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Comparative Study
. 2015 May 18;10(5):e0127278.
doi: 10.1371/journal.pone.0127278. eCollection 2015.

Inflammasome priming is similar for francisella species that differentially induce inflammasome activation

Affiliations
Comparative Study

Inflammasome priming is similar for francisella species that differentially induce inflammasome activation

Mohammed G Ghonime et al. PLoS One. .

Abstract

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 Francisella activate the inflammasome is incompletely understood, particularly regarding the relative importance of priming versus activation steps. To better understand these events we compared Francisella 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 Francisella strains (step 1), followed by ATP (step 2), we found no difference in IL-18 release, p20 caspase-1 release and ASC oligomerization between Francisella strains (F. novicida, F. holarctica -LVS and F. tularensis 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, Francisella strains LVS and Schu S4 were impaired in inflammasome triggering compared to F. novicida. Thus, observed differences in inflammasome activation by F. novicida, LVS and Schu S4 depend not on differences in priming but rather on their propensity to trigger the primed inflammasome.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Difference in inflammasome activation by Francisella between rapid and overnight models.
Overnight model; white bars. Primary human monocytes were left untreated (NT) or infected with 100 MOI of different strains of Francisella for 16 h and IL1B mRNA (A), mature IL-1β (B) and IL-18 (C) release in response to F. novicida (Fn), F. tularensis-LVS (LVS), SchuS4 (S4) and F. novicida FPI mutants mglA and iglC was measured. Priming model; black bars. When monocytes were infected with Francisella for only 30 min, a second signal (5 mM ATP for 30 min) was required to induce IL-18 processing and release. NT—not infected by Francisella (D, E). Monocytes stimulated with F. novicida in the priming model or overnight model were tested for IL-18 release by immunoblot and for ASC in cell lysates and oligomeric ASC in the insoluble cell pellet by ASC immunoblot (F). Data represent mean ± SEM, n ≥ 3 independent experiments. * p<0.05. ND—not statistically different. Blots are representative of repeated experiments.
Fig 2
Fig 2. Francisella priming inflammasome is TLR2 dependent.
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), E. coli 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 (A). IL-18 release from human monocytes primed for 30 min with 1 μg/ml of LPS from E. coli, F. novicida (Fn) and F. holarctica-LVS (LVS) and stimulated with ATP (5 mM) for 30 min (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 F. novicida (Fn) for 30 min and activated with ATP (5 mM) for 30 min (C). IL-18 release by monocytes preloaded for 30 min with (5 and 10 μM) CU-CPT and incubated with F. novicida (Fn) for 16 h (D). Data represent mean ± SEM, n = 3 independent experiments. * p<0.05. White bars—overnight model, black bars—rapid priming model.
Fig 3
Fig 3. Inflammasome priming by Francisella is independent of bacteria internalization.
Human monocytes were pretreated for 30 min with latrunculin A (Latr) (200 nM) and cytochalasin D (Cyto D) (5 μg/ml) and infected with F. novicida for 16 h. CFU of internalized bacteria (A) and IL-18 release (B) were counted. Monocytes treated with latrunculin and cytochalasin D as in A, B and then primed with Francisella for 30 min followed by ATP (5mM) for 30 min were analyzed for IL-18 in cell culture media by ELISA (C). Data represent mean ± SEM, n = 3 independent experiments. * p<0.05. White bars—overnight model, black bars—rapid priming model. ND—not statistically different.
Fig 4
Fig 4. Francisella priming is dependent on K+ efflux.
Human monocytes were pretreated with K+ (100mM) or glybenclamide (50, 100 and 200 μM) for 30 min and then infected with F. novicida. IL-18 release was measured either 16 h post-infection (A, B) or 30 min followed by 30 min of ATP stimulation (C,D). Cell culture media, extract and pellet were immunoblotted for IL-18, caspase-1 and ASC (E). Data represent mean ± SEM, n = 3 independent experiments. * p<0.05. White bars—overnight model, black bars—rapid priming model.
Fig 5
Fig 5. Monocyte priming by Francisella is ERK dependent.
Human monocytes were infected for 16 h with live or killed F. novicida (Fn) and IL-18 release was measured by ELISA (A). Human monocytes were primed with live or killed F. novicida for 30 min followed by ATP stimulation for 30 min and release of IL-18 was measured by ELISA (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 F. novicida (Fn) for 30 min followed by ATP stimulation for 30 min (C). Monocytes, primed with live (L) or heat killed (HK) F. novicida (Fn) and F. tularensis SchuS4 (S4) for 30 min were lysed and cell lysate was analyzed for ERK phosphorylation. LPS from E. coli was used as a positive control (D). 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.

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