26 research outputs found
Investigating Inflammasome Activation in Response to Legionella Pneumophila and its Application to Other Bacterial Pathogens
The mucosal surfaces of metazoan organisms provide niches for colonization by commensal microbes. However, these barrier surfaces also encounter pathogens. Therefore, sentinel immune cells must be capable of distinguishing between pathogenic and non-pathogenic organisms to tailor appropriate immune responses. Virulent microorganisms often uniquely possess mechanisms for accessing the host cell cytosol. Therefore, to detect pathogens, innate immune cells encode cytosolic receptors, which recognize conserved, pathogen-associated molecular patterns. Many mammalian cytosolic receptors activate the inflammasome, a multi-protein complex that activates the host enzyme caspase-1. Caspase-1 mediates IL-1 family cytokine release and a pro-inflammatory form of cell death, which are important for host defense. The canonical inflammasome activates caspase-1, but recent studies have shown that a related enzyme, caspase-11, contributes to inflammasome activation. However, it remains unclear if caspase-11 mediates inflammasome responses against bacteria that use virulence-associated secretion systems to deliver bacterial products into the host cytosol. Additionally, humans encode two orthologs of caspase-11, caspase-4 and caspase-5, and it is unclear if either enzyme contributes to inflammasome activation in primary macrophages. Furthermore, the bacterial ligands that trigger inflammasome activation in human cells are poorly defined. Legionella pneumophila, which causes pneumonia, uses a specialized secretion system to access the host cytosol to establish a replicative niche in both murine and human cells. Therefore, we investigated the host and bacterial requirements for inflammasome activation in response to L. pneumophila, and we interrogated if these requirements are conserved for the response against other Gram-negative bacterial pathogens. Our studies demonstrate that caspase-11 contributes to IL-1 release and cell death in response to bacterial pathogens in murine macrophages, and we find that inflammasome activation requires the presence of virulence-associated secretion systems. Using neutralizing antibodies, we show that IL-1α and IL-1β have distinct roles in pulmonary defense against L. pneumophila in vivo. Through siRNA knockdown studies, we demonstrate that human caspase-4 has a conserved role in inflammasome activation in response to multiple Gram-negative bacterial pathogens. Finally, using bacterial mutants, we show that flagellin is a trigger for inflammasome activation in human macrophages. Overall, our studies help define the mechanism by which host cells initiate defense against bacterial pathogens
Caspase-11 Activation in Response to Bacterial Secretion Systems That Access the Host Cytosol
Inflammasome activation is important for antimicrobial defense because it induces cell death and regulates the secretion of IL-1 family cytokines, which play a critical role in inflammatory responses. The inflammasome activates caspase-1 to process and secrete IL-1β. However, the mechanisms governing IL-1α release are less clear. Recently, a non-canonical inflammasome was described that activates caspase-11 and mediates pyroptosis and release of IL-1α and IL-1β. Caspase-11 activation in response to Gram-negative bacteria requires Toll-like receptor 4 (TLR4) and TIR-domain-containing adaptor-inducing interferon-β (TRIF)-dependent interferon production. Whether additional bacterial signals trigger caspase-11 activation is unknown. Many bacterial pathogens use specialized secretion systems to translocate effector proteins into the cytosol of host cells. These secretion systems can also deliver flagellin into the cytosol, which triggers caspase-1 activation and pyroptosis. However, even in the absence of flagellin, these secretion systems induce inflammasome activation and the release of IL-1α and IL-1β, but the inflammasome pathways that mediate this response are unclear. We observe rapid IL-1α and IL-1β release and cell death in response to the type IV or type III secretion systems of Legionella pneumophila and Yersinia pseudotuberculosis. Unlike IL-1β, IL-1α secretion does not require caspase-1. Instead, caspase-11 activation is required for both IL-1α secretion and cell death in response to the activity of these secretion systems. Interestingly, whereas caspase-11 promotes IL-1β release in response to the type IV secretion system through the NLRP3/ASC inflammasome, caspase-11-dependent release of IL-1α is independent of both the NAIP5/NLRC4 and NLRP3/ASC inflammasomes as well as TRIF and type I interferon signaling. Furthermore, we find both overlapping and non-redundant roles for IL-1α and IL-1β in mediating neutrophil recruitment and bacterial clearance in response to pulmonary infection by L. pneumophila. Our findings demonstrate that virulent, but not avirulent, bacteria trigger a rapid caspase-11-dependent innate immune response important for host defense
IL-1R signaling enables bystander cells to overcome bacterial blockade of host protein synthesis
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
Human caspase-4 mediates noncanonical inflammasome activation against gram-negative bacterial pathogens
AP-3 promotes survival upon <i>Salmonella</i> Typhimurium infection.
<p>WT CD57BL/6J and congenic pearl (pe) mice were infected orally with 10<sup>8</sup> 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 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 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 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<sup>+</sup> 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
Neutrophils and Ly6C<sup>hi</sup> monocytes collaborate in generating an optimal cytokine response that protects against pulmonary <i>Legionella pneumophila</i> infection
<div><p>Early responses mounted by both tissue-resident and recruited innate immune cells are essential for host defense against bacterial pathogens. In particular, both neutrophils and Ly6C<sup>hi</sup> monocytes are rapidly recruited to sites of infection. While neutrophils and monocytes produce bactericidal molecules, such as reactive nitrogen and oxygen species, both cell types are also capable of synthesizing overlapping sets of cytokines important for host defense. Whether neutrophils and monocytes perform redundant or non-redundant functions in the generation of anti-microbial cytokine responses remains elusive. Here, we sought to define the contributions of neutrophils and Ly6C<sup>hi</sup> monocytes to cytokine production and host defense during pulmonary infection with <i>Legionella pneumophila</i>, responsible for the severe pneumonia Legionnaires’ disease. We found that both neutrophils and monocytes are critical for host defense against <i>L</i>. <i>pneumophila</i>. Both monocytes and neutrophils contribute to maximal IL-12 and IFNγ responses, and monocytes are also required for TNF production. Moreover, natural killer (NK) cells, NKT cells, and γδ T cells are sources of IFNγ, and monocytes direct IFNγ production by these cell types. Thus, neutrophils and monocytes cooperate in eliciting an optimal cytokine response that promotes effective control of bacterial infection.</p></div