Temporal and Spatial Dynamics of Inflammasome Activation After Ischemic Stroke

Background: The inflammasome represents a highly pro-inflammatory mechanism. It has been identified that inflammasome was activated after ischemic stroke. However, the impact of inflammasomes on stroke outcomes remains contradictory. The participating molecules and the functioning arena of post-stroke inflammasome activation are still elusive. Methods: In the present study, blood samples from stroke patients were collected and analyzed with flow cytometry to evaluate the correlation of inflammasome activation and stroke outcomes. A stroke model was established using male C57/Bl6 mice with transient middle cerebral artery occlusion (tMCAO, 1 h). The dynamics of inflammasome components, cell type, and location of inflammasome activation and the therapeutic effects of inhibiting post-stroke inflammasome executors were evaluated. Results: We found that a high level of inflammasome activation might indicate detrimental stroke outcomes in patients and mice models. Post-stroke inflammasome activation, especially NLRP3, cleaved Caspase-1, cleaved Caspase-11, IL-1β, IL-18, and GSDMD, peaked at 3–5 days and declined at 7 days with the participation of multiple components in mice. Macrophage that infiltrated into the ischemic lesion was the main arena for post-stroke inflammasome activation among myeloid cells according to the data of mice. Among all the members of the Caspase family, Caspase-1 and −11 served as the main executing enzymes. Inhibiting Caspase-1/−11 signaling efficiently suppressed DAMPs-induced macrophage inflammasome activation and displayed neuroprotection to stroke models including infarct size (Control: 48.05 ± 14.98; Cas1.i: 19.34 ± 12.21; Cas11.i: 21.43 ± 14.67, P < 0.001) and neurological deficit score (0 d-Control: 2.20 ± 0.63; 0 d-Cas1.i: 2.20 ± 0.63; 0 d-Cas11.i: 2.20 ± 0.63; 1 d-Control: 2.50 ± 0.53; 1 d-Cas1.i: 1.50 ± 0.71; 1 d-Cas11.i: 2.00 ± 0.67; 2 d-Control: 2.30 ± 0.48; 2 d-Cas1.i: 1.30 ± 0.48; 2 d-Cas11.i: 1.50 ± 0.53; 3 d-Control: 2.00 ± 0.67; 3 d-Cas1.i: 1.20 ± 0.42; 3 d-Cas11.i: 1.30 ± 0.48, P < 0.001). Conclusions: Taken together, inflammasome activation played a detrimental role in stroke pathology. Targeting post-stroke inflammasome executing enzymes fitting in the dynamics of macrophages might obtain potential and efficient therapeutic effects.

Among all the components of inflammasomes, the dispatchers that take part in the process of inflammasome activation vary according to the disease settings (9,10). Components that participate in post-stroke inflammasome activation are to be explored. Inflammasome formation has been found in diverse cell kinds including dendritic cells, neutrophils, and macrophages (11)(12)(13), but we still need to determine which cell type the inflammasome mainly activates after a stroke. The executors in acute ischemic stroke remain elusive. Therefore, the current study first identified the main participants of inflammasome activation in stroke models and then explored the therapeutic effects targeting the identified targets.
In this study, we aim to affirm the therapeutic value of inflammasome suppression, identifying the participants in poststroke inflammasome activation and the arena of inflammasome formation. The protective effects of targeting inflammasome executing enzymes were also explored.

Ethical Statement
The clinical and the animal experimental studies were approved by the Medical Ethics Committee of the Third Affiliated Hospital of Sun Yat-Sen University and the Animal Care and Use Committee of Sun Yat-Sen University, respectively. All participants gave their informed consent according to the principles illustrated in the Declaration of Helsinki.

Patients
In this study, 26 stroke patients recruited in the Third Affiliated Hospital of Sun Yat-Sen University from July 2018 to October 2019 consecutively had an independently documented primary stroke event in combination with confirmed magnetic resonance imaging (MRI) evidence showing ischemic stroke. The inclusion and exclusion criteria have been published previously (14). Age (mean 64 and 95% CI 52-75), gender (female:male = 9:17), and scores on the National Institute stroke scale (NIHSS) of patients were recorded. A total of 40 age-matched healthy community dwellings was recruited as healthy controls (HCs). Patient demographics including co-morbidities were summarized in Table 1.

MRI Scanning and Infarct Volume Analysis of Patients
Magnetic resonance imaging (MRI) was performed within 24 h of admission using 1.5-or 3.0-T magnetic resonance imaging (Signa; GE Medical Systems, Milwaukee, WI, USA). In this study, the diffusion-weighted imaging (DWI) spin-echo planar sequence included 20 contiguous axial oblique slices (b = 0 and 1000 s/mm 2 isotropically weighted; repetition time/echo time, 6000/60.4 ms; acquisition matrix, 128 × 128; slice thickness, 5 mm; interslice gap, 1 mm; field of view, 24 cm). DWI lesions in 26 patients were measured with Analyze 7.0 software (Analyze Direct, KS). Cerebral infarct sizes were assessed by the largest infarct diameter determined on the image demonstrating the largest lesion (15). MRI scans of patients were assessed by experienced neurologist Zhengqi Lu, who was blinded to the patients' clinical features. All images were interpreted with the same window settings, same types of monitors, and lighting conditions. Animals C57/Bl6 wild type mice (age 8-12 weeks and weight 18-25 g) were purchased from Guangdong Medical Laboratory Animal Center (Guangzhou, China) and housed in a humidity-and temperature-controlled animal facility in Sun Yat-sen University with a 12-h light-dark cycle for at least 1 week before induction of ischemic stroke. Food and water were freely accessible. All the experimental protocols were approved by the Animal Care and Use Committee of Sun Yat-sen University following the Guide for the Care and Use of Laboratory Animals. A total of 71 male mice were used in the transient middle cerebral artery occlusion (tMCAO, 1 h) model, including 11 mice that were excluded from further assessments due to death after ischemia or unsuccessful induction of stroke. There were 18 male mice used for primary macrophage-enriched culture.

Animal Experiments Statement
The animal research has been written up in accordance with the ARRIVE (Animal Research: Reporting in vivo Experiments) guidelines.

Murine Model of Transient Focal Ischemia
A focal ischemic stroke model was induced in mice with transient middle cerebral artery occlusion (tMCAO) as previously described (16). Briefly, mice were anesthetized with 1.5% isoflurane in the air under spontaneous breathing. A midline neck incision was made, and soft tissues were retracted gently over the trachea. The left common carotid artery (CCA) was isolated and ligated temporarily. A permanent knot was placed on the distal part of the external carotid artery (ECA), and a loose temporal knot was placed on both the proximal part of ECA. A tight temporal knot was placed on the internal carotid artery (ICA). ECA was cut between the permanent knot and the temporal knot, and a filament was inserted into ECA and directed to the middle cerebral artery (MCA) through ICA after loosening the temporal knot on ICA. Once the filament insertion into the MCA was confirmed, the loose temporal knot on ECA was tightened and the temporal knot on CCA was loosened. Filament insertion into the ICA was maintained for 60 min and followed by reperfusion. Sham-operated animals underwent the same anesthesia and surgical procedures but were not subjected to arterial occlusion. Core body temperatures were maintained with a heating pad. Regional cerebral blood flow (rCBF) during the surgery was measured by laser Doppler flowmetry. Mice with <70% reduction of blood flow in the ischemic core or those that died during surgery were excluded from further analysis.

Flow Cytometric Analysis
Blood samples were collected from AIS patients and healthy controls. A lysis buffer (Roche) was applied to samples for 10 min to exclude red blood cells (RBC). Single cells then were stained with surface markers of CD11c-PE (1:400, Biolegend), CD11b-PE-Cy7 (1:400, Biolegend), CD66b-PerCP-Cy5.5 (1:400, Biolegend), and CD14-APC-Cy7 (1:400, Biolegend). Cells were then fixed and permeabilized. Antibodies of anti-NLRP3 (Rabbit, abcam) and anti-IL1β (goat, R&D system) were then applied to cells overnight. After washing, cells were incubated with secondary antibodies of anti-rabbit-Alexa Flour 488 and antigoat-Alexa Flour 488 for 30 min. Cells were then subjected to analysis after wash. Blood and brain were extracted from animals at indicated time points. Brains were dissected, and ipsilateral hemispheres were collected after saline perfusion. Each hemisphere was subjected to 0.25% trypsin-EDTA (Thermo Fisher) digestion at 37 • C for 25 min. Brain tissue was then pressed through a cell strainer (70 µm, Thermo Fisher). Brain cells were separated from myelin debris by centrifugation in 30/70% Percoll solution (GE healthcare). Brain cells at the interface were collected, washed with PBS, and subjected to further staining. Red blood cell lysis buffer (Sigma Aldrich) was applied to get rid of red blood cells in the blood that were subjected to further staining.
Flow cytometric analysis was performed using a fluorescenceactivated cell sorter flow cytometer (BD Biosciences), and data were analyzed using FlowJo X 10.0.7r2 software.

Primary Macrophage-Enriched Culture
Primary macrophage-enriched cultures were prepared from the bone marrow of 8-to 10-week-old C57/Bl6 mice. Monocytes were isolated using a mouse monocyte isolation kit (STEMCELL Technologies) according to the manufacturer's instructions. Monocytes were then cultured in the presence of macrophage colony-stimulating factor (MCSF, 20 ng/ml) for 7 days in vitro to induce macrophage.

Preparation of Danger Associated Molecular Patterns (DAMPs)
DAMPs were collected from the ipsilateral brain 3 days after ischemic stroke using methods previously described (17). Briefly, mice were sacrificed and perfused with sterile iced saline. The ipsilateral brain was extracted and digested with 2 ml of 0.25% Trypsin for 20 min at 37 • C. For each hemisphere, 5 ml of RPMI1640 containing 10% FBS was applied to stop digestion. Samples were then centrifuged, and the supernatant was collected. The supernatant was then pressed first through a 0.22 µm filter to exclude any cell component. The solution of DAMPs were then treated to macrophage for inflammasome induction.

Statistical Analysis
Results were presented as mean ± standard deviation (SD). GraphPad Prism software (version 8.0; La Jolla, CA) was used for statistical analyses. The two-tailed Student's t test was used for the comparison of the two groups. The differences in means among multiple groups were analyzed using one-way analysis of variance (ANOVA) for continuous variables with normal distribution. Linear regression was used to analyze the correlation between two sets of continuous variables. When ANOVA showed significant differences, pair-wise comparisons between means were tested by post hoc Bonferroni tests. In all analyses, P ≤ 0.05 was considered statistically significant.

High Level of Inflammasome Product Indicates Detrimental Stroke Outcomes
To confirm the impact of inflammasome activation on stroke outcomes, blood samples from 26 stroke patients and 40 sexand age-matched healthy controls ( Table 1) were collected and inflammasome markers NLRP3 and IL-1β were analyzed. As is reported, the level of IL-1β (inflammasome product) increased at 1 day after AIS (HC: 6,064.85 ± 1,735.25; AIS patients: 1,1345.56 ± 1,5130.49, P < 0.05) ( Figure 1A). Expression of NLRP3 (receptor of inflammasome) increased in a part of patients ( Figure 1B). Nevertheless, it is not a significant result. Regression analysis revealed that Mean fluorescent intensity (MFI) of IL-1β (1 day) positively correlated with infarct size (R 2 = 0.3552, P = 0.0013) (Figure 1C). MFI of NLRP3 (1 day) correlated somewhat with the 7-day NIHSS score (R 2 = 0.1505, P = 0.0502) ( Figure 1D). The results indicated that inflammasome activation may be correlated with detrimental stroke outcomes, indicating the rationale for studying post-stroke inflammasome activation as a therapeutic target.

DISCUSSION
It is widely accepted that post-stroke inflammation promotes secondary tissue damage in the ischemic brain. Inflammasome activation represents a potent pro-inflammatory mechanism. The current study confirmed the occurrence of inflammasome activation after stroke and increased infarct volume. The dynamics of inflammasome components and the arena of inflammasome activation have been identified. Therapeutic effects of suppressing the main executors (Caspase-1 and−11) of post-stroke inflammasome activation was also explored.
The potential of targeting inflammasome in stroke therapy has been studied since its discovery. Among inflammasome components, NLRP3 is the most understood especially in aseptic inflammation. Mounting evidence has indicated that the NLRP3 inflammasome played a prominent role in the pathogenesis and progression of ischemic stroke (4). Inhibiting the NLRP3 inflammasome could be a promising therapeutic strategy (5,24). However, contradictory ideas exist. Lemarchand, E. and colleagues reported that NLRP3 did not contribute to the extension of ischemic brain injury after ischemic stroke, as no protection to stroke was observed when applying NLRP3 inhibitor or knocking out NLRP3 in mice (6). We showed that the inflammasome product IL-1β elevated after AIS in both patients and mice models, which indicated the occurrence of inflammasome activation. Nevertheless, no expression alteration of NLRP3 level was detected in patients, which revealed that the function of NLRP3 might be negligible in post-stroke inflammasome activation. Indeed, the data on human specimens may not be directly paralleled with those in mice, since they have been obtained only in the blood of patients. Moreover, we found that besides NLRP3, other receptors, such as NLRC4, participated in post-stroke inflammasome formation in mice models, which indicates that NLRP3 is not the only path for inflammasome activation.
Our results revealed that post-stroke inflammasome activation involved multi-players, including various receptors, executors, and subsequent products. Among all the components, executing enzymes play a check-point-like role. Whichever receptors detect the dangerous signals would activate the downstream executing enzymes for further procession. It seems that the executors of inflammasome vary depending on the disease pathology (9,10). It has been reported that it was Caspase-8 that played a decisive role in multiple sclerosis (10), whereas our data suggested that it was Caspase-1/-11 that represented the primary executors in post-stroke inflammasome activation. Since Caspase-1 and −11 keep the gate of poststroke inflammasome activation, targeting the enzymes for inflammasome inhibition may be more efficient than the receptors. Indeed, treatment of Caspase-1/-11 inhibitor after ischemic stroke offered favorable neuroprotection.
Inflammasome activates primarily in myeloid cells. On the other hand, several kinds of brain cells have been shown to display inflammasome formation (e.g., neuron, astrocyte, and endothelial cells) (11)(12)(13). Our results suggested that it was macrophages that endured the most robust inflammasome activation after stroke among myeloid cells since over half of the inflammasome activating cells in the stroke brain were macrophages. Moreover, inflammasome activation in macrophages occurred primarily within the stroke lesion. Orienting the cell type and location of inflammasome activation provides clues for the improvement of therapeutic efficiency. However, we lack the data on the stroke lesion in patients, which needs further improvement. Our data suggested that fitting the time point for macrophage infiltration and increasing BBB entry of inflammasome suppressors may favor their therapeutic effects.
In conclusion, we found that inflammasome activated mainly in macrophages that infiltrated into stroke lesion and played a detrimental role in post-stroke inflammation. Suppressing inflammasome activation by targeting the checkpoint executing enzymes Caspase-1/-11 displayed promising therapeutic effects.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author/s.

ETHICS STATEMENT
The animal study was reviewed and approved by The animal experimental studies were approved by the Animal Care and Use Committee of Sun Yat-Sen University. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.

AUTHOR CONTRIBUTIONS
DL designed and performed the experiments, collected and analyzed data, and drafted the manuscript. MH performed animal experiments and collected data. BZ, YL QZ, and XM contributed to the experimental design and revised the manuscript. WC and ZL designed and supervised the study and critically revised the manuscript. All authors read and approved the final manuscript.