ATF3 triggers M2 macrophage polarization to protect against pulp inflammation through WNT4 regulation

Abstract

Pulpitis is a common inflammatory oral disease that can lead to pulp necrosis. The aim of this study is to investigate the expression and regulatory mechanisms of ATF3, a potential therapeutic marker, in pulpitis. A mouse pulpitis model with different degrees of inflammation is established, and the expression of ATF3 in pulpitis is explored. The histological features of healthy pulp and pulpitis are analyzed by HE staining, and classical inflammatory factors are detected by immunohistochemistry (IHC). In an in vitro study, we investigate the role of ATF3 in the regulation of WNT4 transcription and explore the effects of the ATF3/WNT4 axis on the polarization of RAW264.7 macrophages, the inflammatory response and the osteogenic differentiation of human dental pulp stem/stromal cells (hDPSCs). Our results show that ATF3 is expressed at low levels in inflamed pulp tissues; overexpression of ATF3 reduces the area of pulp necrosis, decreases the level of pro-inflammatory factors, and promotes macrophage polarization toward the M2 type. Furthermore, we reveal that ATF3 binds to the WNT4 promoter region and positively regulates the expression of WNT4 and that ATF3 downregulates M1 markers and increases the expression of M2 markers by regulating WNT4 expression. In addition, ATF3 promotes the osteogenic differentiation of dental pulp stem cells. In summary, this study reveals that ATF3 promotes M2 macrophage polarization by regulating WNT4, which in turn inhibits pulpal inflammatory responses and promotes the osteogenic differentiation of dental pulp stem cells. These findings suggest that ATF3 may be a potential target for pulpitis treatment.

1. Introduction

Pulpitis is an inflammatory oral disease that typically occurs in pulpal tissues and is caused by caries, trauma, and chemical irritation, with caries-borne bacterial infection being the most common etiological factor [1]. Caries is one of the most prevalent diseases worldwide, with recent reports indicating that more than 90% of adults suffer from caries [2]. Bacterial invasion of the dental pulp is a dynamic process of inflammatory injury resulting from direct bacterial damage to virulence factor damage to activate the immune response [3]. Pulpitis usually progresses from a reversible to an irreversible stage, and even in the early stages of microbial infection, bacterial components can spread through dentin tubules and cause a localized inflammatory response in the pulp [4]. If left untreated, pulpitis can progress to pulp necrosis and even severe apical periodontitis [5]. In addition, oral inflammation is a common factor that induces other systemic diseases, such as cardiovascular disease and diabetes mellitus, which seriously affect people′s quality of life and general health [6]. Therefore, the pathogenesis and treatment of endodontic inflammation has been a hot topic of research, so it is crucial to explore the molecular mechanism of endodontic inflammation development to study and find therapeutic targets.

The inflammatory response is an important defense mechanism that stimulates the repair of damage to pulpal tissues and alleviates the degree of inflammation and tissue damage [7]. Among them, the role of macrophages in pulpitis has received increasing attention. Macrophages, as remarkable plastic cells, can be transformed into different subtypes of cells, such as M1-type macrophages with pro-inflammatory effects and M2-type macrophages with anti-inflammatory effects, and these two specific phenotypes are the functional responses of macrophages to stimuli and signals released from the microenvironment in which they are located, a process referred to as macrophage polarization [8,9]. M2-type macrophages play a significant role in the development and repair of endodontic tissues in the presence of Th2 lymphocytes, IL-4, IL-13, TLRs, and glucocorticoids; exhibit only weak antigen-presenting capacity; and downregulate the expression of immune responses by secreting inhibitory cytokines (IL-10, TGF-β, IL-RA, etc.), thereby regulating the balance of the body′s immunity [10]. In the late inflammatory response, M2-type macrophages can stimulate the proliferation and survival of major inflammatory immune cells in dental pulp tissues through immune modulation, exerting the immunomodulatory effects of phagocytosis and killing, antigen presentation, and cytokine secretion in the pulp microenvironment and promoting the repair and regeneration of local tissues in the organism [11]. Therefore, the regulation of macrophages in the dental pulp microenvironment facilitates the repair of damage to dental pulp tissues.

In the present study, we found for the first time that the activating transcription factor ATF3 was aberrantly expressed in pulpitis and that the ATF3/WNT4 axis promoted the osteogenic differentiation of dental pulp stem cells, suggesting a novel regulatory mechanism in pulp inflammation and repair.