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 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

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

Caspase-8 regulates a functionally important subset of LPS-induced genes.

<p>RNA was extracted from B6, <i>Ripk3</i>^<i>-/-</i> and <i>Ripk3</i>^<i>-/-</i><i>Casp8</i>^<i>-/-</i> BMDMs following 6 hours of LPS treatment (100 ng/mL) and RNA-seq was performed. (A) Schematic of analysis of the data obtained from RNA-seq indicating figure panel where the results of each type of analysis is shown. (B) Principal Component Analysis (PCA) of filtered, normalized gene set displaying PC1 (95.8% of variance) against PC2 (1.5% of variance). (C) Hierarchical clustering by Pearson correlation of differentially expressed LPS-responsive caspase-8-dependent genes. Columns represent genotype and rows represent individual genes. Colored to indicate expression levels based on Z-scores. (D) GO enrichment performed in DAVID showing Biological Process terms enriched in cluster 2 from (C). Number of genes in each group are denoted above bars. Genes in cluster 1 did not show significant enrichment for any Biological Process terms. (E) Differential expression of select genes from cluster 2 and fold change (LPS-treated <i>Ripk3</i>^<i>-/-</i><i>Casp8</i>^<i>-/-</i> vs LPS-treated <i>Ripk3</i>^<i>-/-</i> BMDMs). Dotted line represents 1.5-fold cutoff. (F) GSEA showing enrichment in cytokine signaling from the KEGG MSigDb canonical pathways collection 2 comparing B6 and <i>Ripk3</i>^<i>-/-</i><i>Casp8</i>^<i>-/-</i> LPS-treated BMDMs. GO, gene ontology; DAVID, database for annotation visualization and integrated discovery; GSEA, gene set enrichment analysis; KEGG, Kyoto Encyclopedia of Genes and Genome; NES, normalized enrichment score; FDR, false discovery rate. <i>R3C8</i> = <i>Ripk3</i>^<i>-/-</i><i>Casp8</i>^<i>-/-</i>. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005910#ppat.1005910.s003" target="_blank">S3</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005910#ppat.1005910.s004" target="_blank">S4</a> Figs.</p

Caspase-8 catalytic activity is required for maximal TLR-induced cytokine production.

<p>(A-F) Indicated BMDMs were pretreated with the pan-caspase inhibitor zVAD-fmk (A-C), QVD-oph (D) or IETD-fmk (F) for 1hr prior to 6 hr stimulation with LPS (100 ng/mL). TNF (F), IL12p40 (A, E, F) and IL-6 (A, E, F) were measured by ELISA, IL-1β was measured by flow cytometry (B, C) and cytotoxicity was measured by lactate dehydrogenase release (LDH) (E). All inhibitors were used at 100 μM. ** <i>p</i> < 0.01, *** <i>p</i> < 0.001, **** <i>p</i> < 0.0001. Student’s unpaired two-tailed <i>t</i>-test. Representative of 4 or more independent experiments. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005910#ppat.1005910.s006" target="_blank">S6 Fig</a>.</p

Model for the role of caspase-8 in inflammatory cytokine gene expression.

<p>TLR stimulation activates signaling complexes that mediate NF-κB and MAPK activation, which is required for induction of inflammatory cytokine gene expression. TLR stimulation also has the potential to induce programmed necrosis via RIPK3, or apoptosis via activation of caspase-8. Cleaved caspase-8 homodimers mediate apoptosis, whereas caspase-8/cFLIP heterodimers normally protect TLR-stimulated cells from programmed necrosis. Our data support a model whereby the caspase-8/cFLIP heterodimer also plays an important role in mediating inflammatory cytokine gene expression.</p

Caspase-8 self-cleavage is necessary for apoptosis but not cytokine responses.

<p>(A) Schematic of strategy used to generate <i>Casp8</i>^<i>DA/DA</i> mice using CRISPR/Cas9. GuideRNA is in blue. (B) <i>Casp8</i>^<i>+/+</i>, <i>Casp8</i>^<i>DA/+</i>, <i>Casp8</i>^<i>DA/DA</i> and <i>Ripk3</i>^<i>-/-</i><i>Casp8</i>^<i>-/-</i> BMDMs were infected with YopJ-deficient (ΔYopJ) and wild type <i>Yersinia</i> and caspase-8 processing was measured by western analysis. (C) BMDMs were pretreated with GSK’ 872 or vehicle control 1 hr prior to infection with <i>Yersinia</i>. Cytotoxicity was measured by LDH release 4 hrs post-infection. (D) Cleaved caspase-3 was measured by flow cytometry in BMDMs 2 hrs post-indicated treatments. (E-G) <i>Casp8</i>^<i>+/+</i>, <i>Casp8</i>^<i>DA/+</i>, <i>Casp8</i>^<i>DA/DA</i> and <i>Ripk3</i>^<i>-/-</i><i>Casp8</i>^<i>-/-</i> BMDMs were treated with PAMPs and cytokine production was measured after 6 hrs by flow cytometry. Representative flow plots of IL-12p40 production in response to LPS (100 ng/mL) (E), quantification of percentage of IL-12p40⁺ in response to LPS (100 ng/mL), Pam3CSK4 (1 μg/mL) or CpG (1 μg/mL) (F), IL-1β⁺ and IL-6⁺ in response to LPS (100 ng/mL) (G) (H) BMDMs were treated with LPS (100 ng/mL), Pam3CSK4 (1 μg/mL) or CpG (1 μg/mL) for 6 hrs and TNF production was measured by ELISA. * <i>p</i> < 0.05, ** <i>p</i> < 0.01, *** <i>p</i> < 0.001, **** <i>p</i> < 0.0001. Student’s unpaired two tailed <i>t</i>-test. Representative of 3–5 independent experiments.</p

Caspase-8 regulates optimal cytokine expression independently of RIPK3 deficiency.

<p><i>B6</i>, <i>Mlkl</i>^<i>-/-</i> and <i>Mlkl</i>^<i>-/-</i><i>Casp8</i>^<i>-/-</i> BMDMs were treated with LPS (100 ng/mL), Pam3CSK4 (1 μg/mL) or CpG (1 μg/mL) for 6hrs. IL-6 (A), IL-12p40 (B) and TNF (C) release was assayed by ELISA. *** <i>p</i> < 0.001 by Student's unpaired two-tailed <i>t</i>-test. Representative of two independent experiments.</p