Apical periodontitis (AP) is characterized by sustained local inflammation around the root apex, which results in periapical alveolar bone loss (1). Exogenous bacteria activate the host immune system, leading to the recruitment of immune cells such as neutrophils, macrophages and T cells around the root apex (2). These immune cells secrete inflammatory chemokines and cytokines to modulate the inflammatory process (3). The infection is not completely obliterated by the host defense; thus, a sustained chronic inflammatory immune microenvironment involving bacterial virulence, immune cells, chemokines and cytokines is established ( Figure 1A ) (4).

Inflammation can disrupt physiological bone homeostasis around the root apex and result in inflammatory bone loss (2, 4). The balance between different subsets of immune cells could influence the outcome of infectious inflammatory osteolytic lesions (5). Activated T cells (such as Th1 and Th17) and other immune cells can highly express RANKL or release proinflammatory cytokines to promote osteoclastogenesis (6). Simultaneously, the innate immunoregulatory response against inflammation is mediated by other subsets of immune cells such as regulatory T cells (Tregs), which lead to the dampening of inflammatory and osteoclastogenic pathways (7). Many studies have successfully altered inflammatory bone loss by modulating the reaction of immune cells (8, 9).

As an important signaling pathway to curb the immune response and minimize inflammatory tissue damage, immune checkpoint molecules (ICs) expressed on immune cells actively participate in the maintenance of immune homeostasis (10). ICs play a central role in regulating cancer development, autoimmune diseases, etc. (11, 12). Moreover, recent reports suggest that ICs also participate in the regulation of inflammatory diseases (11). Our previous study demonstrated the overexpression of PD-1 and LAG-3 in periapical lesions, which suggests a potential role of ICs in inflammatory bone loss (13). In addition to the well-known ICs such as PD-1 and LAG-3, V-domain immunoglobulin suppressor of T cell activation (VISTA), which shows considerable homology with PD-1 and PD-L1, is another molecule that has recently attracted considerable interest (14). In contrast to PD-1 or LAG-3, which are mainly expressed on T cells, VISTA is more widely expressed on both myeloid cells and CD4⁺ and CD8⁺ T cells, acting not only as a receptor but also as a ligand. VISTA is involved in peripheral tolerance and mediates the function of immune cells in an unknown manner (15). A VISTA deficiency leads to a spontaneous proinflammatory phenotype in mice (16). The blockade of VISTA by specific in vivo antibodies augments the severity of autoimmune and inflammatory diseases such as asthma mouse models (17) and psoriasis mouse models (18). However, the in vivo function of ICs in a mouse experimental apical periodontitis model has not yet been elucidated, so we decided to investigate the role of VISTA in periapical diseases.

In the present study, we investigated the expression of VISTA in human AP tissue and a mouse experimental apical periodontitis (MAP) model. Moreover, VISTA was blocked using an in vivo anti-VISTA antibody in mice, and the alteration of inflammatory bone loss in the MAP model was investigated.

VISTA has been reported in some inflammatory and autoimmune diseases (23). In this study, we first demonstrated that VISTA is involved in the development of periapical periodontitis (AP). The present study shows that VISTA is expressed on CD3⁺ T cells and CD11b⁺ myeloid cells in human inflammatory periapical tissues. In mouse experimental apical periodontitis (MAP), VISTA-positive cells were not detected in the early stages (D7, D14) but gradually increased in the later stages (D21, D28). CD3⁺ VISTA⁺ T cells and CD11b⁺ VISTA⁺ myeloid cells were also detected in MAP. The specific in vivo blockade of VISTA aggregated bone erosion and immune cell accumulation around the mandibular apex at D21 and D28. These results suggest that VISTA plays a role in the development of AP and might act as a negative regulatory molecule.

The present study first demonstrated the participation of VISTA in exogenous bacteria-induced inflammatory bone disease. In the MAP model, the pulp chamber was perforated and left exposed to the mouse native oral microbial species, then the bone loss around the tooth apex was successfully induced over a period of time (20, 21, 24). As an important immune checkpoint protein, the role and function of VISTA has been mainly investigated in tumors and autoimmune or noninfectious inflammatory diseases (23). VISTA is highly expressed in tumor tissues (25–27), autoimmune disease lesions in systemic lupus erythematosus (18), ConA-induced mouse hepatitis (28), chemical agent-induced ear inflammation (29) and in the synovium of CAIA-induced arthritis (30). VISTA is mainly expressed on T cells and myeloid cells, but the expression patterns of VISTA vary depending on the disease.

Apical periodontitis is a type of inflammatory osteolytic disease (1). Inflammation is a defense mechanism of the host to kill and clear invasive pathogens. However, flood inflammation will cause uncontrolled bone tissue damage (5). To control overt immune responses thus maintaining immunological homeostasis and limiting damage by inflammation, essential intrinsic mechanisms of the host may inhibit excessive inflammation (31). Immune checkpoint molecules (ICs) are crucial for the maintenance of immune homeostasis in the host (12). VISTA expressed on myeloid antigen-presenting cells (APCs) combines with an unidentified receptor on CD4⁺ and CD8⁺ T cells to suppress their proliferation and cytokine production (15), and VISTA also suppresses the cytokine secretion of myeloid cells (32–34). VISTA expressed on CD4⁺ T cells limits T cell activation in a cell-intrinsic manner (25). In the present study, CD3⁺VISTA⁺ T cells and CD11b⁺VISTA⁺ myeloid cells were detected at D21 and D28 but not at D7 or D14 in MAP. The bone loss in MAP appeared to slow down in the later stage, the bone loss volume increased slowly from D21 to D28, which was consistent with previously reported results (35). These collective results suggest that the host invokes the expression of VISTA possibly as an intrinsic negative regulator to avoid excessive tissue damage during the development of MAP.

Regulatory T cells (Tregs) are innate, basic and crucial cells that suppress inflammation and regulate the immune response (36). Tregs have been previously reported to participate in the development of AP (37) and MAP (38). Consistent with these reports, we found that Tregs were expressed in AP. In MAP, we barely detected Tregs in MAP at D7 and D14. The number of Tregs was increased at D21 and D28, and the expression pattern was similar to that of VISTA. Tregs might coordinate VISTA-positive T cells and myeloid cells to restrain the development of MAP.

It has been indicated that the development of inflammatory bone loss can be mediated by modulating the intrinsic suppressing system (39). Bone loss in MAP was alleviated through the adoptive transfer of Tregs (8). In contrast, the blockade of Tregs induced heavier bone loss in experimental periodontitis (40). However, it is not clear whether the blockade of ICs could influence MAP. Antagonistic targeting of VISTA has been proven to be a unique activated avenue for immune cells (41). The blockade of VISTA may exacerbate the extent of inflammatory diseases by augmenting the proinflammatory role of T cells and myeloid cells (17, 18, 32, 42). Therefore, it is interesting to investigate whether anti-VISTA treatment affects the progression of MAP. The results showed that the blockade of VISTA worsened the extent of MAP at D21 and D28. It has been reported that the blockade of VISTA results in the accumulation of inflammatory cells in diseases targeting tissues (16, 29, 34). The present study also confirmed that the blockade of VISTA enhanced the recruitment of CD3⁺ T cells and CD11b⁺ myeloid cells (including F4/80⁺ macrophages). VISTA seems to be a pleiotropic cell checkpoint in the regulation of cytokines and chemokines (41), the deficiency of VISTA would result in the accumulation of cytokines and chemokines in mice (16, 18). In the present study, the elevated CXCL12 in anti-VISTA- treated group might contributed to the recruitment of immune cells at D28 in MAP. We also found IL-6 and TNF-α were upregulated in anti-VISTA group compared with the control group, which might indicate the more active inflammatory process in the treated group. The number of FOXP3⁺ Tregs increased slightly (not statistically significant), which might represent feedback from the exacerbated inflammatory microenvironment of MAP. Until now the cellular and molecular mechanisms of VISTA-mediated immune regulation are still not clear. Recent studies have presented several possible candidates such as VSIG3 (43). PSGL-1 (44) in human samples. However, more precise pathway of the regulative function of VISTA is still under investigation.

The present study revealed the potential negative regulatory mechanism of VISTA on inflammatory bone loss. The potential over-activation of VISTA of current anti-inflammatory drug may be underestimated and warrant explored. Furthermore, the VISTA-Ig has been applied on oxazolone-induced allergic mice dermatitis model and ameliorated the symptoms (45). Also, agonists of VISTA can reduce T cell response, prevent development of GVHD (46) and experimental asthma in mice model (17). These results provided a potential therapeutic implication by over-activating VISTA for therapeutic of inflammatory bone loss, which warrant further exploration in the future.

In conclusion, our study first demonstrated that VISTA might play a negative role in the development of AP, and that the development of AP might be affected by regulating VISTA-positive cells. Our study provides an increased understanding of AP and may inspire additional therapies for AP.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The studies involving human participants were reviewed and approved by the Medical Ethics Committee of the School and Hospital of Stomatology at Wuhan University. The patients/participants provided their written informed consent to participate in this study. The animal study was reviewed and approved by the Experimental Animal Ethics Committee of the School and Hospital of Stomatology at Wuhan University.

FY contributed to the conception and design, data collection, data analysis and drafting the manuscript. YZ helped to collect data. ZC contributed to the conception and design. LZ reviewed the manuscript and managed the project. All authors contributed to the article and approved the submitted version.

This work was funded by grants from National Natural Science Foundation of China (81974148, 81771064) to LZ and (81420108011) to ZC.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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