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Selective NLRP3 (Pyrin Domain–Containing Protein 3) Inflammasome Inhibitor Reduces Brain Injury After Intracerebral Hemorrhage

Selective NLRP3 (Pyrin Domain–Containing Protein 3) Inflammasome Inhibitor Reduces Brain Injury... Selective NLRP3 (Pyrin Domain–Containing Protein 3) Inflammasome Inhibitor Reduces Brain Injury After Intracerebral Hemorrhage Honglei Ren, MD; Ying Kong, MD; Zhijia Liu, MD; Dongyun Zang, MD; Xiaoxia Yang, MD; Kristofer Wood, BS; Minshu Li, PhD; Qiang Liu, MD, PhD Background and Purpose—Intracerebral hemorrhage (ICH) is a devastating disease without effective treatment. As a key component of the innate immune system, the NOD-like receptor (NLR) family, NLRP3 (pyrin domain–containing protein 3) inflammasome, when activated after ICH, promotes neuroinflammation and brain edema. MCC950 is a potent, selective, small-molecule NLRP3 inhibitor that blocks NLRP3 activation at nanomolar concentrations. Here, we examined the effect of MCC950 on brain injury and inflammation in 2 models of ICH in mice. Methods—In mice with ICH induced by injection of autologous blood or bacterial collagenase, we determined the therapeutic potential of MCC950 and its mechanisms of neuroprotection. Results—MCC950 reduced IL-1β (interleukin-1β) production and attenuated neurodeficits and perihematomal brain edema after ICH induction by injection of either autologous blood or collagenase. In mice with autologous blood-induced ICH, the protection of MCC950 was associated with reduced leukocyte infiltration into the brain and microglial production of IL-6. MCC950 improved blood–brain barrier integrity and diminished cell death. Notably, the protective effect of MCC950 was abolished in mice depleted of either microglia or Gr-1 myeloid cells. Conclusions—These results indicate that the NLRP3 inflammasome inhibitor, MCC950, attenuates brain injury and inflammation after ICH. Hence, NLRP3 inflammasome inhibition is a potential therapy for ICH that warrants further investigation. Visual Overview—An online visual overview is available for this article. (Stroke. 2018;49:184-192. DOI: 10.1161/ STROKEAHA.117.018904.) Key Words: brain edema cell death inflammation neuroprotection ◼ ◼ ◼ ntracerebral hemorrhage (ICH) is a devastating, often fatal As a critical component of the innate immune response Iaftermath of stroke, but not generally amenable to treat- to tissue injury, activation of the NOD-like receptor (NLR) 1–5 ment. Emerging evidence has demonstrated that a stroke- NLRP3 (pyrin domain–containing protein 3) inflamma- incited inflammatory cascade accelerates the formation of some leads to the cleavage of pro–IL (interleukin)-1β and edema that surrounds hematomas, exacerbates the mass effect, pro–IL-18, as well as the subsequent release of biologically 4–8 and amplifies cell death. After the ictus of ICH, blood com- active IL-1β, IL-18, and other soluble mediators of inflamma- ponents, including leukocytes, enter the brain and activate tion. Recent evidence indicates that the ICH-induced activa- resident immune cells such as microglia. Subsequently, the tion of NLRP3 inflammasome can amplify the inflammatory infiltrating leukocytes and activated microglia boost the local response by releasing IL-1β and promoting neutrophil infiltra- 9–11 production of proinflammatory factors. Together with cell tion, thereby exacerbating brain edema. Although previous death products, these catastrophic events amplify blood–brain studies provide mechanistic insights into NLRP3 as a promis- barrier (BBB) disruption and destroy surrounding tissues, ing target for restricting neuroinflammation and brain edema contributing to the development of perihematomal edema and after ICH, they do not reflect clinical application because they 4–8 the aggravated mass effect. Therefore, brain inflammation involve either knockout models or use of pharmacological has emerged as a major modifiable determinant now targeted agents with limited potency and nonspecificity preceding ICH 7 9–11 for the development of novel ICH treatment. induction. For clinical translation of prior experimental Received July 31, 2017; final revision received November 12, 2017; accepted November 13, 2017. From the Department of Neurology, Tianjin Neurological Institute, Tianjin Medical University General Hospital, China (H.R., Y.K., Z.L., X.Y., M.L., Q.L.); Department of Neurology, Barrow Neurological Institute, St. Joseph’s Hospital and Medical Center, Phoenix, AZ (K.W., M.L., Q.L.); and Department of Neurosurgery, Tianjin Huanhu Hospital, China (D.Z.). The online-only Data Supplement is available with this article at http://stroke.ahajournals.org/lookup/suppl/doi:10.1161/STROKEAHA. 117.018904/-/DC1. Correspondence to Qiang Liu, MD, PhD, Department of Neurology, Tianjin Neurological Institute, Tianjin Medical University General Hospital, China. E-mail [email protected] © 2017 The Authors. Stroke is published on behalf of the American Heart Association, Inc., by Wolters Kluwer Health, Inc. This is an open access article under the terms of the Creative Commons Attribution Non-Commercial-NoDerivs License, which permits use, distribution, and reproduction in any medium, provided that the original work is properly cited, the use is noncommercial, and no modifications or adaptations are made. Stroke is available at http://stroke.ahajournals.org DOI: 10.1161/STROKEAHA.117.018904 184 Ren et al NLRP3 Inhibitor Reduces Hemorrhagic Brain Injury 185 outcomes, preclinical testing in relevant models is mandatory. Behavioral Assessment However, this option has been precluded by the lack of selec- Behavioral assessment was conducted at days 1 and 3 after ICH using the modified Neurological Severity Score together with corner-turning tive, potent NLRP3 inflammasome inhibitors. test to comprehensively evaluate motor, sensory, reflex, and balance Now, we have devised a means of clinically relevant testing 21–24 functions, as previously described. The range of scores for modi- in an experimental ICH model by using MCC950, a selective, fied Neurological Severity Score is from 0 to 18, defined as follows: small-molecule NLRP3 inflammasome inhibitor with proven a score of 13 to 18 indicates severe injury, 7 to 12 indicates moderate high potency in vitro and in vivo. We hypothesized that the injury, and 1 to 6 indicates mild injury. Mice were given 1 point if they failed to perform a task. The corner-turning test assesses sensorimotor pharmacological inhibition of NLRP3 inflammasome by and postural asymmetries. In this process, each mouse is allowed to administration of MCC950 would reduce inflammation and proceed into a corner with an angle of 30° and then must turn right brain edema after ICH induced by the injection of autologous or left. Each mouse repeated this procedure for 10×, with at least 30 s blood or collagenase. between trials. The percentage of right turns is then calculated. Materials and Methods Magnetic Resonance Imaging Details of materials and experimental procedures are available Lesion volume and perihematomal edema were quantified at day 3 13,16 from the online-only Data Supplement. This article adheres to the after ICH using a 7-T small animal MRI, as previously described. AHA Journals implementation of the Transparency and Openness T2-weighted image sequence and susceptibility-weighted image Promotion Guidelines. sequence were scanned to detect lesion volume and hematoma volume, respectively. Details are described in the online-only Data Supplement. Animals All animal experiments were approved by the Committee on the Brain Water Content Measurement Ethics of Animal Experiments of Barrow Neurological Institute Water contents were measured at day 3 after ICH, as previously (Phoenix, AZ). All experiments were conducted in accordance 25 described. In brief, without perfusion, brains were removed from with the National Institutes of Health (Bethesda, MD) Guide mice and divided into 3 parts: the ipsilateral, the contralateral, and for the Care and Use of Laboratory Animals, and experiments the cerebellum. The brain samples were weighed immediately on an were designed, performed, and reported according to the Animal electronic balance to obtain wet weights, then dried for 24 hours at Research: Reporting In Vivo Experiments guidelines (https:// 100°C, and weighed again to obtain dry weights. The following for- www.nc3rs.org.uk/arrive-guidelines). Male C57BL/6 mice, 8 to mula was used to calculate the brain water content: (wet weight−dry 10 weeks old (20–25 g), were purchased from the Charles River weight)/wet weight×100%. Laboratories (Wilmington, MA). Female mice were not used to avoid any influences of sex steroids. All mice were housed in ani- mal facilities under a standardized light–dark cycle and had free Flow Cytometry + int access to food and water. All animal surgeries were performed To quantify cells expressing NLRP3, microglia (CD11b CD45 ), + high + under anesthesia. Animals were randomly divided and assigned neutrophils (CD11b CD45 Ly6G ), monocyte/macrophages (CD1 + high + + high + + + to experimental groups using a random number list generated by 1b CD45 Ly6C ), CD4 T cells (CD45 CD3 CD4 ), CD8 T high + + high − + Microsoft Excel 2013. cells (CD45 CD3 CD8 ), B cells (CD45 CD3 CD19 ), NK cells high − + (CD45 CD3 NK1.1 ), cytokines produced by microglia, and cell apoptosis (Annexin V ) in the brain, we isolated cellular components Induction of ICH in Mice from brain tissue to perform flow cytometry analysis as we previously ICH was induced in mice by injection of autologous blood or bac- 23,26 described. Details appear in the online-only Data Supplement. 12–16 terial collagenase, as we previously described. Details of ICH induction are given in the online-only Data Supplement. Real-Time Polymerase Chain Reaction At day 3 after ICH, total RNA was extracted from brain tissue in the Study Design and Drug Administration ipsilateral hemisphere for real-time polymerase chain reaction analy- A total of 302 male C57BL/6 mice (Charles River Laboratories), sis as described in the online-only Data Supplement. 8 to 10 weeks old, were used in this study. In autologous blood model, the mortality rate was 8.5% (20 of total 236) and exclu- sion rate was 16.7% (36 of total 236). In the collagenase model, Assessment of BBB Permeability the mortality rate was 13.3% (8 of total 60) and exclusion rate was Evans Blue dye (Sigma, St. Louis, MO) extravasation assays were 20% (12 of total 60). Six wild-type mice were used for in vitro conducted at day 3 after ICH as a tracer to measure BBB permeabil- experiments. MCC950 was dissolved in phosphate-buffered saline, 16,27 ity, as previously described. as previously described. Mice were given MCC950 at a dose of 10 mg/kg by intraperitoneal injection at indicated time points after ICH. Mice that received an equal volume of vehicle (phosphate- Western Blots buffered saline) only were designated as controls. For the microglia At day 3 after ICH, Western blots analyzed for NLRP3 inflamma- depletion experiment, PLX3397 (Selleckchem, Houston, TX) was some components and tight junction protein expression in the ipsi- given by oral gavage at 40 mg/kg for 21 days before ICH induc- lateral cerebral hemisphere, as we previously described. Details are 16,18 tion. Drug treatment was continued until the end of experiments. given in the online-only Data Supplement. Subsequently, anti-mouse Gr-1 monoclonal antibody (MAb- RB6-8C5; BioXcell, West Lebanon, NH) was given by intraperi- Statistical Analysis toneal injection 1 day before and 1 day after ICH surgery at 250 + 19,20 µg per mouse to deplete Gr-1 myeloid cells in vivo. In mice We determined each sample size by power analysis using a signifi- subjected to microglia depletion, no mice were excluded. More than cance level of α=0.05 with 80% power to detect statistical differ- 90% of microglia were depleted in mice receiving PLX3397. In ences. SAS 9.1 software (SAS Institute Inc, Cary, NC) was used for mice subjected to Gr-1 cell depletion, the exclusion rate was 5.9% power analysis and sample size calculations. Data were analyzed by (1 of total 17 because of death after surgery). The depletion of Gr-1 investigators blinded to experimental treatments. Data are shown cells was verified by sampling circulating blood, and ≈ 90% of Gr-1 as mean±SD. SPSS 19.0 software was used to compare differences between 2 groups where appropriate. One-way ANOVA followed by cells were depleted in all mice included. 186 Stroke January 2018 the Tukey post hoc test or 2-way ANOVA with multiple compari- days starting immediately after ICH induction (Figure 1A). sons followed by Bonferroni post hoc test was used for comparison of Neurological function was evaluated by using modified multigroup data. P values <0.05 are considered significant. Neurological Severity Score and corner-turning tests at days 1 and 3 after ICH. Lesion volume, perihematomal edema, Results and brain water content were measured at day 3 after ICH. MCC950 Attenuates Brain Injury and Compared with vehicle recipients, we found that MCC950- Improves Long-Term Outcome After ICH treated mice had significantly reduced neurodeficits, lesion vol- umes, and perihematomal edema after ICH (Figure 1B and 1C). To determine whether the NLRP3 inflammasome inhibitor, MCC950 reduced brain water content after ICH (Figure I in the MCC950, affects brain injury after ICH, we examined neuro- online-only Data Supplement). In addition, MCC950 reduces deficits, lesion volume, and perihematomal edema in ICH mice receiving MCC950 or a phosphate-buffered saline vehicle. ICH neurodeficits until day 28 after ICH induction (Figure 1D), was induced by injection of autologous blood or collagenase. suggesting that NLRP3 inflammasome inhibition can provide Mice received MCC950 (10 mg/kg) or vehicle for 3 consecutive long-term benefit after ICH. Of note, the benefit of MCC950 to Figure 1. MCC950 attenuates brain injury and improves long-term outcome after intracerebral hemorrhage (ICH). ICH was induced in C57BL/6 mice by injection of autologous blood or collagenase. A, Flow chart illustrates MCC950 administration and experimental design. Mice received daily intraperitoneal (IP) injections of MCC950 (10 mg/kg) or an equal volume of phosphate-buffered saline (PBS) vehicle for 3 consecutive days starting immediately after ICH induction. B, Neurological tests were performed to evaluate the motor, sensory, and balance functions in mice receiving vehicle or MCC950 at days 1 and 3 after injection of autologous blood (left) or collagenase (right). C, T2-weighted image (T2WI) sequences were scanned to assess lesion volume at day 3 after ICH induced by injection of autologous blood (left) or collagenase (right), as outlined in red. Susceptibility-weighted sequences were assessed for hematoma lesion volume, visible in yellow regions. Quantification of lesion volume and perihematomal edema in mice receiving MCC950 or vehicle at day 3 after ICH induced by injection of autologous blood (left) or collagenase (right). n=8 mice per group. D, Mice received vehicle or MCC950 at a dose of 10 mg/kg by intraperitoneal injection. The assessments of modified Neurological Severity Score (mNSS) score and corner test were performed at days 7, 14, and 28 after ICH induced by injection of collagenase. n=10 per group. Data are presented as mean±SD. *P<0.05, **P<0.01. Ren et al NLRP3 Inhibitor Reduces Hemorrhagic Brain Injury 187 reduce neurodeficits and brain edema was restricted to within 24 cellular components as brain-infiltrating leukocytes and hours after ICH (Figure II in the online-only Data Supplement). microglia in the brains of ICH mice (Figure 3A). At day 3 post-ICH, the numbers of brain-infiltrating leukocytes, + high + neutrophils (CD11b CD45 Ly6G ), monocyte/macro- MCC950 Inhibits the Activation of + high + + high phages (CD11b CD45 Ly6C ), CD4 T cells (CD45 NLRP3 Inflammasome Components + + + high + + CD3 CD4 ), and CD8 T cells (CD45 CD3 CD8 ) were and IL-1β Production After ICH reduced in ICH mice receiving MCC950 compared with The effect of MCC950 on NLRP3 inflammasome activation those in vehicle-treated controls (Figure 3B and 3C). Of and IL-1β production was examined in brain tissues of ICH note, MCC950 reduced the infiltration of CD4 T cells, mice. At day 3 after ICH, we found that MCC950 reduced CD8 T cells and neutrophils until day 7 after ICH (Figure the mRNA expression of NLRP3 inflammasome components IV in the online-only Data Supplement). Further, microg- (NLRP3/Caspase-1/ASC) and IL-1β (Figure 2A). In addition, the protein expression of NLRP3, caspase-1, and IL-1β in the lia cell numbers decreased as did microglial expression of factor IL-6 (Figure 3D). In contrast, the microglial expres- brain was suppressed by MCC950 treatment (Figure 2B and 2C). Of interest, MCC950 does not affect lipopolysaccha- sion of IL-10 and TGF-β (transforming growth factor-β) was increased in ICH mice receiving MCC950 (Figure 3D). rides-induced production IL-1β and TNF-α (tumor necrosis factor-α) from splenocytes (Figure III in the online-only Data These results indicate that MCC950 can reduce brain inflam- Supplement). These results demonstrate that MCC950 effec- mation and cause a shift in microglia phenotype toward an tively inhibits the activation of NLRP3 inflammasome compo- anti-inflammatory status. nents and IL-1β production in the brain after ICH. MCC950 Preserves BBB Integrity and MCC950 Reduces Leukocyte Infiltration Reduces Cell Death After ICH and Production of Proinflammatory BBB disruption after ICH contributes to vasogenic edema Factors by Microglia After ICH and the expansion of perihematomal edema. To measure the Next, we determined the impact of MCC950 on brain inflam- effect of MCC950 on BBB disruption after ICH, we exam- mation after ICH. Using flow cytometry, we examined such ined Evans Blue dye extravasation and the expression of Figure 2. MCC950 inhibits NOD-like receptor (NLR) family, NLRP3 (pyrin domain-containing protein 3) inflammasome activation and IL (interleukin)-1β expression. Intracerebral hemorrhage (ICH) was induced in C57BL/6 mice by injection of autologous blood. Mice received daily intraperitoneal (IP) injections of MCC950 (10 mg/kg) or an equal volume of vehicle for 3 consecutive days starting immediately after ICH induction. At day 3 after ICH or sham surgery, brain tissues were harvested for the extraction of mRNA and protein. A, The mRNA expression levels of the NLRP3 inflammasome components and IL-1β after ICH. B, Western blot images show the expression of NLRP3, IL-1β, and caspase-1 in the brains of indicated groups of mice receiving sham plus vehicle, ICH plus vehicle, or ICH plus MCC950. C, Data points show the protein expression levels of NLRP3, IL-1β, and caspase-1 in the brains of indicated groups of mice receiving sham plus vehicle, ICH plus vehicle, or ICH plus MCC950. n=6 mice per group. Data are presented as mean±SD. *P<0.05, **P<0.01. 188 Stroke January 2018 Figure 3. MCC950 reduces leukocyte infiltration and microglia production of proinflammatory factors after intracerebral hemor - rhage (ICH). ICH was induced in C57BL/6 mice by injection of autologous blood. Mice received daily intraperitoneal (IP) injections of MCC950 (10 mg/kg) or an equal volume of vehicle for 3 consecutive days starting immediately after ICH induction. At day 3 after ICH, immune cells were isolated from brain tissues of ICH mice receiving MCC950 or vehicle. A, Gating strategy of brain-infiltrating + + + + + + − + − + immune cells including CD4 T cells (CD3 CD4 ), CD8 T cells (CD3 CD8 ), B cells (CD3 CD19 ), NK cells (CD3 NK1.1 ), monocyte/ + high + + high + + int macrophages (CD11b CD45 Ly6C ), neutrophils (CD11b CD45 Ly6G ), and microglia (CD11b CD45 ) and their expression of IL (interleukin)-6, TNF-α (tumor necrosis factor-α), TGF-β (transforming growth factor-β), and IL-10. FMO, fluorescence minus one. B , Data points show counts of brain-infiltrating leukocytes in the brains of ICH mice receiving indicated treatment. C , Data points show counts of microglia in the brains of ICH mice receiving indicated treatment. D, Data points show the counts of microglia expressing IL-6, TNF-α, TGF-β, and IL-10 in the brains of ICH mice receiving indicated treatment. n=6 mice per group. Data are presented as mean±SD. *P<0.05, **P<0.01. tight junction proteins. At day 3 after ICH, MCC950 signifi- Benefit of MCCC950 Against ICH Involves cantly ameliorated leakage of the dye (Figure 4A), denoting Microglia and Gr-1 Myeloid Cells a reduction of BBB permeability. MCC950 also preserved Because microglia are the predominant cell subset expressing expression of the tight junction proteins (claudin-5 and NLRP3 inflammasome after ICH (Figure V in the online-only ZO-1; Figure 4B). In addition, MCC950 reduced numbers of Data Supplement), we sought to understand to what extent Annexin V–expressing cells in the brain at day 3 after ICH, microglia may contribute to the protective effect of MCC950. suggesting a decrease in cell death (Figure 4C). Together, The survival of microglia depends on signaling through these results demonstrate that MCC950 preserves BBB integ- CSF1R (colony-stimulating factor 1 receptor); therefore, rity and reduces cell death after ICH. we used a CSF1R inhibitor, PLX3397, to deplete microglia Ren et al NLRP3 Inhibitor Reduces Hemorrhagic Brain Injury 189 Figure 4. MCC950 treatment preserves blood–brain barrier (BBB) integrity and reduces cell death after intracerebral hemorrhage (ICH). ICH was induced in C57BL/6 mice by injection of autologous blood. Mice received daily intraperitoneal (IP) injections of MCC950 (10 mg/kg) or an equal volume of vehicle for 3 consecu- tive days starting immediately after ICH induction. At day 3 after ICH, brain tis- sues were prepared for measurements of BBB integrity and cell death. A, Histology images show that MCC950 decreased Evens Blue dye leakage compared with vehicle (phosphate-buffered saline [PBS]) treatment. B, Western blot images show expression of the tight junction proteins, claudin-5, and ZO-1, in the brains of ICH mice receiving MCC950 or vehicle. C, Flow cytometry plots and summarized results show percentages of Annexin V cells in the brains of ICH mice receiv- ing MCC950 or vehicle. n=6 mice per group. Data are presented as mean±SD. *P<0.05, **P<0.01. 29 + + before ICH induction (Figure 5A). Consistent with our pre- An anti–Gr-1 mAb (RB6-8C5) was used to deplete Gr-1 viously described PLX3397 treatment, the present result was a myeloid cells from mice before ICH induction (Figure 6A). + int + loss of >90% microglia (CD11b CD45 ) from mice undergo- We monitored the counts of Gr-1 cells in circulating blood ing ICH. Of interest, we found that the benefit of MCC950 at 24 hours before the first injection of anti–Gr-1 mAb treatment was abolished in ICH mice receiving PLX3397 and 48 hours after the second injection of anti–Gr-1 mAb (Figure 5B and 5C), suggesting that microglia participate in (Figure 6B). Consistent with a previous report, anti– + + the beneficial effect of MCC950 after ICH. Gr-1 mAb treatment depleted ≈90% of Gr-1 myeloid cells Because NLRP3 inflammasome was clearly expressed in (Figure 6B), that is, mainly neutrophils and monocytes. Of + + Gr-1 myeloid cells, consisting mainly of neutrophils and note, the depletion of Gr-1 myeloid cells diminished the monocytes that are known contributors to BBB disruption beneficial effects of MCC950 after ICH (Figure 6C and 6D), 30,31 and brain inflammation, we therefore determined whether suggesting that the protection of MCC950 also involves + + Gr-1 myeloid cells also add to the benefit of MCC950. Gr-1 myeloid cells. Figure 5. Microglia contribute to the benefit of MCC950 after intracerebral hemorrhage (ICH). A, Schematic diagram illustrates drug administration and experimental design. C57BL/6 mice received oral gavage of PLX3397 (40 mg/kg) for 21 days and continuously until the experiment ended. ICH was induced in PLX3397-treated mice by injection of autologous blood. Thereafter, these mice received daily injections of MCC950 (10 mg/kg, intraperitoneal [IP]) or an equal volume of vehicle for 3 consecutive days starting immediately after ICH induction. At day 3 after ICH, neurodeficits and brain water content were assessed. B, Quantification of modified Neurological Severity Score (mNSS) and corner-turning test scores in indicated groups of ICH mice. C, Quantification of brain water content in indicated groups of ICH mice. n=6 per group. Data are presented as mean±SD. 190 Stroke January 2018 Figure 6. Gr-1 myeloid cells contribute to the benefit of MCC950 after intracerebral hemorrhage (ICH). A, Schematic diagram illustrates drug administration and experimental design. C57BL/6 mice received anti–Gr-1 mAb 1 day before and 1 day after ICH surgery. ICH was induced by injection of autologous blood. Thereafter, these mice received daily injections of MCC950 (10 mg/kg, intraperitoneal [IP]) or an equal volume of vehicle for 3 consecutive days starting immediately after ICH induction. At day 3 after ICH, neurodeficits and brain water content were assessed. B, Data points show the counts of circulating Gr-1 cells in mice before ICH induction, or mice receiving anti–Gr-1 mAb or IgG control at day 3 after ICH. C, Summarized results of modified Neurological Severity Score (mNSS) and corner-turning test scores in ICH mice receiving indicated treatments. D, Data points show brain water content in ICH mice receiving indicated treatments. n=8 mice per group. Data are presented as mean±SD. 9–11 advancing previous findings, our study for the first time Discussion provides efficacy data depicting pharmacological inhibition This study provides the first evidence that the NLRP3 inflam- by NLRP3 inflammasome in the setting of ICH in vivo. masome inhibitor, MCC950, attenuates hemorrhagic brain Other than MCC950, several small molecules inhibit the injury. MCC950 significantly reduced neurodeficits and NLRP3 inflammasome. For example, glyburide inhibited perihematomal edema in 2 mouse models of ICH induced IL-1β production at micromolar concentrations in response by injection of autologous blood or bacterial collagenase. to the activation of NLRP3 and was effective in reducing MCC950 treatment was sufficient to reduce the resulting leu- edema formation. Purinergic 2X7 receptor antagonist (blue kocyte infiltration, microglial production of proinflammatory brilliant G) also diminished NLRP3 inflammasome activation factors, BBB disruption, and cell death after ICH. In addition, and lessened hemorrhagic brain injury. However, because of the benefit of MCC950 protection from ICH was diminished the limited potency and nonspecific nature of those agents, in mice subjected to depletion of microglia or Gr-1 myeloid the extent to which pharmacologically selective targeting by cells (neutrophils and monocytes). Together, these results sug- gest that MCC950 has potential therapeutic value for reducing NLRP3 inflammasome can impact ICH injury is still unclear. However, results from this study provide new evidence that ICH injury. selective NLRP3 inflammasome inhibition offers the benefit Mechanistic studies have shown an essential role for the NLRP3 inflammasome in brain injury and neuroinflamma- of reducing ICH injury and improving long-term outcome, 9–11 which is an essential step for bringing NLRP3 inflammasome- tion after ICH. The activation of NLRP3 inflammasome facilitates caspase-1 activation and IL-1β processing, lead- targeted therapies from the bench into clinical application. Reportedly, NLRP3 might present certain advantages over ing to the amplification of the inflammatory response, which culminates in the expansion of perihematomal edema, and the use of biological inhibitors of IL-1β. Because MCC950 9–11 thereby exacerbating hemorrhagic brain injury. As a small does not block the major antimicrobial inflammasome NLRC4 or NLRP1, specific targeting of NLRP3 will not result in molecule, the NLRP3 inflammasome inhibitor, MCC950, was recently found to selectively inhibit NLRP3 inflammasome the complete blockade of IL-1β in vivo, and antimicrobial formation and reduce pyroptosis and IL-1β signaling. In the responses may remain intact. In support of this view, we found present study, our results confirmed the ability of MCC950 that MCC950 does not affect lipopolysaccharides-induced to effectively inhibit the activation of NLRP3 inflammasome production IL-1β and TNF-α from splenocytes. Considering 35–37 components and IL-1β production in experimental ICH. In MCC950 may the immunosuppression that follows stroke, Ren et al NLRP3 Inhibitor Reduces Hemorrhagic Brain Injury 191 6. Kuramatsu JB, Huttner HB, Schwab S. Advances in the management have the advantage of less immunosuppressive effects than of intracerebral hemorrhage. 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Selective NLRP3 (Pyrin Domain–Containing Protein 3) Inflammasome Inhibitor Reduces Brain Injury After Intracerebral Hemorrhage

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© 2017 The Authors.
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0039-2499
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10.1161/STROKEAHA.117.018904
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Abstract

Selective NLRP3 (Pyrin Domain–Containing Protein 3) Inflammasome Inhibitor Reduces Brain Injury After Intracerebral Hemorrhage Honglei Ren, MD; Ying Kong, MD; Zhijia Liu, MD; Dongyun Zang, MD; Xiaoxia Yang, MD; Kristofer Wood, BS; Minshu Li, PhD; Qiang Liu, MD, PhD Background and Purpose—Intracerebral hemorrhage (ICH) is a devastating disease without effective treatment. As a key component of the innate immune system, the NOD-like receptor (NLR) family, NLRP3 (pyrin domain–containing protein 3) inflammasome, when activated after ICH, promotes neuroinflammation and brain edema. MCC950 is a potent, selective, small-molecule NLRP3 inhibitor that blocks NLRP3 activation at nanomolar concentrations. Here, we examined the effect of MCC950 on brain injury and inflammation in 2 models of ICH in mice. Methods—In mice with ICH induced by injection of autologous blood or bacterial collagenase, we determined the therapeutic potential of MCC950 and its mechanisms of neuroprotection. Results—MCC950 reduced IL-1β (interleukin-1β) production and attenuated neurodeficits and perihematomal brain edema after ICH induction by injection of either autologous blood or collagenase. In mice with autologous blood-induced ICH, the protection of MCC950 was associated with reduced leukocyte infiltration into the brain and microglial production of IL-6. MCC950 improved blood–brain barrier integrity and diminished cell death. Notably, the protective effect of MCC950 was abolished in mice depleted of either microglia or Gr-1 myeloid cells. Conclusions—These results indicate that the NLRP3 inflammasome inhibitor, MCC950, attenuates brain injury and inflammation after ICH. Hence, NLRP3 inflammasome inhibition is a potential therapy for ICH that warrants further investigation. Visual Overview—An online visual overview is available for this article. (Stroke. 2018;49:184-192. DOI: 10.1161/ STROKEAHA.117.018904.) Key Words: brain edema cell death inflammation neuroprotection ◼ ◼ ◼ ntracerebral hemorrhage (ICH) is a devastating, often fatal As a critical component of the innate immune response Iaftermath of stroke, but not generally amenable to treat- to tissue injury, activation of the NOD-like receptor (NLR) 1–5 ment. Emerging evidence has demonstrated that a stroke- NLRP3 (pyrin domain–containing protein 3) inflamma- incited inflammatory cascade accelerates the formation of some leads to the cleavage of pro–IL (interleukin)-1β and edema that surrounds hematomas, exacerbates the mass effect, pro–IL-18, as well as the subsequent release of biologically 4–8 and amplifies cell death. After the ictus of ICH, blood com- active IL-1β, IL-18, and other soluble mediators of inflamma- ponents, including leukocytes, enter the brain and activate tion. Recent evidence indicates that the ICH-induced activa- resident immune cells such as microglia. Subsequently, the tion of NLRP3 inflammasome can amplify the inflammatory infiltrating leukocytes and activated microglia boost the local response by releasing IL-1β and promoting neutrophil infiltra- 9–11 production of proinflammatory factors. Together with cell tion, thereby exacerbating brain edema. Although previous death products, these catastrophic events amplify blood–brain studies provide mechanistic insights into NLRP3 as a promis- barrier (BBB) disruption and destroy surrounding tissues, ing target for restricting neuroinflammation and brain edema contributing to the development of perihematomal edema and after ICH, they do not reflect clinical application because they 4–8 the aggravated mass effect. Therefore, brain inflammation involve either knockout models or use of pharmacological has emerged as a major modifiable determinant now targeted agents with limited potency and nonspecificity preceding ICH 7 9–11 for the development of novel ICH treatment. induction. For clinical translation of prior experimental Received July 31, 2017; final revision received November 12, 2017; accepted November 13, 2017. From the Department of Neurology, Tianjin Neurological Institute, Tianjin Medical University General Hospital, China (H.R., Y.K., Z.L., X.Y., M.L., Q.L.); Department of Neurology, Barrow Neurological Institute, St. Joseph’s Hospital and Medical Center, Phoenix, AZ (K.W., M.L., Q.L.); and Department of Neurosurgery, Tianjin Huanhu Hospital, China (D.Z.). The online-only Data Supplement is available with this article at http://stroke.ahajournals.org/lookup/suppl/doi:10.1161/STROKEAHA. 117.018904/-/DC1. Correspondence to Qiang Liu, MD, PhD, Department of Neurology, Tianjin Neurological Institute, Tianjin Medical University General Hospital, China. E-mail [email protected] © 2017 The Authors. Stroke is published on behalf of the American Heart Association, Inc., by Wolters Kluwer Health, Inc. This is an open access article under the terms of the Creative Commons Attribution Non-Commercial-NoDerivs License, which permits use, distribution, and reproduction in any medium, provided that the original work is properly cited, the use is noncommercial, and no modifications or adaptations are made. Stroke is available at http://stroke.ahajournals.org DOI: 10.1161/STROKEAHA.117.018904 184 Ren et al NLRP3 Inhibitor Reduces Hemorrhagic Brain Injury 185 outcomes, preclinical testing in relevant models is mandatory. Behavioral Assessment However, this option has been precluded by the lack of selec- Behavioral assessment was conducted at days 1 and 3 after ICH using the modified Neurological Severity Score together with corner-turning tive, potent NLRP3 inflammasome inhibitors. test to comprehensively evaluate motor, sensory, reflex, and balance Now, we have devised a means of clinically relevant testing 21–24 functions, as previously described. The range of scores for modi- in an experimental ICH model by using MCC950, a selective, fied Neurological Severity Score is from 0 to 18, defined as follows: small-molecule NLRP3 inflammasome inhibitor with proven a score of 13 to 18 indicates severe injury, 7 to 12 indicates moderate high potency in vitro and in vivo. We hypothesized that the injury, and 1 to 6 indicates mild injury. Mice were given 1 point if they failed to perform a task. The corner-turning test assesses sensorimotor pharmacological inhibition of NLRP3 inflammasome by and postural asymmetries. In this process, each mouse is allowed to administration of MCC950 would reduce inflammation and proceed into a corner with an angle of 30° and then must turn right brain edema after ICH induced by the injection of autologous or left. Each mouse repeated this procedure for 10×, with at least 30 s blood or collagenase. between trials. The percentage of right turns is then calculated. Materials and Methods Magnetic Resonance Imaging Details of materials and experimental procedures are available Lesion volume and perihematomal edema were quantified at day 3 13,16 from the online-only Data Supplement. This article adheres to the after ICH using a 7-T small animal MRI, as previously described. AHA Journals implementation of the Transparency and Openness T2-weighted image sequence and susceptibility-weighted image Promotion Guidelines. sequence were scanned to detect lesion volume and hematoma volume, respectively. Details are described in the online-only Data Supplement. Animals All animal experiments were approved by the Committee on the Brain Water Content Measurement Ethics of Animal Experiments of Barrow Neurological Institute Water contents were measured at day 3 after ICH, as previously (Phoenix, AZ). All experiments were conducted in accordance 25 described. In brief, without perfusion, brains were removed from with the National Institutes of Health (Bethesda, MD) Guide mice and divided into 3 parts: the ipsilateral, the contralateral, and for the Care and Use of Laboratory Animals, and experiments the cerebellum. The brain samples were weighed immediately on an were designed, performed, and reported according to the Animal electronic balance to obtain wet weights, then dried for 24 hours at Research: Reporting In Vivo Experiments guidelines (https:// 100°C, and weighed again to obtain dry weights. The following for- www.nc3rs.org.uk/arrive-guidelines). Male C57BL/6 mice, 8 to mula was used to calculate the brain water content: (wet weight−dry 10 weeks old (20–25 g), were purchased from the Charles River weight)/wet weight×100%. Laboratories (Wilmington, MA). Female mice were not used to avoid any influences of sex steroids. All mice were housed in ani- mal facilities under a standardized light–dark cycle and had free Flow Cytometry + int access to food and water. All animal surgeries were performed To quantify cells expressing NLRP3, microglia (CD11b CD45 ), + high + under anesthesia. Animals were randomly divided and assigned neutrophils (CD11b CD45 Ly6G ), monocyte/macrophages (CD1 + high + + high + + + to experimental groups using a random number list generated by 1b CD45 Ly6C ), CD4 T cells (CD45 CD3 CD4 ), CD8 T high + + high − + Microsoft Excel 2013. cells (CD45 CD3 CD8 ), B cells (CD45 CD3 CD19 ), NK cells high − + (CD45 CD3 NK1.1 ), cytokines produced by microglia, and cell apoptosis (Annexin V ) in the brain, we isolated cellular components Induction of ICH in Mice from brain tissue to perform flow cytometry analysis as we previously ICH was induced in mice by injection of autologous blood or bac- 23,26 described. Details appear in the online-only Data Supplement. 12–16 terial collagenase, as we previously described. Details of ICH induction are given in the online-only Data Supplement. Real-Time Polymerase Chain Reaction At day 3 after ICH, total RNA was extracted from brain tissue in the Study Design and Drug Administration ipsilateral hemisphere for real-time polymerase chain reaction analy- A total of 302 male C57BL/6 mice (Charles River Laboratories), sis as described in the online-only Data Supplement. 8 to 10 weeks old, were used in this study. In autologous blood model, the mortality rate was 8.5% (20 of total 236) and exclu- sion rate was 16.7% (36 of total 236). In the collagenase model, Assessment of BBB Permeability the mortality rate was 13.3% (8 of total 60) and exclusion rate was Evans Blue dye (Sigma, St. Louis, MO) extravasation assays were 20% (12 of total 60). Six wild-type mice were used for in vitro conducted at day 3 after ICH as a tracer to measure BBB permeabil- experiments. MCC950 was dissolved in phosphate-buffered saline, 16,27 ity, as previously described. as previously described. Mice were given MCC950 at a dose of 10 mg/kg by intraperitoneal injection at indicated time points after ICH. Mice that received an equal volume of vehicle (phosphate- Western Blots buffered saline) only were designated as controls. For the microglia At day 3 after ICH, Western blots analyzed for NLRP3 inflamma- depletion experiment, PLX3397 (Selleckchem, Houston, TX) was some components and tight junction protein expression in the ipsi- given by oral gavage at 40 mg/kg for 21 days before ICH induc- lateral cerebral hemisphere, as we previously described. Details are 16,18 tion. Drug treatment was continued until the end of experiments. given in the online-only Data Supplement. Subsequently, anti-mouse Gr-1 monoclonal antibody (MAb- RB6-8C5; BioXcell, West Lebanon, NH) was given by intraperi- Statistical Analysis toneal injection 1 day before and 1 day after ICH surgery at 250 + 19,20 µg per mouse to deplete Gr-1 myeloid cells in vivo. In mice We determined each sample size by power analysis using a signifi- subjected to microglia depletion, no mice were excluded. More than cance level of α=0.05 with 80% power to detect statistical differ- 90% of microglia were depleted in mice receiving PLX3397. In ences. SAS 9.1 software (SAS Institute Inc, Cary, NC) was used for mice subjected to Gr-1 cell depletion, the exclusion rate was 5.9% power analysis and sample size calculations. Data were analyzed by (1 of total 17 because of death after surgery). The depletion of Gr-1 investigators blinded to experimental treatments. Data are shown cells was verified by sampling circulating blood, and ≈ 90% of Gr-1 as mean±SD. SPSS 19.0 software was used to compare differences between 2 groups where appropriate. One-way ANOVA followed by cells were depleted in all mice included. 186 Stroke January 2018 the Tukey post hoc test or 2-way ANOVA with multiple compari- days starting immediately after ICH induction (Figure 1A). sons followed by Bonferroni post hoc test was used for comparison of Neurological function was evaluated by using modified multigroup data. P values <0.05 are considered significant. Neurological Severity Score and corner-turning tests at days 1 and 3 after ICH. Lesion volume, perihematomal edema, Results and brain water content were measured at day 3 after ICH. MCC950 Attenuates Brain Injury and Compared with vehicle recipients, we found that MCC950- Improves Long-Term Outcome After ICH treated mice had significantly reduced neurodeficits, lesion vol- umes, and perihematomal edema after ICH (Figure 1B and 1C). To determine whether the NLRP3 inflammasome inhibitor, MCC950 reduced brain water content after ICH (Figure I in the MCC950, affects brain injury after ICH, we examined neuro- online-only Data Supplement). In addition, MCC950 reduces deficits, lesion volume, and perihematomal edema in ICH mice receiving MCC950 or a phosphate-buffered saline vehicle. ICH neurodeficits until day 28 after ICH induction (Figure 1D), was induced by injection of autologous blood or collagenase. suggesting that NLRP3 inflammasome inhibition can provide Mice received MCC950 (10 mg/kg) or vehicle for 3 consecutive long-term benefit after ICH. Of note, the benefit of MCC950 to Figure 1. MCC950 attenuates brain injury and improves long-term outcome after intracerebral hemorrhage (ICH). ICH was induced in C57BL/6 mice by injection of autologous blood or collagenase. A, Flow chart illustrates MCC950 administration and experimental design. Mice received daily intraperitoneal (IP) injections of MCC950 (10 mg/kg) or an equal volume of phosphate-buffered saline (PBS) vehicle for 3 consecutive days starting immediately after ICH induction. B, Neurological tests were performed to evaluate the motor, sensory, and balance functions in mice receiving vehicle or MCC950 at days 1 and 3 after injection of autologous blood (left) or collagenase (right). C, T2-weighted image (T2WI) sequences were scanned to assess lesion volume at day 3 after ICH induced by injection of autologous blood (left) or collagenase (right), as outlined in red. Susceptibility-weighted sequences were assessed for hematoma lesion volume, visible in yellow regions. Quantification of lesion volume and perihematomal edema in mice receiving MCC950 or vehicle at day 3 after ICH induced by injection of autologous blood (left) or collagenase (right). n=8 mice per group. D, Mice received vehicle or MCC950 at a dose of 10 mg/kg by intraperitoneal injection. The assessments of modified Neurological Severity Score (mNSS) score and corner test were performed at days 7, 14, and 28 after ICH induced by injection of collagenase. n=10 per group. Data are presented as mean±SD. *P<0.05, **P<0.01. Ren et al NLRP3 Inhibitor Reduces Hemorrhagic Brain Injury 187 reduce neurodeficits and brain edema was restricted to within 24 cellular components as brain-infiltrating leukocytes and hours after ICH (Figure II in the online-only Data Supplement). microglia in the brains of ICH mice (Figure 3A). At day 3 post-ICH, the numbers of brain-infiltrating leukocytes, + high + neutrophils (CD11b CD45 Ly6G ), monocyte/macro- MCC950 Inhibits the Activation of + high + + high phages (CD11b CD45 Ly6C ), CD4 T cells (CD45 NLRP3 Inflammasome Components + + + high + + CD3 CD4 ), and CD8 T cells (CD45 CD3 CD8 ) were and IL-1β Production After ICH reduced in ICH mice receiving MCC950 compared with The effect of MCC950 on NLRP3 inflammasome activation those in vehicle-treated controls (Figure 3B and 3C). Of and IL-1β production was examined in brain tissues of ICH note, MCC950 reduced the infiltration of CD4 T cells, mice. At day 3 after ICH, we found that MCC950 reduced CD8 T cells and neutrophils until day 7 after ICH (Figure the mRNA expression of NLRP3 inflammasome components IV in the online-only Data Supplement). Further, microg- (NLRP3/Caspase-1/ASC) and IL-1β (Figure 2A). In addition, the protein expression of NLRP3, caspase-1, and IL-1β in the lia cell numbers decreased as did microglial expression of factor IL-6 (Figure 3D). In contrast, the microglial expres- brain was suppressed by MCC950 treatment (Figure 2B and 2C). Of interest, MCC950 does not affect lipopolysaccha- sion of IL-10 and TGF-β (transforming growth factor-β) was increased in ICH mice receiving MCC950 (Figure 3D). rides-induced production IL-1β and TNF-α (tumor necrosis factor-α) from splenocytes (Figure III in the online-only Data These results indicate that MCC950 can reduce brain inflam- Supplement). These results demonstrate that MCC950 effec- mation and cause a shift in microglia phenotype toward an tively inhibits the activation of NLRP3 inflammasome compo- anti-inflammatory status. nents and IL-1β production in the brain after ICH. MCC950 Preserves BBB Integrity and MCC950 Reduces Leukocyte Infiltration Reduces Cell Death After ICH and Production of Proinflammatory BBB disruption after ICH contributes to vasogenic edema Factors by Microglia After ICH and the expansion of perihematomal edema. To measure the Next, we determined the impact of MCC950 on brain inflam- effect of MCC950 on BBB disruption after ICH, we exam- mation after ICH. Using flow cytometry, we examined such ined Evans Blue dye extravasation and the expression of Figure 2. MCC950 inhibits NOD-like receptor (NLR) family, NLRP3 (pyrin domain-containing protein 3) inflammasome activation and IL (interleukin)-1β expression. Intracerebral hemorrhage (ICH) was induced in C57BL/6 mice by injection of autologous blood. Mice received daily intraperitoneal (IP) injections of MCC950 (10 mg/kg) or an equal volume of vehicle for 3 consecutive days starting immediately after ICH induction. At day 3 after ICH or sham surgery, brain tissues were harvested for the extraction of mRNA and protein. A, The mRNA expression levels of the NLRP3 inflammasome components and IL-1β after ICH. B, Western blot images show the expression of NLRP3, IL-1β, and caspase-1 in the brains of indicated groups of mice receiving sham plus vehicle, ICH plus vehicle, or ICH plus MCC950. C, Data points show the protein expression levels of NLRP3, IL-1β, and caspase-1 in the brains of indicated groups of mice receiving sham plus vehicle, ICH plus vehicle, or ICH plus MCC950. n=6 mice per group. Data are presented as mean±SD. *P<0.05, **P<0.01. 188 Stroke January 2018 Figure 3. MCC950 reduces leukocyte infiltration and microglia production of proinflammatory factors after intracerebral hemor - rhage (ICH). ICH was induced in C57BL/6 mice by injection of autologous blood. Mice received daily intraperitoneal (IP) injections of MCC950 (10 mg/kg) or an equal volume of vehicle for 3 consecutive days starting immediately after ICH induction. At day 3 after ICH, immune cells were isolated from brain tissues of ICH mice receiving MCC950 or vehicle. A, Gating strategy of brain-infiltrating + + + + + + − + − + immune cells including CD4 T cells (CD3 CD4 ), CD8 T cells (CD3 CD8 ), B cells (CD3 CD19 ), NK cells (CD3 NK1.1 ), monocyte/ + high + + high + + int macrophages (CD11b CD45 Ly6C ), neutrophils (CD11b CD45 Ly6G ), and microglia (CD11b CD45 ) and their expression of IL (interleukin)-6, TNF-α (tumor necrosis factor-α), TGF-β (transforming growth factor-β), and IL-10. FMO, fluorescence minus one. B , Data points show counts of brain-infiltrating leukocytes in the brains of ICH mice receiving indicated treatment. C , Data points show counts of microglia in the brains of ICH mice receiving indicated treatment. D, Data points show the counts of microglia expressing IL-6, TNF-α, TGF-β, and IL-10 in the brains of ICH mice receiving indicated treatment. n=6 mice per group. Data are presented as mean±SD. *P<0.05, **P<0.01. tight junction proteins. At day 3 after ICH, MCC950 signifi- Benefit of MCCC950 Against ICH Involves cantly ameliorated leakage of the dye (Figure 4A), denoting Microglia and Gr-1 Myeloid Cells a reduction of BBB permeability. MCC950 also preserved Because microglia are the predominant cell subset expressing expression of the tight junction proteins (claudin-5 and NLRP3 inflammasome after ICH (Figure V in the online-only ZO-1; Figure 4B). In addition, MCC950 reduced numbers of Data Supplement), we sought to understand to what extent Annexin V–expressing cells in the brain at day 3 after ICH, microglia may contribute to the protective effect of MCC950. suggesting a decrease in cell death (Figure 4C). Together, The survival of microglia depends on signaling through these results demonstrate that MCC950 preserves BBB integ- CSF1R (colony-stimulating factor 1 receptor); therefore, rity and reduces cell death after ICH. we used a CSF1R inhibitor, PLX3397, to deplete microglia Ren et al NLRP3 Inhibitor Reduces Hemorrhagic Brain Injury 189 Figure 4. MCC950 treatment preserves blood–brain barrier (BBB) integrity and reduces cell death after intracerebral hemorrhage (ICH). ICH was induced in C57BL/6 mice by injection of autologous blood. Mice received daily intraperitoneal (IP) injections of MCC950 (10 mg/kg) or an equal volume of vehicle for 3 consecu- tive days starting immediately after ICH induction. At day 3 after ICH, brain tis- sues were prepared for measurements of BBB integrity and cell death. A, Histology images show that MCC950 decreased Evens Blue dye leakage compared with vehicle (phosphate-buffered saline [PBS]) treatment. B, Western blot images show expression of the tight junction proteins, claudin-5, and ZO-1, in the brains of ICH mice receiving MCC950 or vehicle. C, Flow cytometry plots and summarized results show percentages of Annexin V cells in the brains of ICH mice receiv- ing MCC950 or vehicle. n=6 mice per group. Data are presented as mean±SD. *P<0.05, **P<0.01. 29 + + before ICH induction (Figure 5A). Consistent with our pre- An anti–Gr-1 mAb (RB6-8C5) was used to deplete Gr-1 viously described PLX3397 treatment, the present result was a myeloid cells from mice before ICH induction (Figure 6A). + int + loss of >90% microglia (CD11b CD45 ) from mice undergo- We monitored the counts of Gr-1 cells in circulating blood ing ICH. Of interest, we found that the benefit of MCC950 at 24 hours before the first injection of anti–Gr-1 mAb treatment was abolished in ICH mice receiving PLX3397 and 48 hours after the second injection of anti–Gr-1 mAb (Figure 5B and 5C), suggesting that microglia participate in (Figure 6B). Consistent with a previous report, anti– + + the beneficial effect of MCC950 after ICH. Gr-1 mAb treatment depleted ≈90% of Gr-1 myeloid cells Because NLRP3 inflammasome was clearly expressed in (Figure 6B), that is, mainly neutrophils and monocytes. Of + + Gr-1 myeloid cells, consisting mainly of neutrophils and note, the depletion of Gr-1 myeloid cells diminished the monocytes that are known contributors to BBB disruption beneficial effects of MCC950 after ICH (Figure 6C and 6D), 30,31 and brain inflammation, we therefore determined whether suggesting that the protection of MCC950 also involves + + Gr-1 myeloid cells also add to the benefit of MCC950. Gr-1 myeloid cells. Figure 5. Microglia contribute to the benefit of MCC950 after intracerebral hemorrhage (ICH). A, Schematic diagram illustrates drug administration and experimental design. C57BL/6 mice received oral gavage of PLX3397 (40 mg/kg) for 21 days and continuously until the experiment ended. ICH was induced in PLX3397-treated mice by injection of autologous blood. Thereafter, these mice received daily injections of MCC950 (10 mg/kg, intraperitoneal [IP]) or an equal volume of vehicle for 3 consecutive days starting immediately after ICH induction. At day 3 after ICH, neurodeficits and brain water content were assessed. B, Quantification of modified Neurological Severity Score (mNSS) and corner-turning test scores in indicated groups of ICH mice. C, Quantification of brain water content in indicated groups of ICH mice. n=6 per group. Data are presented as mean±SD. 190 Stroke January 2018 Figure 6. Gr-1 myeloid cells contribute to the benefit of MCC950 after intracerebral hemorrhage (ICH). A, Schematic diagram illustrates drug administration and experimental design. C57BL/6 mice received anti–Gr-1 mAb 1 day before and 1 day after ICH surgery. ICH was induced by injection of autologous blood. Thereafter, these mice received daily injections of MCC950 (10 mg/kg, intraperitoneal [IP]) or an equal volume of vehicle for 3 consecutive days starting immediately after ICH induction. At day 3 after ICH, neurodeficits and brain water content were assessed. B, Data points show the counts of circulating Gr-1 cells in mice before ICH induction, or mice receiving anti–Gr-1 mAb or IgG control at day 3 after ICH. C, Summarized results of modified Neurological Severity Score (mNSS) and corner-turning test scores in ICH mice receiving indicated treatments. D, Data points show brain water content in ICH mice receiving indicated treatments. n=8 mice per group. Data are presented as mean±SD. 9–11 advancing previous findings, our study for the first time Discussion provides efficacy data depicting pharmacological inhibition This study provides the first evidence that the NLRP3 inflam- by NLRP3 inflammasome in the setting of ICH in vivo. masome inhibitor, MCC950, attenuates hemorrhagic brain Other than MCC950, several small molecules inhibit the injury. MCC950 significantly reduced neurodeficits and NLRP3 inflammasome. For example, glyburide inhibited perihematomal edema in 2 mouse models of ICH induced IL-1β production at micromolar concentrations in response by injection of autologous blood or bacterial collagenase. to the activation of NLRP3 and was effective in reducing MCC950 treatment was sufficient to reduce the resulting leu- edema formation. Purinergic 2X7 receptor antagonist (blue kocyte infiltration, microglial production of proinflammatory brilliant G) also diminished NLRP3 inflammasome activation factors, BBB disruption, and cell death after ICH. In addition, and lessened hemorrhagic brain injury. However, because of the benefit of MCC950 protection from ICH was diminished the limited potency and nonspecific nature of those agents, in mice subjected to depletion of microglia or Gr-1 myeloid the extent to which pharmacologically selective targeting by cells (neutrophils and monocytes). Together, these results sug- gest that MCC950 has potential therapeutic value for reducing NLRP3 inflammasome can impact ICH injury is still unclear. However, results from this study provide new evidence that ICH injury. selective NLRP3 inflammasome inhibition offers the benefit Mechanistic studies have shown an essential role for the NLRP3 inflammasome in brain injury and neuroinflamma- of reducing ICH injury and improving long-term outcome, 9–11 which is an essential step for bringing NLRP3 inflammasome- tion after ICH. The activation of NLRP3 inflammasome facilitates caspase-1 activation and IL-1β processing, lead- targeted therapies from the bench into clinical application. Reportedly, NLRP3 might present certain advantages over ing to the amplification of the inflammatory response, which culminates in the expansion of perihematomal edema, and the use of biological inhibitors of IL-1β. Because MCC950 9–11 thereby exacerbating hemorrhagic brain injury. As a small does not block the major antimicrobial inflammasome NLRC4 or NLRP1, specific targeting of NLRP3 will not result in molecule, the NLRP3 inflammasome inhibitor, MCC950, was recently found to selectively inhibit NLRP3 inflammasome the complete blockade of IL-1β in vivo, and antimicrobial formation and reduce pyroptosis and IL-1β signaling. In the responses may remain intact. In support of this view, we found present study, our results confirmed the ability of MCC950 that MCC950 does not affect lipopolysaccharides-induced to effectively inhibit the activation of NLRP3 inflammasome production IL-1β and TNF-α from splenocytes. Considering 35–37 components and IL-1β production in experimental ICH. In MCC950 may the immunosuppression that follows stroke, Ren et al NLRP3 Inhibitor Reduces Hemorrhagic Brain Injury 191 6. Kuramatsu JB, Huttner HB, Schwab S. Advances in the management have the advantage of less immunosuppressive effects than of intracerebral hemorrhage. 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Stroke – Pubmed Central

Published: Jan 1, 2018

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