ST2 Deletion Increases Inflammatory Bone Destruction in Experimentally Induced Periapical Lesions in Mice Milena Velickovic, DDS,* Nada Pejnovic, MD, PhD,* Slobodanka Mitrovic, MD, PhD,* Gordana Radosavljevic, MD, PhD,* Ivan Jovanovic, MD, PhD,* Tatjana Kanjevac, DDS, PhD, † Nemanja Jovicic, MD,* and Aleksandra Lukic, DDS, PhD ‡ Abstract Introduction: ST2 is a member of the interleukin (IL)-1 re- ceptor family, and IL-33 is its natural ligand. ST2 signaling promotes Th2 immune response in allergy, autoimmunity, and chronic inflammatory disorders, but its role in the path- ogenesis of periapical lesions is unknown. The purpose of this study was to investigate whether ST2 gene deletion affects the development of experimentally induced periap- ical lesions in mice. Methods: Pulps of mandibular molars from wild-type (WT) and ST2 knockout (ST2 – / – ) BALB/c mice were exposed and left open to the oral environment. After death, hemi-mandibles were isolated and prepared for histologic, immunohistochemical, and flow cytometric analysis. Results: The expression of IL-33 and its receptor ST2 was higher in periapical lesions in WT mice compared with normal root apices (both P < .05). The increased peri- apical bone loss observed in ST2 – / – mice was associated with enhanced influx of neutrophils, CD3+ CXCR3+ Th1 cells, and CD3+ CCR6+ Th17 cells and increased number of tartrate-resistant acid phosphatase+ osteoclasts (all P < .05). Furthermore, periapical lesions in ST2 – / – mice con- tained increased percentages of T cells expressing inter- feron-g, IL-17, tumor necrosis factor-a, and IL-6 (all P < .05). In comparison with WT mice, CD3+ receptor acti- vator of nuclear factor kappa B ligand+ T cells were increased, whereas CD3+ osteoprotegerin+ T cells were decreased in the lesions of ST2 – / – mice (both P < .05). Conclusions: ST2 deletion increases inflammatory bone loss in experimental periapical lesions in mice, which is associated with enhanced Th1/Th17 cell mediated periap- ical immune responses and increased osteoclastogenesis. (J Endod 2015;41:369–375) Key Words Bone loss, inflammation, osteoclasts, periapical lesions, RANKL, ST2, Th1/Th17 cells P eriapical lesions develop in response to chronic stimulation caused by microorgan- isms that invade and destroy the dental pulp (1). The host’s response appears to be similar to the response to bacterial infections elsewhere in the body, with the additional feature of the resorption of alveolar bone that surrounds the dental root apex (2). Oste- oclasts are multinuclear tartrate-resistant acid phosphatase (TRAP) positive cells that are principally responsible for bone resorption, which differentiation and maturation are regulated by the balance between the receptor activator of nuclear factor kappa B ligand (RANKL) and its soluble decoy receptor osteoprotegerin (OPG) (3, 4). Th1- and Th17- derived cytokines together with interleukin (IL)-1, IL-6, and tumor necrosis factor (TNF)- a promote chronic inflammation and bone resorption, whereas Th2-derived cytokines and T-regulatory cells (Tregs) are responsible for the healing processes (5–11). The ST2 gene, a member of the IL-1 receptor family (IL-1R), encodes a shorter sol- uble form and a transmembrane full-length form, sST2 and ST2L, respectively (12). ST2L is stable and selective marker of Th2 cells (13). Natural ligand for ST2L is IL-33, which is released on necrotic cell death during infection and inflammation. IL-33 is mainly ex- pressed in the nucleus of fibroblasts and endothelial and epithelial cells (14) and potently enhances Th2 immune response (15). IL-33/ST2 signaling has a protective role in Th1/ Th17 cell mediated inflammatory diseases such as experimental autoimmune encephalo- myelitis, fulminant hepatitis, and type 1 diabetes (16–18). The aim of this study was to investigate whether the lack of ST2 signaling affects inflammatory bone destruction in experimentally induced periapical lesions in mice. Materials and Methods Mice IL-33 receptor knockout mice (ST2 –/– mice) on the BALB/c background were generated by a targeted disruption of mouse ST2 gene (19). ST2 –/– mice were provided by Dr McKenzie (University of Cambridge, United Kingdom). We used 6- to 8-week-old male wild-type (WT) and ST2 –/– mice for the induction of periapical lesions. All animals were maintained in our animal facilities under standard laboratory conditions and received humane care. Experiments were approved by the Animal Ethics Committee of the Faculty of Medical Sciences, University of Kragujevac, Serbia. Induction of Periapical Lesions Mice were anesthetized with ketamine hydrochloride (60 mg/kg body weight) and xylazine (10 mg/kg body weight) by intraperitoneal injection. Mandibular molar pulps were exposed by using a high-speed electric dental handpiece (W&H Dentalwerk, Bur- moos, Austria) (20). For histologic, morphometric, and immunohistochemical analyses, periapical lesions were induced by pulp exposure of the mandibular first molar pulps. For flow cytometric analysis, mice were subjected to both first and second mandibular molar pulp exposure. Mice were killed 14 and 28 days after lesion induction. Immunohistochemistry Paraffin-embedded sections of periapical lesions and normal root apices from WT mice were deparaffinized and incubated with primary rabbit anti-IL-33 antibody (sc- 98660; Santa Cruz Biotechnology, Santa Cruz, CA; 1:100), followed by visualization by using Expose Rb specific HRP/DAB detection IHC kit (Abcam, Cambridge, UK). From the *Center for Molecular Medicine, Kragujevac, Serbia; † Department of Preventive and Pediatric Dentistry, Fac- ulty of Medical Sciences, University of Kragujevac, Kragujevac, Serbia; and ‡ Department of Endodontics, Faculty of Medical Sci- ences, University of Kragujevac, Kragujevac, Serbia. Address requests for reprints to Dr Aleksandra Lukic, Fac- ulty of Medical Sciences, University of Kragujevac, Svetozara Markovica 69, 34000 Kragujevac, Serbia. E-mail address: miodrag.lukic@medf.kg.ac.rs 0099-2399/$ - see front matter Copyright ª 2015 American Association of Endodontists. http://dx.doi.org/10.1016/j.joen.2014.11.017 Basic Research—Biology JOE — Volume 41, Number 3, March 2015 ST2 Deletion Increases Bone Loss in Periapical Lesions 369