All-Trans Retinoic Acid Modulates The Immune Status of M2 Macrophages in Experimental Periodontitis Induced by Porphyromonas Gingivalis in Mice

Background: M2 macrophages are important innate immune cells that participate in the pathogenesis of periodontitis. The effect of all-trans retinoic acid (ATRA) on the immune status of M2 macrophages in periodontitis has not been reported. Methods: An experimental model of periodontitis was established in mice by oral administration of Porphyromonas gingivalis, and then ATRA or vehicle was administered orally to model mice every other day (P.g+ATRA and P.g+CMC mice, respectively). Flow cytometry was used to analyze the numbers of F4/80+CD206+ M2 macrophages in the gingiva, spleen, and peritoneal lavage uid (PLF). M2 macrophage–related cytokines were quantied by real-time reverse transcription-polymerase chain reaction. Results: Compared with P.g+CMC mice, P.g+ATRA mice showed a signicantly reduced cemento-enamel junction to alveolar bone crest (CEJ-ABC) distance. The percentage of F4/80+CD206+ M2 macrophages in gingiva, PLF and spleen in model mice increased after ATRA treatment. The mRNA expression levels of M2 macrophage –related cytokines (IL-10, TGF-b1 and Arg-1) in gingiva, PLF and spleen of P.g+ATRA mice were higher than those of P.g+CMC mice. Conclusions: These results suggested that ATRA modulates the immune status of M2 macrophages and provides protection against periodontitis by enhancing M2 activation.


Introduction
Periodontitis is a disease involving the destruction of periodontal supporting tissue caused by unattached subgingival plaque and its metabolites in the gingival sulcus. Periodontitis is characterized by the loss of attachment, alveolar bone resorption, and periodontal pocket formation. The host immune response, including both innate and adaptive immune responses, is initiated against periodontal pathogens and plays an important role in the occurrence, development, and prognosis of periodontitis [1]. Resident macrophages in gingival tissue secrete large numbers of proin ammatory cytokines and chemokines after interactions with periodontal microorganisms. The macrophages participate in the phagocytosis and in ammation of periodontal pathogenic microorganisms and regulate alveolar bone resorption [2].
Macrophages are heterogeneous cells with strong plasticity and functional diversity that play key roles in both innate and adaptive immunity. Macrophages can be divided into many types based on their activated phenotypes and functions, including the classically and alternatively activated macrophages, designated as M1 and M2 macrophages, respectively. The phenotypic variations in macrophages induced by activation are reversible. M2 macrophages are activated by Th2 cytokines, such as interleukin (IL)-4, IL-13, and IL-10 [3,4], and secrete anti-in ammatory cytokines such as IL-10 and IL-1 receptor antagonists, as well as high levels of mannose receptor (CD206), arginase-1 (Arg-1), chitinase-3-like protein 1 (CHI3L1), and resistin-like molecule alpha (RELMα). M2 macrophages play important roles in the repair and remodeling of tissue damage caused by pathogenic microorganisms in the later stages of in ammation [5,6]. Intraperitoneal injection of M2 macrophages was reported to reduce experimental colitis in mice via an IL-10-dependent mechanism [7]. In a study in an experimental periodontitis model, adiponectin was suggested to participate in M2 macrophage polarization and host response by regulating the JMJD3-IRF4 axis, thus alleviating alveolar bone resorption [8]. Therefore, regulation of macrophage polarization to M2 macrophages may represent a new strategy for the treatment of periodontitis.
All-trans retinoic acid (ATRA) is an active metabolite of vitamin A that has a wide range of immunomodulatory effects and plays important roles in immune cell differentiation and activation as well as maintenance of the immune system. Studies showed that ATRA can be used in the treatment of a wide range of autoimmune diseases [9]. ATRA also regulates dendritic cells and reduces their antigen presentation ability, which further stimulate the T cells to respond to Th2 [10]. In addition, the combination of ATRA and IL-4 signi cantly increased the expression of arginase-1 (Arg-1), which is a marker of M2 activation [11]. However, there have been no studies on the effects of ATRA on the immune status of M2 macrophages in periodontitis. In this study, we examined the effects of ATRA on the immune status of M2 macrophages in periodontitis using a mouse model of periodontitis.

Mice
The experiments were performed in 7-week-old speci c pathogen free C57BL/6 female mice weighing 18 g, with intact dentition and no caries or periodontal disease. Mice were obtained from the Animal Center of Jinzhou Medical University. Animals were kept in individual cages with ad libitum access to food and water and fed adaptively for 1 week before establishment of the periodontitis model. The experimental procedures and animal treatment protocols were approved by the Ethics Committee of Jinzhou Medical University.
Periodontitis model was induced in mice by oral infection with 10 9 P. gingivalis W83, suspended in 100 μl sterile phosphate-buffered saline (PBS) containing 2% CMC, twice a day for 7 days. Sham mice were administered 100 μl of PBS containing 2% CMC by a micropipette for 7 days. Mice in the P.g+ATRA group were intra-gastrically administered 200 μL of ATRA per mouse dissolved in 1% CMC solution at 0.25 mg/ μl every other day from day 0 to day 42, and mice in the P.g+CMC and Sham group were intra-gastrically administered 200 μl of 1%CMC solution per mouse. All mice were killed by asphyxiation with CO 2 on day 42 and subjected to subsequent analyses described below.

Bone loss measurement
The right maxilla was dissected out and the soft tissue was removed by soaking in 3% hydrogen peroxide for 6 h, followed by boiling for 5 min to remove the residual soft tissue; the tissues were then stained with 0.1% methylene blue. The distance from the cement enamel junction (CEJ) of the maxillary rst molar to the alveolar bone crest (ABC) of the third molar (CEJ-ABC distance) was measured under a stereomicroscope at 32× magni cation. The mesial as well as the buccal and palatal molar surfaces were measured in each tooth, and the average values of 12 sites were determined. The total CEJ-ABC distance of the 12 sites in each group was calculated. The bone resorption rate of the experimental group (%) was calculated as = (total CEJ-ABC distance of the experimental group − total CEJ-ABC distance of the Sham group) / total CEJ-ABC distance of the Sham group.

Histological examination
Staining with hematoxylin and eosin (H&E) was performed to visualize the pathological damage of periodontal tissue. The molar segment of the left maxilla was washed with phosphate buffer, xed with 4% paraformaldehyde for 48 h, and decalci ed in 10% neutral ethylenediamine tetra acetic acid (EDTA; Solarbio). After dehydration in an alcohol series and para n embedding, the samples were cut into proximal and distal sections (5 μm thick). The sections were then stained with H&E and examined under a dissecting microscope at 100× magni cation.

Flow cytometric analysis
Isolation of gingival mononuclear cells The isolated periodontal tissue was washed with PBS and then added to 1 mL of PBS containing 2% FCS, 2 mg/mL collagenase II (Invitrogen, CA, USA), 1 mg/mL DNase I (Solarbio), and 1 mg/mL hyaluronidase (Sigma-Aldrich) in centrifuge tubes. Next, 20 μL of 0.5 M EDTA was added, and the tubes were incubated horizontally in a water bath preheated at 37°C with shaking at 200 rpm for 60 min. After centrifugation at 400 xg, 4°C for 8 min, the supernatant was discarded and the pellet was passed through a 200 mesh sieve. The ltered cell suspension was then centrifuged at 320 xg, 4°C for 8 min, and the supernatant was discarded. The pellet was resuspended in 200 μL of 2% fetal bovine serum (FBS) in PBS (FBS-PBS) and the cell numbers were counted.

Extraction of peritoneal lavage uid
Under aseptic conditions, the abdominal cavity of mice was opened and injected with 5 mL of precooled 2% FBS-PBS gently over a period of 2-3 min. After holding for an additional 5 min, the peritoneal lavage uid was absorbed and lavaged again. The uid was collected into 15-mL centrifuge tubes and the tubes were centrifuged at 300 × g, 4°C for 8 min. The supernatant was discarded; the pellet was resuspended in 200 μL of 2% FBS-PBS and the cell numbers were counted.

Preparation of single cell suspensions of spleen cells
The spleens of mice were removed aseptically according to routine procedures, rinsed with 1 mL of PBS, ground on a 200-mesh stainless steel screen, and ltered through a 30-μm nylon mesh to remove agglomerated cells. The cell suspension was centrifuged at 300xg, 4°C for 5 min, and then the supernatant was aspirated. Next, 1 mL red blood cell lysate was added to the precipitated cell mass.
RPMl1640 medium containing 10% FBS and 1% penicillin-streptomycin was then added to terminate the reaction. After centrifugation at 300 xg, 4°C for 5 min, the supernatant was discarded. The pellet was then washed twice with PBS and the supernatant was discarded. Cell viability was determined by trypan blue staining, and the cells were counted.
To Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) For qRT-PCR, total RNA was extracted from gingival tissues, peritoneal cells, and splenocytes using TRIzol reagent (Vazyme Biotechnology Co. Ltd, Nanjing, China) in accordance with the manufacturer's protocol.
The quality and quantity of RNA were measured using a spectrophotometer.

Statistical analysis
The data are expressed as the mean ± standard deviation (SD) and were analyzed using GraphPad Prism 22.0 software. The results were analyzed by one-way analysis of variance (ANOVA) and Student's t test.
In all analyses, P < 0.05 was taken to indicate statistical signi cance.

ATRA prevented the body weight loss in mice induced by P. gingivalis infection
To examine the effects of ATRA on the mouse model of periodontitis established by infection with P. gingivalis, we rst compared the changes in body weight between the P.g+ATRA, P.g+CMC, and Sham groups. The mice in the P.g+CMC group weighed signi cantly less than those in the Sham group on day 21 (P < 0.01, Figure 1). The body weights of the mice in the P.g+ATRA group began to show a signi cant upward trend on day 21 compared with mice in the P.g+CMC group, which continued until the animals were sacri ced on day 42 (P < 0.01, Figure 1). These data suggest that ATRA can improve the changes in body weight in periodontitis mice, possibly by ameliorating the effects of periodontitis.
ATRA reduced alveolar bone loss induced by P. gingivalis infection To examine the effects of ATRA on periodontitis in mice infected with P. gingivalis, we measured the CEJ-ABC distance in the P.g+ATRA, P.g+CMC, and Sham groups. The CEJ-ABC distance was signi cantly increased in the P.g+CMC mice compared with the distance in the Sham mice (P < 0.01, Figure 2a, b). However, the CEJ-ABC distance in P.g+ATRA mice was greatly reduced compared with the distance in the P.g+CMC mice (P < 0.01). No signi cant difference was observed in alveolar bone resorption between the P.g+ATRA mice and Sham mice (P < 0.05, Figure 2b).

ATRA attenuated periodontal lesions induced by P. gingivalis
Histological analysis with H&E staining indicated that the epithelium in the sulcus was intact in the Sham mice; there was no in ammatory cell in ltration in gingival epithelium and the periodontal ligament ber showed an orderly and regular arrangement ( Figure 3). In contrast, the P.g+CMC mice showed in ammatory cell in ltration and collagen ber degeneration and destruction. We also detected in ltration of large numbers of in ammatory cells and loss of attachment. ATRA treatment resulted in reduced in ammatory cell in ltration and attachment losses in P. gingivalis-infected periodontal tissues. Moreover, there was no apparent alveolar bone resorption in P.g+ATRA mice compared with that observed in P.g+CMC mice.

ATRA increased the proportion of M2 macrophages during P. gingivalis infection
To clarify the role of ATRA in the immune status of M2 macrophages in mouse periodontitis, the percentages of F4/80 + CD206 + cells (M2 macrophages) in gingivae, peritoneal cavity, and spleen were analyzed by ow cytometry. The results showed that the proportions of F4/80 + CD206 + M2 macrophages in gingival mononuclear cells, peritoneal lavage uid (PLF), and spleen were signi cantly decreased in the P.g+CMC mice compared with levels in the Sham mice (P < 0.01, Figure 4). In contrast, the P.g+ATRA mice showed signi cantly higher proportions of F4/80 + CD206 + M2 macrophages in gingival mononuclear cells, PLF, and spleen compared with levels in the P.g+CMC mice (P < 0.01, Figure 4).
ATRA upregulated the mRNA expression of M2 macrophage-related cytokines in periodontitis qRT-PCR analyses were performed to determine the mRNA expression levels of M2 macrophage-related cytokines in gingivae, PLF, and spleen. The levels of IL-10 and TGF-b1 mRNAs in gingivae and PLF were signi cantly increased in the P.g+CMC mice compared with levels in the Sham mice (P < 0.01, Figure 5a, b). Arg1 mRNA expression levels in gingival tissue were signi cantly higher in the P.g+CMC mice than in the Sham mice (P < 0.01, Figure 5a), but there were no signi cant differences in the PLF and spleen (P > 0.05, Figure 5b, c). The levels of IL-10, TGF-b1, and Arg-1 mRNAs in the gingivae, PLF, and spleen were signi cantly higher in the P.g+ATRA mice than in the P.g+CMC mice (P < 0.01, Figure 5ac).

Discussion
The clinical treatment of periodontitis currently includes plaque removal, surgical treatment, auxiliary laser, drug therapy, and other treatments. However, plaque removal and periodontal surgery have limited e cacy in the management of periodontitis [12]. Given the critical role of the immune system in the occurrence, development, and prognosis of periodontitis, effective regulation of the immune response is critical to enable it to play a protective role against periodontitis. Macrophages are involved in the development and persistence of in ammatory responses and the protection against periodontitis [13]. M2 macrophages are immunomodulatory cells that are characterized by the expressions of c-MAF, Arginase-1, CD14, CD163, mannose receptor (CD206), IL-10 and TGF-β1 [14]. Our study results indicate that ATRA upregulated the immune status of M2 macrophages in a mouse model of periodontitis and effectively inhibited periodontal tissue destruction. ATRA increased the proportions of F4/80 + CD206 + M2 macrophages in gingival tissue, spleen, and PLF and upregulated the expression of IL-10, TGF-b1, and Arg-1 mRNAs, which may have a protective effect against periodontitis.
Some studies have shown that P. gingivalis inhibits the production of α-ketoglutaric acid by M2 macrophages, thus maintaining the high in ammatory state of the periodontal microenvironment [15]. Miao et al.[16] reported that the proportion of M2 macrophages was signi cantly smaller in a mouse model of periodontitis induced by P. gingivalis compared with the healthy control group. These results imply that M2 macrophages are involved in maintaining periodontal tissue homeostasis in the periodontal microenvironment. Consistent with the above results, we found that the proportion of M2 macrophages in the gingival tissue of mice with periodontitis decreased in the model mice compared with the Sham group, while the mRNA expression levels of M2 macrophage-related cytokines IL-10, TGF-b1, and Arg1 increased. Although the levels of anti-in ammatory factors increased in mice with periodontitis, periodontal attachment loss and alveolar crest resorption were observed, which may have been because the e cacy of the anti-in ammatory cytokines secreted by M2 macrophages was insu cient to resist the tissue destruction caused by in ammation with the progression of in ammatory periodontitis. These results indicate a weak macrophage-mediated immune response in periodontitis.
Fotinoset al. [17] reported that the autoimmune reaction caused by severe periodontitis resulted in the accumulation of large numbers of immune cells, such as macrophages, in the periodontal tissue that increasing demyelination and inhibiting the progression of in ammation. Zhuang et al. [19] reported that the proportion of CD206 + M2 macrophages in gingival tissue increased, the number of osteoclasts decreased, and bone resorption decreased following injection of CCL2 into the gingivae of mice orally infected with P. gingivalis.
ATRA has a number of biological roles in cell proliferation and differentiation, embryogenesis, immunity, and metabolism [20]. ATRA was reported to induce the secretion of M2 macrophage chemokines in both C57BL/6 (B6) and BALB/c mice by inhibiting the expression of pro-in ammatory cytokines, nitric oxide synthase (NOS2), and NO and promoted the transition of macrophages from M1 to M2 in the two mouse strains [10]. Feng et al. [3] reported that ATRA reduced the expression of CD80, CD86 and CCR7 in splenic macrophages, increased the expression of CD209, promoted the polarization of macrophages to M2 macrophages, enhanced the production of IL-10 in M2 macrophages and inhibited the secretion of IL-12 and TNF-α, which further alleviated the progression of idiopathic thrombocytopenic purpura. Our previous studies con rmed that ATRA regulates the imbalance of Th17/Treg cells and improves periodontitis caused by P. gingivalis. These activities were likely mediated by ATRA inhibiting the response of Th17 cells, enhancing the response of Treg cells, and reducing the expression of RANKL in CD4 + T cells, thus regulating the Th17/Treg imbalance [21]. In the present study, oral administration of ATRA in a P.
gingivalis-induced periodontitis mouse model resulted in an increase in F4/80 + CD206 + M2 macrophages; there was no obvious alveolar bone resorption, and the mRNA expression levels of M2 macrophage-related factors IL-10, TGF-b1, and Arg1 in gingival tissue increased. Similar results were observed in the spleen and PLF. Therefore, ATRA not only modulates the immune state of M2 macrophages in the periodontal region, but also regulates the systemic immune state.
ATRA has been reported to activate JAK1/3 by regulating the binding of IL-4 with its receptor IL-4R. Phosphorylated JAK1/3 then recruits signal transduction and transcriptional activator 6 (STAT6) to promote the gene expression of Arg1, a key gene for tissue repair in M2 macrophages [22]. Lee et al. [23] reported that ATRA activates Arg-1 by upregulating the transcription of biosynthetic rate-limiting enzyme Raldh2, thereby promoting wound healing. ATRA was reported to induce the expression of TIMP1 in human alveolar M2 macrophages and regulate tissue remodeling by inhibiting the degradation of the extracellular matrix by various matrix metalloproteinases [24] . Retinoic acid nuclear receptor RAR-b is highly expressed in peritoneal macrophages, and retinoic acid response elements are also present in the gene regulatory regions of speci c GATA6 transcription factors expressed in peritoneal macrophages. ATRA regulates the expression of Arg-1-speci c genes in peritoneal macrophages through GATA6 transcription factors [25,26]. Taken together, the above results imply that ATRA affects the polarization of macrophages through a variety of signaling pathways.
In conclusion, the results of this study demonstrate that oral administration of ATRA modulated the immune status of M2 macrophages, affected the local oral microenvironment, and improved the in ammatory state in an experimental mouse model of P. gingivalis-induced periodontitis. Treatment with ATRA reduced both alveolar bone loss and the systemic immune response. These observations provide a basis for studying the in uence of periodontitis on innate immune function, as well as the relationships between periodontitis, systemic health, and immune regulation of periodontitis.
Declarations WJZ and LYW conceived the study. WJZ drafted the manuscript. LYW revised the manuscript. Flow cytometry was completed by WLY. P.gingivalis W83 culture and mouse periodontitis model was completed by XQG and NG. Reverese transcription-quantitative PCR was performed by WJZ. All authors have read and approved the manuscript.
Consent to participate All authors listed in the article have approved the manuscript that is enclosed.
Consent to publish The manuscript is approved by all authors for publication.
Informed consent All authors agree and give consent for the publication.

Figure 2
Evaluation of alveolar bone resorption in experimental periodontitis. (a, b) Alveolar bone resorption was analyzed by measuring the distance from the CEJ to the ABC on both the buccal and palatal molar surfaces. Representative images are shown (32× magni cation) (a). Values are shown as the mean ± SD of three mice per group. *P < 0.05, **P < 0.01 (b). (c) Alveolar bone resorption rate. a: P < 0.01 vs. Sham group; b: P < 0.01 vs. P.g+CMC group.