NLRP3 Exacerbate NETosis-Associated Neuroinflammation in an LPS-Induced Inflamed Brain

Abstract

Neutrophil extracellular traps (NETs) exert a novel function of trapping pathogens. Released NETs can accumulate in inflamed tissues, be recognized by other immune cells for clearance, and lead to tissue toxicity. Therefore, the deleterious effect of NET is an etiological factor, causing several diseases directly or indirectly. NLR family pyrin domain containing 3 (NLRP3) in neutrophils is pivotal in signaling the innate immune response and is associated with several NET-related diseases. Despite these observations, the role of NLRP3 in NET formation in neuroinflammation remains elusive. Therefore, we aimed to explore NET formation promoted by NLRP3 in an LPS-induced inflamed brain. Wild-type and NLRP3 knockout mice were used to investigate the role of NLRP3 in NET formation. Brain inflammation was systemically induced by administering LPS. In such an environment, the NET formation was evaluated based on the expression of its characteristic indicators. DNA leakage and NET formation were analyzed in both mice through Western blot, flow cytometry, and in vitro live cell imaging as well as two-photon imaging. Our data revealed that NLRP3 promotes DNA leakage and facilitates NET formation accompanied by neutrophil death. Moreover, NLRP3 is not involved in neutrophil infiltration but is predisposed to boost NET formation, which is accompanied by neutrophil death in the LPS-induced inflamed brain. Furthermore, either NLRP3 deficiency or neutrophil depletion diminished pro-inflammatory cytokine, IL-1β, and alleviated blood-brain barrier damage. Overall, the results suggest that NLRP3 exacerbates NETosis in vitro and in the inflamed brain, aggravating neuroinflammation. These findings provide a clue that NLRP3 would be a potential therapeutic target to alleviate neuroinflammation.

Keywords: Brain; Inflammation; Intravital microscopy; NLR family pyrin domain-containing 3 protein; Neutrophil extracellular traps.

Figure 1. NLRP3 promotes DNA leakage from neutrophils.

(A) Scheme of live cell imaging in WT and NLRP3 KO mice neutrophils in the presence of SYTOX and LPS. Given conditions were treated with LPS (1 µg/ml) and SYTOX-orange (red, 5 µM). Live imaging was performed from 1–3 h after LPS stimulation. (B-D) SYTOX staining indicated DNA leakage with bright orange fluorescence. (B) Relative SYTOX signal with time progression (C) quantitative comparison at the endpoint at 3 h after LPS stimulation. (D) Fluorescence SYTOX-orange (red) staining images of WT and NLRP3 KO neutrophils at 2 h after LPS stimulation (scale bars=50 µm). (E, F) The number of SYTOX-positive cells in WT and NLRP3 KO neutrophils after 3 h of LPS stimulation by flow cytometry. (E) Representative LPS-treated neutrophils in WT and NLRP3 KO group (F) quantitative demonstration of membrane-cleaved cells. All experiments were independently repeated at least 3 times. ^**p<0.01, ^***p<0.001.

Figure 2. NLRP3 elevates NET formation and induces neutrophil death.

(A) Representative fluorescent images of NET formation by staining with SYTOX-orange (red), CitH3, and nuclei (blue) in WT and NLRP3 KO neutrophils stimulated with 1 µg/ml of LPS for 3 h (B) quantitative demonstration of NET formation with SYTOX and CitH3 co-localization (scale bars=50 µm). (C-E) Viability test of WT and NLRP3 KO neutrophils after stimulation with 1 µg/ml of LPS for 3 h. (C) Representative and (D) quantitative comparison of annexin V and PI double-positive (dead) neutrophils in WT and NLRP3 KO neutrophils by flow cytometry. (E) Annexin V and PI double-negative live neutrophils under LPS stimulation. All results represent findings from at least 3 independent experiments. ^*p<0.05, ^**p<0.01.

Figure 3. NLRP3 deficiency does not affect the amount of neutrophil infiltration in neuroinflammation.

(A, B) Comparison of the number of infiltrated neutrophils in the brain post 6 h and 24 h of 2.5 mg/kg LPS injection performed intravenously in WT mice. Infiltrated Neutrophils in the brain were sorted using CD11b and Ly6G staining (B) quantitative demonstration by flow cytometry. (C) Flow cytometry analysis of the number of infiltrated neutrophils in the brains of WT and NLRP3 KO at 24 h following LPS injection (D) quantitative comparison of infiltrated neutrophil number. All data are representative of experiments repeated more than thrice. ^**p<0.01.

Figure 4. NLRP3 accelerates NET formation in the inflamed brain.

(A) Scheme of experimental design in vivo. (B) Representative cropped western blots of CitH3 and β-actin with proteins extracted from the whole brain tissues. (C) CitH3 production, as analyzed from Western blots done in triplicate (B) was normalized over β-actin in WT and NLRP3 KO brain. (D) Representative Immunohistochemistry images of brain sections showing NET formation in LPS-injected WT and NLRP3 KO mice. CitH3 (red) and MPO were used as NET indicators, and DAPI (blue) for nuclei staining (scale bar=100 µm). (E-H) Extracellular expression of CitH3 and MPO in Ly6G⁺ neutrophils derived from the brain and blood of WT and NLRP3 KO mice after LPS injection. (E) Representative flow cytometry analysis of Ly6⁺CitH3⁺MPO⁺ co-expression in brain neutrophils; NET formation (F) quantitative evaluation of WT and NLRP3 KO. (G) Representative (H) quantitative comparison of NET formation from blood neutrophils; circulating NETs in WT and NLRP3 KO after LPS injection. (I) Representative and (J) quantitative comparison of annexin V and PI double-positive (dead) neutrophils in WT and NLRP3 KO brain infiltrated neutrophils via flow cytometry. All results shown are from 3 independent experiments. ^*p<0.05, ^**p<0.01, ^****p<0.0001.

Figure 5. Two-photon deep tissue observation of diminished NET formation in NLRP3-deficient mice brains.

(A) Representative images of NET formation obtained from two-photon imaging of WT and NLRP3 KO mice. Mice were injected with 2.5 mg/kg LPS 24 h prior to imaging. NET components CitH3, stained with Alexa-594 anti CitH3 Ab (red) and Alexa 488-conjugated NE, blood vessels were labeled with WGA (blue). Quantitative analysis of NETs in the brain of mice systemically injected with LPS; (B) area of CitH3 and (C) of NE staining (scale bar=50 µm). Data represent findings from experiments repeated thrice. ^*p<0.05.

Figure 6. NLRP3-deficiency attenuates neuroinflammation in inflamed mice brains.

(A) Representative images of IHC obtained from WT and NLRP3 KO mice brains by staining with ZO-1 and Claudin-5. Mice were injected with 2.5 mg/kg LPS 24 h before harvest and perfused with PBS. BBB tight junction-associated proteins, ZO-1 and Claudin-5, were labeled in 10 µm sectioned brain tissues. Quantitative analysis of ZO-1 and Claudin-5 expression in the brain; (B) area of Claudin-5 and (C) of ZO-1 staining (scale bar=50 µm). Inflammatory cytokine level was evaluated with whole brain lysates after 24 h of 2.5 mg/kg LPS injection in WT and NLRP3 KO groups. (D) TNF-α and (E) IL-1β concentration were detected with ELISA. Data represent findings from experiments repeated at least 3 times. ^*p<0.05, ^**p<0.01.

Figure 7. NET inhibition via neutrophil depletion alleviates neuroinflammation in the inflamed brain.

Mice were treated with 4 mg/kg of anti-Ly6G twice; Anti-Ly6G was administered 24 h prior to LPS injection. As a second injection, anti-Ly6G was administered simultaneously with 2.5 mg/kg of LPS. Mice brains were harvested 24 h after LPS injection. (A) Representative IHC images of staining with ZO-1 and Claudin-5 were obtained from anti-Ly6G-treated WT mice. Quantitative analysis of ZO-1 and Claudin-5 expression in the brains of mice treated with only LPS and LPS with anti-Ly6G; (B) area of Claudin-5 and (C) of ZO-1 staining (scale bar=50 µm). (D) IL-1β and (E) TNF-α expression levels were detected with ELISA. Data represent findings from experiments repeated thrice. ^*p<0.05, ^***p<0.001.