microscopy of HEK cells and HEK cGAS* cells loaded with calcein and added to HEK STING cells for 4 h. b, Co-culturing was performed as in a and after 0–8 h HEK STING cells were analysed by fluorescence microscopy for STING aggregation in nine independent visual fields. A dot-blot diagram correlating calcein-positive HEK STING cells with STING aggregate formation is presented (y = 1.27_x, R_2 = 0.85). AU, arbitrary units. c, HEK STING cells were co-cultured with HEK cGAS* in the presence of CBX as indicated for 4 h and studied for STING activation in nine independent visual fields. Data are presented as mean and s.e.m. d, IRF3 phosphorylation in HEK STING cells with HEK cells, with HEK cGAS* cells or with CMA in the presence or absence of CBX (100 μM, 150 μM and 200 μM) (3 h). e, The scrape loading technique. f, Fluorescence images of wounded HEK STING cells incubated with nothing, cGAMP(2′-5′) or cGAMP(2′-5′) with 150 μM CBX. HEK STING cells with CMA served as control (dashed line, scratch margins; arrows, STING complexes). One representative experiment out of two independent experiments is shown (a-d, f). *P < 0.05, ***P < 0.001.
Figure 4. Connexin 43 and 45 mediate cGAMP(2′-5′) transfer in HEK STING cells
a, Fluorescence microscopy of HEK STING CX43/45WT and HEK STING CX43/45DKO cells left untreated, stimulated with CMA or co-cultured with calcein-loaded HEK cGAS* cells (ratio HEK/HEK STING:HEK cGAS* = 1:0.25) after 8 h. b, Phosphorylation of IRF3 in HEK STING CX43/45WT and HEK STING CX43/45DKO cells left untreated, co-incubated with HEK cGAS* cells or stimulated with CMA for 4 h (asterisk indicates nonspecific band). c, HEK STING CX43/45WT and HEK STING CX43/45DKO cells were co-cultured with HEK cGASlow cells (ratios from 1:0.5 to 1:0.0312 HEK STING CX43/45WT or HEK STING CX43/45DKO cells:cGASlow) transfected with pIFN-β-GLuc and luciferase activity was assessed after 20 h. Mean and s.e.m. (biological duplicates) of one representative experiment out of three independent experiments is shown. d, Fluorescence microscopy of HEK STING CX43/45DKO cells co-cultured with HEK cGAS* cells and stimulated with CMA, transfected with empty vector (pCI) or an expression vector for murine CX45 (mmCX45, 20 h). e, Co-cultures from d were analysed for phosphorylation of IRF3. One representative experiment out of two independent experiments is shown (a, b, d, e). *P < 0.05, **P < 0.01.
Figure 5. Vaccina virus triggers STING-dependent antiviral immunity in bystander cells
a, b, HEK cGASlow cells or HEK cells were infected with MVA–GFP, washed and then loaded onto HEK STING cells for 8 h. a, Confocal microscopy of HEK STING cells co-incubated with MVA-infected HEK cGAS cells (arrows, STING activation in bystander cells). b, Quantification of STING activation in response to MVA-infected HEK cGASlow cells or MVA-infected HEK cells. c, HEK cells or HEK cGASlow cells were MVA-infected or left untreated, added onto HEK cells or HEK STING cells and studied for IRF3 phosphorylation (viral particles per ml: ++ = 3.2 × 107; + = 1.6 × 107). d, e, Experiments as in a in the presence or absence of CBX 150 μM (d) or using HEK STING CX43/45WT and HEK STING CX43/45DKO cells as responder cells (e). f, HEK cGAS* cells were co-incubated with MEFs and after 14 h mouse Ifnb mRNA, mouse Cxcl10 mRNA and mouse Irf7 mRNA were assessed by qPCR. g, h, HEK cGAS* cells were co-incubated with MEFs and after 12 h vaccinia virus was added (multiplicity of infection (m.o.i.): 2-0.5). Twenty-four hours later cell survival was analysed. Visual fields of one representative experiment are depicted (g, right panel) and mean and s.e.m. of three independent experiments are summarized (h). VV, vaccinia virus. One representative experiment out of two (a, b, e) or three (c, d) independent experiments are shown or mean and s.e.m. of n = 5 independent experiments is presented (f). *P < 0.05, **P < 0.01, ***P < 0.001.
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