MCC950, a selective NLPR3 inflammasome inhibitor, improves neurologic function and survival after cardiac arrest and resuscitation

Abstract

Background: Cardiac arrest (CA) is associated with high morbidity and mortality, even after spontaneous circulation is re-established. This dire situation is partly due to post-CA syndrome for which no specific and effective intervention is available. One key component of post-CA syndrome is sterile inflammation, which affects various organs including the brain. A major effector of sterile inflammation is activated NLRP3 inflammasome, which leads to increased release of interleukin (IL)-1β. However, how NLRP3 inflammasome impacts neuroinflammation and neurologic outcome after CA is largely undefined.

Methods: Mice were subjected to a potassium-based murine CA and cardiopulmonary resuscitation (CPR) model. MCC950 was used to suppress activation of NLRP3 inflammasome after CA/CPR. Levels of protein and mRNA were examined by Western blotting and quantitative PCR, respectively. Immunologic changes were assessed by measuring cytokine expression and immune cell compositions. CA outcomes, including neurologic deficits, bacterial load in the lung, and survival rate, were evaluated.

Results: Using our CA/CPR model, we found that NLRP3 inflammasome was activated in the post-CA brain, and that pro-inflammatory cytokine levels, including IL-1β, were increased. After treatment with MCC950, a potent and selective NLRP3 inflammasome inhibitor, mice exhibited improved functional recovery and survival rate during the 14-day observational period after CA/CPR. In line with these findings, IL-1β mRNA levels in the post-CA brain were significantly suppressed after MCC950 treatment. Interestingly, we also found that in MCC950- vs. vehicle-treated CA mice, immune homeostasis in the spleen was better preserved and bacterial load in the lung was significantly reduced.

Conclusions: Our data demonstrate that activation of NLRP3 inflammasome could be a key event shaping the post-CA immuno- and neuro-pathology, and identify this pathway as a unique and promising therapeutic target to improve outcomes after CA/CPR.

Keywords: CPR; Immunosuppression; Inflammasome; Ischemia/reperfusion; NLRP3; Neuroinflammation; Sterile inflammation.

Fig. 1

Levels of cytokines and NLRP3 inflammasome components in the brain after CA/CPR. Mice were subjected to sham or CA/CPR surgery. Brain cortical samples were collected on day 1 or day 3 after CA/CPR. a Cytokines in the post-CA brain. The mRNA levels of tumor necrosis factor alpha (TNF-α), interleukin 1beta (IL-1β), and transforming growth factor beta (TGF-β), were measured by qRT-PCR. b, c NLRP3 inflammasome components in the post-CA brain. b The protein levels of NLRP3 and ASC were evaluated by Western blotting. β-actin was used as a loading control. c The mRNA levels of NLRP3 and IL-18 in the brain on day 3 after CA/CPR. The mean values in sham samples were set to 1.0. Data are presented as mean ± SEM (n = 4/group). *p < 0.05; **p < 0.01; ***p < 0.001

Fig. 2

Post-CA activation of NLRP3 inflammasome was suppressed in the brain of MCC950-treated mice. a Mice were subjected to sham or CA/CPR surgery. Mice received intraperitoneal injections of MCC950 (treated) or vehicle (control) daily for 3 consecutive days starting at 15 minutes after resuscitation (day 0). On day 3 post-CA, cortex samples were collected for Western blotting analysis. b–d Quantification of Western blotting. Intensities of each band were measured and normalized to β-actin. The mean values in sham samples were set to 1.0 (except for cleaved caspase-1). Data are presented as mean ± SEM (n = 4/group). *p < 0.05; **p < 0.01; ***p < 0.001

Fig. 3

Functional outcome after CA/CPR was improved in MCC950-treated mice. a Schematic diagram of the experimental design. Mice were subjected to CA/CPR or sham surgery. After 15-min reperfusion, mice received intraperitoneal (ip) injection of MCC950 (treated) or vehicle (control), followed by daily injection for 2 days. Behavioral tests were performed at the indicated time points. Data from sham-operated mice served as baseline references. b–d Functional outcome assessment on day 3 after CA/CPR included b neurologic scores, c rotarod test (two mice from the vehicle group were not subjected to this test due to severe sickness), and d open field test. e Survival rate. Data are presented as median or mean ± SEM (n = 5–10/group). *p < 0.05; **p < 0.01

Fig. 4

CA-induced inflammatory cytokine expression in the brain was attenuated in MCC950-treated mice. Mice were subjected to sham or CA/CPR surgery. After 15-min reperfusion, mice received intraperitoneal injection of MCC950 (treated) or vehicle (control), followed by daily injection for 2 days. On day 3 post-CA, brains were collected for qRT-PCR and flow cytometry analyses. a Expression of cytokines. The mRNA levels (n = 4/group) of IL-1β, TNF-α, TGF-β, and IL-10 were measured by qRT-PCR. b Leukocyte infiltration into the post-CA brain. Single-cell suspensions were prepared from whole brains and subjected to flow cytometric analysis (n = 5/group). Infiltrating leukocytes (CD45^+hi) and CD45^+hiCD11b⁺ cells with their subsets, monocytes/macrophages (CD45^+hiCD11b⁺F4/80⁺), and neutrophils (CD45^+hiCD11b⁺Ly6G⁺) are quantified and the representative dot plots are also shown here. Data are presented as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ns not significant

Fig. 5

Post-CA immune homeostasis and bacterial load in the lung in MCC950-treated mice. Mice were subjected to sham or CA/CPR surgery. After 15 minutes reperfusion, mice received intraperitoneal injection of MCC950 (treated) or vehicle (control), followed by daily injection for 2 days. All analyses were performed on day 3 after CA/CPR. a Immune cell populations in the spleen. The following leukocyte subpopulations are shown here: T cells (CD45⁺CD3⁺), B cells (CD45⁺ CD19⁺), neutrophils (CD45⁺CD11b⁺Ly6G⁺), and natural killer cells (NK; CD45⁺CD3^-NK1.1⁺). b Spleen weight. c Bacterial load in the lung. Representative images illustrate the colonies on blood agar plates. Data are presented as mean ± SEM (n = 5/group). *p < 0.05; **p < 0.01; ***p < 0.001