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 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

CD8⁺ T cell cytotoxicity mediates pathology in the skin by inflammasome activation and IL-1β production

<div><p>Deregulated CD8+ T cell cytotoxicity plays a central role in enhancing disease severity in several conditions. However, we have little understanding of the mechanisms by which immunopathology develops as a consequence of cytotoxicity. Using murine models of inflammation induced by the protozoan parasite leishmania, and data obtained from patients with cutaneous leishmaniasis, we uncovered a previously unrecognized role for NLRP3 inflammasome activation and IL-1β release as a detrimental consequence of CD8+ T cell-mediated cytotoxicity, ultimately resulting in chronic inflammation. Critically, pharmacological blockade of NLRP3 or IL-1β significantly ameliorated the CD8+ T cell-driven immunopathology in leishmania-infected mice. Confirming the relevance of these findings to human leishmaniasis, blockade of the NLRP3 inflammasome in skin biopsies from leishmania-infected patients prevented IL-1β release. Thus, these studies link CD8+ T cell cytotoxicity with inflammasome activation and reveal novel avenues of treatment for cutaneous leishmaniasis, as well as other of diseases where CD8+ T cell-mediated cytotoxicity induces pathology.</p></div

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

Treatment of mice with NLRP3 inhibitors dampens the immunopathology caused by CD8 T cells.

<p>WT C57BL/6 mice were infected with <i>L</i>. <i>major</i> in the ear, and 2 weeks later mice were co-infected with 2×10⁵ PFU of LCMV Armstrong strain by i.p. injection. Ten days post LCMV infection mice were treated with MCC950, glyburide or vehicle; (a and b) ear thickness was assessed weekly. Five weeks post infection with <i>L</i>. <i>major</i>, mice were euthanized and the lesions were digested and the (c and d) frequency of neutrophils in the skin was determined directly ex vivo by flow cytometry. (e and f) Number of parasites in the skin was determined at 5 weeks post infection with <i>L</i>. <i>major</i>. Results in mice are data from one experiment with 5 mice per group. <i>*p</i> ≤ <i>0</i>.<i>05</i>, *<i>*p<0</i>.<i>01</i> or ***<i>p</i> ≤ <i>0</i>.<i>001</i></p

Increased IL-1β in <i>L</i>. <i>braziliensis</i> lesions is dependent on CD8 T cell cytotoxicity.

<p>RAG-/- mice were infected with <i>L</i>. <i>braziliensis</i> in the ear, and reconstituted with CD8 T cells or did not receive cells and (a) the course of infection was monitored and representative images of lesions are shown. At 7 weeks post infection mice were euthanized and (b) mRNA levels for <i>IL1a</i> and <i>IL1b</i> were assessed. mRNA data is represented as a fold change (FC) over expression in naïve mice. At 7 weeks post infection, lesions were also digested and used for flow cytometric analysis. Depicted are (c) representative histogram and (d) bar graph of intracellular staining for IL-1β. (e) Ears were cultured for 48 hours and IL-1β release was measured in the supernatants by ELISA. RAG-/- mice were infected with <i>L</i>. <i>braziliensis</i> in the ear, and reconstituted with either WT or perforin-/- CD8 T cells or did not receive cells and (f) course of infection was monitored. At 7 weeks post infection mice were euthanized, lesions were digested and used for flow cytometric analysis. Depicted are (g) representative histogram and (h) bar graph of intracellular staining for IL-1β. Representative data from one of three or more independent experiments (n = 3 to 5 mice per group) with similar results are presented. <i>*p ≤ 0</i>.<i>05</i> or <i>***p ≤ 0</i>.<i>001;</i> ns, non-significant</p

Differential induction of innate immune signaling by genetically related parasite species.

<p>a) Microarray-based expression profiling of human foreskin fibroblasts (HFF) infected with <i>Neospora caninum</i> (NcLiv isolate) or <i>Toxoplasma</i> (GT1, Prugniaud or VEG strains). Heat map shows hierarchical clustering analysis of 822 genes differentially regulated relative to uninfected cells by ≥2-fold (FDR≤5%), in any of these experiments. Each row in heatmap represents average of duplicate (NcLiv and GT1) or triplicate arrays (VEG, PRU, uninf). Color pattern on heatmaps represents column Z-score. b) A cluster of 66 genes (Fig. 1a, asterisk) induced by <i>Neospora</i> but not any strain of <i>Toxoplasma</i>, including several well-known type I interferon response genes (arrows). c) Gene Ontology (GO) enrichment analysis of 66 <i>Neospora</i>-induced genes. Bar graph shows fold enrichment for top five GO Biological Process terms enriched at <i>P</i>≤0.05 and represented by ≥5 genes. Number of genes and GO term name is shown at the right of each bar. d) Fluorescence micrographs of uninfected HFF cells, or cells infected with <i>Neospora</i> or <i>Toxoplasma</i>, then challenged with GFP-tagged vesicular stomatitis virus. Representative images are shown. Experiment was repeated three times with similar results.</p

Immunopathology caused by CD8 T cells is IL-1β-dependent.

<p>RAG-/- mice were infected with <i>L</i>. <i>braziliensis</i> in the ear, and reconstituted with CD8 T cells or did not receive cells and at 2 weeks post infection mice were treated with (a) anti-IL-1R mAb, (b) anti-IL-1β mAb (c) anti-IL-1α mAb or (f) anakinra; ear thickness was assessed weekly. (d) 4 weeks or (g) 6 weeks post infection mice were euthanized and lesions were digested and used for flow cytometric analysis of intracellular IFN-γ and GzmB on CD8 T cells. (e and h) Parasite burden in the lesions. Graphs are data combined from 2 independent experiments (n = 3 to 5 mice per group in each experiment). C57BL/6 mice were infected with <i>L</i>. <i>major</i> in the ear, and 2 weeks later mice were co-infected with 2×10⁵ PFU of LCMV Armstrong strain by i.p. injection. Ten days post LCMV infection mice were treated with anakinra or were left untreated; (i) ear thickness was assessed weekly. Five weeks post infection with <i>L</i>. <i>major</i>, mice were euthanized and the (j) number of parasites in the skin was determined and lesions were digested and used for flow cytometric analysis of intracellular IFN-γ, and GzmB on (k) CD4 T cells or (l) CD8 T cells. Graphs are data from 2 independent experiments (n = 5 mice per group) with similar results are presented. <i>*p ≤ 0</i>.<i>05</i>, <i>**p ≤ 0</i>.<i>01</i> or <i>***p ≤ 0</i>.<i>001;</i> ns, non-significant</p