Fig 2: GBPs Mediate the Release of DN to Activate Cytosolic DNA-Sensing Pathways(A) pJB908 plasmid extracted from cytosolic fractions of macrophages challenged with L. pneumophila (pJB908) WT or ⊿sdhA strains at 6 hpi.(B) pJB908 plasmid extracted from cytosolic fractions of macrophages challengedwith WT L. pneumophila (pJB908) at3hpiinthe presence or absence of IFNβ 20 hr pre-treatment.(C) Ifnb transcriptional induction from WT and L. pneumophila ⊿sdhA-infected macrophages of the indicated strains measured by qRT-PCR at 4 hpi.(D) Cell death of B6, Casp11−/− and Casp1−/−Casp11−/− macrophages when challenged with L. pneumophila ⊿sdhA.(E) Representative images of ⊿sdhA L. pneumophila and ASC immunofluorescence by antibody staining at 7 hpi. Images were taken with 20× lens; scale bar, 10 μm.(F) Left: quantification of ASC-positive staining gated within regions of interest (ROIs), defined as regions of positive anti-L. pneumophila signal. Right: quantification of ASC-positive cellsfrom total numberofcells enumerated via Hoechst staining. Each symbol is a technical replicate. One representative experiment out of 3 is shown.(G)Whole-cell lysate and precipitated supernatant from L. pneumophila-infected cells were probed by western blot for a p17 cleavage product of IL-1β Representative western blot on the left; quantification from n = 3 on the right.

Fig 3: 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-1β 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 β 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 4: TRIF determines inflammatory output of Fas through RIP1K activation(A) Levels of CXCL1 produced by macrophages of indicated genotype in response to FasL over time. Data points indicate individual mice as source of isolated macrophages.(B) IκBα and total and phosphorylated p65 over time in whole-cell lysates from B6, Cd14−/−, and Trif−/− macrophages after FasL stimulation.(C, E, and F) Relative IFNB and ISG15 mRNA levels normalized to ACTB in response to FasL over time in indicated neutrophils and macrophages.(D) Total and phosphorylated forms of RIPK1 over time in whole-cell lysates from B6, Cd14−/−, and Trif−/− macrophages after FasL stimulation.(G) Spleen levels of IFN-β 4 h post-mFasL injection of 0.3 μg/mL; each point represents an individual mouse.qPCR and ELISA data are presented as the means ± SD for duplicate wells from three or more independent experiments. Data from western blotting are representative of three or more independent experiments. ANOVA was used for comparison between groups: ns (p > 0.05), *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.