NLRP3 inflammasome: key mediator of neuroinflammation in murine Japanese encephalitis

Abstract

Background: Japanese Encephalitis virus (JEV) is a common cause of acute and epidemic viral encephalitis. JEV infection is associated with microglial activation resulting in the production of pro-inflammatory cytokines including Interleukin-1 β (IL-1β) and Interleukin-18 (IL-18). The Pattern Recognition Receptors (PRRs) and the underlying mechanism by which microglia identify the viral particle leading to the production of these cytokines is unknown.

Methodology/principal findings: For our studies, we have used murine model of JEV infection as well as BV-2 mouse microglia cell line. In this study, we have identified a signalling pathway which leads to the activation of caspase-1 as the key enzyme responsible for the maturation of both IL-1β and IL-18 in NACHT, LRR and PYD domains-containing protein-3 (NLRP3) dependent manner. Depletion of NLRP3 results in the reduction of caspase-1 activity and subsequent production of these cytokines.

Conclusion/significance: Our results identify a mechanism mediated by Reactive Oxygen Species (ROS) production and potassium efflux as the two danger signals that link JEV infection to caspase-1 activation resulting in subsequent IL-1β and IL-18 maturation.

Figure 1. JEV induces the production of IL-1β and IL-18 in vivo.

Infection was carried out in BALB/c mice with 5×10⁵ PFU of JEV Intravenously. Brain samples were isolated from mock-infected control (C) as well JEV infected mice after 3 d, 5 d and 7 d post infection. (A–C) Cytokine levels were estimated using CBA. The graphs represent the levels of pro-inflammatory cytokines, TNF-α, CCL2 and IL-6 in pg/ml from protein homogenates isolated from infected and mock-infected mice brain (D–E) qRT-PCR analysis was carried out from total RNA isolated from mock-infected and JEV infected mice brains on all the time points and levels of IL-1β as well as IL-18 mRNA were measured. Graphs represent fold change in mRNA values with respect to mock-infected control normalized to 18 s rRNA internal control. (F–G) ELISA study was carried out to measure the levels of mature IL-1β and IL-18 cytokine from JEV infected as well as uninfected brain samples. Graphs represent the cytokine levels in pg/ml in mock-infected control as well as infected brain samples. Data represent mean ± SEM of 5 animals in each group. Statistical differences were evaluated using one way ANOVA with Bonferroni's post hoc test. *, **, Statistical differences in comparison to mock-infected control values (* p<0.05; ** p<0.01).

Figure 2. JEV induces the production of IL-1β and IL-18 in vitro.

BV-2 microglial cells were infected with 5 MOI of JEV for different time points. LPS+ATP condition was used as a positive control for qRT-PCR, ELISA and caspase-1 activity studies. (A) Cytokine analysis from mock-infected control and JEV infected BV-2 cells was carried out after 6 h of JEV infection using cytokine bead array. Graphs represent the levels of pro-inflammatory cytokines, TNF- α, CCL2 and IL-6 after JEV infection with respect to mock-infected control condition. (B–C) qRT-PCR studies on total RNA isolated from uninfected cells as well as on BV-2 cells infected with JEV for 3 h. IL-1β and IL-18 mRNA levels are represented in terms of fold change with respect to mock-infected control normalized to 18 s rRNA internal control. (D–E) Levels of mature IL-1β and IL-18 cytokines upon JEV infection with respect to mock-infected control condition were measured by ELISA. Graphs represent the fold change values in JEV infected cells with respect to mock-infected control condition. Data represent mean ± SEM from 3 independent experiments performed in duplicate. Statistical differences were evaluated using one way ANOVA with Bonferroni's post hoc test. *, **, Statistical differences in comparison to mock-infected control values (* p<0.05; ** p<0.01).

Figure 3. JEV induces caspase-1 activation in vivo and in vitro.

Infection was carried out in BALB/c mice with 5×10⁵ PFU of JEV Intravenously. Brain samples were isolated from mock-infected control (C) as well JEV infected mice after 3 d, 5 d and 7 d post infection for estimation of caspase-1 activity. (A) Caspase-1 activity is represented as fold change in caspase-1 activity in JEV infected brain with respect to mock-infected brain. Data represent mean ± SEM of 5 animals in each group. (B) Caspase-1 activity was measured in BV-2 cells infected with 5 MOI of JEV after 3 h, 6 h and 12 h of JEV infection over that of mock-infected condition. Caspase-1 activity is represented as fold change with respect to mock-infected condition. Data represent mean ± SEM from 3 independent experiments performed in duplicate. Statistical differences were evaluated using one way ANOVA with Bonferroni's post hoc test. *, **, Statistical differences in comparison to mock-infected control values (* p<0.05; ** p<0.01).

Figure 4. Caspase-1 activity is required for the production of IL-1β and IL-18 during JEV infection.

BV-2 cells were incubated with 5 µM YVAD for 30 min to inhibit caspase-1 activity followed by JEV infection. (A–B) ELISA for IL-1β and IL-18 was carried out in JEV infected BV-2 cells upon caspase-1 inhibition. The cytokine levels were then measured using ELISA and the values are represented in pg/ml. Data represent mean ± SEM from 3 independent experiments performed in duplicate. Statistical differences were evaluated using one way ANOVA with Bonferroni's post hoc test. **, Statistical difference in comparison to mock-infected control values (** p<0.01) and #, Statistical difference with respect to JEV infected condition (p<0.01).

Figure 5. NLRP3 is critical for caspase-1 activity as well as IL-1β and IL-18 production.

Transient knockdown of NLRP3 using 100 nM of NLRP3 SiRNA was carried out in BV-2 microglial cells. Scrambed RNA (ScRNA) was used as a transfection control. This was followed by virus infection with 5 MOI dose for 6 h. (A) NLRP3 mRNA levels were measured by qRT-PCR from total RNA isolated from JEV infected as well as uninfected BV-2 cells. The graph represent a fold change in NLRP3 mRNA with respect to mock-infected control normalised to 18 s rRNA internal control. (B) Caspase-1 activity was also measured in NLRP3 knockdown condition upon JEV infection. Graph represents fold change in caspase-1 activity in different conditions with respect to mock-infected control. (C–D) In order to measure IL-1β and IL-18 production, ELISA was then carried out in NLRP3 knockdown (SiRNA+JEV) condition with respect to JEV infected sample. Graph represents cytokine levels in pg/ml. Data represent mean ± SEM from 3 independent experiments performed in duplicate. Statistical differences were evaluated using one way ANOVA with Bonferroni's post hoc test. *, **, Statistical difference in comparison to mock-infected control values (* p<0.05, ** p<0.01) and #, Statistical difference with respect to JEV infected condition (p<0.01).

Figure 6. Generation of ROS is critical for caspase-1 activity and subsequent IL-1β and IL-18 maturation.

BV-2 cells were incubated with 1 µM of DPI for inhibition of ROS generation. (A) Representative FACS plot showing intracellular ROS production, after 4 h of JEV infection and representation of the Mean Fluorescent Intensities (M.F.I) (right graph) in mock-infected control (C), JEV+DPI and JEV alone condition. (B) Caspase-1 activity measured after 6 h of JEV infection in presence or absence of DPI. Graph represents fold change in caspase-1 activity with respect to mock-infected control. (C–D) ELISA study showing the levels of mature IL-1β and IL-18 in JEV+DPI condition with respect to JEV alone infected sample. Graph represents cytokine levels in pg/ml. For Potassium efflux study, BV-2 cells were incubated with 50 mM KCl for 20 min in order to study the requirement of K⁺ efflux for caspase-1 activity and its downstream effects. Data represent mean ± SEM from 3 independent experiments performed in duplicate. Statistical differences were evaluated using one way ANOVA with Bonferroni's post hoc test. *, **, Statistical difference in comparison to mock-infected control values (*p<0.05, ** p<0.01) and #, Statistical difference with respect to JEV infected condition (p<0.01).

Figure 7. Potassium efflux is required for caspase-1 activity and subsequent inflammation upon JEV infection.

(A) Caspase-1 activity was measured in presence of KCl upon JEV infection. Graph represents caspase-1 activity in JEV infected cells incubated with KCl with respect to untreated JEV alone condition. (B–C) ELISA study showing the levels of IL-1β and IL-18 in KCl treated condition upon JEV infection over that of JEV alone condition. Graph represents IL-1β and IL-18 levels in pg/ml. Data represent mean ± SEM from 3 independent experiments performed in duplicate. Statistical differences were evaluated using one way ANOVA with Bonferroni's post hoc test. *, **, Statistical difference in comparison to mock-infected control values (*p<0.05, ** p<0.01) and #, Statistical difference with respect to JEV infected condition (p<0.01).

Figure 8. Schematic showing the signalling pathway leading to IL-1β and IL-18 production upon JEV infection in microglia.

NLRP3 is the key PRR involved in the identification of JEV intracellularly in a microglia. The NLRP3 interacts with an adaptor molecule, ASC and recruits pro-caspase-1 forming a biochemical complex termed as inflammasome. In response to JEV, NLRP3 cleaves pro-caspase-1 into its active form, caspase-1. Caspase-1 then cleaves the inactive pro-forms of IL-1β and IL-18 to their mature forms which are then secreted out. ROS and K⁺ efflux are the host derived danger signals that are critical for the formation of NLRP3 biochemical complex during JEV infection.