Xi-Lei Liu ,Dong-Dong Sun ,Mu-Tian Zheng,Xiao-Tian LiHan-Hong Niu,Lan Zhang,Zi-Wei ZhouHong-Tao RongYi WangJi-Wei WangGui-Li YangXiao LiuFang-Lian ChenYuan Zhou,Shu Zhang,Jian-Ning Zhang

Abstract Neuroinflammation and the NACHT,LRR,and PYD domains-containing protein 3 inflammasome play crucial roles in secondary tissue damage following an initial insult in patients with traumatic brain injury (TBI).Maraviroc,a C-C chemokine receptor type 5 antagonist,has been viewed as a new therapeutic strategy for many neuroinflammatory diseases.We studied the effect of maraviroc on TBI-induced neuroinflammation.A moderate-TBI mouse model was subjected to a controlled cortical impact device.Maraviroc or vehicle was injected intraperitoneally 1 hour after TBI and then once per day for 3 consecutive days.Western blot,immunohistochemistry,and TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling) analyses were performed to evaluate the molecular mechanisms of maraviroc at 3 days post-TBI.Our results suggest that maraviroc administration reduced NACHT,LRR,and PYD domains-containing protein 3 inflammasome activation,modulated microglial polarization from M1 to M2,decreased neutrophil and macrophage infiltration,and inhibited the release of inflammatory factors after TBI.Moreover,maraviroc treatment decreased the activation of neurotoxic reactive astrocytes,which,in turn,exacerbated neuronal cell death.Additionally,we confirmed the neuroprotective effect of maraviroc using the modified neurological severity score,rotarod test,Morris water maze test,and lesion volume measurements.In summary,our findings indicate that maraviroc might be a desirable pharmacotherapeutic strategy for TBI,and C-C chemokine receptor type 5 might be a promising pharmacotherapeutic target to improve recovery after TBI.

Key Words:C-C chemokine receptor type 5 (CCR5) antagonist;high mobility group protein B1 (HMGB1);maraviroc;M1 microglia;nuclear factor-κB pathway;NACHT,LRR,and PYD domains-containing protein 3 (NLRP3) inflammasome;neuroinflammation;neurological function;neurotoxic reactive astrocytes;traumatic brain injury

Introduction

Traumatic brain injury (TBI) can result in neurological disorders or death and remains a heavy burden to families and society worldwide.However,there are currently no effective treatment guidelines to mitigate the brain damage caused by TBI (Maas et al.,2017).The primary injury occurs immediately after the initial insult and can lead to cerebral contusion,cranial hematoma,and axonal injury.A variety of secondary events follow the initial insult and comprise oxidative stress,blood-brain barrier (BBB) disruption,neuroinflammation,and apoptosis (Corps et al.,2015;Johnson et al.,2018;Ismail et al.,2020).The pathogenesis of TBI is regulated by the immune system and neuroinflammation,including innate and adaptive immunity,resident microglial activation,cytokine release,and inflammasome activation(Jassam et al.,2017).

Microglia and peripheral immunocytes move to the core of the initial insult to defend against pathogens and therefore contribute to secondary injury after TBI (Corps et al.,2015).Microglia polarize into two phenotypes:classically activated M1 microglia and alternatively activated M2 microglia (Jassam et al.,2017;Simon et al.,2017).The M1 phenotype releases proinflammatory mediators that aggravate brain tissue damage.In contrast,alternatively activated M2 microglia aid brain recovery by secreting anti-inflammatory factors (Wang et al.,2013;Hu et al.,2015).In addition,it is well established that nucleotide-binding oligomerization domain-like receptor family activation is a trigger of cell pyroptosis and leads to poor outcomes after brain injury(O’Brien et al.,2020).NACHT,LRR,and PYD domains-containing protein 3 (NLRP3) has been studied in a number of acute central nervous system(CNS) disorders (Ren et al.,2018;Xu et al.,2018a;Sun et al.,2019;Chen et al.,2020).The NLRP3 inflammasome consists of the sensor protein NLRP3,the apoptosis-associated speck-like protein adapter,and the precursor enzyme pro-caspase-1.Interleukin (IL)-1β,IL-18,and the amino terminus of gasdermin-D are cleaved by activated caspase-1 from inactive proisomers to their active forms.These proteins ultimately lead to cell disruption accompanied by rapid secretion of proinflammatory cytokines (Franke et al.,2021).Thus,reduction of activation of the NLRP3 inflammasome and M1 microglia might be a promising therapeutic strategy for TBI.

Recent studies reported that CNS injury and disease could stimulate polarization of astrocytes into two different phenotypes,termed neurotoxic A1 reactive astrocytes and neuroprotective A2 reactive astrocytes,which express complement C3 and protein S100-A10,respectively (Diaz-Castro et al.,2019;Miyamoto et al.,2020;Peng et al.,2020).The astroglial transition from a resting state to the neurotoxic A1 reactive astrocyte state is activated by IL-1α,tumor necrosis factor (TNF),and complement C1q,which are released by microglia and cause the death of neuronal cells.A2 astrocytes are activated by ischemia-hypoxia and may play a neuroprotective role in CNS diseases(Liddelow et al.,2017).A1 astrocytes that highly express complement C3 were able to kill neurons and oligodendrocytes in CNS diseases by releasing verylong-chain fatty acid acyl chains and free fatty acids (Escartin et al.,2021).However,the A1 and A2 astrocyte polarization theories remain controversial.The authors of a review of reactive astrocytes argued that it is not appropriate to simply divide reactive astrocytes into A1 and A2 phenotypes and suggested that astrocytes in CNS diseases should be termed ‘reactive astrocytes’ (Escartin et al.,2021).Although nomenclature and definitions of reactive astrocytes need to be clarified,inhibiting the activation of neurotoxic astrocytes is important to the survival of neurons.

Maraviroc was the first C-C chemokine receptor type 5 (CCR5) antagonist licensed by the U.S.Food and Drug Administration and has been viewed as a new therapeutic strategy to treat neuroinflammatory diseases such as multiple sclerosis,Rasmussen encephalitis,and HIV-associated neurocognitive disorders (Martin-Blondel et al.,2016).According to recent studies,knockdown or pharmacological blocking of CCR5 enhanced motor function,strengthened learning and memory,decreased cognitive decline,and reduced lesion area and hippocampal neuron loss after stroke and TBI (Joy et al.,2019;Friedman-Levi et al.,2021).In addition,

in vivo

and

in vitro

experiments showed that CCR5 blockade decreases peripheral immune cell and microglia trafficking to the lesion region and exerts a protective effect by attenuating neuroinflammation (Glass et al.,2005;Rosi et al.,2005;Arberas et al.,2013).Taken together,these results suggested that CCR5 may be beneficial to patients suffering cerebral damage.Nevertheless,whether maraviroc alleviates TBI-induced microglial polarization and inflammasome activation has not been studied.Therefore,we hypothesized that maraviroc protects against TBI by suppressing NLRP3 inflammasome activation and modulating microglial and astrocyte polarization.

Methods

Animals

All experimental animal protocols were reviewed and approved by the Animal Care and Use Committee of Tianjin Medical University General Hospital,Tianjin,China,on January 20,2020 (approval No.IRB2020-DW-19) and conducted in strict accordance with international laws and National Institutes of Health policies,including the Guide for the Care and Use of Laboratory Animals (8ed.,2011).Male specific-pathogen-free C57BL/6J mice (8–10 weeks old,22–25 g) used in the study were supplied by Beijing Vital River Laboratory Animal Technology Co.,Ltd.,Beijing,China (license No.SCXK(Jing) 2021-0006).The mice were housed in the animal facilities at 20 ± 2°C and 45% humidity under a 12-hour light/dark cycle with food and water

ad libitum

.All 60 mice were divided into three equal groups at random using the random number table method (

n

=20/group):(1) sham group,(2) TBI +vehicle group,and (3) TBI+maraviroc group.The experimental results were obtained by a researcher who was blinded to the experimental states and treatment.

TBI model

A controlled cortical impact (CCI) device (eCCI-6.3 device,Custom Design&Fabrication,Inc.,Sandston,VA,USA) was used on the mice to induce the experimental TBI model.Briefly,intraperitoneal injection of 1% sodium pentobarbital solution (6 mL/kg,Beijing Solarbio Science &Technology Co.,Ltd.,Beijing,China) was used to deeply anesthetize the mice.Then,the mice were placed in a stereotactic apparatus (RWD Life Science Co.,Ltd.,Shenzhen,China).Subsequently,a 3.0-mm-diameter hole was made centered between bregma and lambda and lateral to the sagittal suture on the right parietal bone.The skull cap was then removed gently to ensure that the dura was intact.The moderate TBI model was induced by CCI with the following parameters:depth of 1.8 mm,velocity of 4.5 m/s,and duration time of 200 ms.After TBI,the scalp was immediately closed with 6-0 silk sutures.Each mouse was placed on a heating pad to recover from the anesthesia,and then each mouse was housed in an individual cage.Mice in the sham group were anesthetized and had the skull cap removed without CCI.

Drug administration

Maraviroc (Selleck Chemicals LLC,Houston,TX,USA) was dissolved in 5%dimethyl sulfoxide,40% polyethylene glycol 300,and 5% Tween 80 in saline,and 20 mg/kg maraviroc was injected intraperitoneally 1 hour after CCI and daily for the next 3 days.Mice receiving vehicle received identical proportions of dimethyl sulfoxide,polyethylene glycol 300,and Tween 80 in saline by intraperitoneal injection 1 hour after CCI or the sham operation.The dose of maraviroc (20 mg/kg) used in the study was selected in accordance with previous reports (Joy et al.,2019;Friedman-Levi et al.,2021).

Behavioral assessment Modified neurological severity scores

Short-term neurological function was assessed by the modified neurological severity score (mNSS) as shown in

Additional Table 1

,which comprises tests to evaluate reflexes,alertness,coordination,and motor abilities.The mNSS was used to evaluate neurological function at 1,3,7,and 14 days post-TBI (

n

=10/group).Lower scores imply better neurological outcomes.

Rotarod test

Motor coordination and balance were determined by the rotarod test(Sacks et al.,2018).The mice (

n

=10/group) were placed on moving rotarod equipment (RWD Life Science Co.,Ltd.) and tested using the rotarod protocol(Xu et al.,2018b).The mice received a trial at a slow speed (4 r/min) to familiarize them with the test and four consecutive trials with acceleration(from 4 to 40 r/min) to record baseline latency on the day before TBI.Data were collected at 1,3,7,and 14 days post-TBI.Each trial ended when the mouse fell off the rod or after a maximum of 5 minutes.

Morris water maze

The spatial learning and memory abilities of the mice were measured using a Morris water maze (MWM) 15–21 days post-TBI (Ran et al.,2020).The MWM pool consisted of a stainless steel cylindrical pool (122 cm in diameter and 51 cm in height) with a submerged hidden platform (10 cm in diameter).The MWM apparatus was filled with water 22 ± 2°C and dyed white with nontoxic paint.The experiment was separated into two consecutive phases:a training phase of 15–20 days and a spatial memory test phase of 21 days.In the probe phase,there were 4 trials of the latency test each day,with 90 seconds for each test.The mice (

n

=10/group) were placed in each quadrant of the pool in turn,from the first to the fourth quadrant.Then,they were allowed to seek the hidden platform for 90 seconds and to stay on the platform for 5 seconds.If a mouse did not find the platform within 90 seconds,it was placed on the platform for 15 seconds,and the time was recorded as 90 seconds.In the test phase,the mice were placed diagonally opposite the platform quadrant with the platform removed and allowed to find the platform site for 90 seconds.The platform crossing times,escape latency times,swimming traces,and the training phase were recorded and analyzed with a video tracking system (EthoVision XT 13,Noldus Information Technology,Wageningen,the Netherlands).

Western blotting

The mice (

n

=5/group) were euthanized 3 days post-CCI.To prevent contamination of experimental results by blood proteins,the mice were transcardially perfused with cold phosphate-buffered saline (PBS).Brain samples were removed as previously reported (Chen et al.,2021) and homogenized with a triturator in ice-cold radioimmunoprecipitation assay buffer (Beijing Solarbio Science &Technology Co.,Ltd.) with protease and phosphatase inhibitors (Beijing Solarbio Science &Technology Co.,Ltd.) for 30 minutes.After centrifugation at 12,000 r/min at 4°C for 10 minutes,the supernatants of homogenates were collected,4× loading buffer was added,and the samples were heated for 10 minutes at 95°C.Protein concentration was measured using a BCA (bicinchoninic acid) Protein Assay Kit (Thermo Fisher Scientific,Waltham,MA,USA).Equivalent protein (8 μg) was resolved using 10–15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels.Then,the resolved proteins were transferred onto presoaked polyvinylidene fluoride membranes(MilliporeSigma,Burlington,MA,USA) by electroblotting,and the blots were incubated with 5% skim milk dissolved in Tris-buffered saline-Tween 20 at room temperature for 2 hours.Afterward,the membranes were incubated at 4°C for 24 hours with the primary antibodies shown in

Table 1

.The membranes were washed three times with Tris-buffered saline-Tween 20 buffer and subsequently submerged in the appropriate horseradish peroxidase-conjugated secondary antibody (1:5000,all from ZSGB-Bio,Beijing,China) at room temperature for 1 hour as follows:goat anti-rabbit (Cat# ZB-2301,RRID:AB_2747412),goat anti-mouse (Cat# ZB-2305,RRID:AB_2747415),goat anti-rat (Cat# ZB-2307),rabbit anti-goat (Cat# ZB-2306,RRID:AB_2868454).Protein bands were visualized with an enhanced chemiluminescence system (MilliporeSigma).The protein expression level was determined by ImageJ software (version 1.8.0,National Institutes of Health,Bethesda,MD,USA;Schneider et al.,2012) and was standardized to that of β-actin.

Immunohistochemical staining

At 3 days post-CCI,the mice (

n

=5/group) were deeply anesthetized and killed by transcardiac perfusion with PBS followed by 4% paraformaldehyde.The brains were carefully removed and submerged overnight in 4%paraformaldehyde.The brains were moved to 15% and then 30% sucrose solutions over 24 hours at 4°C to dehydrate the tissue.After dehydration,the brains were cut into 5-mm sections as previously reported (Yan et al.,2020).The brain sections were immersed in optimal cutting compound temperature medium (Sakura Finetek USA,Torrance,CA,USA) and cut into 8-μm coronal sections using a cryostat microtome (CM1950,Leica Biosystems,Nu?loch,Germany).The coronal sections were rinsed with PBS for 10 minutes to remove the optimal cutting compound temperature medium and then were permeabilized and blocked with 0.2% Triton X-100 (MilliporeSigma) and 3%bovine serum albumin for 1.5 hours.The sections were incubated overnight at 4°C with the primary antibodies shown in

Table 1

.After washing with PBS,the sections were immersed in the corresponding Alexa Fluor-conjugated IgG(1:500,all from Thermo Fisher Scientific) for 1 hour at room temperature as follows:donkey anti-rabbit IgG,Alexa Fluor 488 (Cat# A-21206),donkey anti-rabbit IgG,Alexa Fluor 555 (Cat# A-31572),donkey anti-mouse IgG,Alexa Fluor 488 (Cat# A-21202),donkey anti-mouse IgG,Alexa Fluor 555 (Cat#A-31570),donkey anti-rat IgG,Alexa Fluor 488 (Cat# A-21208),donkey anti-goat IgG,Alexa Fluor Plus 555 (Cat# A-32816).Finally,4′,6-diamidino-2-phenylindole (Abcam,Cambridge,UK) was applied to counterstain the nuclei.A fluorescence microscope (IX73,Olympus Corporation,Tokyo,Japan) was used to take micrographs of every slice.We captured five fields of view for every section and for each sample in the same regions of the pericontusional cortex.The number of cells and the fluorescence intensity were determined using ImageJ software (Schneider et al.,2012).

Table 1 ｜ Primary antibody used in this study

Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining

We determined apoptosis of neurons in the brain tissue sections at 3 days post-TBI using an In Situ Cell Death Detection Kit (Roche,Basel,Switzerland).The mouse brain slices (

n

=5/group) were rinsed with PBS and incubated with 0.2% Triton X-100 and 3% bovine serum albumin for 1.5 hours.Each section was stained with rabbit anti-neuronal nuclear protein antibody shown in

Table 1

overnight at 4°C.The sections were warmed to room temperature for 30 minutes and subsequently washed with PBS three times.Then,the sections were incubated with Alexa Fluor 555 donkey anti-rabbit IgG (Cat#A-31572,Thermo Fisher Scientific) and fixed with TUNEL reaction solution for 60 minutes at 37°C.The counterstaining of nuclei was conducted with 4′,6-diamidino-2-phenylindole for 5 minutes.

Hematoxylin and eosin staining and measurement of lesion volume

Lesion volume was detected as previously described (Xu et al.,2018a).After the MWM test,the mice (

n

=6/group) were euthanized,the brains were removed and embedded in paraffin,and 5-μm coronal sections were sliced at intervals of 120 μm (approximately 25 sections/brain).For hematoxylin and eosin (H&E) staining,the sections were deparaffinized with xylene and an alcohol gradient,and the sections were counterstained with H&E (Beijing Solarbio Science &Technology Co.,Ltd.) for 5 minutes.Then,the sections were dried and mounted with neutral balsam (Beijing Solarbio Science &Technology Co.,Ltd.) followed by observation using a light microscope (IX73,Olympus Corporation).The volume of each tissue lesion was calculated by measuring the area of the lesion in the contralateral and ipsilateral hemispheres using ImageJ software.Then,the lesion volume (%) was calculated as follows:(interval distance × lesion volume of each section)/area of the contralateral hemisphere × 100.

Statistical analysis

No statistical methods were used to predetermine sample sizes;however,our sample sizes are similar to those reported in previous publications (Xu et al.,2018a).All data are based on at least three independent experiments.Measurement data are shown as the mean ± standard deviation (SD).All statistical analyses were performed using SPSS 22.0 software (IBM Corp.Released 2013.IBM SPSS Statistics for Windows,Version 22.0.Armonk,NY:IBM Corp.).To analyze the neurobehavioral evaluation data,two-way analysis of variance followed by Tukey’s

post hoc

test was performed.Other multigroup comparisons were performed using one-way analysis of variance followed by Tukey’s

post hoc

test.

P

<0.05 was deemed statistically significant.

Results

Administration of maraviroc improves neurological functions after traumatic brain injury

All experimental designs are shown in

Figure 1A

.To investigate whether administration of maraviroc is protective in mice with TBI,we conducted mNSS tests,rotarod tests,and MWM tests.In the mNSS test,animals in the TBI+vehicle group had much higher scores than the sham group at any time point measured.Maraviroc treatment significantly alleviated the impairment of motor abilities between the third and seventh days post-TBI (

Figure 1B

),and mice in all groups reached full spontaneous recovery at 14 days post-TBI.The rotarod test results showed that the mice in the TBI+vehicle group had the worst motor coordination and balance,and mice in the TBI+maraviroc group displayed significant improvement on the third and seventh days post-TBI (

Figure 1C

).In the MWM tests,the escape latency of the TBI+vehicle group was more than the sham group,and administration of maraviroc decreased the escape latency compared with the TBI+vehicle group on days 19 and 20 (

Figure 1D

).There was no significant difference in the swim speed of all groups (

Figure 1G

).Once we removed the hidden platform at 21 days post-TBI to evaluate the number of crossings,a significant decrease in crossing number was observed in the TBI+maraviroc group compared with the TBI+vehicle group (

Figure 1E

).Furthermore,the TBI+vehicle group spent less time in the target region than the sham group,while maraviroc treatment ameliorated this phenomenon (

Figure 1F

and I).The mice in the maraviroc group traveled a shorter distance while searching for the platform compared with those in the TBI+vehicle group (

Figure 1H

).

Administration of maraviroc enhances tissue preservation after traumatic brain injury

At 21 days post-TBI,H&E staining of brain tissue showed that the sham group had no gross lesion to the cortex,while noticeable damage was observed in the TBI+vehicle group.The TBI+maraviroc group had more tissue preservation than the TBI+vehicle group (

Figure 1J

and

K

).

Administration of maraviroc regulates microglial polarization and reduces neutrophil and macrophage infiltration after traumatic brain injury

Microglia convert from the resting type into the M1 and M2 phenotypes after TBI;this process plays a vital role in the neuroinflammatory response (Long et al.,2020).To determine the effect of maraviroc on microglial polarization,coimmunofluorescence staining of a panmicroglial marker (ionized calciumbinding adapter molecule 1 [Iba-1]),an M1 microglial marker (inducible nitric oxide synthase [iNOS]),and an M2 microglial marker (macrophage mannose receptor 1 [CD206]) was performed to determine the shifts in microglial polarization 3 days after TBI.Maraviroc administration remarkably decreased the number of Iba-1-positive cells that also expressed iNOS and increased the expression of CD206 in the perilesional area,indicating an M1-to-M2 microglial transition (

Figure 2A–D

).Furthermore,western blot analysis at 3 days post-TBI in the lesioned cortex showed that iNOS expression was inhibited and CD206 expression was significantly increased after maraviroc administration.However,the protein expression level of Iba-1 in tissues from mice in the maraviroc treatment group did not significantly differ from that in mice in the vehicle treatment group (

Figure 2E–H

).In addition,immunofluorescence staining showed the accumulation of adhesion G protein-coupled receptor E1 (cell surface glycoprotein F4/80 [F4/80])-positive and lymphocyte antigen 6G (Ly-6G)-positive cells in the pericontusional region in the TBI+vehicle group compared with that in the TBI+maraviroc group at 3 days post-TBI (

Figure 3A–D

).

Figure 1 ｜ Neuroprotective effect of maraviroc on neurological function and cortical lesion size after traumatic brain injury.

Figure 2 ｜ Effect of maraviroc on microglial activation and polarization in the perilesional cortex area of mice after traumatic brain injury.

Figure 3 ｜ Effect of maraviroc on neutrophil and macrophage infiltration after traumatic brain injury.

Figure 4 ｜ Effect of maraviroc on the HMGB1/NF-κB pathway and inflammatory cytokines in the perilesional cortex after traumatic brain injury.

Administration of maraviroc inhibits the HMGB1/NF-κB pathway and alters the inflammatory response in the pericontusional cortex after traumatic brain injury

High mobility group protein B1 (HMGB1) translocation and release have been shown to activate microglia and exacerbate neuroinflammation induced by TBI (Paudel et al.,2020).The nuclear factor kappa B (NF-κB) pathway is related to high expression levels of HMGB1 and the subsequent release of inflammatory factors.The HMGB1/NF-κB pathway may play a critical role in the pathological process of TBI (Chen et al.,2018).Western blot analysis illustrated that the expression levels of HMGB1 and NF-κB p65 in the lesioned cortex were significantly increased at 3 days after TBI (

Figure 4A

,

E

,and

F

).In contrast,administration of maraviroc effectively decreased HMGB1 and NF-κB p65 protein expression compared with vehicle treatment after TBI.Moreover,a western blot assay of proinflammatory cytokine levels showed that maraviroc treatment significantly inhibited the expression of these inflammatory factors compared with vehicle treatment after TBI (

Figure

4A–D

).Immunofluorescence staining further demonstrated that the TBI +maraviroc group had a significantly reduced percentage of cells with nuclei that stained positive for NF-κB p65 compared with that in the TBI+vehicle group (

Figure 4G

and

H

).

Administration of maraviroc suppressed NLRP3 inflammasome activation after traumatic brain injury

Western blotting and immunofluorescence were conducted to determine the NLRP3 inflammasome expression levels among different groups.The TBI +vehicle group had elevated levels of NLRP3,caspase-1 p20,apoptotic speckcontaining protein,IL-18,IL-1β,and gasdermin-D compared with the sham group (

Figure 5A–G

).The TBI+maraviroc group had significantly decreased protein levels of the NLRP3 inflammasome compared with the TBI+vehicle group.In addition,immunofluorescence analysis revealed that the elevated caspase-1 p20 immunoreactivity in the pericontusional cortex was greatly alleviated by maraviroc administration compared with vehicle administration(

Figure 5H

and

I

).

Administration of maraviroc decreased the activation of neurotoxic reactive astrocytes

We estimated whether maraviroc treatment suppressed neurotoxic reactive astrocytes using immunohistochemical and western blot assays.The expression levels of glial fibrillary acidic protein (GFAP) were significantly increased in the TBI+vehicle group in the ipsilateral hemisphere compared with the sham group (

P

=0.0091),but there was no difference between the TBI+vehicle group and the TBI+maraviroc group (

P

=0.3987;

Figure 6A

and

B

).However,we observed that the protein levels of the neurotoxic reactive astrocyte-associated marker complement C3 were elevated in the vehicletreated group compared with the sham group and decreased after maraviroc administration (

Figure 6C

).Coimmunofluorescence revealed that the maraviroc treatment group exhibited a notable decline in the number of C3-positive and GFAP-positive astrocytes compared with the vehicle treatment group in the lesioned cortex 3 days post-TBI (

Figure 6D

and

E

).

Administration of maraviroc protects neurons against traumatic brain injury-induced neuronal apoptosis

Excessively activated inflammatory responses,NLRP3 inflammasomes,and A1 astrocytes are closely related to the prevalence of apoptosis (Liu et al.,2013;Roth et al.,2014;Liddelow et al.,2017;Skelly et al.,2019).We estimated the effect of maraviroc on neural cell death at 3 days post-TBI,and western blotting analyses were performed to quantify apoptotic cells.Maraviroc administration decreased the levels of cleaved caspase-3 and the apoptosis regulator BAX compared with vehicle administration (

Figure 7A

and

B

).In addition,double staining with the TUNEL assay and neuronal nuclear protein revealed many more apoptotic cells in the vehicle treatment group than in the sham group,but maraviroc treatment significantly decreased the apoptotic index compared with vehicle treatment (

Figure 7C–E

).

Figure 5 ｜ Effect of maraviroc on the expression of the NLRP3 inflammasome after traumatic brain injury.

Figure 6 ｜ Effect of maraviroc on neurotoxic reactive astrocyte activation in the pericontusional cortex of mice with traumatic brain injury.

Figure 7 ｜ Effect of maraviroc on neuronal apoptosis at 3 days after traumatic brain injury.

Discussion

The major points of our present study are as follows:administration of maraviroc,an U.S.Food and Drug Administration-approved drug,alleviated neurological deficits and resulted in neurological function recovery after TBI;maraviroc treatment enhanced tissue preservation after TBI;maraviroc treatment regulated microglial polarization,reduced neutrophil and macrophage infiltration and NLRP3 inflammasome activation,and inhibited the HMGB1/NF-κB pathway and subsequent release of inflammatory factors after TBI;and maraviroc treatment inhibited neuronal apoptosis and reduced complement C3 and caspase-3 expression levels.

Neuroinflammation exerts a vital effect on the physiological process of TBI (Morganti-Kossmann et al.,2019).At the early stage of TBI,resident microglia are activated,and peripheral neutrophils are recruited to the perilesional cortex.Subsequently,chemokine signaling causes the recruitment and infiltration of immunocytes into the lesioned cortex(Jassam et al.,2017).Meanwhile,inflammatory cytokines,such as TNF-α,IL-1β,and IL-6,are released by these immunocytes.Excessive posttraumatic neuroinflammation contributes to secondary brain damage and neuronal cell death in the perilesional cortex and hippocampus and exacerbates neurological dysfunctions (Morganti-Kossmann et al.,2019).Microglia rapidly respond to brain injury and are then recruited to the pericontusional cortex and release inflammatory cytokines,ultimately resulting in axonal injury and neuronal cell death after TBI (Witcher et al.,2015).Moreover,activated microglia polarize from the proinflammatory M1 subtype to the anti-inflammatory M2 subtype to regulate neuroinflammation (Wang et al.,2013;Hu et al.,2015).M1 microglia infiltrate lesioned cortex areas at 7 days poststroke,and M2 microglia are the main subtype present at 3 days poststroke (Xiong et al.,2016).Ample evidence indicates that knocking out or pharmacologically inhibiting CCR5 suppresses the inflammatory response by alleviating leukocyte,T cell,and macrophage infiltration and by promoting M2 macrophage activation (Glass et al.,2005;Rosi et al.,2005;Arberas et al.,2013;Long et al.,2020).Our study demonstrated that maraviroc could decrease neutrophil and macrophage infiltration and proneuroinflammatory cytokine release.Furthermore,we expand the notion that maraviroc treatment encourages a shift from M1 microglia toward M2 microglia to inhibit progressive inflammation and the destruction of the lesioned cortex 3 days post-TBI.

NLRP3 inflammasome activation leads to cleavage of the precursor form of caspase-1,the release of IL-1β and IL-18,and the induction of neuronal degradation (Feng et al.,2021).Recently,the proinflammatory effects of the NLRP3 inflammasome in stroke,multiple sclerosis,Alzheimer’s disease,Parkinson’s disease,and TBI have been described (Heneka et al.,2013;Ren et al.,2018;Malhotra et al.,2020;Kwon et al.,2021).Thus,the therapeutic strategy targeting the NLRP3 inflammasome could potentially improve neurological outcomes after TBI.As shown in our studies,the expression levels of pyroptosis-related proteins were higher in the lesioned cortex of mice subjected to TBI.In contrast,maraviroc treatment significantly decreased the levels of these proteins.HMGB1,an endogenous damage-associated molecule widely expressed in microglia,exerts a proinflammatory effect(Lee et al.,2014).Binding of HMGB1 to toll-like receptor 4 and advanced glycosylation end product-specific receptor activates p38 and NF-κB to amplify the inflammatory response.Recent studies have shown that activation of the HMGB1/toll-like receptor 4 or HMGB1/advanced glycosylation end product-specific pathways results in the activation of NF-κB to exacerbate the inflammatory cascade (Crews et al.,2013;Jia et al.,2019).In addition,several studies have illustrated that activation of NLRP3 is triggered by HMGB1,which leads to pyroptosis-mediated cell death in endothelial cells and acute pancreatitis (Jia et al.,2019;Wu et al.,2021).Our study demonstrates that maraviroc mitigates the protein levels of HMGB1 and NF-κB in the perilesional cortex at 3 days post-TBI and is the first study,to our knowledge,that links CCR5 receptor inhibition to the HMGB1/NF-κB/NLRP3 pathway.

Astrocytes are widely distributed in the mammalian CNS and perform numerous essential functions.Astrocytes undergo a process called astrogliosis to become“reactive astrocytes”in reaction to CNS injury (Zamanian et al.,2012).Previous studies reported that reactive astrocytes restrict neuroinflammation,BBB repair,neuronal protection,and neurologic function recovery (Sofroniew,2015;Almad and Maragakis,2018;G?bel et al.,2020).However,reactive astrocytes can exert negative effects,such as aggravating inflammation or interfering with axon growth (Silver and Miller,2004).Recently,a study demonstrated that reactive astrocytes were polarized into neurotoxic reactive astrocytes (A1 astrocytes) and neuroprotective reactive astrocytes (A2 astrocytes) in response to neuroinflammation and ischemia,respectively (Liddelow et al.,2017).IL-1α,TNF-α,and complement C1q are released by activated microglia,causing the activation of A1 astrocytes in CNS injuries and diseases,such as Alzheimer’s disease,Parkinson’s disease,stroke,and TBI (Goetzl et al.,2018;Yun et al.,2018;Clark et al.,2019;Cao et al.,2021).A1 astrocytes lose their fundamental functions and exert neurotoxic functions,such as inducing the death of neuronal cells and mature oligodendrocytes (Liddelow et al.,2017).Neurotoxic reactive astrocytes that highly expressed complement C3 play neurotoxic roles in CNS diseases by releasing very-long-chain fatty acid acyl chains and free fatty acids (Escartin et al.,2021).Targeting neurotoxic reactive astrocytes may be a potential approach to promote the preservation of neuronal cells.In our study,we demonstrated that maraviroc had no effect on the activation of astrocytes or GFAP expression in the ipsilateral hemisphere.However,maraviroc induced a significant reduction in complement C3,which is a neurotoxic reactive astrocyte marker,in the pericontusional cortex 3 days postinjury.Double immunofluorescence staining of GFAP and complement C3 confirmed that maraviroc inhibits A1 astrocyte activation.The NF-κB signaling pathway is involved in physiopathological processes after TBI,such as neuroinflammatory reactivity in astrocytes and microglia and cell survival.Furthermore,downregulation of NF-κB and upregulation of phosphatidylinositol 3-kinase/protein kinase B regulates the shift from the A1 to the A2 phenotype (Xu et al.,2018b).Notably,our results suggest that maraviroc might suppress neurotoxic reactive astrocyte alterations by regulating the NF-κB pathway.To the best of our knowledge,our study illustrates for the first time that maraviroc exerts a neuroprotective role by modulating neurotoxic reactive astrocyte activation and reducing neuronal cell loss.

Maraviroc is the primary CCR5 antagonist licensed by the U.S.Food and Drug Administration and has been considered a novel therapy in various neuroinflammatory diseases except for human immunodeficiency virus treatment.A study demonstrated that CCR5 plays a crucial role in HIV infection and pathogenesis because CCR5delta32,a mutant allele of the CCR5 gene,confers relative resistance to HIV infection (Dean et al.,1996).The identification of CCR5delta32 promoted the discovery and development of CCR5 inhibitors,such as maraviroc (Xu et al.,2014).Ample evidence has confirmed that maraviroc contributes to neurological function recovery after CNS injuries.A recent study demonstrated that people carrying the CCR5delta32 mutation have better cognitive function after stroke (Joy et al.,2019).However,the neuroprotective role of maraviroc in TBI mediated by inhibition of NLRP3 inflammasome activation has not been revealed to date.Our results first confirmed that administration of maraviroc attenuated neuroinflammation by regulating microglial polarization and reducing neutrophil infiltration,inflammatory cytokine production,and the activation of the HMGB1/NF-κB/NLRP3 pathway after TBI.Furthermore,maraviroc administration protected neuronal cells against apoptosis by decreasing the expression of caspase-3 and BAX.Maraviroc administration also inhibited neurotoxic reactive astrocyte activation and the caspase-3 pathway to exert antiapoptotic effects.

Neurologic dysfunction,including short-term neurologic dysfunction and long-term cognitive dysfunction,is common after brain injury,and more than half of patients with TBI experience TBI-induced chronic cognitive impairment(Rabinowitz and Levin,2014).TBI induces the apoptosis of hippocampal neurons,which is responsible for cognitive deficits in the chronic postinjury phase (Yang et al.,2016).Our findings reveal that maraviroc treatment promotes the survival of neurons by inhibiting neuroinflammation,caspase-3 expression,and neurotoxic reactive astrocyte activation and improves cognitive function recovery after TBI.

Our study had some limitations.We only focused on the potential antiinflammatory effects of maraviroc without investigating its effects on BBB leakage and endothelial dysfunction following TBI.Further studies are required to determine the effects of maraviroc on BBB function after TBI.

In summary,our study provides compelling evidence that maraviroc could attenuate neuroinflammation and regulate the polarization of microglia and astrocytes after TBI via pharmacological blockade of the CCR5 receptor.Thus,the CCR5 receptor might be a promising pharmacotherapeutic target after TBI.

Acknowledgments:

The authors are grateful to Ying Li,Lei Zhou,Hao Liang,Weiyun Cui and Li Liu from the Tianjin Neurological Institute for providing technical support.

Author contributions:

JNZ,SZ and YZ designed the experiments.XLL,DDS,MTZ and HHN carried out the experiments.MTZ and HHN analyzed the experimental results.XL and DDS wrote the manuscript.XL,LZ,YW,ZWZ,HTR,JWW,GLY,XL and FLC took part in the experiments and proposed some suggestions.All authors approved the final version of this paper.

Conflicts of interest:

The authors have no conflict of interest to declare.

Availability of data and materials:

All data generated or analyzed during this study are included in this published article and its supplementary information files.

Open access statement:

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Additional file:

Modified Neurological Severity Scoring (mNSS).