Multi-Omics Profiling Identifies Microglial Annexin A2 as a Key Mediator of NF-κB Pro-inflammatory Signaling in Ischemic Reperfusion Injury
2024; Elsevier BV; Linguagem: Inglês
10.1016/j.mcpro.2024.100723
ISSN1535-9484
AutoresXibin Tian, Wuyan Yang, Wei Jiang, Zhen Zhang, Junqiang Liu, Haijun Tu,
Tópico(s)Immune Response and Inflammation
Resumo•Multi-omics reveals immune-related events in cerebral ischemic reperfusion.•Anxa2 is exclusively upregulated in microglia in response to OGD/R.•Anxa2 knockdown reduces inflammatory response and apoptosis induced by OGD/R.•Anxa2 knockdown inhibits NF-κB signaling activation triggered by OGD/R.•Microglial Anxa2 knockdown with OGD/R treatment alleviated neuronal death. Cerebral stroke is one of the leading causes of mortality and disability worldwide. Restoring the cerebral circulation following a period of occlusion and subsequent tissue oxygenation leads to reperfusion injury. Cerebral ischemic reperfusion (I/R) injury triggers immune and inflammatory responses, apoptosis, neuronal damage, and even death. However, the cellular function and molecular mechanisms underlying cerebral I/R-induced neuronal injury are incompletely understood. By integrating proteomic, phosphoproteomic, and transcriptomic profiling in mouse hippocampi after cerebral I/R, we revealed that the differentially expressed genes and proteins mainly fall into several immune inflammatory response–related pathways. We identified that Annexin 2 (Anxa2) was exclusively upregulated in microglial cells in response to cerebral I/R in vivo and oxygen-glucose deprivation and reoxygenation (OGD/R) in vitro. RNA-seq analysis revealed a critical role of Anxa2 in the expression of inflammation-related genes in microglia via the NF-κB signaling. Mechanistically, microglial Anxa2 is required for nuclear translocation of the p65 subunit of NF-κB and its transcriptional activity upon OGD/R in BV2 microglial cells. Anxa2 knockdown inhibited the OGD/R-induced microglia activation and markedly reduced the expression of pro-inflammatory factors, including TNF-α, IL-1β, and IL-6. Interestingly, conditional medium derived from Anxa2-depleted BV2 cell cultures with OGD/R treatment alleviated neuronal death in vitro. Altogether, our findings revealed that microglia Anxa2 plays a critical role in I/R injury by regulating NF-κB inflammatory responses in a non-cell-autonomous manner, which might be a potential target for the neuroprotection against cerebral I/R injury. Cerebral stroke is one of the leading causes of mortality and disability worldwide. Restoring the cerebral circulation following a period of occlusion and subsequent tissue oxygenation leads to reperfusion injury. Cerebral ischemic reperfusion (I/R) injury triggers immune and inflammatory responses, apoptosis, neuronal damage, and even death. However, the cellular function and molecular mechanisms underlying cerebral I/R-induced neuronal injury are incompletely understood. By integrating proteomic, phosphoproteomic, and transcriptomic profiling in mouse hippocampi after cerebral I/R, we revealed that the differentially expressed genes and proteins mainly fall into several immune inflammatory response–related pathways. We identified that Annexin 2 (Anxa2) was exclusively upregulated in microglial cells in response to cerebral I/R in vivo and oxygen-glucose deprivation and reoxygenation (OGD/R) in vitro. RNA-seq analysis revealed a critical role of Anxa2 in the expression of inflammation-related genes in microglia via the NF-κB signaling. Mechanistically, microglial Anxa2 is required for nuclear translocation of the p65 subunit of NF-κB and its transcriptional activity upon OGD/R in BV2 microglial cells. Anxa2 knockdown inhibited the OGD/R-induced microglia activation and markedly reduced the expression of pro-inflammatory factors, including TNF-α, IL-1β, and IL-6. Interestingly, conditional medium derived from Anxa2-depleted BV2 cell cultures with OGD/R treatment alleviated neuronal death in vitro. Altogether, our findings revealed that microglia Anxa2 plays a critical role in I/R injury by regulating NF-κB inflammatory responses in a non-cell-autonomous manner, which might be a potential target for the neuroprotection against cerebral I/R injury. Cerebral stroke is a devastating and debilitating cerebrovascular disease, that causes high disability and mortality, and has emerged as one of the major public health issues around the world (1Jia J. Deng J. Jin H. Yang J. Nan D. Yu Z. et al.Effect of Dl-3-n-butylphthalide on mitochondrial Cox7c in models of cerebral ischemia/reperfusion injury.Front. Pharmacol. 2023; 141084564Crossref Scopus (1) Google Scholar, 2Liu J. Luo Q. Ke J. Zhang D. Xu Y. Liao W. et al.Enriched environment attenuates ferroptosis after cerebral ischemia/reperfusion injury via the HIF-1alpha-ACSL4 pathway.Oxid. Med. 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Wang Y. et al.Annexin A2 modulates ROS and impacts inflammatory response via IL-17 signaling in polymicrobial sepsis mice.PLoS Pathog. 2016; 12: e1005743Crossref PubMed Scopus (55) Google Scholar). Anxa2 deficiency exacerbates neutrophil infiltration, neuroinflammation, and long-term neurological outcomes after traumatic brain injury (33Liu N. Jiang Y. Chung J.Y. Li Y. Yu Z. Kim J.W. et al.Annexin a2 deficiency exacerbates neuroinflammation and long-term neurological deficits after traumatic brain injury in mice.Inter. J. Mol. Sc. 2019; 20: 6125Crossref PubMed Scopus (20) Google Scholar). Moreover, recombinant annexin A2 binds to Toll-like receptor 4 in leukocytes and inhibits leukocyte brain infiltration, neuroinflammation, and neuronal cell death after traumatic brain injury (34Liu N. Han J. Li Y. Jiang Y. Shi S.X. Lok J. et al.Recombinant annexin A2 inhibits peripheral leukocyte activation and brain infiltration after traumatic brain injury.J. Neuroinflammation. 2021; 18: 173Crossref PubMed Scopus (9) Google Scholar). However, the cellular and molecular mechanisms underlying the role of Anxa2 in modulating microglial activation in cerebral I/R pathogenesis remain poorly investigated. Nuclear factor-kappa B (NF-κB) is a transcription factor widely associated with inflammatory responses following ischemia and other neuroinflammatory disorders (23Simmons L.J. Surles-Zeigler M.C. Li Y. Ford G.D. Newman G.D. Ford B.D. Regulation of inflammatory responses by neuregulin-1 in brain ischemia and microglial cells in vitro involves the NF-kappa B pathway.J. Neuroinflammation. 2016; 13: 237Crossref PubMed Scopus (94) Google Scholar). Genetic and pharmacological studies targeting NF-κB-activating IKK showed that inhibiting NF-κB generally benefits stroke recovery (35Iadecola C. Anrather J. The immunology of stroke: from mechanisms to translation.Nat. Med. 2011; 17: 796-808Crossref PubMed Scopus (1847) Google Scholar, 36Zeng J. Wang Y. Luo Z. Chang L.C. Yoo J.S. Yan H. et al.TRIM9-Mediated resolution of neuroinflammation confers neuroprotection upon ischemic stroke in mice.Cell Rep. 2019; 27: 549-560.e6Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). Activated microglia can enable different inflammatory pathways, including the NF-κB signaling pathway. It can trigger the release of many pro-inflammatory mediators, such as TNF-α, IL-1β, and IL-6, which lead to inflammatory reactions. The production of inflammatory factors can aggravate the damage of neighboring neurons and result in tissue injury. Therefore, we examined whether Anxa2 regulates microglial activation and targets these signaling molecules or pathways after cerebral I/R. In this study, we performed combinatorial analyses of proteome, phosphoproteome, and transcriptome and identified Anxa2 as a crucial molecule for regulating the immune-inflammatory response in microglia. Loss of Anxa2 expression impairs gene expression signatures associated with inflammation in microglia. Anxa2 is required for microglial activation and the production of pro-inflammatory factors via the NF-κB signaling pathway by facilitating the nuclear translocation of the p65 subunit. Importantly, loss of Anxa2 attenuates neuronal death in response to oxygen-glucose deprivation and reoxygenation (OGD/R) in a non-cell-autonomous manner. Thus, our findings reveal a critical role of microglial Anxa2 in regulating the inflammatory response, suggesting that Anxa2 may be a potential therapeutic target for cerebral ischemia. All animal experiments were performed with approval from the Hunan University Animal Ethics Committee (No. SYXK [Xiang] 2023-0010). In this study, adult C57BL/6J male mice (8 weeks old) weighing 22 to 26 g were randomly divided into the sham or I/R groups. Mice were kept in standard conditions, with sterile cages, bedding, water, and food, with 12 h of light and 12 h of dark cycle. The cerebral I/R model was induced by extracranial intraluminal middle cerebral artery occlusion (MCAO), as previously described (37Uluc K. Miranpuri A. Kujoth G.C. Akture E. Baskaya M.K. Focal cerebral ischemia model by endovascular suture occlusion of the middle cerebral artery in the rat.J. Vis. Exp. 2011; 5: 1978Google Scholar). Briefly, 8 weeks old mice were anesthetized with 5% isoflurane and maintained with 1% isoflurane (RWD Life Science, R511-22) in an oxygen/air mixture by using a gas anesthesia mask (RWD Life Science, R580SRWD). Under the operating stereo microscope, the left common carotid artery, the external carotid artery (ECA), and the internal carotid artery were sequentially exposed. The ECA was ligated with a 5–0 silk suture, and a 2 cm length of silicon-rubber-coated monofilament (RWD Life Science, MSMC24B104PK50) was inserted from the ECA through the internal carotid artery up to the bifurcation of the left middle cerebral artery and anterior cerebral artery to block the circulation in the left middle cerebral artery territory. After 90 min of occlusion, blood flow was restored by the withdrawal of the inserted filament (Reperfusion). The sham-operated mice underwent the same experimental procedures except for the filament insertion. Cerebral blood flow was monitored using a Laser Speckle Imaging System (RWD Life Science, RFLSI III) during MCAO and reperfusion. Mice were sacrificed 24 h post-reperfusion. For proteomic and transcriptomic analyses, nine hippocampi from the sham or MCAO/R group were analyzed by three biological replicate MS runs (3 hippocampi were pooled per MS run). Five percent (40 μg) of the pooled samples were used for whole proteomic analysis, and the remaining 95% (760 μg) were subjected to phosphoproteomic profiling. For BV2 cell transcriptomic analysis, three biological replicates were used to compare genes in each stable knockdown cell line. Each biological replicate comprises three technical replicates (1 μg of total RNA). For all omics analyses, the differentially expressed genes (DEGs)/proteins (DEPs, p-value <0.05; fold-change 1.2 [upregulated]) were subjected to Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. Protein extraction, trypsin digestion, and tandem mass tag (TMT) labeling were performed as described previously (38Jiang W. Zhang P. Yang P. Kang N. Liu J. Aihemaiti Y. et al.Phosphoproteome analysis identifies a synaptotagmin-1-associated complex involved in ischemic neuron injury.Mol. Cell Proteomics. 2022; 21100222Abstract Full Text Full Text PDF Scopus (4) Google Scholar). After cerebral I/R, hippocampi from the ipsilateral hemispheres were grounded in liquid nitrogen and then resuspended with four volumes of lysis buffer (8 M urea, 1% protease inhibitor mix). The mixture was sonicated three times on ice with a high-intensity ultrasonic processor. Tissue debris was removed by centrifugation at 12,000g at 4 °C for 10 min. Finally, the supernatant was collected, and the protein concentration was determined with a BCA kit according to the manufacturer's instructions. For digestion, the protein solution was reduced with 5 mM DTT for 30 min at 56 °C and alkylated with 11 mM iodoacetamide for 15 min at room temperature in the dark. Then, 100 mM TEAB was added to decrease the urea concentration below 2 M. Finally, trypsin was added at a 1:50 trypsin-to-protein mass ratio for the first digestion overnight and at a 1:100 ratio for the second digestion for 4 h. For TMT labeling, peptides were desalted with a Strata X C18 SPE column (Phenomenex) and vacuum dried. They were then reconstituted in 0.5 M TEAB and processed according to the manufacturer's protocol. Briefly, one unit of TMT reagent was thawed and reconstituted in acetonitrile. The peptide mixtures were then incubated for 2 h at room temperature and pooled, desalted, and dried by vacuum centrifugation. Phosphopeptide enrichment was performed following our previously published method (38Jiang W. Zhang P. Yang P. Kang N. Liu J. Aihemaiti Y. et al.Phosphoproteome analysis identifies a synaptotagmin-1-associated complex involved in ischemic neuron injury.Mol. Cell Proteomics. 2022; 21100222Abstract Full Text Full Text PDF Scopus (4) Google Scholar). Phosphopeptide mixtures were suspended using immobilized metal affinity chromatography (IMAC) microsphere suspensions with vibration in loading buffer (50% acetonitrile/6% TFA). The IMAC microspheres were collected by centrifugation, washed with 50% acetonitrile/6% trifluoroacetic acid, and again with 30% acetonitrile/0.1% trifluoroacetic acid. The enriched phosphopeptides were eluted with 10% ammonium hydroxide from the IMAC microspheres and analyzed with liquid chromatography-tandem mass spectrometry (LC–MS/MS). LC-MS/MS analysis and data search were performed according to our previous methods (38Jiang W. Zhang P. Yang P. Kang N. Liu J. Aihemaiti Y. et al.Phosphoproteome analysis identifies a synaptotagmin-1-associated complex involved in ischemic neuron injury.Mol. Cell Proteomics. 2022; 21100222Abstract Full Text Full Text PDF Scopus (4) Google Scholar). The peptides were dissolved in 0.1% formic acid (solvent A) and loaded onto a homemade reversed-phase analytical column (15 cm length, 75 μm i.d.). The gradient was comprised of an increase from 6% to 23% solvent B (0.1% formic acid in 98% acetonitrile) over 26 min, 23% to 35% in 8 min, and climbing to 80% in 3 min, then holding at 80% for the last 3 min, all at a constant flow rate of 400 nl/min on an EASY-nLC 1000 UPLC system. The peptides were subjected to an NSI source followed by tandem mass spectrometry (MS/MS) in a Q Exactive TM Plus (Thermo) coupled online to UPLC. The electrospray voltage of 2.0 kV was applied, the m/z scan range from 350 to 1800 was used for the full scan, and intact peptides were detected in the Orbitrap at a resolution of 70,000. Peptides were then selected for MS/MS using the NCE setting of 28, and the fragments were detected in the Orbitrap at a resolution of 17,500. A data-dependent procedure was alternated with one MS scan followed by 20 MS/MS scans with 15.0 s dynamic exclusion. Automatic gain control was set at 50,000, and the fixed first mass was set at 100 m/z. The resulting MS/MS data were processed using the MaxQuant search engine (v.1.5.2.8). Tandem mass spectra were searched against the SwissProt Mouse database concatenated with the reverse decoy database (released February 2018; 16,964 sequences). Carbamidomethyl on cysteines was specified as a fixed modification. Acetylation on the N-terminal of proteins and oxidation on methionine were set as variable modifications for the proteomic analysis. N-terminal protein acetylation, oxidation on methionine, and phosphorylation on serine, threonine, and tyrosine were specified as variable modifications for the phosphoproteomic analysis. Trypsin/P was specified as a cleavage enzyme, allowing up to four missing cleavages. The mass tolerance for precursor ions was set as 20 ppm in the first search and 5 ppm in the main search, and the mass tolerance for fragment ions was set as 0.02 Da. FDR was adjusted to 40. After cerebral I/R, ipsilateral hippocampi were dissected rapidly, washed with PBS, immediately snap-frozen in liquid nitrogen, and stored at −80 °C until use. Total RNA was extracted using Trizol reagent (Invitrogen) according to the manufacturer's protocol. cDNA library generation and RNA sequencing were performed by NovoGene, and the details were described previously (39Zhang X. Zhou Q. Zou W. Hu X. Molecular mechanisms of developmental toxicity induced by graphene oxide at predicted environmental concentrations.Environ. Sci. Technol. 2017; https://doi.org/10.1021/acs.est.7b01922Crossref Scopus (155) Google Scholar). The clean data was aligned by Tophat 2 to the reference genome of the species which was derived from the Ensembl database (http://www.ensembl.org/) and annotated according to the collected information from UniProtKB, Ensembl, gene ontology (GO), KEGG, and eggNOG. Finally, based on the number of sequences aligned to the gene, the expression level was calculated using HTSeq 0.6.1p2. After data normalization, DEGs and DEPs between I/R versus sham groups were used for subsequent bioinformatical analyses. The GO and KEGG enrichment analyses were performed with the 'clusterProfiler' package (v. 4.4.4) with a p-value threshold set to 0.05. The hierarchical cluster analysis was conducted by the 'pheatmap' package (v. 1.0.8) in RStudio (v. 4.2.1). The principal component analysis (PCA) analysis was performed using TBtools (v. 1.108). For gene network analysis, DEGs were input into STRING (https://string-db.org/) database to obtain the inner interaction, and the gene-gene network was visualized by Cytoscape software (v. 3.9.1). CytoHubba plug-in (v. 0.1) was used to predict the important gene and subnetwork based on the topological algorithms of Maximal Clique Centrality. HEK293T cells and BV2 microglial cells were maintained in Dulbecco's modified Eagle's medium (DMEM; Corning) supplemented with 10% fetal bovine serum (FBS; Gibco) and 1% penicillin-streptomycin antibiotics (Thermo Fisher Scientific) in a humidified 5% CO2 incubator at 37 °C. To generate Anxa2 knockdown cell lines, lentiviral particles were prepared according to a previously described protocol (40Tang Q. Liu M. Liu Y. Hwang R.D. Zhang T. Wang J. NDST3 deacetylates α-tubulin and suppresses V-ATPase assembly and lysosomal acidification.EMBO J. 2021; 40: e107204Crossref PubMed Scopus (6) Google Scholar). HEK293T cells were cotransfected with either Anxa2-targeting shRNA (Sigma, TRCN00000110695) or a control shRNA expressed from the pLKO.1 vector (Addgene, 10878) with the pSPAX2 packaging plasmid (Addgene, 12260) and the pMD2.G envelope plasmid (Addgene, 12259) using Lipofectamine 2000 (Thermo Fisher Scientific, 11668019) in Opti-MEM medium. Tissue culture supernatants were collected 72 h after transfection and filtered through a 0.45 μm PVDF membrane filter. The harvested lentivirus was aliquoted and stored at −80 °C for later use. For all the lentivirus infections, BV2 cells were plated at 2 × 104 cells per well in 6-well plates overnight, and the
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