Increased autophagic sequestration in adaptor protein-3 deficient dendritic cells limits inflammasome activity and impairs antibacterial immunity

<div><p>Bacterial pathogens that compromise phagosomal membranes stimulate inflammasome assembly in the cytosol, but the molecular mechanisms by which membrane dynamics regulate inflammasome activity are poorly characterized. We show that in murine dendritic cells (DCs), the endosomal adaptor protein AP-3 –which optimizes toll-like receptor signaling from phagosomes–sustains inflammasome activation by particulate stimuli. AP-3 independently regulates inflammasome positioning and autophagy induction, together resulting in delayed inflammasome inactivation by autophagy in response to <i>Salmonella</i> Typhimurium (STm) and other particulate stimuli specifically in DCs. AP-3-deficient DCs, but not macrophages, hyposecrete IL-1β and IL-18 in response to particulate stimuli <i>in vitro</i>, but caspase-1 and IL-1β levels are restored by silencing autophagy. Concomitantly, AP-3-deficient mice exhibit higher mortality and produce less IL-1β, IL-18, and IL-17 than controls upon oral STm infection. Our data identify a novel link between phagocytosis, inflammasome activity and autophagy in DCs, potentially explaining impaired antibacterial immunity in AP-3-deficient patients.</p></div

AP-3 is required for optimal transcriptional activation of pro-IL-1β and some NLRs after particulate LPS priming.

<p>BMDCs from WT or pearl (pe) mice were untreated or stimulated with LPS or LPS beads for 2 h (<b>A</b>-<b>C</b>) or 3 h (<b>D</b>, <b>E</b>). <b>A-C</b>. cDNA generated from isolated RNA was analyzed by RT-PCR. Shown are mRNA levels of: <b>A</b>, NLRC1, NLRC2, NLRP3; <b>B</b>, pro-IL-1β, pro-IL-18; and <b>C</b>, NLRC4, pro-caspase-1 and ASC. Data from three independent experiments were normalized to the average of five housekeeping genes, and the ΔΔCt values were calculated and represented as mean ± SD fold induction of mRNA in stimulated cells relative to unstimulated cells. <b>D</b>, <b>E.</b> Cell pellets were lysed and fractionated by SDS-PAGE, and NLRP3, NLRC4, pro-caspase-1 (pro-casp. 1), pro-IL-1β and ASC were detected by immunoblotting. <b>D</b>, representative blots. <b>E</b>, quantification of band intensities represented as mean ± SD fold induction in stimulated cells relative to unstimulated cells for pro-IL-1β (<i>top</i>) and NLRP3 (<i>bottom</i>), normalized to tubulin levels. Two or more fold induction was considered significant. *p< 0.05; **p<0.01. See also <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006785#ppat.1006785.s008" target="_blank">S1 Table</a></b>.</p

AP-3 limits inflammasome sequestration and autophagy induction after STm infection or alum stimulation.

<p><b>(A</b>, <b>B)</b>. WT and pearl (pe) BMDCs expressing ASC-GFP were infected with flagellin-expressing STm (stimulates NLRC4) for 30 or 60 min. Cells were then permeabilized for 1 min with 50 μg/ml digitonin or throughout labeling with 0.1% saponin as indicated, washed, and incubated with mouse anti-GFP and allophycocyanin (APC)-conjugated anti-mouse antibodies. Cells were analyzed by flow cytometry, gating on GFP⁺ cells (R1). <b>A</b>. Shown are representative dot plots of transduced WT and pe DCs indicating gated region based on GFP fluorescence and side scatter (SSC, <i>left panels</i>), and representative histogram plots indicating GFP (<i>middle panels</i>) or APC (anti-GFP) fluorescence (<i>right panels</i>). <i>Black lines</i>, WT; <i>blue lines</i>, pe; <i>dotted lines</i>, secondary antibody alone. <b>B</b>. The ratio of mean fluorescence intensity (MFI) values for anti-GFP signal in digitonin-treated DCs relative to saponin-treated DCs is shown from 4 independent experiments. (<b>C-L)</b>. WT and pearl (pe) BMDCs that were non-transduced (-) or transduced with lentiviruses encoding non-target (ctrl) or either of two ATG7-specific shRNAs or ATG-5- or LC3b-specific shRNAs were infected with STm (<b>C-E</b>) or primed for 3 h with soluble LPS and stimulated with alum (<b>F-L</b>). (<b>C-E</b>) Cell supernatants collected 2 h after Stm infection were assayed for IL-1β by ELISA. <b>C.</b> Representative experiment. <b>D</b>. Data from 3 independent experiments were normalized to IL-1β values from cells treated with non-target shRNA and presented as fold induction. <b>E</b>. IL-1β values for pearl BMDC treated with non-target or ATG7 shRNAs from 3 independent experiments are shown as percent of values for WT DCs treated with the same shRNAs. (<b>F-L</b>) Cell pellets collected 4 h after alum stimulation were lysed, fractionated by SDS-PAGE and immunoblotted for caspase-1 and tubulin. (<b>F, H</b>). Representative immunoblots, showing pro-caspase-1 (pro-casp.-1) and mature p10 (casp.-1 p10) bands. (<b>G, I</b>) Quantification of band intensities for caspase-1 p10 normalized to pro-caspase-1 and tubulin from three independent experiments are shown as caspase-1 fold change relative to unstimulated (-) WT cells (mean ± SD). (<b>J, K</b>) Data from three independent experiments were normalized to caspase-1 values from untreated cells and presented as fold increase. (<b>L</b>). Caspase-1 values for pearl BMDC treated with non-target, ATG5, ATG7 or LC3b shRNAs from 3 independent experiments are shown as percent of values for WT DCs treated with the same shRNAs. Data in all panels represent mean ± SD. **p<0.01; ***p<0.001. See also <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006785#ppat.1006785.s007" target="_blank">S7 Fig</a></b>.</p

AP-3 promotes survival upon <i>Salmonella</i> Typhimurium infection.

<p>WT CD57BL/6J and congenic pearl (pe) mice were infected orally with 10⁸ STm (+ STm) or received PBS as a control (naïve). (<b>A, B).</b> Mouse survival was assessed over 12 days (<b>A</b>; n = 5) or 7 days (<b>B</b>; n = 11 each mouse type; surviving mice were euthanized on day 7). (<b>C-E).</b> Peyer patches, MLN and spleens were harvested 5 days post-infection, homogenized and plated to measure bacterial load. CFUs were normalized to tissue weight (expressed as CFU/ g of tissue). Data are pooled from three independent experiments. Dotted lines, background (threshold value from uninfected mice); solid lines, geometric means of values above background. *p<0.05; **p<0.01; ***p<0.001. See also <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006785#ppat.1006785.s001" target="_blank">S1</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006785#ppat.1006785.s002" target="_blank">S2</a> Figs</b>.</p

AP-3 is required for perinuclear inflammasome positioning and limits autophagy induction after <i>Salmonella</i> Typhimurium infection in DCs.

<p><b>(A-C).</b> WT and pearl (pe) BMDCs expressing ASC-GFP were infected with flagellin-expressing mCherry-STm (stimulates NLRC4). Cells were fixed 1 h after infection, labeled with DAPI and analyzed by fluorescence microscopy. <b>A.</b> Representative images showing ASC speck (green) relative to STm (red) and nucleus (blue) in four infected WT and pearl DCs each. <b>B.</b> Quantification of perinuclear (within a radius of one μm from the nucleus) and non-perinuclear ASC specks in 20 cells per cell type in each of four independent experiments. <b>C.</b> Quantification of ASC speck distance to the nucleus in 15 cells per cell type in each of three independent experiments. <b>(D</b>, <b>E)</b>. WT and pearl BMDCs were infected with STm, and endogenous LC3 (and actin as a control) was detected by immunoblotting fractionated cell lysates at the indicated time points. <b>D.</b> Representative blot with positions of molecular weight markers (MW) indicated at left. <b>E</b>. Quantification of LC3-II band intensities from three independent experiments, expressed as fold increase relative to unstimulated cells and normalized to LC3-I and β-actin levels. (<b>F</b>-<b>I).</b> WT and pearl BMDCs expressing ASC-GFP alone or with mCherry-LC3 were infected with STm and analyzed by live fluorescence imaging (for LC3) or fixed immunofluorescence microscopy (for p62) 1 h later. <b>F.</b> Representative images showing ASC speck (green) and either LC3 puncta (red, <i>left panels</i>) or endogenous p62 puncta (red) and nuclei (blue; <i>right panels</i>) in infected cells. Corresponding DIC images show nuclear position. <b>G, H.</b> Quantification of LC3 (<b>G</b>) or p62 (<b>H</b>) puncta within a radius of 0.5 μm from the ASC speck (representative image shown at right) in 15 cells per cell type in each of 3 independent experiments. <b>I</b>, Quantification of total p62 puncta normalized to cell area. Data represent mean ± SD. Scale bar: 10 μm.**p<0.01; ***p<0.001. See also <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006785#ppat.1006785.s005" target="_blank">S5</a></b>and <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006785#ppat.1006785.s006" target="_blank">S6</a> Figs</b>.</p

Mutagenesis of TLR9 Tyr870 results in impaired receptor maturation.

<p>(A, B) <i>Tlr9</i>^<i>-/-</i> BMDCs expressing HA-tagged TLR9 WT, Y870F, or Y870A were untreated (-) or treated for 18 h with 1 mg/ml CpG. (A) Cells were lysed and analyzed by Western blotting for HA expression relative to Erk as a loading control. A representative blot from 3 experiments is shown at the left. Right, the 160 kDa and 80 kDa bands were quantified, and the amount of active receptor was calculated by normalizing the amount of processed receptor (80 kDa) to the amount of unprocessed receptor (160 kDa). Shown is the average from 3 experiments. (B) CpG-induced TNFα and IL-6 levels from WT or mutant TLR9-expressing BMDC cultures were normalized to the amounts of processed (active) receptor as determined in Fig 4A. N.D. indicates not determined, as there is no mature Y870A receptor to which cytokine can be normalized. Note, CpG-induced cytokine levels in Y870A-expressing cells were essentially no different from unstimulated samples, as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0200913#pone.0200913.g002" target="_blank">Fig 2C</a>. * indicates p-value < 0.05 by Bonferroni post-test. (C) WT bone marrow was uninfected (left) or transduced with either WT or Y870A TLR9, and then differentiated to BMDCs. Cells were stimulated for 0, 60 or 180 min with CpG as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0200913#pone.0200913.g003" target="_blank">Fig 3B</a>, and cleavage of overexpressed TLR9 to the 80kD form in these cells was assessed by immunoblotting for HA (relative to total Erk as a loading control) as in (A). Shown is a representative of 3 experiments.</p

TLR9 tyrosine 870 is conserved across TLRs and their adaptors.

<p>Mouse TLRs, except TLR1, TLR6, and TLR12, have a tyrosine in the box 1 region of the TIR domain. N-terminal and C-terminal amino acid positions are noted to the left and to the right respectively. Note the aspartic acid residues in Mal that are underlined were found to be potentially involved in the dimerization surface in the crystal structure [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0200913#pone.0200913.ref015" target="_blank">15</a>].</p

Y870A TLR9 is not retained in the ER or Golgi.

<p><i>Tlr9</i>^<i>-/-</i> bone marrow was transduced with doxycycline inducible WT or Y870A HA-tagged TLR9, and differentiated towards DCs. Three days later, protein expression was induced by doxycycline (0.5μg/ml) treatment, and BMDCs were fixed and permeabilized at 4 hours post-Doxycycline treatment. The 4 h post-doxycycline fixed cells were labeled for HA and either the late endosome/lysosome marker LAMP1 (A), the ER marker Calreticulin (B), or the Golgi marker GM130 (C). In all cases, nuclei were labeled by DAPI. Each panel shows an image of both WT and Y870A-expressing BMDCs for each individual label, a merged image (Merged), and a colocalization image in which areas of overlap of the two markers, after thresholding by the method of Costes [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0200913#pone.0200913.ref024" target="_blank">24</a>], is indicated by the white areas (“co-loc. pixels”). HA colocalization with each organelle marker is expressed both as a Pearson’s correlation coefficient and by the percent of manually thresholded HA-labeled structures that overlap with the marker. *, p<0.01; **, p<0.005; ***, p<0.0001.</p

Expression of TLR9 Y870A inhibits ligand induced cytokine secretion by endogenous TLR9 in a dominant-negative manner.

<p>(A) 293T cells were co-transfected with HA- and GFP-tagged forms of TLR9 as WT (WT) or Y870A (A) variants as indicated. HA-tagged TLR9 was immunoprecipitated using an antibody to HA, and interaction with GFP-tagged TLR9 was assessed by immunobloting for both HA and GFP in both the lysates (input) and the immunoprecipitates (IP: HA). Immunoblotting for Erk served as a control. Results are representative of at least 3 independent experiments. (B) WT (i.e., TLR9 sufficient) C57BL/6 bone marrow was transduced with retrovirus expressing HA-tagged WT or Y870A TLR9 or empty vector as a control, and then differentiated to DCs. BMDCs were stimulated for 3 hours with CpG, and then supernatants were harvested and TNFα and IL-6 were measured by ELISA. Bar graphs are representative of at least 3 independent experiments each performed in triplicates. * indicates p-value < 0.05 by Bonferroni post-test. (C) These same transduced BMDCs were stimulated with CpG for various times, and lysates were subjected to Western blot analysis for the presence of HA-tagged TLR9, phosho-Erk or total Erk. Blots are representative of 3 experiments. (D) Quantification from a representative experiment of phospho-Erk normalized to total Erk.</p

Y870A TLR is consumed by autophagy.

<p><i>Tlr9</i>^<i>-/-</i> bone marrow was transduced with doxycycline inducible WT or Y870A HA-tagged TLR9, and differentiated towards DCs. Three days later, protein expression was induced by doxycycline (0.5μg/ml) treatment, and BMDCs were fixed and permeabilized at 4- and 24- hours post-doxycycline treatment. The 4 h post-doxycycline fixed cells were labeled for HA and the autophagosome markers SQSTM1 / p62 (A) or LC3B (B), and then by species-specific Alexa568- and Alexa488-conjugated secondary antibodies. The 24 h post-doxycycline fixed cells were labeled for HA and LAMP1 (C). In all cases, nuclei were labeled by DAPI. Each panel shows an image of both WT and Y870A-expressing BMDCs for each individual label, a merged image (Merged), and a colocalization image in which areas of overlap of the two markers after thresholding by the method of Costes is indicated by the white areas (“co-loc. pixels”). HA colocalization with each organelle marker is expressed both as a Pearson’s correlation coefficient and by the percent of manually thresholded HA-labeled structures that overlap with the marker. *, p<0.01; **, p<0.005; ***, p<0.0001.</p