Fig 2: Autophagy downregulates activation of the inflammasome by P. aeruginosa. (A) BMDMs from wild-type mice (Vav1-atg7+/+) or mice with a conditional deletion of Atg7 in bone marrow-derived cells (Vav1-atg7-/-) were infected with PA103?U?T and autophagy quantified. Panels show immunoblots for indicated proteins in cells infected as shown. Graphs show secretion of active IL1B and TNF from basal and infected BMDMs as shown. Columns are means of 3 independent determinations; error bars are SEM. * indicates a significant difference between the levels in BMDMs from WT or Vav1-atg7-/- mice, P < 0.05. n.s., not significant. (B) As (A), but in cells depleted of Atg5 by siRNA knockdown as shown. (C) As (A) but in cells depleted of Lc3b by siRNA knockdown as shown and infected as indicated. ** indicates a significant difference between the levels in BMDMs from Lc3b siRNA-treated cells and control, P < 0.01. ((D)to F) Immunoblots of key proteins (including the ATG12&z.vrecto;ATG5 conjugate) of (A) to (C) depleted by knockout or knockdown as shown, with TUBB5/ß-tubulin as control. C is control. D, BMDMs from WT or Vav1-atg7 knockout animals as shown probed for ATG7. (E) and (F), BMDMs treated with siRNA or control siRNA to Atg5 (E) and Lc3b (F).

Fig 3: (See previous page.) Mitochondrial DNA release following infection and requirement for mitochondria for inflammasome activation by P. aeruginosa. (A) and (B), qPCR analysis of cytosolic mitochondrial DNA (mtDNA) relative to nuclear DNA in macrophages pretreated (A) with Mito-TEMPO (500 µM) or 3-MA (10 mM) or control or Lc3b siRNA (B) and infected with PA103?U?T (MOI 25) for 4 h or uninfected (Basal) as shown. Columns show means of 3 independent determinations; error bars are SEM. (C) Mitochondrial content of J774A.1 cells exposed to ethidium bromide (EtBr) at the indicated concentration (ng/ml) measured by qPCR (normalized to untreated cells; upper panel) and immunoblot for the mitochondrial protein ATPIF1 (lower panel) at low and high exposure time; TUBB5 is shown as a loading control. (D) Mitochondrial content of control or ethidium bromide-treated J774A.1 cells (?J774A.1) assayed by flow cytometry of MitoTracker Green stained cells. (E) Flow cytometry of J774A.1 and ?°J774A.1 cells left uninfected (Basal) or infected with PA103?U?T (MOI 25) for 4 h and stained with MitoSOX Red. (F) J774A.1 cells grown in the absence or presence of 500 ng/ml ethidium bromide (EtBr) were left untreated (basal) or infected with PA103?U?T (MOI 25) for 4 h and analyzed as described in Figure 1A. *** indicates significant differences between the levels in the presence and absence of the EtBr (500 ng/ml), P < 0.001. All data are representative of results from 2 or 3 independent experiments.

Fig 4: Manipulating in vitro and in vivo levels of autophagy modulates inflammasome activation following P. aeruginosa infection. (A) The panels show representative protein gel blots of LC3B isoforms and TUBB5 as a loading control following infection and rapamycin (50 µg/ml) (Rap) or resveratrol (50 µg/ml) (Res) treatment as indicated. Graphs are means (with SEM as error bars) of IL1B secretion in the same experiments. *** indicates significant difference between the levels in the presence and absence of rapamycin or resveratrol during infection, P < 0.001. (B) Levels of IL1B (upper panel) and cytosolic mitochondrial DNA (lower panel) in cells transfected with expression plasmid for murine Atg5 (pUNO1-mAtg5) or control plasmid (pUC19) infected as shown. (C) Flow cytometry of BMDMs transfected with plasmids as indicted and infected as shown and stained for mitochondrial reactive oxygen with mitososx. (D to G), results from intraperitoneal infection of female C57BL/6 mice with PA103?U?T treated with rapamycin (R), or 3-methyl adenine as indicated. (D) Levels of LC3B-I and -II in recovered peritoneal cells following infections and treatments as shown; lower panel shows mean (± SEM) numbers of LC3B containing puncta per cell as described in Fig. S1B. (E) Levels (n = 3) of IL1B and TNF (error bars are SEM) in the blood (left panels) and peritoneal washings (right panels) before and 6 h after infection with the indicated treatments. * and ** and *** indicate significant differences from the levels in infected animals with no pretreatment, P < 0.05, 0.01 and 0.001 respectively. (F) Protein concentration in peritoneal fluid following infection and treatments shown. (G) Bacterial colony counts per ml of recovered fluid from the peritoneal cavity with the treatments as shown. Columns are means of triplicate determinations, error bars SEM. (D to G) Representative data from 2 independent in vivo experiments. Other experiments were repeated 2 or 3 times.

Fig 5: Infected BMDMs undergo mitophagy. (A) Representative confocal images of BMDMs infected with PA103?U?T (MOI 25 for 4 h) and stained for LC3B (green), and mitochondrial ATPIF1 (Mito, red); nuclei are stained blue with DAPI. Coloc shows areas of colocalization of the mitochondrial and LC3B staining in gray. Overlay shows the merged LC3B and mitochondrial signals; colocalizing areas shown as yellow. Scale bar = 5 µm. Repeated 3 times. ((B)to E) Electron micrographs of autophagosomes of untreated BMDM (B) or infected with PA103?U?T(MOI 25) for 4 h (C) to (E). Arrowhead indicates autophagosome with enclosing double membrane, M indicate mitochondria. Scale bar is 500 nm. Representative image from > 10 infected cells. (F) Immunoblot of BMDMs infected at a MOI of 25 with PA103?U?T for the indicated times probed for PINK1. Full-length and cleaved PINK1 are labeled. The blot was reprobed for TUBB5 as a loading control. (G) As (F) but with infections and treatments as shown and is representative of 2 independent experiments.