Volume 45, Issue 1, Pages 106-118 (July 2016) Loss of the DNA Damage Repair Kinase ATM Impairs Inflammasome-Dependent Anti- Bacterial Innate Immunity  Saskia F. Erttmann, Anetta Härtlova, Marta Sloniecka, Faizal A.M. Raffi, Ava Hosseinzadeh, Tomas Edgren, Reza Rofougaran, Ulrike Resch, Maria Fällman, Torben Ek, Nelson O. Gekara  Immunity  Volume 45, Issue 1, Pages 106-118 (July 2016) DOI: 10.1016/j.immuni.2016.06.018 Copyright © 2016 Elsevier Inc. Terms and Conditions

Immunity 2016 45, 106-118DOI: (10.1016/j.immuni.2016.06.018) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 1 Atm−/− Mice Are More Susceptible to Pulmonary S. pneumoniae Infection in a Manner Independent of Defects in the Adaptive Immune System (A) Visualization of bacterial burden in WT, Atm+/−, and Atm−/− mice and their corresponding lungs by IVIS 5 days after intranasal inoculation with bioluminescent S. pneumoniae-Xen10. (B) Bioluminescence (RLU) -based quantification of bacterial burden in lungs of WT, Atm+/−, and Atm−/− mice. (C) Morbidity of WT, Atm+/−, and Atm−/− mice after intranasal infection with S. pneumoniae-Xen10 (2 × 107/mouse). (D) ELISA analysis of extracellular IL-1β, TNF-α, IL-6, and total IgG in lungs of WT and Atm−/− mice 5 days post S. pneumoniae-Xen10 infection. Data in (A)–(D) are representative of three independent experiments each with 3–9 mice per genotype and are shown as mean ± SEM. ∗p < 0.05 (one-way ANOVA followed by Bonferroni post-test). (E) Visualization of bacterial burden in representative lungs of WT, Atm+/−, Rag2−/−, and Atm+/−Rag2−/− mice by IVIS 3 days after intranasal inoculation with bioluminescent S. pneumoniae-Xen10. (F–J) WT, Atm+/−, Rag2−/−, and Atm+/−Rag2−/− mice infected intranasally with (2 × 107/mouse) S. pneumoniae-Xen10 monitored for percentage weight loss (F), clinical severity (G), and bacterial burden (H) as well as extracellular IL-1β (I), TNF-α (J), and total IgG (K) in lungs 3 days post infection. Each experiment represents 4 mice per genotype and are shown as mean ± SEM. ∗p < 0.05, ∗∗∗p < 0.001; (one-way ANOVA followed by Bonferroni post-test). Immunity 2016 45, 106-118DOI: (10.1016/j.immuni.2016.06.018) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 2 Susceptibility of Atm−/− Mice to S. pneumoniae Infection Is Consistent with Inflammasome Defects (A) Visualization of bacterial burden in WT, Atm+/−, Asc−/−, and Atm+/−Asc−/− mice and their corresponding lungs by IVIS 3 days after intranasal inoculation with S. pneumoniae-Xen10 (2 × 107/mouse). (B) Bioluminescence (RLU)-based quantification of bacterial burden in lungs of WT, Atm+/−, Asc−/−, and Atm+/−Asc−/− mice. (C and D) ELISA analysis of IL-1β (C) and TNF-α (D) in lungs of WT, Atm+/−, Asc−/−, and Atm+/−Asc−/− mice 3 days post S. pneumoniae-Xen10 infection. Each experiment represents 4 to 5 mice per genotype. Data are shown as mean ± SEM. ∗p < 0.05; (one-way ANOVA followed by Bonferroni post-test). Immunity 2016 45, 106-118DOI: (10.1016/j.immuni.2016.06.018) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 3 Cells from AT Patients Exhibit Defects in Inflammasome-Dependent Cytokine Response (A–E) ELISA analysis of IL-1β and IL-6 response in monocyte-derived macrophages from healthy subjects and AT patients that were first primed with LPS (for 4.5 hr) then infected either with S. pneumoniae (MOI 30) (A), S. Typhimurium (MOI 10) (B), or P. aeruginosa (MOI 20) (C) or stimulated with ATP (D) or nigericin (E) for indicated duration. (F) ELISA analysis of IL-1β in PBMCs from healthy subjects and AT patients that were first primed with LPS, heat-killed L. monocytogenes (HK L.m.), or Pam3CSK4 (Pam), then stimulated with ATP. (G) ELISA analysis of IL-6 in healthy or AT PBMCs 6 hr after stimulation with Pam. Data are shown as mean ± SEM. Immunity 2016 45, 106-118DOI: (10.1016/j.immuni.2016.06.018) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 4 ATM Deficiency Impairs Caspase-1 and IL-1β Processing and Secretion WT and Atm−/− bone marrow-derived macrophages (BMDMs) primed (or not) with LPS (500 ng/ml) for 4.5 hr and then stimulated with ATP (5 mM) for 15, 30, or 60 min, S. Typhimurium (MOI 10) for 1 hr or S. pneumoniae (MOI 100) for 4 hr. (A–C) ELISA analysis of IL-1β secretion. (D–F) Immunoblot analysis of cell lysates (CL) and supernatants (SN) for Caspase-1, IL-1β processing. (G–I) Measurement of cell cytotoxicity by lactate dehydrogenase (LDH) release assay. Results are representative of more than three independent experiments. Data are shown as mean ± SEM. ∗∗p < 0.01, ∗∗∗p < 0.001; (one-way ANOVA followed by Bonferroni post-test). Please see Figures S1–S3. Immunity 2016 45, 106-118DOI: (10.1016/j.immuni.2016.06.018) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 5 ATM Kinase Activity Is Required for the Efficient Assembly of ASC-Caspase-1 Complexes (A–E) Microscopic visualization of ASC (red) and active Casp-1 (green) foci in WT and Atm−/− BMDMs primed with LPS (500 ng/ml) then stimulated with ATP (5 mM) for 30 min (A) or S. Typhimurium (MOI 10) for 1 hr (C). Corresponding percentages of BMDMs containing ASC and active Caspase-1 foci after stimulation with ATP (B) or infection with S. Typhimurium (D). Close up view of representative ASC+Caspase-1 foci highlighted in square depicted in the far right of panels in (C). Scale bar represents 10 μM. Determination of percentage cell counts in (B) and (D) by enumerating at least 300 cells per sample. Average radius determination of ASC+Casp-1 foci from at least 50 representative cells by the Nikon Imaging Software NIS-Elements (E). (F) WT and Atm−/− intraperitoneal macrophages primed with LPS (500 ng/ml) for 4.5 hr, then stimulated with ATP (5 mM) and analyzed biochemically for ASC complex formation, Casp-1 and IL-1β processing. (G) WT BMDMs pre-treated (or not) with ATM inhibitor KU-55933 (KU, 5 μM) for 12 hr, primed with LPS (500 ng/ml), then stimulated with ATP (5 mM) for 30 min and analyzed biochemically for ASC complexes formation, Casp-1, and IL-1β processing. Results are representative of at least three experiments. Data are shown as mean ± SEM. ∗∗p < 0.01 (one-way ANOVA followed by Bonferroni post-test). Immunity 2016 45, 106-118DOI: (10.1016/j.immuni.2016.06.018) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 6 Oxidative Stress Contributes to Reduced Caspase-1 Activation in Atm−/− Cells (A) Flow cytometric analysis of resting WT and Atm−/− BMDMs stained with the mitochondrial ROS-specific marker MitoSOX Red. (B) Mean fluorescence intensity (MFI) of cells in (A). (C) Experimental outline for data in (D), (E), (G)–(I). Normal condition: BMDMs primed with LPS for 4.5 hr then stimulated with ATP (5 mM) for 30 min. Pre-priming: BMDMs primed with LPS in the presence of N-acetylcysteine (NAC; 25 mM) or catalase (CAT; 100 U/ml) for 4.5 hr before ATP stimulation. Post-priming: BMDMs primed with LPS for 4 hr 20 min, then treated with NAC or catalase for 10 min followed by stimulation with ATP for 30 min. (D and E) Immunoblot for Caspase-1 and IL-1β processing and IL-1β ELISA assays in LPS-primed WT BMDMs that were treated with NAC or CAT and stimulated with ATP as indicated in (C). (F) Immunoblot for Caspase-1 and IL-1β processing and IL-1β ELISA assays in WT BMDMs LPS-primed for 4.5 hr and then stimulated with ATP in the presence of indicated H2O2 concentrations. (G–I) Immunoblot for Caspase-1 and IL-1β processing in (G) WT or Atm−/− BMDMs LPS-primed for 4.5 hr then stimulated with ATP for 30 min in the presence or absence of NAC. (H) WT BMDMs pre-treated (or not) with KU-55933 (5 μM) for 12 hr, LPS-primed for 4.5 hr then stimulated with ATP for 30 min in the presence or absence of NAC. (I) WT and Gp91phox−/− BMDMs pre-treated (or not) with KU-55933 (5 μM) for 12 hr, LPS-primed for 4.5 hr then stimulated with ATP for 30 min. Data are representative of two to three independent experiments. Data are shown as mean ± SEM. ∗∗∗p < 0.001; (one-way ANOVA followed by Bonferroni post-test). Please see Figures S4 and S5. Immunity 2016 45, 106-118DOI: (10.1016/j.immuni.2016.06.018) Copyright © 2016 Elsevier Inc. Terms and Conditions

Figure 7 Oxidative Stress Contributes to Impaired Inflammasome Complex Assembly in Atm−/− Cells (A) Microscopic analysis of active Caspase-1 (green) and ASC (red) foci in LPS-primed WT and Atm−/− BMDMs infected with S. Typhimurium (MOI 30) for 1 hr in the presence or absence of NAC. (B) Percentage of LPS-primed BMDMs containing active Casp-1 and ASC foci following infection with S. Typhimurium in the presence or absence of NAC. (C) LPS-primed WT and Atm−/− BMDMs infected with S. Typhimurium in the presence or absence of H2O2 (50 μM), then analyzed biochemically for ASC complex formation, Casp-1, and IL-1β processing. (D) ELISA analysis of TNF-α secretion by LPS-primed WT and Atm−/− BMDMs infected with S. Typhimurium in the presence or absence of H2O2. (E) Biochemical analysis of ASC oligomerization in WT BMDMs incubated with CAT during (pre-prime) or after LPS priming (post-prime) followed by ATP stimulation. (F) ASC oligomerization in WT and Atm−/− BMDMs incubated with CAT after LPS priming then infected with S. Typhimurium (MOI 10) for 1 hr. (G) ASC oligomerization in WT and Gp91phox−/− BMDMs primed with LPS then infected with S. Typhimurium (MOI 10) for 1 hr. Results are representative of at least two independent experiments. Data are shown as mean ± SEM. ∗∗p < 0.01; (one-way ANOVA followed by Bonferroni post-test). Please see Figure S6. Immunity 2016 45, 106-118DOI: (10.1016/j.immuni.2016.06.018) Copyright © 2016 Elsevier Inc. Terms and Conditions