Citation: Yong, J.; Gröger, S.; von Bremen, J.; Meyle, J.; Ruf, S. Immunorthodontics: PD-L1, a Novel Immunomodulator in Cementoblasts, Is Regulated by HIF-1α under Hypoxia. Cells 2022, 11, 2350. https://doi.org/10.3390/ cells11152350 Academic Editors: Li Xiao and Takeo Tsutsui Received: 1 July 2022 Accepted: 28 July 2022 Published: 30 July 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). cells Article Immunorthodontics: PD-L1, a Novel Immunomodulator in Cementoblasts, Is Regulated by HIF-1α under Hypoxia Jiawen Yong 1,2,3, * , Sabine Gröger 1,2 , Julia von Bremen 1 , Joerg Meyle 2 and Sabine Ruf 1 1 Department of Orthodontics, Faculty of Medicine, Justus Liebig University Giessen, 35392 Giessen, Germany; sabine.e.groeger@dentist.med.uni-giessen.de (S.G.); julia.v.bremen@dentist.med.uni-giessen.de (J.v.B.); sabine.ruf@dentist.med.uni-giessen.de (S.R.) 2 Department of Periodontology, Faculty of Medicine, Justus Liebig University Giessen, 35392 Giessen, Germany; joerg.meyle@dentist.med.uni-giessen.de 3 Stomatology Hospital, School of Stomatology, Zhejiang University School of Medicine, Zhejiang Provincial Clinical Research Center for Oral Diseases, Key Laboratory of Oral Biomedical Research of Zhejiang Province, Cancer Center of Zhejiang University, Hangzhou 310003, China * Correspondence: jiawen.yong@dentist.med.uni-giessen.de or yongjiawen.ggl@gmail.com; Tel.: +49-641-99-46131 Abstract: Recent studies have revealed that hypoxia alters the PD-L1 expression in periodontal cells. HIF-1α is a key regulator for PD-L1. As hypoxia presents a hallmark of an orthodontically induced microenvironment, hypoxic stimulation of PD-L1 expression may play vital roles in immunorthodon- tics and orthodontically induced inflammatory root resorption (OIIRR). This study aims to investigate the hypoxic regulation of PD-L1 in cementoblasts, and its interaction with hypoxia-induced HIF-1α expression. The cementoblast (OCCM-30) cells (M. Somerman, NIH, NIDCR, Bethesda, Maryland) were cultured in the presence and absence of cobalt (II) chloride (CoCl 2 ). Protein expression of PD-L1 and HIF-1α as well as their gene expression were evaluated by Western blotting and RT-qPCR. Immunofluorescence was applied to visualize the localization of the proteins within cells. The HIF-1α inhibitor (HY-111387, MedChemExpress) was added, and CRISPR/Cas9 plasmid targeting HIF-1α was transferred for further investigation by flow cytometry analysis. Under hypoxic conditions, cementoblasts undergo an up-regulation of PD-L1 expression at protein and mRNA levels. Silencing of HIF-1α using CRISPR/Cas9 indicated a major positive correlation with HIF-1α in regulating PD-L1 expression. Taken together, these findings show the influence of hypoxia on PD-L1 expression is modulated in a HIF-1α dependent manner. The HIF-1α/PD-L1 pathway may play a role in the immune response of cementoblasts. Thus, combined HIF-1α/PD-L1 inhibition could be of possible therapeutic relevance for OIIRR prevention. Keywords: cementoblasts; hypoxia; PD-L1; HIF-1α; orthodontic; immunorthodontics 1. Introduction Orthodontic tooth movement (OTM) takes place as a direct consequence of connective tissue remodeling within the periodontium which occurs through a localized inflamma- tory response induced by external mechanical forces [1] when using removable or fixed orthodontic appliances [2]. The compressive force results in an altered homeostasis of the periodontal ligament (PDL) cells and the host immune response [3]. This orthodontic force causes capillary vasodilatation within the blood vessels of the PDL, in turn resulting in migration of immune cells and the expression of various cytokines such as GAS-6 [4]. These soluble cytokines regulate the process of cementum resorption, so-called orthodontic- induced inflammatory root resorption (OIIRR), in response to orthodontic force [5]. Ce- mentum seems to be able to protect the tooth root from OIIRR [6]. PDL cells (mainly cementoblasts) are capable of repairing resorption pits with new cementum [7,8] and play an immunomodulatory role under hypoxic conditions [9]. Cells 2022, 11, 2350. https://doi.org/10.3390/cells11152350 https://www.mdpi.com/journal/cells