Fig 2: Chromosome 3 GBPs Are Involved in Restriction of Legionella Bacterium during Pneumonia(A) Mice were infected with 1 × 107 L. pneumophila bacteria oropharyngeally. Rectal temperature of mice at various time points post-infection. Each dot represents an animal; results are pooled from 2 experiments.(B) Serum CXCL1 was measured by ELISA. Each dot represents an animal; results are pooled from 2 experiments in which tissues were harvested between 4 and 6 hr post-infection.(C) Lung IL-1a and IL-1ß were measured by ELISA. Each dot represents an animal; results are pooled from 2 experiments in which tissues were harvested between 12 and 18 hr post-infection.(D and E) Legionella colony-forming units from whole-lung tissue; the geometric mean is shown. Each dot represents an animal; results are pooled from 2 experiments. Animals were infected with (D) WT or ?sdhA bacterium and (E) WT bacterium.(F) Whole-lung tissue was extracted from B6, ifnar-/-, and Ifnb-/- animals in the absence of bacterial challenge. Each symbol represents one mouse. mRNA transcripts for indicated Gbps were quantified by qRT-PCR.(G) Body temperature of mice infected with 1 × 107 L. pneumophila bacteria oropharyngeally. Each dot represents an animal; results are pooled from 2 experiments.(H and I) Lung IL-1 a and IL-1 ß measured by ELISA. Each dot represents an animal; results are pooled from 2 experiments in which tissues were harvested between 4 and 6 hpi (H) or 12 and 18 hpi (I).(J) Legionella colony-forming units from whole-lung tissue of B6 and ifnar-/- animals.(K) Serum CXCL1 was measured by ELISA. Each dot represents an animal; results are pooled from 2 experiments in which tissues were harvested between 4 and 6 hpi.

Fig 3: Endogenous GBP Expression and Rate of Cell Death Are Controlled by a Narrow Window of Low-Dose IFN Signaling(A) Macrophages were challenged with L. pneumophila ?sdhA, and macrophage death was measured by PI incorporation. Left: representative kinetics of cell death. Right: average cell death at 6 hpi; N = 3.(B) lfnb-/- macrophages were treated for 20 hr with recombinant IFNß. Cells were then challenged with L. pneumophila ?sdhA, and macrophage death was measured by PI incorporation.(C and D) Immunoblot (C) and qRT-PCR (D) after 20 hr of treatment of Ifnb-/ macrophages with low-dose IFNß. The dotted black line indicates steady-state expression of genes of interest in B6 macrophages.(E and F) B6 and Ifnb-/-BMDMs were stimulated with recombinant IFNß for 8 hr. Immunoblot shown in (E). Quantification of band intensity over GAPDH shown in (F). The dotted red line indicates the threshold of antibody detection on the LI-COR Biosciences. Representative of 3 experiments.See Figure S3.

Fig 4: Constitutive type I interferon (IFN-I) signaling is required for initiation of necroptosis. a, b Propidium iodide incorporation as a readout of cytotoxicity, measured every 15 min following LPS/zVAD treatment of BMDMs of indicated genotype. c Western blot of indicated proteins of resting BMDMs from B6 and Ifnb−/− with or without overnight interferon treatment (2.5 IU IFNβ/ml). d Treatment scheme for overnight or 1 h treatment of BMDMs with 2.5 IU/ml of recombinant IFNβ. e Viability of B6 and Ifnb−/− BMDMs measured 6 h after LPS /zVAD treatment. f Treatment scheme for overnight or 1 h antibody treatment of B6 BMDMs. g Western blot for indicated proteins from B6 BMDMs treated with antibodies as in (f) before stimulation with 200 IU/ml IFNβ for 30 min. h, i Propidium iodide incorporation of B6 BMDMs treated with LZ and antibody pre-treatment for 36 h (h) or 1 h (i) as in (f). j Propidium iodide incorporation over 22 h of B6 BMDMs treated with LPS/zVAD or TNF (50 ng/ml)/zVAD (50 μM) and Ifnb−/− BMDMs treated with TNF/zVAD. k, l Cytotoxicity measurement at 7 h (k) or 20 h (l) from B6 and Ifnb−/− BMDMs treated with TNF/zVAD with IFN pre-treatment as in (d). All LPS/zVAD treatments were: LPS (10 ng/ml) and zVAD (50 μM/ml). In all cases of IFNβ pre-treatment (overnight or 1 h), the IFNβ is washed away before addition of experimental conditions. Time point cytotoxicity quantification ± SD from three independent experiments compared using student two tailed t-test: ns non-significant (p > 0.05); *p < 0.05; **p < 0.01; ***p < 0.001. All western blot and kinetic cytotoxicity data are representative of three or more experiments. See related supplementary Figure 1

Fig 5: Def6 deficiency inhibits endogenous expression of IFN-ß and ISG genes via downregulation of PKR in osteoblasts.(A–B) ELISA analysis of IFN-ß level in mouse serum (A) and in CD45 negative calvarial osteoblast cell cultures (B) from WT and Def6-/- mice (n = 5). (C) ALP staining at day 7 and (D) Alizarin red staining at day 21 (left panel) and its quantification (right panel) of WT and Ifnar1-/- (IFNaßR KO) calvarial osteoblast differentiation in osteogenic medium. (E) qPCR analysis of mRNA relative expression of osteoblast marker genes and ISGs during WT and Ifnar1-/- (IFNaßR KO) calvarial osteoblast differentiation process. (F) Immunoblot analysis of PKR expression in WT and Def6-/- calvarial osteoblasts. p38 was used as a loading control. (G) Immunoblot analysis of PKR expression after knockdown of PKR by PKR-LNA induced gene silencing in ST2 cells. p38 was used as a loading control. (H–J) qPCR analysis of mRNA expression of Ifnb and ISGs (H), ALP staining, scale bar: 500 µm (I) and qPCR analysis of mRNA expression of osteoblastic genes (J) in ST2 cells with or without PKR knockdown. (K) Immunoblot analysis of PKR expression after overexpression of PKR by retroviral transduction for 24 hr. p38 was used as a loading control. (L) qPCR analysis of mRNA expression of Ifnb, ISGs and osteoblast markers. Data are mean ± SD. *p<0.05. **p<0.01. n.s., not statistically significant.Figure 6—source data 1.Source data for Figure 6.