Tyrosine dephosphorylation of ASC modulates the activation of the NLRP3 and AIM2 inflammasomes

The inflammasome is an intracellular multi-protein complex that orchestrates the release of the pro-inflammatory cytokines IL-1β and IL-18, and a form of cell death known as pyroptosis. Tyrosine phosphorylation of the inflammasome sensors NLRP3, AIM2, NLRC4, and the adaptor protein, apoptosis-associated speck-like protein (ASC) has previously been demonstrated to be essential in the regulation of the inflammasome. By using the pharmacological protein tyrosine phosphatase (PTPase) inhibitor, phenylarsine oxide (PAO), we have demonstrated that tyrosine dephosphorylation is an essential step for the activation of the NLRP3 and AIM2 inflammasomes in human and murine macrophages. We have also shown that PTPase activity is required for ASC nucleation leading to caspase-1 activation, IL-1β, and IL-18 processing and release, and cell death. Furthermore, by site-directed mutagenesis of ASC tyrosine residues, we have identified the phosphorylation of tyrosine Y60 and Y137 of ASC as critical for inflammasome assembly and function. Therefore, we report that ASC tyrosine dephosphorylation and phosphorylation are crucial events for inflammasome activation

Inflammasome Priming Is Similar for <i>Francisella</i> Species That Differentially Induce Inflammasome Activation

<div><p>Inflammasome activation is a two-step process where step one, priming, prepares the inflammasome for its subsequent activation, by step two. Classically step one can be induced by LPS priming followed by step two, high dose ATP. Furthermore, when IL-18 processing is used as the inflammasome readout, priming occurs before new protein synthesis. In this context, how intracellular pathogens such as <i>Francisella</i> activate the inflammasome is incompletely understood, particularly regarding the relative importance of priming versus activation steps. To better understand these events we compared <i>Francisella</i> strains that differ in virulence and ability to induce inflammasome activation for their relative effects on step one vs. step two. When using the rapid priming model, i.e., 30 min priming by live or heat killed <i>Francisella</i> strains (step 1), followed by ATP (step 2), we found no difference in IL-18 release, p20 caspase-1 release and ASC oligomerization between <i>Francisella</i> strains (<i>F</i>. <i>novicida</i>, <i>F</i>. <i>holarctica</i> –LVS and <i>F</i>. <i>tularensis</i> Schu S4). This priming is fast, independent of bacteria viability, internalization and phagosome escape, but requires TLR2-mediated ERK phosphorylation. In contrast to their efficient priming capacity, <i>Francisella</i> strains LVS and Schu S4 were impaired in inflammasome triggering compared to <i>F</i>. <i>novicida</i>. Thus, observed differences in inflammasome activation by <i>F</i>. <i>novicida</i>, LVS and Schu S4 depend not on differences in priming but rather on their propensity to trigger the primed inflammasome.</p></div

Inflammasome priming by <i>Francisella</i> is independent of bacteria internalization.

<p>Human monocytes were pretreated for 30 min with latrunculin A (Latr) (200 nM) and cytochalasin D (Cyto D) (5 μg/ml) and infected with <i>F</i>. <i>novicida</i> for 16 h. CFU of internalized bacteria (<b>A</b>) and IL-18 release (<b>B</b>) were counted. Monocytes treated with latrunculin and cytochalasin D as in A, B and then primed with <i>Francisella</i> for 30 min followed by ATP (5mM) for 30 min were analyzed for IL-18 in cell culture media by ELISA (<b>C</b>). Data represent mean ± SEM, n = 3 independent experiments. * p<0.05. White bars—overnight model, black bars—rapid priming model. ND—not statistically different.</p

<i>Francisella</i> priming inflammasome is TLR2 dependent.

<p>Human monocytes were left untreated (NT) or primed for 30 min with TLR2, 4, 5, 6, 9 ligands: Pam3CysSK4 (P3C) (100 ng/ml), Malp2 (M2) (100 ng/ml), PolyI:C (PIC) (10 μg/ml), <i>E</i>. <i>coli</i> endotoxin (LPS) (1 μg/ml), flagellin (Fla) (100 ng/ml) or CpG (10 μg/ml), and then stimulated with ATP (5 mM) for additional 30 min and IL-18 release was measured by ELISA (<b>A</b>). IL-18 release from human monocytes primed for 30 min with 1 μg/ml of LPS from <i>E</i>. <i>coli</i>, <i>F</i>. <i>novicida</i> (Fn) and <i>F</i>. <i>holarctica</i>-LVS (LVS) and stimulated with ATP (5 mM) for 30 min (<b>B</b>). IL-18 release by monocytes, pretreated with TLR2/1 inhibitor CU-CPT (5 and 10 μM) or TLR4 inhibitor RS-LPS (1 μg/ml) for 30 min and then primed with <i>F</i>. <i>novicida</i> (Fn) for 30 min and activated with ATP (5 mM) for 30 min (<b>C</b>). IL-18 release by monocytes preloaded for 30 min with (5 and 10 μM) CU-CPT and incubated with <i>F</i>. <i>novicida</i> (Fn) for 16 h (<b>D</b>). Data represent mean ± SEM, n = 3 independent experiments. * p<0.05. White bars—overnight model, black bars—rapid priming model.</p

Monocyte priming by <i>Francisella</i> is ERK dependent.

<p>Human monocytes were infected for 16 h with live or killed <i>F</i>. <i>novicida</i> (Fn) and IL-18 release was measured by ELISA (<b>A</b>). Human monocytes were primed with live or killed <i>F</i>. <i>novicida</i> for 30 min followed by ATP stimulation for 30 min and release of IL-18 was measured by ELISA (<b>B</b>). IL-18 release from monocytes pretreated with UO126 (20 μM), AG126 (10 μM) or DPI (50 μM) for 30 min and then primed with <i>F</i>. <i>novicida</i> (Fn) for 30 min followed by ATP stimulation for 30 min (<b>C</b>). Monocytes, primed with live (L) or heat killed (HK) <i>F</i>. <i>novicida</i> (Fn) and <i>F</i>. <i>tularensis</i> SchuS4 (S4) for 30 min were lysed and cell lysate was analyzed for ERK phosphorylation. LPS from <i>E</i>. <i>coli</i> was used as a positive control (<b>D</b>). Data represent mean ± SEM, n = 3 independent experiments. * p<0.05. ND—not statistically different. Blots are representative of repeated experiments. White bars—overnight model, black bars—rapid priming model.</p

Chimeric HCMV/HSV-1 and Δγ134.5 oncolytic herpes simplex virus elicit immune mediated antigliomal effect and antitumor memory

Malignant gliomas are the most common primary brain tumor and are characterized by rapid and highly invasive growth. Because of their poor prognosis, new therapeutic strategies are needed. Oncolytic virotherapy (OV) is a promising strategy for treating cancer that incorporates both direct viral replication mediated and immune mediated mechanisms to kill tumor cells. C134 is a next generation Δγ134.5 oHSV-1 with improved intratumoral viral replication. It remains safe in the CNS environment by inducing early IFN signaling which restricts its replication in non-malignant cells. We sought to identify how C134 performed in an immunocompetent tumor model that restricts its replication advantage over first generation viruses. To achieve this we identified tumors that have intact IFN signaling responses that restrict C134 and first generation virus replication similarly. Our results show that both viruses elicit a T cell mediated anti-tumor effect and improved animal survival but that subtle difference exist between the viruses effect on median survival despite equivalent in vivo viral replication. To further investigate this we examined the anti-tumor activity in immunodeficient mice and in syngeneic models with re-challenge. These studies show that the T cell response is integral to C134 replication independent anti-tumor response and that OV therapy elicits a durable and circulating anti-tumor memory. The studies also show that repeated intratumoral administration can extend both OV anti-tumor effects and induce durable anti-tumor memory that is superior to tumor antigen exposure alone