Inflammasomes as microbial sensors

Members of the Nod-like receptor family and the adaptor ASC assemble into multiprotein platforms, termed inflammasomes, to mediate the activation of caspase-1 and subsequent secretion of IL-1Β and IL-18. Recent studies have identified microbial and endogenous molecules as well as possible mechanisms involved in inflammasome activation.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/69174/1/611_ftp.pd

<i>Shigella</i> Type III Secretion Protein MxiI Is Recognized by Naip2 to Induce Nlrc4 Inflammasome Activation Independently of Pkcδ

<div><p>Recognition of intracellular pathogenic bacteria by members of the nucleotide-binding domain and leucine-rich repeat containing (NLR) family triggers immune responses against bacterial infection. A major response induced by several Gram-negative bacteria is the activation of caspase-1 via the Nlrc4 inflammasome. Upon activation, caspase-1 regulates the processing of proIL-1β and proIL-18 leading to the release of mature IL-1β and IL-18, and induction of pyroptosis. The activation of the Nlrc4 inflammasome requires the presence of an intact type III or IV secretion system that mediates the translocation of small amounts of flagellin or PrgJ-like rod proteins into the host cytosol to induce Nlrc4 activation. Using the <i>Salmonella</i> system, it was shown that Naip2 and Naip5 link flagellin and the rod protein PrgJ, respectively, to Nlrc4. Furthermore, phosphorylation of Nlrc4 at Ser533 by Pkcδ was found to be critical for the activation of the Nlrc4 inflammasome. Here, we show that Naip2 recognizes the <i>Shigella</i> T3SS inner rod protein MxiI and induces Nlrc4 inflammasome activation. The expression of MxiI in primary macrophages was sufficient to induce pyroptosis and IL-1β release, which were prevented in macrophages deficient in Nlrc4. In the presence of MxiI or <i>Shigella</i> infection, MxiI associated with Naip2, and Naip2 interacted with Nlrc4. siRNA-mediated knockdown of Naip2, but not Naip5, inhibited <i>Shigella</i>-induced caspase-1 activation, IL-1β maturation and Asc pyroptosome formation. Notably, the Pkcδ kinase was dispensable for caspase-1 activation and secretion of IL-1β induced by <i>Shigella</i> or <i>Salmonella</i> infection. These results indicate that activation of caspase-1 by <i>Shigella</i> is triggered by the rod protein MxiI that interacts with Naip2 to induce activation of the Nlrc4 inflammasome independently of the Pkcδ kinase.</p></div

<i>Shigella</i> induces Naip2-dependent Asc pyroptosome formation in macrophages.

<p>WT (<b>A–D</b>), <i>Asc</i>^−/− (<b>A–D</b>), Naip2-deficient (siRNA) or Naip5-deficient (siRNA) (<b>C</b>) BMDM were infected with <i>Shigella</i> WT or S325 mutant for up to 90 min (<b>A</b>–<b>D</b>). Cells were fixed and analyzed by confocal microscopy (<b>A</b>–<b>D</b>) and the percentage of cells containing Asc pyrotopsomes was evaluated (<b>D</b>). Caspase-1 activation was detected using FLICA reagent (<b>A</b>) (green), Asc localization with anti-Asc antibody (red in <b>A</b>, green in <b>B</b> and <b>C</b>) and nuclei with DAPI (<b>A</b>) (blue). Arrows denotes Asc pyroptosomes. * p<0.01. Results represent mean ± SD and are representative of three independent experiments.</p

Expression of <i>Shigella</i> rod protein MxiI induces activation of the Nlrc4 inflammasome in macrophages.

<p>WT or <i>Nlrc4</i>^−/− BMDMs were nucleofected with MSCV-IRES-GFP (GFP) or MSCV-IRES-GFP encoding <i>Shigella</i> MxiI (MxiI-GFP). After 20 hrs, the percentage of GFP-positive viable cells in the total cell population was analyzed by fluorescence microscopy (<b>A</b>) and the production of IL-1β in cell free supernatants by ELISA (<b>B</b>). * p<0.0001. (<b>A</b> and <b>B</b>) Results represent mean ± SD and are representative of three independent experiments.</p

Pkcδ is not required for inflammasome activation caused by <i>Shigella</i> infection.

<p>(<b>A</b>) BMDMs from WT and <i>Prkcd</i>^−/− mice were stimulated with LPS and the expression of Pkcδ was evaluated by immunoblotting. (<b>B–D</b>) BMDMs from WT and <i>Prkcd</i>^−/− mice were infected with <i>Shigella</i> WT or S325 <i>Shigella</i> mutant (<b>B–E</b>) or <i>Salmonella</i> (<b>C and E</b>) at a bacteria/macrophage ratio of 10∶1 for various time points (<b>B</b>) or at the indicated bacteria/macrophage ratio (<b>C</b>) for 1 hr, or with indicated bacteria/macrophage ratios for 2 hrs (<b>D</b>) or 30 min (<b>E</b>). The production of cytokines in cell free supernatant was analyzed by ELISA (<b>B–D</b>) and the activation of caspase-1 was evaluated by detecting cleaved caspase-1 (p20) by immunoblotting (<b>E</b>). *p<0.02. Results represent mean ± SD. Results are representative of at least three independent experiments.</p

<i>Shigella</i> MxiI interacts with Naip2 and promotes the interaction of Naip2 with Nlrc4.

<p>(A)T7-tagged MxiI was co-expressed with HA-tagged Naip2 or Naip5 or control empty vector in caspase-1-deficient BMDMs. Cell lysates were immunoprecipitated with anti-T7 antibody and the interaction between MxiI and Naip2/5 was analyzed by immunoblotting with anti-HA antibody. (B) T7-tagged Nlrc4 or empty vector was co-expressed with HA-tagged Naip2, Naip5, or empty vector in caspase-1-deficient BMDMs. After 16 hrs cells were infected with <i>Shigella</i> WT or S325 mutant for 2 hr at a bacteria/macrophage ratio of 10∶1. Cell lysates were immunoprecipitated with anti-T7 beads and the interaction between Nlrc4 and Naip2/5 was analyzed by immunoblotting with anti-HA antibody. (C) T7-tagged MxiI or empty vector was co-expressed with HA-tagged Naip2. Cell lysates were immunoprecipitated with anti-HA beads and the interaction of Naip2 with MxiI and endogenous Nlrc4 was analyzed by immunoblotting with anti-T7 or anti-Nlrc4 antibody. (A–C) Results are representative of three independent experiments.</p