Gao et al. Journal of Neuroinflammation (2021) 18:118 https://doi.org/10.1186/s12974-021-02171-6 RESEARCH Open Access Downregulation of CD151 restricts VCAM-1 mediated leukocyte infiltration to reduce neurobiological injuries after experimental stroke Ceshu Gao1†, Wangyue Jia2†, Wendeng Xu1, Qiong Wu2* and Jian Wu1* Abstract Background: Translational failures in anti-adhesion molecule therapies after stroke reveal the necessity of developing new strategies that not only interrupt leukocyte recruitment but also consider the inhibition of endothelial cell inflammation, verification of therapeutic time window, and normal function maintenance of circulating leukocytes. Our study focused on the potential therapeutic value of CD151 downregulation in improving current anti-adhesion molecule therapies. Methods: Lentivirus intracerebroventricular administration was conducted to inhibit the CD151 expression and observe its functional influence on neurological injuries and outcomes. Then, immunohistochemistry and myeloperoxidase activity assessment were performed to explore the effects of CD151 expression on neutrophil and monocyte recruitment after rat cerebral ischemia. Primary rat brain microvascular endothelial cells were subjected to oxygen glucose deprivation and reoxygenationtoelucidatetheunderlyingworking mechanisms between CD151 and VCAM-1. Results: The CD151 downregulation remarkably reduced neurological injuries and improved neurological outcomes, which were accompanied with reduced neutrophil and monocyte infiltration after the CD151 downregulation. The VCAM-1 expression was remarkably decreased among the adhesion molecules on the endothelial cell responsible for neutrophil and monocyte infiltration. The activation of p38 MAPK and NF-κB pathways was restricted after the CD151 downregulation. p38 MAPK and NF-κB inhibitors decreased the VCAM-1 expression, and p38 acted as an upstream regulator of NF-κB. However, CD151 downregulation did not directly influence the neutrophil and monocyte activation. Conclusions: Overall, CD151 regulated the expression of adhesion molecules. It also played a critical role in suppressing VCAM-1-mediated neutrophil and monocyte infiltration via the p38/NF-κB pathway. This study possibly provided a new basis for improving current anti-adhesion molecule therapies. Keywords: CD151, VCAM-1, Endothelial cell, Neutrophil, Monocyte, Stroke * Correspondence: [email protected]; [email protected] †Ceshu Gao and Wangyue Jia contributed equally to this work. 2MOE Key Laboratory of Bioinformatics, School of Life Sciences, Tsinghua University, Beijing 100084, China 1Department of Neurology, Beijing Tsinghua Changgung Hospital, School of Clinical Medicine, Tsinghua University, Beijing 102218, China © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Gao et al. Journal of Neuroinflammation (2021) 18:118 Page 2 of 16 Background approved by the Institutional Animal Care and Use Leukocyte infiltration after cerebral ischemia is linked to Committee at Tsinghua University. The animals were secondary injury after ischemic stroke [1]. It is also asso- placed in a house with a 12-h light/dark cycle condition, ciated with a poor prognosis in clinical trials and experi- a temperature of 23 °C ± 3 °C, a relative humidity of 50– mental animal studies [2, 3]. Neutrophils and monocytes 60%, and ad libitum access to water and chow. All the are among the major immune cells that infiltrate after rats were randomly assigned to different groups. All the cerebral ischemia and their infiltration peaks within 3 experiments followed a double-blind method. days after cerebral ischemia [4]. As the base of leukocyte infiltration, the blockade of adhesion molecules respon- Middle cerebral artery occlusion (MCAO) model in rats sible for the leukocyte-endothelial cell interaction has A rat experimental ischemic stroke model was established led to promising neurological outcomes [5]. However, as previously described [11]. After lentivirus intracerebro- anti-adhesion molecule therapies fail to meet the clinical ventricular administration and 7-day normal breeding, the needs. The translational failures of these therapies indi- weight of rats naturally increased. The adult rats (295– cate the need for the development of reliable potential 305 g) were initially anesthetized with 5% isoflurane and targets and the verification of optimal therapeutic time maintained with 2–2.5% isoflurane during surgery. The window [6]. body temperature of the rats was maintained at 37 °C ± New evidence suggests the emerging role of tetraspa- 0.5 °C by using a surface heating pad. Experimental cere- nin in the interaction of immune and endothelial cells. bral ischemia was induced by inserting a silicone-coated As a member of the tetraspanin family, CD151 is known filament (Cinontech, China) into the right common ca- as a modulator of the activities of different transmem- rotid artery to block the middle cerebral artery (MCA). brane protein families; it also functions as a partner of After 2 h MCAO, the filament was withdrawn to restore adhesion molecules during chronic inflammation [7]. the blood flow. Sham-operated rats were subjected to the CD151, CD9, VCAM-1, and ICAM-1 form tetraspanin- same procedure, but the filament was inserted into the ca- enriched microdomains (TEMs) [8]. Activated endothe- rotid artery without blocking the MCA. lial cells actually assemble preexisting TEMs to form endothelial docking structures instead of relying on the Lentivirus construction, intracerebroventricular (ICV) unstructured interaction of adhesion molecules and li- administration, and effectiveness measurement gands [9, 10]. Thus, adhesion molecules (ICAM-1, The lentivirus for expressing the CD151 shRNA (LV VCAM-1, and E-selectin) respond to ligands presented CD151 shRNA) and the scramble lentivirus with an inert by leukocytes and are regulated by CD151 on endothelial random shRNA sequence (LV Vehicle) were purchased cells in the early stage. Therefore, the involvement of from GenePharma Corporation (Shanghai, China). Both CD151 in adhesion molecule regulation enriches our un- the two lentiviruses expressed GFP tag. The lentivirus derstanding of the interaction between endothelial cells shRNA information is presented in detail in Table S1. and leukocytes and may further explain the current fail- The effectiveness of different modes of lentivirus CD151 ures in anti-adhesion molecule therapies. However, stud- shRNA transfection was confirmed through Western ies have yet to clarify whether the role of tetraspanin, blot and RT-PCR (Additional file: Figure S1). especially CD151, in TEMs is valuable enough to bridge The rats were divided into three groups, namely, lenti- the translational gap in terms of anti-adhesion molecule virus vehicle injection and sham surgery (LV Vehicle + therapies in ischemic stroke. Sham), lentivirus vehicle injection and MCAO (LV Ve- This work aimed to investigate the therapeutic value hicle + MCAO), and lentivirus CD151 shRNA injection of CD151 downregulation and its function in leukocyte and MCAO (LV CD151 shRNA + MCAO). infiltration after cerebral ischemia. Herein, we specific- The rats were transfected with the lentivirus through ally focused on the changes in the expression of TEM ICV injection 7 days before the MCAO surgery. Briefly, components and other adhesion molecules in endothe- they were anesthetized with 2% sodium pentobarbital lial cells in neutrophil and monocyte recruitment after (60–80 mg/kg) and placed in a stereotaxic apparatus experimental stroke. (Cinontech, China). The ICV injection was performed using a 10-μl syringe, and coordinates were 2 mm lateral Methods to the bregma, 1.5 mm posterior to the bregma, and 4–5 Animals mm deep from the dura. The rats received either 8 μlof A total of 239 specific pathogen-free male Sprague- LV CD151 shRNA (1 × 109 TU/ml) or 8 μl of LV vehicle Dawley rats were included, and a mortality of around 6% (1 × 109 TU/ml) at a rate of 0.5 μl/min. After 5 min, the was observed. The rats weighing 250–260 g were first needle was slowly withdrawn to prevent reflux. purchased from the Beijing Vital River Laboratory Ani- The GFP expression, which indicated successful lenti- mal Technology Co. Experimental protocols were virus infection in vivo was detected with LSM880 Gao et al. Journal of Neuroinflammation (2021) 18:118 Page 3 of 16 Airyscan (Zeiss, Germany) in the cryosections of the A forelimb placing test was carried out to assess the brain tissues 7 days after