Francisella tularensis reveals a disparity between human and mouse NLRP3 inflammasome activation

Abstract

Pathogen-triggered activation of the inflammasome complex leading to caspase-1 activation and IL-1β production involves similar sensor proteins between mouse and human. However, the specific sensors used may differ between infectious agents and host species. In mice, Francisella infection leads to seemingly exclusive activation of the Aim2 inflammasome with no apparent role for Nlrp3. Here we examine the IL-1β response of human cells to Francisella infection. Francisella strains exhibit differences in IL-1β production by influencing induction of IL-1β and ASC transcripts. Unexpectedly, our results demonstrate that Francisella activates the NLRP3 inflammasome in human cells. Francisella infection of THP-1 cells elicits IL-1β production, which is reduced by siRNA targeting of NLRP3. Moreover, in reconstituted 293T cells, Francisella triggers assembly of the NLRP3 inflammasome complex. In addition, inhibitors of reactive oxygen species, cathepsin B, and K(+) efflux pathways, known to specifically influence NLRP3, substantially but not completely impair the Francisella-elicited IL-1β response, suggesting the involvement of another inflammasome pathway. Finally, shRNA targeting of NLRP3 and AIM2 reveals that both pathways contribute to the inflammasome response. Together these results establish NLRP3 as a cytosolic sensor for Francisella in human cells, a role not observed in mouse.

FIGURE 1.

Francisella infection induces IL-1β production in THP-1 cells. A, colony counts after infection at the times are indicated. CFU, colony-forming units. B, secretion of TNFα and IL-6 at 24 h in THP-1 is shown. uninf, uninfected. C, IL-1β (left panel) was measured at 24 h post-infection, and a Western blot (right panel) of IL-1β from cell lysate combined with concentrated culture supernatants (34, pro-IL-1β; 17, active IL-1 β) in THP-1 is shown. D, quantitative real-time PCR of the indicated mRNAs at 24 h post-infection in THP-1 is shown. E and F, conditions were as for C and D but with PMA-treated THP-1. Data are shown as the mean ± S.E. for panels B, C, and E (n = 3–4) and the mean ± S.D. for one representative experiment for panels D and F. *, p < 0.05; **, p < 0.005.

FIGURE 2.

Francisella infection induces assembly of the NLRP3-inflammasome complex. A and B, HEK293T cells were transfected with plasmids encoding human procaspase-1, pro-IL-1β, ASC, and the indicated NLR followed by infection with either LVS or U112. At 24 h post-infection, cells were fixed and stained for myc-ASC to monitor the recruitment of ASC to inflammasome specks. A, shown are images (20× magnification) representative for each condition. B, the percentage of ASC-positive cells (red) with formation of a single perinuclear speck (ASC-aggregates) is shown as the mean ± S.E. for two-three independent experiments. More than 500 cells were manually counted for each experimental condition (*, p < 0.05). C, co-localization of human myc-ASC (red) and NLRP3 (green) is shown. One representative image of two-three independent transfections is shown for NLRP3 inflammasome after F. novicida U112 infection.

FIGURE 3.

Human NLRP3 inflammasomes are responsive to Francisella infection. A, an invasion and replication assay of HEK293T cells infected with Francisella LVS or U112 strains is shown. CFU, colony-forming units. B, HEK293T cells were transiently transfected with plasmids encoding human procaspase1, pro-IL-1β, ASC, and NLRP3. At 4 h post-transfection, cells were infected with LVS or U112, and the level of IL-1β secretion was measured at 24 h post-infection. Data shown are the mean ± S.E. of duplicate samples from 3–4 independent experiments (*, p < 0.05; **, p < 0.01).

FIGURE 4.

NLRP3 is involved in the IL-1β response to Francisella infection. A, PMA-treated THP-1 cells were transfected with the indicated siRNA and incubated for 36 h, and NLRP3 mRNA was measured by quantitative PCR (left panel). NLRP3 protein expression was determined by a Western blot using 100 nm NLRP3 siRNA, and band intensities were compared with ImageJ, normalized to GAPDH (% relative expression) (right panel). B, PMA-treated THP-1 cells were transfected with the indicated siRNA (100 nm) followed by infection with F. novicida U112 strain. At 24 h post-infection, IL-1β (left panel) and TNFα levels (right panel) were measured in culture supernatants. C, PMA-treated THP-1 cells were transfected with indicated siRNA followed by infection with either U112 (left panel) or LVS (right panel) strains of Francisella. At 24 h post-infection IL-1β was measured in culture supernatants. Data are shown as the mean ± S.E. of duplicate samples from at least three independent experiments (*, p < 0.05).

FIGURE 5.

ROS, cathepsin B, and K⁺ efflux inhibitors reduce IL-1β production after Francisella infection. A–C, PMA-treated THP-1 cell were infected with U112 after a 30-min preincubation with inhibitors at the indicated concentrations. Secretion of IL-1β and TNFα was measured at 24 h post-infection. Cytokine production is shown as a percentage of the untreated control. A, ROS inhibitors. B, cathepsin B inhibitors. z-FA-FMK, benzyloxycarbonyl-FA-fluoromethyl ketone. C, K⁺ efflux inhibitor. D, PMA-treated THP-1 cell were transfected with endotoxin-free plasmid DNA (1 μg) after a 30-min preincubation with ROS inhibitors at the indicated concentrations. IL-1β secretion was measured at 24 h post-transfection. TNFα was also measured in two experiments with a decrease of ∼60% for ammonium pyrrolidinedithiocarbamate (APDC) and 0–30% for N-acetylcysteine (NAC) relative to untreated DNA-transfected controls (data not shown). For all panels, the mean ± S.E. for three independent experiments is shown.

FIGURE 6.

Silencing of either AIM2 or NLRP3 reduces IL1β secretion. PMA-treated THP-1 cells were transiently transduced with retroviral shRNA constructs targeting the indicated gene or a nonspecific (NS), 29-mer control. After infection with U112 (m.o.i. = 100), production of IL-1β was measured at 24 h post-infection. Results are the means ± S.E. for triplicate samples from three experiments. None of the differences observed between specific shRNAs reached statistical significance (p < 0.05).