Autophagy proteins are not universally required for phagosome maturation

<p>Phagocytosis plays a central role in immunity and tissue homeostasis. After internalization of cargo into single-membrane phagosomes, these compartments undergo a maturation sequences that terminates in lysosome fusion and cargo degradation. Components of the autophagy pathway have recently been linked to phagosome maturation in a process called LC3-associated phagocytosis (LAP). In this process, autophagy machinery is thought to conjugate LC3 directly onto the phagosomal membrane to promote lysosome fusion. However, a recent study has suggested that ATG proteins may in fact impair phagosome maturation to promote antigen presentation. Here, we examined the impact of ATG proteins on phagosome maturation in murine cells using FCGR2A/FcγR-dependent phagocytosis as a model. We show that phagosome maturation is not affected in <i>Atg5</i>-deficient mouse embryonic fibroblasts, or in <i>Atg5</i>- or <i>Atg7</i>-deficient bone marrow-derived macrophages using standard assays of phagosome maturation. We propose that ATG proteins may be required for phagosome maturation under some conditions, but are not universally required for this process.</p

P2X₇R-KO mice generated less inflammatory cytokines and neutrophil infiltration in the early stage of Ad infection.

<p>The wild-type (C57/B6) and the P2X₇R-KO mice were infected with Ad (1×10¹¹ vp/mice) by i.n. administration. (A) The mice were sacrificed on day 4 (n = 4) and the lungs were obtained for histological analysis. Representative microscopic sections are shown. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035812#pone.0035812.s003" target="_blank">Figure S3</a> for sections from day 2 and 6. (B) The lung pathological scores were used to compare difference between the wild-type and the P2X₇R-KO mice according to the criteria described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035812#s4" target="_blank">Materials and Methods</a>. Data are expressed as mean ± SD. (C and D) Twenty four hours after Ad administration BALF was collected from the wild-type (n = 10) and the P2X₇R-KO mice (n = 8). IL-1β and IL-6 in the BALF were analyzed by ELISA (C). Differential cell counts were performed with the cell fraction of BALF (D). Data are expressed as mean ± SD.</p

Ad infection of epithelial cell and macrophage co-culture induced secretion of IL-1β and IL-18.

<p>(A and B) MLE and J774 mono-culture as well as MLE-J774 co-culture were infected with Ad. IL-1β (A) and IL-18 (B) secretion was measured by ELISA assay using the media 24 h after infection. Data are expressed as mean ± SD. (C) MLE-J774 co-culture was infected with 0, 2, 20, or 100 MOI of Ad and IL-1β in the medium was measured 24 h after infection. Data are expressed as mean ± SD. (D) MLE-J774 co-culture was infected with Ad and medium was collected at designated time for Western blot analysis following immunoprecipitation. (E) Co-cultures of different ratio of MLE and J774 were established and IL-1β was measured in the medium 24 h after Ad infection. Data are expressed as mean ± SD. (F) A human lung epithelial cell line, A549, instead of MLE was used to establish the co-culture with J774 macrophages and infected with Ad. IL-1β was measured 24 h after infection. Data are expressed as mean ± SD.</p

Treatment with oATP inhibited inflammatory responses in Ad infected MLE-Raw co-culture.

<p>The MLE-Raw co-culture was infected with Ad for 24 h in the presence of oATP at the designated concentrations. (A), The amount of NO produced in the culture was measured from the medium using Griess reagent. Data are expressed as mean ± SD. (B), The MLE-Raw co-culture was infected with Ad and ROS positive cells from were counted by flow cytometry after treating with 3-(p-aminophenyl) fluorescein (APF) without gating for specific cell types. Shown is a representative FACS histogram of three independent experiments with the percentage of ROS positive population. (C and D) Mouse IL-6 and KC, respectively, were measured from the medium using commercially available ELISA kits. Data are expressed as mean ± standard deviation (SD).</p

IPMK plays a role in SopB-mediated Akt activation.

<p>(A) HeLa cells were treated with control or IPMK siRNA for 48 h. Cells were infected with wild type <i>S</i><i>. Typhimurium</i> for 30 min. As a control, cells were uninfected. Akt activation was determined by immunoblotting the cell lysates with a phospho-specific anti-Ser473 Akt antibody. Pan-Akt antibodies were used to ensure equal protein loading. (B) Wild type (ES10) or IPMK knockout (ES2 and ES5) embryonic stem cells were infected with wild type or ΔSopB mutant <i>S</i><i>. Typhimurium</i> for 30 min. As a control, cells were uninfected. Where indicated, cells were treated with 100 µM LY294002 for 30 min. prior to infection. Akt activation was determined by immunoblotting the cell lysates with a phospho-specific anti-Ser473 Akt antibody. Pan-Akt antibodies were used to ensure equal protein loading. (C) Western blot results of ES10 and ES5 from B were analyzed by estimating the intensities of protein bands with the ImageJ software. Shown on the graph are the relative and normalized expression levels of phospho-Akt ± SD for three separate experiments, calculated as outlined in the Materials and Methods. The p-values from one-way ANOVA analysis are shown.</p

Intranasal Ad infection in mice caused ARDS and fatality but inhibition of P2X₇R and caspase-1 enhanced survival.

<p>Ad was intranasally administered to mice with matching age and gender. The mice were monitored and body weight was measured each day. The survival curves were generated based on the humane end point of 20% weight loss and the comparison was made by the log rank test. (A) The wild-type (C57/B6) mice were infected with Ad at two dosages, 5×10¹⁰ or 1×10¹¹ vp/mouse. The mice that received 5×10¹⁰ vp (n = 5) showed no apparent symptoms and sustained only slight weight loss but recovered within a few days. The mice that received 1×10¹¹ vp (n = 5) showed ARDS like symptoms and continuously lost their body weight to reach the experimental humane end point. In the subsequent <i>in vivo</i> experiments Ad dosage of 1×10¹¹ vp/mouse was used. (B and C) The wild-type (n = 12) and the P2X₇R-KO mice (n = 13) were intranasally infected with Ad. The individual body weight on day 3, when all the subject mice were still alive, was presented as the relative retained body weight. The bold line represents the average body weight. (D and E) Similar to B and C, but the wild-type (n = 6) and the caspase-1-KO mice (n = 6) were compared. (F) The wild-type mice (n = 15/group) were treated with A438079 (300 µmol/kg), z-YVAD-fmk (10 mg/kg), or apyrase (4 U/mice) on day 0 and day 1of Ad administration and their survival curve was generated.</p

Co-cultures with primary macrophages from P2X₇R and caspase-1 knockout mice secreted less IL-1β.

<p>(A) The co-culture was established with MLE and peritoneal macrophages obtained from wild-type (C56BL/6) mice and infected with Ad with or without oATP (200 µM) or A438079 (100 µM). IL-1β was measured from the medium 24 h after infection. Data are expressed as mean ± SD. (B–D), Peritoneal macrophages were collected from and P2X₇R (B), caspase-1 (C), or NLRP3 (D) knockout mice and the co-cultures were established with MLE. IL-1β was measured from the medium 24 h (18 h and 24 h for NLRP3-KO) after Ad infection and compared to the wild-type counterpart. Data are expressed as mean ± SD.</p

Inhibition of P2X₇R and caspase-1 reduced IL-1β secretion in the Ad infected co-culture.

<p>(A and B) MLE-J774 co-culture was infected with Ad in the presence of oATP (0∼400 µM) or z-YVAD-fmk (0∼50 µM) and IL-1β secretion was measured in the medium. Data are expressed as mean ± SD. (C) The co-culture of MLE and a P2X₇R deficient J774 cell line (ATPR) was establish and IL-1β in the medium was measured 24 h after Ad infection. Data are expressed as mean ± SD. (D) MLE-J774 co-culture was treated with a P2X₇R specific inhibitor, A438079 (0∼200 µM) at the designated concentration before Ad infection and IL-1β was measured from the medium 24 h after infection. Data are expressed as mean ± SD.</p

SopB-mediated Akt activation in HeLa cells is only partially sensitive to PI3-Kinase inhibitor LY294002.

<p>(A) HeLa cells were treated with different concentrations of LY294002 (1 µM to 100 µM) for 30 min. Cells were then infected with wild type <i>S</i><i>. Typhimurium</i> for 30 min or incubated with 100 ng/mL EGF for 5 min. As controls, cells were either uninfected or infected with Δ<i>sopB</i> mutant <i>S</i><i>. Typhimurium</i> for 30 min. Akt activation was determined by immunoblotting the cell lysates with a phospho-specific anti-Ser473 Akt antibody. Pan-Akt antibodies were used to ensure equal protein loading. (B) Western blot results from A were analyzed by estimating the intensities of protein bands with the ImageJ software. Shown on the graph are the relative and normalized expression levels of phospho-Akt ± SD induced by wild type <i>S</i><i>. Typhimurium</i> or EGF for three separate experiments, calculated as outlined in the Materials and Methods. Asterisks indicate that the percent value is significantly different from the control (P < 0.05).</p

SopB-mediated Akt activation at <i>Salmonella</i> invasion ruffles is PI(3,4) P₂/PI(3–5) P₃-dependent.

<p>(A) HeLa cells were infected with wild type or Δ<i>sopB</i> mutant <i>S</i><i>. Typhimurium</i> and fixed at 30 min p.i. Cells were examined by epifluorescence microscopy after co-staining for activated Akt with a phospho-specific (Ser473) antibody, actin with fluorescently-labeled phalloidin and bacteria with DAPI. Insets are enlarged from boxed areas. (B) HeLa cells were transiently transfected with GFP-PH-Akt for 16 h prior to <i>Salmonella</i> infection. Cells were infected and fixed as in A. Cells were co-stained for actin with fluorescently-labeled phalloidin and bacteria with a polyclonal antibody to <i>S</i><i>. Typhimurium</i>. Insets are enlarged from boxed areas. (C) HeLa cells were transiently transfected with GFP or PTEN-A4-YFP for 16 h prior to <i>Salmonella</i> infection. Cells were infected with wild type <i>S</i><i>. Typhimurium</i> and fixed as in A. Cells were co-stained for actin with fluorescently-labeled phalloidin, Akt with a phospho-specific (Ser473) antibody and bacteria with a polyclonal antibody to <i>S</i><i>. Typhimurium</i>. Insets are enlarged from boxed areas. Size bars, 10 µm. (D) The percentage of phospho-Akt⁺<i>Salmonella</i> invasion ruffles from C was quantified (n ≥ 50). Averages ± SD for three separate experiments are shown. Asterisk indicates that the percent value is significantly different from the control (P < 0.001) as determined by one-way ANOVA analysis. (E) HeLa cells were transiently transfected with GFP-Akt and PTEN-A4-YFP or GFP for 16 h prior to infection. Cells were uninfected, infected as in A, or incubated with 100 ng/mL EGF for 5 min. Where indicated, cells were treated with 100 µM LY294002 for 30 min prior to infection. Akt activation was determined by immunoblotting the cell lysates with a phospho-specific anti-Ser473 Akt antibody. GFP antibodies were used to ensure equal Akt transfection.</p