A type I IFN-dependent DNA damage response regulates the genetic program and inflammasome activation in macrophages
- Weill Cornell Medical College, United States
- Washington University School of Medicine, United States
- National Institute of Environmental Health Sciences, United States
Figures
Figure 1 with 2 supplements
ATM and DNA-PKcs-dependent DDR activation in macrophages.
(A–C) Western blotting for γ-H2AX, H2AX, phosphorylated KAP-1 (p-KAP-1) and KAP-1 in whole cell lysates from (A) wild type (WT) BMDMs after no stimulation (-) or stimulation (+) with LPS, IFN-γ, or …
Figure 1—figure supplement 1
ATM is efficiently deleted in BMDM.
Southern blot analysis of genomic DNA digested with KpnI and hybridized to the 3’ ATM probe from BMDMs from AtmC/C, Atm-/-and Scid:AtmC/C:Lyz2Cre/+ mice. Arrows indicate the conditional (C) and …
Figure 1—figure supplement 2
L. monocytogenes infection induces G1 arrest.
Flow cytometric analysis of BrdU incorporation (y axis) and DNA content (7AAD; x axis) was performed in Lyz2Cre/+ and Scid: AtmC/C: Lyz2Cre/+ BMDMs after L. monocytogenes infection for 16 hr. …
Figure 2 with 1 supplement
Activation of DDR by DNA DSBs.
(A) Neutral Comet assay and Olive Tail Moment quantification of BMDMs left untreated (UT) or after infection with L. m. or LPS + IFN-γ for 12 hr. (B) Immunofluorescence for L. m. (green) and γ-H2AX …
Figure 2—figure supplement 1
L.monocytogenes-infected BMDM exhibit discrete γ-H2AX foci.
(A) Additional representative images of immunofluorescence for L. m. (green) and γ-H2AX (red) in WT BMDMs 9 hr. post-infection. Nuclei are revealed by DAPI (blue). (B) Quantification of γ-H2AX foci …
Figure 3
The DDR depends on TLR and interferon signaling cascades.
(A,B) Western blotting for γ-H2AX and H2AX in whole cell lysates from (A) WT or Myd88-/- BMDMs after no infection (-) or infection (+) with L. monoctytogenes (L. m.) for 24 hr. (B) WT BMDMs after no …
Figure 4 with 2 supplements
Optimal DDR depends on type I interferon signaling.
(A) Nitrite concentration in culture supernatants collected from WT and Ifnar1-/- BMDMs after no infection (-) or infection (+) with L. m. for 24 hr. in the presence or absence of IFN-γ. Data are a …
Figure 4—figure supplement 1
Type I IFN does not affect the cell cycle in BMDMs.
Flow cytometric analysis of BrdU incorporation (y axis) and DNA content (7AAD; x axis) was performed in BMDMs left untreated or after stimulation with IFN-β (100 U/mL), bleomycin (1 ug/mL), or …
Figure 4—figure supplement 2
Type I IFN does not impact expression of key DDR factors.
Western blotting for Mre11, Nbs1, Rad50, Ku70, Ku80, ATM, ATR, DNA-PKcs, and H2AX in untreated (-) WT mouse embryonic fibroblasts (MEFs) or BMDMs after no stimulation (-) or stimulation (+) with …
Figure 5
ATM and DNA-PKcs regulate the genetic program of activated macrophages.
(A) Heat map showing genes that are significantly up-regulated (≥4 fold, adj. p-value<0.001) in L. m.-infected Lyz2Cre/+and Scid:Atm C/C:Lyz2Cre/+ BMDMs relative to no infection. Red indicates …
-
Figure 5—source data 1
Gene expression changes induced by L. monocytogenes infection.
Normalized log2 probe values for genes up-regulated (≥4 fold, adj. p-value<0.001) in L. m.-infected Lyz2Cre/+and Scid:Atm C/C:Lyz2Cre/+ BMDMs relative to uninfected cells. The fold-change between infected Scid:Atm C/C:Lyz2Cre/+-and infected Lyz2Cre/+ cells for each gene is also shown.
- https://doi.org/10.7554/eLife.24655.012
Figure 6 with 2 supplements
DDR regulates inflammasome activation.
(A) IL-1β protein concentration as determined by ELISA in supernatants from uninfected (-) and L. m.-infected (+) Scid:AtmC/C:Lyz2Cre/+ and Lyz2Cre/+ BMDMs 12 and 24 hr. post-infection. Data are …
Figure 6—figure supplement 1
Mitochondrial ROS levels are equivalent in DDR-deficient and –sufficient BMDM.
Flow cytometric analysis of mitochondrial superoxide levels in live BMDM from Lyz2Cre/+, AtmC/C: Lyz2Cre/+, and Scid: AtmC/C: Lyz2Cre/+ mice. Superoxide is detected with the fluorogenic dye MitoSOX …
Figure 6—figure supplement 2
Gene expression of inflammasome components.
(A–D) Quantitative RT- PCR analysis of (A) pro Il-1β, (B) Asc, (C) Nlrp3, and (D) Aim2 gene expression in Scid: AtmC/C: Lyz2cre/+and Lyz2cre/+BMDMs infected (+) or not (-) with L. m. Data are a …
Figure 7 with 1 supplement
DNA-PKcs regulates IL-18 production and NK cell activation.
(A) IL-18 protein concentration as determined by ELISA in supernatants from uninfected (-) and L. m.-infected WT and Scid BMDMs 12 and 24 hr. post-infection. Data are mean and SEM of technical …
Figure 7—figure supplement 1
Il18 and Il12 gene expression is equivalent in WT and Scid BMDM after L.m. infection.
(A,B) Quantitative RT- PCR analysis of (A) Pro Il18 and (B) Il12p40 gene expression in WT and Scid BMDM 12 hr post- L. m. infection. Data are a compilation of three independent experiments and …
