key: cord-0023323-bzj7m0od
authors: Yan, Jun; Xu, Weilin; Lenahan, Cameron; Huang, Lei; Wen, Jing; Li, Gaigai; Hu, Xin; Zheng, Wen; Zhang, John H.; Tang, Jiping
title: CCR5 Activation Promotes NLRP1-Dependent Neuronal Pyroptosis via CCR5/PKA/CREB Pathway After Intracerebral Hemorrhage
date: 2021-11-01
journal: Stroke
DOI: 10.1161/strokeaha.120.033285
sha: d7c1f92fc60b57ba526f5379e0d02a9bdc84e753
doc_id: 23323
cord_uid: bzj7m0od

Neuronal pyroptosis is a type of regulated cell death triggered by proinflammatory signals. CCR5 (C-C chemokine receptor 5)-mediated inflammation is involved in the pathology of various neurological diseases. This study investigated the impact of CCR5 activation on neuronal pyroptosis and the underlying mechanism involving cAMP-dependent PKA (protein kinase A)/CREB (cAMP response element binding)/NLRP1 (nucleotide-binding domain leucine-rich repeat pyrin domain containing 1) pathway after experimental intracerebral hemorrhage (ICH). METHODS: A total of 194 adult male CD1 mice were used. ICH was induced by autologous whole blood injection. Maraviroc (MVC)—a selective antagonist of CCR5—was administered intranasally 1 hour after ICH. To elucidate the underlying mechanism, a specific CREB inhibitor, 666-15, was administered intracerebroventricularly before MVC administration in ICH mice. In a set of naive mice, rCCL5 (recombinant chemokine ligand 5) and selective PKA activator, 8-Bromo-cAMP, were administered intracerebroventricularly. Short- and long-term neurobehavioral assessments, Western blot, Fluoro-Jade C, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), and immunofluorescence staining were performed. RESULTS: The brain expression of CCL5 (chemokine ligand 5), CCR5, PKA-Cα (protein kinase A-Cα), p-CREB (phospho-cAMP response element binding), and NLRP1 was increased, peaking at 24 hours after ICH. CCR5 was expressed on neurons, microglia, and astrocytes. MVC improved the short- and long-term neurobehavioral deficits and decreased neuronal pyroptosis in ipsilateral brain tissues at 24 hours after ICH, which were accompanied by increased PKA-Cα and p-CREB expression, and decreased expression of NLRP1, ASC (apoptosis-associated speck-like protein containing a CARD), C-caspase-1, GSDMD (gasdermin D), and IL (interleukin)-1β/IL-18. Such effects of MVC were abolished by 666-15. At 24 hours after injection in naive mice, rCCL5 induced neurological deficits, decreased PKA-Cα and p-CREB expression in the brain, and upregulated NLRP1, ASC, C-caspase-1, N-GSDMD, and IL-1β/IL-18 expression. Those effects of rCCL5 were reversed by 8-Bromo-cAMP. CONCLUSIONS: CCR5 activation promoted neuronal pyroptosis and neurological deficits after ICH in mice, partially through the CCR5/PKA/CREB/NLRP1 signaling pathway. CCR5 inhibition with MVC may provide a promising therapeutic approach in managing patients with ICH.

role in the pathogenesis of secondary brain injury. 3 However, the molecular mechanisms underlying inflammation-induced neuronal cell death are complex and poorly understood in the setting of ICH.

Inflammasome-mediated regulated cell death, namely pyroptosis, is identified as an important mechanism of inflammation-induced neuronal cell death in a variety of neurological diseases. 4 NLRP1 (nucleotide-binding domain leucine-rich repeat pyrin domain containing 1) is an NLR family member. A wealth of literature suggests that NLRP1 inflammasomes are important drivers of the caspase-1 cleavage. The activated caspase-1 subsequently promotes the maturation and release of proinflammatory IL (interleukin)-1β and IL-18 via formation of plasma membrane pores, namely, cell pyroptosis. 5 In the brain, NLRP1 inflammasomes are primarily expressed by neurons. 5 Thus, targeting NLRP-1 inflammasome-mediated neuronal pyroptosis may provide new insight and a theoretical basis for developing an effective treatment for ICH. CCR5 (C-C chemokine receptor 5) is a 7-transmembrane G-protein-coupled receptor that participates in leukocyte recruitment to areas of tissue damage during inflammatory responses and has been shown to be a viable target of anti-inflammatory therapy. Maraviroc (MVC)-an Food and Drug Administration-approved selective CCR5 antagonist for patients with HIV-improved outcomes in animal models of immune-mediated acute and chronic tissue inflammation. 6 Recent studies have also demonstrated that CCR5 inhibition promoted early recovery of motor function after traumatic brain injury and cerebral ischemia in rodents. 7 Interestingly, the CREB (cAMP response element binding) protein is reportedly one of the downstream pathway proteins involved in CCR5 signaling. 8 The downregulation of neuronal CCR5 in mice with HIV enhanced the plasticity of cortical neurons and the recovery of learning and memory function by increasing CREB protein level. 8 The cAMP-dependent PKA (protein kinase A) is a vital kinase in CREB activation 9 that phosphorylates CREB at the serine 133 site. The active CREB further activated the gene-related synaptic plasticity responsible for participating in long-term memory formation. 9 Although CREB often promotes antiinflammatory immune responses, 10 its effects on NLRP1 inflammasome have not been explored.

In the current study, we hypothesized that the activation of CCR5 promoted NLRP1-mediated pyroptosis after ICH. MVC-mediated CCR5 inhibition would attenuate neurological deficits and decrease NLRP1-dependent neuronal pyroptosis through activation of the PKA/ CREB signaling pathway after ICH in mice.

The data that support the findings of this study are available from the corresponding author upon reasonable request. Detailed description of the Materials and Methods is provided in Methods in the Supplemental Material. The authors declare that all supporting data are available within the article and the Supplemental Material. We provide the detailed statistics in the Data Set in the Supplemental Material. All experimental protocols and procedures were approved by the Ethics Committee of the Guangxi Medical University, and in accordance with the National Institutes of Health guidelines.

The overall mortality was 5.15% (10/194) . None of the sham-operated mice died in this study. The mortality did not significantly differ among the experimental ICH groups. None of the ICH mice were excluded (Table I  in Figure 1A ).

PKA-Cα and p-CREB-the potential downstream proteins of CCR5-were also elevated temporarily after ICH in a similar pattern to CCR5. NLRP1 inflammasome expression was significantly upregulated at 6 hours Nonstandard Abbreviations and Acronyms

C-C chemokine ligand 5 CCR5

C-C chemokine receptor type 5 CREB cAMP response element binding protein

nucleotide-binding domain leucine-rich repeat pyrin domain containing 1 PKA protein kinase A rCCL5

recombinant C-C chemokine ligand 5 (0.78±0.11 versus 0.46±0.04; P<0.001) and peaked at 72 hours (0.78±0.08 versus 0.46±0.04; P<0.001) after ICH when compared with the sham group ( Figure 1A ). Double immunofluorescence staining revealed that CCR5 was mainly expressed on the neurons, microglia, and astrocytes of the ipsilateral basal cortex at 24 hours after ICH ( Figure 1B ). Figure 2C ) at 72 hours after ICH.

Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining and Fluoro-Jade C staining were each used to assess whether MVC treatment reduced neuronal cell death. Specifically, costaining of neuronal marker, neuronal nuclear (NeuN), with the cleaved caspase-1 was used to assess neuronal pyroptosis 24 hours after ICH. TUNEL-positive neurons (34.83±4.31 versus 3.5±2.17; P<0.001) and Figure 4A ).

In the Morris water maze test, the ICH+vehicle group showed significantly longer swim distance and escape latency compared with the sham group. However, the MVC treatment significantly decreased the escape latency and swim distance on blocks 3 to 5 compared with the ICH+vehicle group (P<0.001; Figure 4A ). In the probe Figure 5A ). In addition, MVC significantly increased protein levels Figure 6B ). Conversely, PKA activator, 8-Bromo-cAMP, reversed the effects of rCCL5 by significantly increasing PKA-Cα expression in the naive+rCCL5+8-Bromo-cAMP group when compared with the naive+rCCL5+vehicle group (1.02±0.19 versus 0.58±0.08; P<0.001; Figure 6B ).

In the present study, we explored the effects of CCR5 antagonist, MVC, on inhibiting NLRP1-mediated pyroptosis through activation of the PKA/CREB signaling pathway in a mouse model of ICH. The novel findings are as follows: (1) the expression of CCR5 and CCL5 and the downstream effectors of PKA Cα, CREB, and NLRP1 were upregulated and peaked at 24 hours after ICH. CCR5 was expressed in neurons, astrocytes, and microglia; (2) intranasal administration of MVC at a dose of 150 μg/kg per day significantly improved short-and long-term neurobehavioral outcomes, reduced neuronal pyroptosis, increased brain expression of PKA-Cα and p-CREB, but decreased expression of NLRP1, ASC, C-caspase-1, N-GSDMD, IL-1β, and IL-18 at 24 hours after ICH. Conversely, the CREB inhibitor reversed these effects of MVC; (3) the activation of brain CCR5 using rCCL5 via intracerebroventricular injection administration resulted in neurological deficits and pyroptosis in naive mice. These detrimental effects in naive mice were abolished significantly by activating brain PKA-Cα using 8-Bromo-cAMP. Collectively, CCR5 activation promotes NLRP1-mediated pyroptosis after ICH, at least partly through the CCR5/PKA/CREB signaling pathways.

Regulated cell death is characterized as any form of cell death caused by an intracellular or extracellular death program, including apoptosis, necroptosis, and pyroptosis. Unlike apoptosis and necroptosis, pyroptosis is a highly inflammatory form of regulated cell death exclusively mediated by cleaved caspase-1. 11 NLRP1 activation promotes inflammasome formation, resulting in the recruitment and activation of caspase-1 via conversion of precursor caspase-1 into cleaved caspase-1. The cleaved caspase-1 further cleaves precursor IL-1β and IL-18 into mature proinflammatory IL-1β and IL-18. 5 Additionally, cleaved caspase-1 is an enzyme specific for cell pyroptosis, which leads to cell membrane pore formation, rapid loss of membrane integrity, and release of proinflammatory intracellular contents. 12 In an animal model of spinal cord injury, the mRNA and protein expression of NLRP1 was significantly elevated from 18 to 24 hours after spinal cord injury. 13 In a cerebral ischemic stroke model, NLRP1 inflammasome expression in the ipsilateral brain tissue increased as early as 1 hour and was maintained at the increased level when measured at 12, 24, and 72 hours after stroke. 14 Consistently, we observed a time-dependent upregulation of endogenous expression of NLRP1 inflammasome in the ipsilateral hemisphere after ICH, which started at 12 hours and peaked at 24 hours after ICH. CCR5-a chemokine receptor-plays an essential role in mediating leukocyte transport during inflammation. CCR5 was widely expressed in cortex, thalamus, striatum, hippocampus, endothelium, and ependyma. 15 Our present data showed that CCR5 was primarily colocalized with neurons, microglia, and astrocytes within the brain, which coincided with previous studies. 15, 16 The CCR5 mRNA and protein levels were increased in brain, spinal cord, and blood plasma at 22 hours after middle cerebral artery occlusion in mice. 17 Consistently, we also observed upregulated protein expression of CCR5 was upregulated at an early stage and peaked at 24 hours after ICH, which accompanied a similar pattern of increased CCL5 ligands, as well as increased protein levels of PKA-Cα, p-CREB, and NLRP1. These results suggested that CCR5 was increased as a response to ICH-induced CCL5 elevation in the acute phase. The upregulation of PKA-Cα and p-CREB may serve as an endogenous neuroprotective mechanism to counteract the deleterious signaling of CCR5 activation and may facilitate homeostatic maintenance in the injured brain. However, such endogenous increases are not sufficient to overcome the effects of CCL5/CCR5 on NLRP1 activation after ICH.

MVC-a novel selective antagonist of CCR5-has been shown to be anti-inflammatory and antiapoptotic in animal models of autoimmune encephalomyelitis and pancreatic cancer. 18, 19 However, the effect of MVC-mediated CCR5 inhibition on neuronal pyroptosis has not been elucidated. In this study, we observed that intranasal administration of MVC 1 hour after ICH significantly improved neurological deficits. Based on our results, the treatment of MVC 1 hour after ICH is effective but not at 24 hours. Further studies are needed to further explore the maximum therapeutic window within 24 hours. In addition, MVC inhibited the expression of brain NLRP1 inflammasome, cleaved caspase-1, IL-1β/IL-18β, and pyroptosis marker, N-GSDMD, after ICH. Importantly, immunohistochemical assays showed that MVC reduced the numbers of degenerating neurons, especially cleaved caspase-1-positive neurons after ICH. The antipyroptotic feature of MVC was additional to its antiapoptotic effect. ICH directly damaged the striatal region, resulting in impaired sensorimotor function. 20 In the present study, a battery of neurobehavioral tests, including modified Garcia, limb placement, and corner turn tests, consistently revealed the sensorimotor deficits in mice at 24 and 72 hours post-ICH, which correlated with the neuronal death and loss of neurons in the perihematomal brain tissues. The striatal-associated motor dysfunction remains as shown in the foot fault test and rotarod test at 1, 2, and 3 weeks after ICH in mice. Furthermore, secondary cerebral ischemia may occur in the hippocampal CA1 region post-ICH. 21 Neurons in the hippocampal CA1 region were sensitive to ischemia and were closely related to learning and memory functions of humans and mammals. Nissl staining showed that MVC treatment delayed neuronal death in the hippocampal CA1 region at day 25 after ICH, which is associated with spatial learning deficits identified by the Morris water maze test. Early inhibition of CCL5/CCR5 signaling provided long-term neuronal protection, leading to improved sensorimotor and cognitive dysfunction in ICH mice. Furthermore, we explored the possible mechanism underlying brain CCR5 activation in NLRP1-mediated pyroptosis after ICH. Previous studies have demonstrated that CCR5 inhibition promoted early recovery of motor function, neuroplasticity, learning, and memory through the CREB signaling pathway in disease models of traumatic brain injury and cerebral ischemia. 7, 8 High expression of local CREB significantly improved motor nerve function after cerebral infarction. 22 Our results demonstrated that CCR5 inhibition using MVC significantly increased expression of phosphorylated PKA-Cα and CREB but downregulated NLRP1, ASC, C-caspase-1, N-GSDMD, IL-1β, and IL-18 at 24 hours after ICH. A potent and selective CREB inhibitor 666-15 could decrease CREB activation. 23 In the present study, the application of 666-15 before ICH induction significantly decreased protein levels of p-CREB within ipsilateral brain tissue and reversed the beneficial effects of MVC on neurobehavioral deficits and pyroptosis after ICH. These findings implicated that CREB phosphorylation may be the downstream signal of MVC-mediated CCR5 inhibition responsible for downregulating the NLRP1-dependent neuronal pyroptosis after ICH.

Interestingly, the administration of 666-15 did not alter the MVC-induced brain PKA-Cα upregulation. Although the biological role of PKA-Cα has not been well-defined, it appears to function as a key kinase in activating CREB. 24 We further investigated the role of PKA-Cα in CCR5-mediated pyroptosis and its relationship to CREB activation. Brain CCR5 activation via intracerebroventricular administration of rCCL5 in naive mice decreased the brain expression of PKA-Cα and phosphorylated CREB but upregulated NLRP1, ASC, C-caspase-1, N-GSDMD, as well as IL-1β and IL-18 at 24 hours after rCCL5 injection, leading to significant neurological deficits. The administration of 8-Bromo-cAMP reversed the detrimental effects of rCCL5mediated CCR5 activation on neurobehavioral function and brain pyroptosis by upregulating the expression of PKA-Cα and p-CREB, suggesting PKA as an upstream signal of CREB activation after ICH. Collectively, our findings reveal that CCR5 activation using rCCL5 promoted pyroptosis, partially through the CCR5/PKA/ CREB/NLRP1 signaling pathway.

There are some limitations in this study. First, the present study only focused on pyroptosis after ICH. However, we cannot exclude the contribution of CCR5 to apoptosis and the mechanism involving the MAPK (mitogen-activated protein kinase) signaling pathway. 8 Second, we cannot rule out the possibility of NLRP3 signaling in CCR5-mediated pyroptosis after ICH. Microglial NLRP3 inflammasome was significantly upregulated in the mouse model of ICH. 25 CREB phosphorylation negatively regulated the expression of NLRP3-ASC-caspase 1 inflammasome. 26 Nevertheless, the NLRP1 inflammasome is mainly expressed in neurons and is essential for pyroptotic neuronal death, 5 whereas the NLRP3 inflammasome appears to be primarily expressed in microglia and is likely responsible for microglial activation. 27 Third, we used only male mice in this study. Sexual dimorphism exists in neuroinflammation and its mechanisms. Previous studies have shown that female mice are less susceptible to mortality and have improved neurobehavioral outcomes compared with male mice in the setting of ICH. 28 At last, the primary antibody omission control is not the true negative control for validating the antibody specificity, which only controls the nonspecific staining of secondary antibody. The 5 key conceptual pillars were proposed by the International Working Group on Antibody Validation in 2016. The knockout validation would be robust technique for confirming the antibody specificity. Further studies are warranted to verify the neuroprotective effects of CCR5 inhibition after experimental ICH in female mice.

The present study showed that CCR5 activation promoted NLRP1-dependent neuronal pyroptosis and neurological deficits, at least, in part, via the CCR5/PKA/CREB signaling pathway after experimental ICH in mice. CCR5 inhibition using MVC may provide a promising therapeutic strategy in the management of patients with ICH.

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Incidence, case fatality, and functional outcome of intracerebral haemorrhage over time, according to age, sex, and ethnic origin: a systematic review and meta-analysis

Molecular pathophysiology of cerebral hemorrhage: secondary brain injury

Fiery cell death: pyroptosis in the central nervous system

The role of neuronal NLRP1 inflammasome in Alzheimer's disease: bringing neurons into the neuroinflammation game

CCR5 blockade for neuroinflammatory diseases-beyond control of HIV

CCR5 Is a therapeutic target for recovery after stroke and traumatic brain injury

CCR5 is a suppressor for cortical plasticity and hippocampal learning and memory

The molecular biology of memory: cAMP, PKA, CRE, CREB-1, CREB-2, and CPEB

The role of the transcription factor CREB in immune function

Cell death pathways in ischemic stroke and targeted pharmacotherapy

GSDMD membrane pore formation constitutes the mechanism of pyroptotic cell death

Heme oxygenase-1 promotes neuron survival through down-regulation of neuronal NLRP1 expression after spinal cord injury

Intravenous immunoglobulin suppresses NLRP1 and NLRP3 inflammasome-mediated neuronal death in ischemic stroke

CCL5-Glutamate cross-talk in astrocyte-neuron communication in multiple sclerosis

Hypoxic-ischemic injury induces macrophage inflammatory protein-1alpha expression in immature rat brain

Experimental stroke induces massive, rapid activation of the peripheral immune system

Maraviroc attenuates the pathogenesis of experimental autoimmune encephalitis

The CCR5 antagonist maraviroc causes remission of pancreatic cancer liver metastasis in nude rats based on cell cycle inhibition and apoptosis induction

Striatal mechanisms underlying movement, reinforcement, and punishment

Melatonin Alleviates intracerebral hemorrhage-induced secondary brain injury in rats via suppressing apoptosis, inflammation, oxidative stress, DNA damage, and mitochondria injury

CREB controls cortical circuit plasticity and functional recovery after stroke

Cerebrolysin ameliorates focal cerebral ischemia injury through neuroinflammatory inhibition via CREB/PGC-1α pathway

PKA-mediated effect of MAS receptor in counteracting angiotensin II-stimulated renal Na+-ATPase

nlrp3 inflammasome in the pathophysiology of hemorrhagic stroke: a review

G-proteincoupled estrogen receptor activation upregulates interleukin-1 receptor antagonist in the hippocampus after global cerebral ischemia: implications for neuronal self-defense

Rifampicin attenuates rotenone-induced inflammation via suppressing NLRP3 inflammasome activation in microglia

Sex differences in gene and protein expression after intracerebral hemorrhage in mice

Ccr1 activation promotes neuroinflammation through ccr1/tpr1/erk1/2 signaling pathway after intracerebral hemorrhage in mice

Fingolimod reduces cerebral lymphocyte infiltration in experimental models of rodent intracerebral hemorrhage

Involvement of spinal CC chemokine ligand 5 in the development of bone cancer pain in rats

Angiogenesis and vasculogenic mimicry are inhibited by 8-Br-cAMP through activation of the cAMP/PKA pathway in colorectal cancer

Inhibition of EZH2 (enhancer of zeste homolog 2) attenuates neuroinflammation via H3k27me3/SOCS3/TRAF6/NF-κB (trimethylation of histone 3 lysine 27/suppressor of cytokine signaling 3/tumor necrosis factor receptor family 6/nuclear factor-κB) in a rat model of subarachnoid hemorrhage

A protocol for stripping and reprobing of Western blots originally developed with colorimetric substrate TMB

RANTES has a potential to play a neuroprotective role in an autocrine/paracrine manner after ischemic stroke

Selective mGluR1 negative allosteric modulator reduces blood-brain barrier permeability and cerebral edema after experimental subarachnoid hemorrhage

Brain ceruloplasmin expression after experimental intracerebral hemorrhage and protection against iron-induced brain injury

Inhibition of PAR-2 attenuates neuroinflammation and improves short-term neurocognitive functions Via ERK1/2 signaling following asphyxia-induced cardiac arrest in rats

Drs Yan, Xu, and Tang conceived the study; Drs Yan and Xu supervised and coordinated all aspects of the work; Drs Yan, Huang, Zhang, and Tang designed the experiments; Dr Yan, C. Lenahan, and Dr Huang wrote the paper; Drs Yan, Xu, Wen, Li, Hu, and Zheng performed the experiments, analyzed the data, and prepared the figures and tables; Drs Wen and Xu contributed with analytical tools.

This study was supported by grants from the National Natural Science Foundation of China (No. 82060225), Guangxi Natural Science Foundation (No. 2020JJA140117 and 2018JJA140243) to Dr Yan.

None.

Expanded Materials and Methods Supplemental Table I  Supplemental I-XVII  Data Set  References 18-38