fmolb-08-660072 April 30, 2021 Time: 20:29 # 1 ORIGINAL RESEARCH published: 07 May 2021 doi: 10.3389/fmolb.2021.660072 Cox4i2 Triggers an Increase in Reactive Oxygen Species, Leading to Ferroptosis and Apoptosis in HHV7 Infected Schwann Cells Bowen Chang1†, Haochen Guan2†, Xueyi Wang1, Zheng Chen1, Wanchun Zhu1, Xiangyu Wei1 and Shiting Li1* 1 Department of Neurosurgery, School of Medicine, Xinhua Hospital, Shanghai Jiao Tong University, Shanghai, China, 2 Department of Nephrology, School of Medicine, Shanghai General Hospital, Shanghai Jiao Tong University, Shanghai, China Emerging evidence suggests that reactive oxygen species (ROS) play a significant role in the pathogenesis of peripheral nerve damage. Our previous study indicated that human herpesvirus 7 (HHV7) induces Bell’s palsy. However, the specific mechanism underlying Edited by: the effects of ROS in HHV7 infection-induced facial nerve damage is unknown. In Fernando Antunes, this study, we established a rat FN model by inoculating an HHV7 virus solution. University of Lisbon, Portugal The facial grading score and LuxolFastBlue (LFB) staining were used to assess the Reviewed by: Ana Cipak Gasparovic, success of the model. Using mRNA-sequencing analysis, we found that the expression Rudjer Boskovic Institute, Croatia of Complex IV Subunit 4 Isoform 2 (Cox4i2) increased in infected Schwann cells (SCs). Ana Sofia Fernandes, CBIOS, Universidade Lusófona Cox4i2 was suggested to increase COX activity, thereby promoting ROS production. Research Center for Biosciences The changes in the endogenous oxidant and antioxidant system were assessed, and and Health Technologies, Portugal the results showed that oxidative stress increased after HHV7 infection in vivo and *Correspondence: in vitro. However, we found that oxidative injury was relieved after the transfection Shiting Li [email protected] of shCox4i2 in HHV7-treated SCs by evaluating cell death, cell proliferation, and the †These authors have contributed ROS level as well as the levels of malondialdehyde (MDA), superoxide dismutase (SOD), equally to this work and glutathione (GSH). Furthermore, we hypothesised that Cox4i2 loss would attenuate HHV7-induced ferroptosis and apoptosis, which are closely related to ROS in SCs. Our Specialty section: This article was submitted to research illustrated that the knockdown of Cox4i2 suppresses HHV7-induced RSC96 Cellular Biochemistry, cell ferroptosis as well as apoptosis via the ERK signalling pathway. Overall, several a section of the journal Frontiers in Molecular Biosciences in vitro and in vivo methods were adopted in this study to reveal the new mechanism of Received: 28 January 2021 ROS-induced and Cox4i2-mediated apoptosis and ferroptosis in HHV7 infected SCs. Accepted: 14 April 2021 Keywords: COX4I2, HHV7, Schwann cells, ferroptosis, apoptosis Published: 07 May 2021 Citation: Chang B, Guan H, Wang X, INTRODUCTION Chen Z, Zhu W, Wei X and Li S (2021) Cox4i2 Triggers an Increase Bell’s palsy is characterised by acute facial nerve paralysis, which frequently leads to peripheral in Reactive Oxygen Species, Leading to Ferroptosis and Apoptosis in HHV7 facial palsy, with a morbidity of 11.5–40.2/100 000 persons every year (Eviston et al., 2015). Infected Schwann Cells. Previous studies have suggested that human herpesvirus 7 (HHV7) infection may contribute to Front. Mol. Biosci. 8:660072. peripheral nerve damage (Wakisaka et al., 2001; Kanerva et al., 2008). In our previous study, doi: 10.3389/fmolb.2021.660072 HHV-7 was found in the epineurium of the facial nerve in patients with Bell’s palsy using Frontiers in Molecular Biosciences| www.frontiersin.org 1 May 2021| Volume 8| Article 660072 fmolb-08-660072 April 30, 2021 Time: 20:29 # 2 Chang et al. Cox4i2 Leading to Ferroptosis metagenomic next-generation sequencing (Chang et al., 2020). no significant difference in bilateral vibrissae movement; 1, the Typically, the reactivated hidden HHV7 infection within the vibrissae movement for the affected side was weaker than that facial nerve has been recognised as the cause of Bell’s palsy. The of the contralateral side; 2, the vibrissae movement disappeared). study of the pathogenesis of HHV7-induced peripheral nerve The position of the apex nasi was observed and scored on a 0– injury is particularly important for the treatment and prevention 1 scale (0, the position of the apex nasi is in the middle; 1, the of Bell’s facial paralysis. position of the apex nasi is tilted to the opposite side). Facial nerve Human herpesvirus 7 belongs to the Herpesviridae family, palsy was represented by a total score of 3 or 4. the Betaherpesvirinae superfamily, and the Roseolovirus genus. Similar to other human herpesviruses, HHV-7 is omnipresent, Cell Culture and HHV7 Infection which leads to a persistent hidden infection that may trigger The Rat Schwann cell line RSC96 was obtained from the additional reactivation or reinfection (Pohl-Koppe et al., American Type Culture Collection (ATCC). The cells were 2001; Agut et al., 2016). Several questions about HHV7 cultured in Dulbecco’s modified Eagle’s medium (DMEM) remain unanswered, especially the mechanism underlying its (Gibco, United States) supplemented with 10% foetal bovine disease causation. serum (Gibco, United States) and 1% penicillin/streptomycin Due to lack of research on HHV7 in peripheral nerve disease, (Sigma-Aldrich, United States) in an incubator at 37◦C in a the pathogenesis of HHV7 in peripheral nerve damage remains humidified 5% CO2 atmosphere. The cells were cultured in six- largely unknown. Peripheral facial paralysis usually results from well plates at a density of 5 × 105 per well. A normal culture damage to the myelin sheath of the facial nerve, which has various medium was used for the control group. For viral infections, causes. Schwann cells (SCs), the main components of the myelin HHV7 was added at a multiplicity of infection (MOI) of 10 until sheath, have been considered principal targets of viruses. In this obvious lesions appeared. All the cells were collected after 72 h study, we established for the first time an animal model of facial for subsequent experiments. paralysis caused by HHV7 and investigated the effects of HHV7 infection on rat SCs. HHV7 mediated increased production LuxolFastBlue Staining of reactive oxygen species (ROS) by upregulating Complex IV LuxolFastBlue (LFB) staining was used to observe the Subunit 4 Isoform 2 (Cox4i2), which resulted in SCs apoptosis demyelination of the nerve tracts, and it was performed and ferroptosis and led to myelin injury of the facial nerve. according to standard techniques (Fujiwara et al., 2017). Briefly, paraffin sections were immersed in a 0.1% LFB reagent for 8–16 h at 60◦C. After this, 0.05% lithium carbonate aqueous solution MATERIALS AND METHODS and 70% alcohol, 0.25% tar violet solution, and glacial acetic acid dyeing solution were used to redye all specimens for 10 min. Animals and Virus Inoculation After rinsing, the sections were observed as dehydrated and Twenty three-weeks-old female Wistar rats weighing 60–80 g transparent under a light microscope. were randomly divided into control and experimental groups after adaptive feeding for 7 days. The rats were treated by Immunofluorescent Staining of Facial scratching the unilateral auricular surface with needle 27 after Nerve Nuclei 3% halothane anaesthesia. The scratching side of auricular On the seventh day after the inoculation of HHV7 infection, surface was inoculated with 0.1 mL HHV7 virus solution [titer the brainstem of rats were collected to evaluate the facial of 1.0 × 106 plaque-forming units (PFU) per mL] in the nerve nucleus. Immunofluorescent staining of the brainstem experimental group, whereas PBS was used in the control group. was performed as described previously (Yokoyama et al., 2006). The blink reflex and vibrissae movement were evaluated after The brain sections were incubated with the primary antibodies 7 days of inoculation. The rats were sacrificed at the end of against anti-Neun (#ab177487, Abcam, United States) and the seventh day. All animal procedures in this study were Anti-CD11b (#66519-1-Ig, Proteintech, United States) at 4◦C reviewed and approved by the Institutional Animal Care and Use overnight followed by incubation with DyLight 488-and DyLight Committee of Xinhua Hospital Affiliated to Shanghai Jiao Tong 649-labelled secondary antibodies. NeuN, which is a marker University School of Medicine. for neurons, was labelled with DyLight 488 to label the facial The Evaluation of Facial Nerve Palsy nucleus. CD11b, a microglial marker that is also expressed by leukocytes, including monocytes, neutrophils, natural killer The model was evaluated by scores of the blink reflex, vibrissae cells, and granulocytes, was labelled with DyLight 649; 40,6- movement, and the position of the apex nasi after the virus diamidino-2-phenylindole (DAPI, Beyotime, China) was used for inoculation. Blink reflex was performed using a 5-mL syringe nuclear staining. equipped with an 18G needle to blow air into the eye at a distance of 2 cm. We observed the movement and closure of the rats’ RNA Extraction, Library Preparation, and eyelids and the degree was graded on a 0–2 scale (0, no significant difference between the two sides; 1, the blink reflex was delayed Quantitative Real-Time Polymerase compared to the unaffected side; 2, the eyelid of the affected Chain Reaction side cannot be closed). Counting the vibrissae movement on Total RNA of rats’ facial nerve tissues and RSC96 cells were both sides of the rats within 30 s and scored on a 0–2 scale (0, extracted per the manufacturer’s instructions (Life Science, Frontiers in Molecular Biosciences| www.frontiersin.org 2 May 2021| Volume 8| Article 660072 fmolb-08-660072 April 30, 2021 Time: 20:29 # 3 Chang et al. Cox4i2 Leading to Ferroptosis United States). The libraries were prepared from cDNA, which of 1 × 105 cells per well. When the cell density reached was established using a cDNA Synthesis Kit (Takara, Japan) approximately 70–80%, the plasmids were transfected into cells and sequenced on an Illumina Hiseq4000 (Illumina Inc., using Lipofectamine 3,000 (Invitrogen, United States) according United States).