International Journal of Molecular Sciences Article Programmed Death-Ligand 2 Deficiency Exacerbates Experimental Autoimmune Myocarditis in Mice Siqi Li 1, Kazuko Tajiri 1,* , Nobuyuki Murakoshi 1, DongZhu Xu 1, Saori Yonebayashi 1, Yuta Okabe 1, Zixun Yuan 1, Duo Feng 1, Keiko Inoue 1, Kazuhiro Aonuma 1, Yuzuno Shimoda 1, Zoughu Song 1, Haruka Mori 1, Honglan Huang 2, Kazutaka Aonuma 1 and Masaki Ieda 1 1 Department of Cardiology, Faculty of Medicine, University of Tsukuba, Tsukuba 305-8575, Japan; [email protected] (S.L.); [email protected] (N.M.); [email protected] (D.X.); [email protected] (S.Y.); [email protected] (Y.O.); [email protected] (Z.Y.); [email protected] (D.F.); [email protected] (K.I.); [email protected] (K.A.); [email protected] (Y.S.); [email protected] (Z.S.); [email protected] (H.M.); [email protected] (K.A.); [email protected] (M.I.) 2 Department of Pathogenobioligy, College of Basic Medical Sciences, Jilin University, Changchun 130012, China; [email protected] * Correspondence: [email protected]; Tel.: +81-29-853-3143 Abstract: Programmed death ligand 2 (PD-L2) is the second ligand of programmed death 1 (PD-1) protein. In autoimmune myocarditis, the protective roles of PD-1 and its first ligand programmed death ligand 1 (PD-L1) have been well documented; however, the role of PD-L2 remains unknown. In this study, we report that PD-L2 deficiency exacerbates myocardial inflammation in mice with experimental autoimmune myocarditis (EAM). EAM was established in wild-type (WT) and PD-L2- deficient mice by immunization with murine cardiac myosin peptide. We found that PD-L2-deficient Citation: Li, S.; Tajiri, K.; Murakoshi, mice had more serious inflammatory infiltration in the heart and a significantly higher myocarditis + N.; Xu, D.; Yonebayashi, S.; Okabe, Y.; severity score than WT mice. PD-L2-deficient dendritic cells (DCs) enhanced CD4 T cell proliferation Yuan, Z.; Feng, D.; Inoue, K.; Aonuma, in the presence of T cell receptor and CD28 signaling. These data suggest that PD-L2 on DCs protects + K.; et al. Programmed Death-Ligand against autoreactive CD4 T cell expansion and severe inflammation in mice with EAM. 2 Deficiency Exacerbates Experimental Autoimmune Keywords: cardio-oncology; EAM; myocarditis; immune checkpoint; PD-L2; autoimmunity Myocarditis in Mice. Int. J. Mol. Sci. 2021, 22, 1426. https://doi.org/ 10.3390/ijms22031426 1. Introduction Academic Editor: Guido Iaccarino The programmed cell death protein 1 (PD-1) pathway has been shown to be an Received: 31 December 2020 attractive target for cancer immunotherapy. Immune checkpoint inhibitors targeting PD- Accepted: 28 January 2021 Published: 31 January 2021 1 or one of its ligands such as the programmed cell death ligand 1 (PD-L1) improves the prognosis of various malignancies [1,2]. However, the clinical benefit provided by Publisher’s Note: MDPI stays neutral these treatments can be accompanied by a unique and distinct spectrum of immune- with regard to jurisdictional claims in related adverse events. Among them, myocarditis is a relatively rare, but potentially life- published maps and institutional affil- threatening cardiovascular side effect; therefore, it has become an emergent problem for iations. clinicians in their daily routines [2,3]. Given these clinical aspects, a better understanding of how disruption of this pathway influences the immune system in the heart is needed. The protective roles of PD-1 and PD-L1 in cardiac immune homeostasis have been well established [4–8]. PD-1−/− mice on a BALB/c background developed autoimmune dilated cardiomyopathy, with the production of autoantibodies against cardiac troponin Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. I[4,5]. PD-1 or PD-L1 deficiency in the autoimmune-prone MRL mice induced lymphocytic + + This article is an open access article myocarditis with massive infiltration of CD4 and CD8 T cells [6,7]. Moreover, adminis- distributed under the terms and tration of anti-PD-1 antibodies exacerbated cardiac myosin peptide-induced experimental conditions of the Creative Commons autoimmune myocarditis (EAM) [9]. However, the role of PD-L2, the second ligand of Attribution (CC BY) license (https:// PD-1, in cardiac autoimmunity remains unknown. creativecommons.org/licenses/by/ In this study, we investigated the effect of PD-L2 deficiency on the development of 4.0/). myocarditis in the EAM model. We found that PD-L2 deficiency caused severe inflamma- Int. J. Mol. Sci. 2021, 22, 1426. https://doi.org/10.3390/ijms22031426 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 2 of 12 istration of anti-PD-1 antibodies exacerbated cardiac myosin peptide-induced experi- mental autoimmune myocarditis (EAM) [9]. However, the role of PD-L2, the second lig- and of PD-1, in cardiac autoimmunity remains unknown. Int. J. Mol. Sci. 2021, 22, 1426 2 of 12 In this study, we investigated the effect of PD-L2 deficiency on the development of myocarditis in the EAM model. We found that PD-L2 deficiency caused severe inflamma- tory infiltration in the heart. PD-L2-deficient dendritic cells (DCs) enhanced CD4+ T cell proliferationtory infiltration in the in thepresence heart. of PD-L2-deficient the T cell receptor dendritic (TCR) cells and (DCs)CD28 enhancedsignaling, CD4suggesting+ T cell thatproliferation PD-L2 on in DCs the presenceprotects agai of thenst T cardiac cell receptor antigen-reactive (TCR) and CD28CD4+ T signaling, cell expansion suggesting and severethat PD-L2 inflammation on DCs protectsin mice with against EAM. cardiac antigen-reactive CD4+ T cell expansion and severe inflammation in mice with EAM. 2. Results 2. Results 2.1. Expression Levels of PD-1 and PD-Ls in the EAM Hearts 2.1. Expression Levels of PD-1 and PD-Ls in the EAM Hearts We first examined the dynamics of inflammatory cells in EAM hearts. The heart in- We first examined the dynamics of inflammatory cells in EAM hearts. The heart flammation peaked at 14 days after the first immunization with cardiac myosin peptide inflammation peaked at 14 days after the first immunization with cardiac myosin peptide as evidenced by an increase in the number of infiltrating CD45+ leukocytes, CD11c+ den- as evidenced by an increase in the number of infiltrating CD45+ leukocytes, CD11c+ dritic cells (DCs), CD4+ T cells, CD11b+Ly6G− monocytes/macrophages, and CD11b+ Ly6G+ dendritic cells (DCs), CD4+ T cells, CD11b+Ly6G− monocytes/macrophages, and CD11b+ + neutrophilsLy6G+ neutrophils (Figure 1). (Figure The 1number). The number of infiltrating of infiltrating CD8 T CD8cells +peakedT cells on peaked day 21 on (Figure day 21 1).(Figure 1). FigureFigure 1. 1. DynamicsDynamics of of inflammatory inflammatory cells cells in in cardiac cardiac myosin myosin peptid peptide-inducede-induced autoimmune autoimmune myocard myocarditis.itis. Quantification Quantification of of inflammatoryinflammatory cells inin thethe hearts hearts obtained obtained from from mice mice with with experimental experimental autoimmune autoimmune myocarditis, myocarditis, represented represented as a percentage as a per- centageof live cells of live at thecells indicated at the indicated time points time (npoints = 10 (n at = each 10 at time each point). time point). The values The values are expressed are expressed as the asmean the mean± standard ± standarderror errorof the of mean. the mean. * p < * 0.05P < 0.05 vs. dayvs. day 0. 0. Next,Next, we we examined examined the the expression expression levels levels of of PD-1, PD-1, PD-L1, PD-L1, and and PD-L2 PD-L2 in in the the hearts hearts of of micemice with with EAM. EAM. The The mRNA mRNA expression expression levels levels of of PD-1, PD-1, PD-L1, PD-L1, and and PD-L2 PD-L2 were were markedly markedly upregulatedupregulated on on day day 14 14 in in the the EAM EAM hearts hearts (Figure (Figure 2A).2A). Particularly, Particularly, PD-L2 gene expression increasedincreased approximately approximately 4000-fold 4000-fold compared compared to to the the control control (Figure (Figure 22A).A). Flow cytometric + + analysesanalyses revealed revealed that that the the expression expression of PD-1, PD-L1, and PD-L2 on CD4+ TT cells, cells, CD8 + TT + cells,cells, and and CD11c CD11c+ DCsDCs peaked peaked around around 14–21 14–21 days days after after the the first first immunization immunization (Figure (Figure 2B).2B). Thus,Thus, the the expression expression levels levels of of PD-L2, PD-L2, as as well well as as PD-1 PD-1 and and PD-L1, PD-L1, were were upregulated upregulated in in the the heartheart during during cardiac cardiac inflammation. inflammation. Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 3 of 12 Int. J. Mol. Sci. 2021, 22, 1426 3 of 12 Figure 2. Expression levels of programmed cell death protein 1 (P (PD-1)D-1) and programmed cell de deathath ligands (PD-Ls) in the experimental autoimmune myocarditis myocarditis (EAM) (EAM) hearts. hearts. ( (AA)) mRNA mRNA expression expression of of PD-1, PD-1, PD PD-L1,-L1, and and PD-L2 PD-L2 in in the hearts obtained from mice withwith EAMEAM (days(days 1414 and and 28) 28) and and control control mice mice (n (n = = 6–7). 6–7). (B (B) Flow) Flow cytometric cytometric analysis analysis of of the the expression expression of of PD-1, PD-L1, or PD-L2 on infiltrating CD4+ T cells, CD8+ T cells, and CD11c+MHC II+ dendritic cells in the hearts obtained PD-1, PD-L1, or PD-L2 on infiltrating CD4+ T cells, CD8+ T cells, and CD11c+MHC II+ dendritic cells in the hearts obtained from mice with EAM on days 0, 7, 14, 21, and 35 after first immunization with cardiac myosin peptide (n = 6 at each time from mice with EAM on days 0, 7, 14, 21, and 35 after first immunization with cardiac myosin peptide (n = 6 at each time point).