Rad50-CARD9 interactions link cytosolic DNA sensing to IL-1β production

Abstract

Double-stranded DNA (dsDNA) in the cytoplasm triggers the production of interleukin 1β (IL-1β) as an antiviral host response, and deregulation of the pathways involved can promote inflammatory disease. Here we report a direct cytosolic interaction between the DNA-damage sensor Rad50 and the innate immune system adaptor CARD9. Transfection of dendritic cells with dsDNA or infection of dendritic cells with a DNA virus induced the formation of dsDNA-Rad50-CARD9 signaling complexes for activation of the transcription factor NF-κB and the generation of pro-IL-1β. Primary cells conditionally deficient in Rad50 or lacking CARD9 consequently exhibited defective DNA-induced production of IL-1β, and Card9(-/-) mice had impaired inflammatory responses after infection with a DNA virus in vivo. Our results define a cytosolic DNA-recognition pathway for inflammation and a physical and functional connection between a conserved DNA-damage sensor and the innate immune response to pathogens.

Figure 1. CARD9 interacts with Rad50

(a) BRET assays of binary interactions of CARD9 with Rad50 fragments, Bcl10 or AIM2 as Rluc and YFP fusion proteins in COS7 cells. The BRET ratios are given as the means + SEMs of two independent experiments. The dashed line indicates the method-specific threshold for a positive protein-protein interaction. (b) BRET saturation experiments using co-transfected fusions of CARD9 with Rad50 zinc hook (fragment B) orAIM2 at the indicated acceptor-to-donor ratio. The specific interaction of CARD9 with Rad50 is demonstrated by the hyperbolic behavior of BRET ratios as a function of the acceptor-to-donor ratio. (c, d) THP1 cell lysates were immunoprecipitated (IP) with anti-CARD9 (c), anti-Rad50 (d), or the respective isotype control antibodies. Immunoprecipitates were separated on one gel and transferred onto a single membrane, which was subsequently cut and used for immunoblot (IB) analysis with anti-Rad50 or CARD9 antibody, as indicated. (e) THP-1 cell lysates were left untreated (−) or were immunodepleted for Rad50 1, 2, or 3 times. Subsequently, bead-immobilized dsDNA (DNA beads) or empty streptavidin beads (Beads only) were incubated with THP-1 cell lysates that were either not treated, or immunodepleted for Rad50 three times. The beads were precipitated, and bound proteins were separated on a gel and blotted onto a single membrane. The membrane was cut and used for western blot analysis with anti-Rad50 or CARD9 antibody, as indicated. The data are representative of at least three independent experiments (c-e).

Figure 2. CARD9 is recruited to cytoplasmic dsDNA-sensing Rad50 complexes

(a-c) Confocal microscopy analysis. (a) BMDCs were left unstimulated (us) or were transfected with ATTO 647N (A647N) fluorescently labeled poly(dG:dC) (DNA) (2.5 μg/ml) for 2 hours and then stained with DAPI as a DNA counterstain and antibodies against Rad50 and CARD9. The arrow in the merged picture indicates a representative cytosolic dsDNA/Rad50/CARD9 complex. (b) BMDCs were transfected with dsDNA, stained, and analyzed as in (a). A dsDNA/Rad50/CARD9 complex (upper left picture, box) was visualized at higher magnifications and in different z-layers (z1 – z5). (c) BMDCs were transfected with dsDNA as in (a) and stained with DAPI and antibodies against Rad50 and Mre11 or Nbs1. Cytosolic dsDNA/Rad50/Mre11 or dsDNA/Rad50/Nbs1 complexes are indicated by arrows. Scale bars represent 5 μm, and the data are representative of at least three independent experiments in which at least 50 individual cells were analyzed per experiment and assay point.

Figure 3. CARD9 and Rad50 are essential for DNA-induced IL-1β production

(a, b) BMDCs from WT and Card9^−/− mice were transfected with dsDNA (1 - 4 μg/ml) of different origins, or stimulated with LPS, CpG, and curdlan plus ATP for 6 hours. IL-1β (a) and IFN-β (b) concentrations in the supernatants were determined. (c, d) BMDCs from WT and Tmem173^−/− mice were transfected with dsDNA or stimulated with CpG + ATP as in (a, b). IL-1β (c) and IFN-β (d) concentrations were determined in the supernatants. (e) BMDCs were transfected with poly(dG:dC) (4 μg/ml) for the indicated time period. (f) BMDCs were transfected with increasing amounts of purified dsDNA from vaccinia virus (VV-DNA) for 16 hours. (g) BMDCs were transfected with the indicated microbial DNA for 16 hours, and the IL-1β levels in the supernatants were determined. (h, i) Dendritic cells were differentiated from murine bone marrow of the indicated genotype; 4-hydroxy-tamoxifen (4-OHT) was added on culture day 5. (h) BMDCs (with or without 4-OHT treatment) were lysed at the indicated time after the addition of 4-OHT. The Rad50 and β-actin protein levels in the cellular lysates were analyzed by western blotting. (i) At 5 days after the addition of 4-OHT, BMDCs were transfected with dsDNA (1 - 4 μg/ml) or stimulated with LPS + ATP and curdlan + ATP, and IL-1β concentrations in the supernatants were determined. The data are represented as the mean + SEM of triplicates. One representative of at least three independent experiments is shown. *p < 0.05, **p < 0.01, ***p < 0.01, Student’s t-test. ND, not detectable.

Figure 4. CARD9 controls dsDNA-mediated NF-κB activity

(a-c) BMDCs from WT or Card9^−/− mice were left untreated (us), transfected with poly(dG:dC) (DNA) (2.5 μg/ml), or stimulated with CpG for 1 hour, and subsequently fixed and stained with DAPI and an anti-p65 or anti-c-Rel antibody. p65 and c-Rel translocation into the nucleus was monitored by confocal microscopy and quantified by determining the frequency of p65- or c-Rel-positive nuclei in at least 100 individual cells. The scale bar represents 5 μm. (d) BMDCs were left untreated, transfected with poly(dG:dC) (DNA) (2.5 μg/ml), or stimulated with CpG for 4 hours, and IL-1β transcript levels were measured by quantitative real-time PCR and normalized to β-actin mRNA levels. The data are shown as the mean + SEM. ***p < 0.001, Student’s t-test. (e, f) WT or Card9^−/− BMDCs were transfected with dsDNA for the indicated time. (e) Immunoblot analysis of cytosolic extracts. The blots were probed with an antibody against phospho-IRF3 (Ser396) or IRF3. (f) Nuclear extracts were immunoblotted with anti-IRF3 and anti-Lamin B antibodies. (g) BMDCs from WT and Tmem173^−/− mice were stimulated and analysed as in (e). (h) WT or Tmem173^−/− BMDCs were left untreated or transfected with dsDNA, and analysed as in (a-c). (i) BMDCs from WT and Tmem173^−/− mice were treated and analysed as in (d).The data are representative of two (i) or three (a-h) independent experiments.

Figure 5. Rad50/CARD9 interactions recruit Bcl10 for IL-1β responses

IL-1β (a) and IFN-β (b) concentrations were measured in the supernatants of BMDCs transfected with dsDNA (1 - 4 μg/ml) of different origins. LPS, CpG, and curdlan plus ATP were used as controls. The data are shown as the mean + SEM. (c) BMDCs were left untreated or transfected with poly(dG:dC) (DNA) (2.5 μg/ml) or stimulated with CpG for 4 hours. IL-1β transcript levels were measured by quantitative real-time PCR and normalized to β-actin mRNA levels. The data are shown as the mean + SEM. (d) BMDCs of the indicated genotypes were transfected with poly(dG:dC) (DNA), or stimulated with LPS+ATP for 8 hours. Caspase-1 p10 protein levels in the tissue culture supernatants were analysed by immunoblot. (e) BMDCs of the indicated genotypes were transfected with poly(dG:dC) (DNA) for 2 hours and analyzed by confocal microscopy following immunofluorescence staining with antibodies against Rad50 and CARD9 or Bcl10; DNA was stained with DAPI. The merged images are shown; the arrows indicate cytosolic dsDNA/Rad50/CARD9, dsDNA/Rad50/Bcl10, or dsDNA/Rad50 complexes. The scale bar represents 5 μm. At least 50 individual cells were analyzed per experiment and assay point. All data are representative of at least three independent experiments. ***p < 0.001, Student’s t-test. ND, not detectable.

Figure 6. Recognition of DNA virus infection by Rad50/CARD9 complexes

(a, b) BMDCs were infected with VV, fixed, and stained with antibodies against Rad50 and CARD9. The cells were analyzed using confocal microscopy; dsDNA was stained with DAPI. The arrows indicate viral-dsDNA/Rad50/CARD9 complexes (a). In (b), the indicated viral-dsDNA/Rad50/CARD9 complex (upper left picture, box) was visualized at higher magnifications and in different z-layers (z1 - z5). Scale bars, 5 μm. One representative of three independent experiments is shown. (c) BMDCs of the indicated genotype were infected with increasing MOIs of VV, and IL-1β, TNF, and IL-6 concentrations were measured in the supernatants. The data are represented as the mean + SEM. *p < 0.05, **p < 0.01, ***p < 0.001, Student’s t-test.

Figure 7. CARD9 controls DNA virus-induced immune responses in vivo

(a) WT and Card9^−/− mice were infected with VV in vivo, and the IL-1β concentrations were measured in the serum after 6 hours. (b) At 8 days post-infection, splenocytes were isolated and stimulated with a VV-specific peptide (B8R) or a control peptide, OVA(257-264), for 5 hours. The percentage of IFN-γ⁺ CD8⁺ T cells was determined by flow cytometry. Circles represent individual mice. The data represent the mean +/− SEM. *p < 0.05, **p < 0.01, Student’s t-test.