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. 2019 Jul 22;98(9):1027–1036. doi: 10.1177/0022034519858063

Activation of the STAT1 Pathway Accelerates Periodontitis in Nos3-/- Mice

W Wei 1,*, X Xiao 1,*, J Li 1, H Ding 1, W Pan 1, S Deng 1, W Yin 1, L Xue 1, Q Lu 1, Y Yue 1, Y Tian 1, M Wang 1, L Hao 1,✉
PMCID: PMC6651763  PMID: 31329047

Abstract

Early studies on the etiology and pathogenesis of hypertension have shown that it has a considerable association with inflammation and the immune response as well as periodontitis. Clinical studies have also shown that hypertension can promote the periodontal tissue destruction caused by periodontitis. However, the underlying mechanisms remain unclear. This study aimed to explore the possible mechanisms of how hypertension aggravates periodontitis. Treatment with or without the signal transducer and activator of transcription 1 (STAT1) inhibitor fludarabine was performed in an endothelial nitric oxide synthase gene knockout-related (Nos3-/-) mouse model with the hypertension phenotype of periodontitis induced by bacteria. Micro–computed tomography, immunohistochemistry, Western blot, quantitative reverse transcription polymerase chain reaction, immunofluorescence, and ELISA were performed. We demonstrated that Nos3-/--related hypertension increases bone resorption and periodontal destruction in periodontitis lesion areas, which can be inhibited by the STAT1 inhibitor. Experimental data also showed that Nos3-/- significantly increased macrophage infiltration and proinflammatory cytokine expression in the periodontitis lesion area, which is dependent on the angiotensin II–induced STAT1 pathway. Inhibition of STAT1 in vivo can decrease the expression of proinflammatory cytokines and macrophage infiltration. Furthermore, data in this study showed that Nos3-/--related hypertension further downregulated the STAT3 anti-inflammatory function and its downstream chemokine expression in a STAT1-dependent manner. By applying RAW 264.7 and L929 cell lines and monocytes isolated from Nos3-/- mice, we confirmed that activation of the STAT1 pathway inhibits STAT3 and its downstream pathway and promotes inflammatory cytokine expression in vitro. Collectively, our current study demonstrated that STAT1 plays an indispensable role in the Nos3-/--related hypertension with aggravation of periodontitis, suggesting that STAT1 may be a key target for the treatment of periodontitis with hypertension.

Keywords: hypertension, inflammation, STAT3 transcription factor, STAT1 transcription factor, macrophage, periodontal diseases

Introduction

As a major cardiovascular disease, hypertension is the leading cause of death (Lawes et al. 2008). Many dietary and lifestyle factors contribute to the chronic inflammation associated with hypertension (Niskanen et al. 2004). As a major factor in the pathogenesis of hypertension, angiotensin II (Ang II) is a critical vasoconstrictor that promotes the renin-angiotensin system (Veerasingham and Raizada 2003) and can activate the signal transducer and activator of transcription 1 (STAT1) pathway through its membrane receptor AT1 (Marrero et al. 1995).

The pathologic feature of periodontal disease is chronic inflammatory lesions in tissues surrounding the teeth (Nakib et al. 2004), manifested as damage to the alveolar bone and connective tissue supporting the teeth (Cochran 2008). Smoking, diabetes, immunosuppression, and certain bacteria are known to cause periodontitis (Borrell and Papapanou 2005). A large-scale clinical study showed an association between hypertension and periodontal disease (Eke et al. 2012). Although a number of studies have found that hypertension can affect bone remodeling in periodontitis and aggravate periodontitis lesions reciprocally (Leong et al. 2014), the mechanism by which hypertension affects periodontitis is still unclear.

STAT1, a member of the signal transducer and activator of transcription protein family, plays a regulatory role in many aspects, such as cell growth, differentiation, inflammation, and apoptosis (Ivashkiv 2000). STAT1 is a critical pathway for regulating the disorders encountered by the immune and inflammatory system. Studies have identified STAT1 as a requisite for Ang II (Lee et al. 2007) stimuli in hypertension. Other members of the STAT family also perform specific functions. For example, STAT3 can play an anti-inflammatory role activated by IL6 (Hodge et al. 2005). STAT proteins have individual functions but simultaneously interact with one another. They also promote the expression of suppressor of cytokine signaling (SOCS) proteins, which in turn negatively regulate specific STAT protein phosphorylation (Yoshimura et al. 2007).

In our previous study, we successfully established a Nos3 knockout (Nos3-/-)–related hypertension mouse model with periodontitis and showed that Nos3-/- mice, which have higher blood pressure and Ang II expression as compared with wild-type mice, can greatly aggravate periodontal bone destruction (Li et al. 2019). As hypertension is associated with immunity and inflammation (Agita and Alsagaff 2017) and Ang II plays an important role in the course of hypertension, we hypothesized that Ang II and its downstream STAT1 pathway may play an active role in periodontitis. We focused on the immune inflammatory cells and explored the mechanism underlying how hypertension affects the development of periodontitis lesions in vivo.

Materials and Methods

For complete Materials and Methods, see the Appendix.

Animals

Four-week-old Nos3-/- mice were acquired from the Model Animal Research Center of Nanjing University for mating and breeding; these mice showed spontaneous hypertension (de Jonge et al. 2002; Li et al. 2004) and upregulation of Ang II (Li et al. 2019). Eight-week-old female mice were divided into 8 groups for the experiment:

  • 1, 2) control groups (C57BL/6J) with or without treatment with the STAT1 inhibitor fludarabine (C and CI)

  • 3, 4) Nos3-/- mice with or without fludarabine treatment (KO and KO I)

  • 5, 6) bacteria-infected mice (C57BL/6J) with or without fludarabine treatment (P and P I)

  • 7, 8) bacteria-infected Nos3-/- mice and the corresponding inhibitor groups (P KO and P KO I)

The experiments in this study were conducted independently in triplicate. This study was approved by the Institutional Animal Care and Use Committee of Sichuan University (program WCCSIRB-D-2015-030). Animals were fed with nutrients and distilled water ad libitum at the State Key Laboratory for Oral Disease.

Bacterial Strain Infection

The mouse model of periodontitis was established according to the previous protocol (Hao et al. 2015). Porphyromonas gingivalis (ATCC: 53978) was grown under anaerobic conditions at 37 °C (Byrne et al. 2009). Sterile 3% (w/v) carboxymethylcellulose (Fisher) was added to an equal volume of bacterial suspension (5 × 1010 bacterial cells per milliliter) and thoroughly stirred to prepare a bacterial mixture. A small dental swab was applied to inoculate the bacteria for 8 d consecutively to induce periodontitis (Pan et al. 2019).

Administration of the STAT1 Inhibitor

After infection of the mice with the bacteria, we treated the inhibition treatment groups with phosphate-buffered saline containing 10 mg/mL of fludarabine (CAT#F2773, Sigma-Aldrich), a specific inhibitor of STAT1 (Feng et al. 2017). Three hundred microliters of the inhibitor solution were applied to each mouse by intraperitoneal injection, and the noninhibitor group was administered with an equal volume of phosphate-buffered saline. The treatment was administered every other day for 4 wk (Tajima et al. 2010).

Statistical Analysis and Data Quantification

The figures are representative of all data (n = 10). Data are represented as the mean ± SD of independent samples. One-way analysis of variance for parametric data and Mann-Whitney U test for nonparametric data were applied for analyzing the data. P values <0.05 or U values >1.96 were considered significant.

Results

Treatment with the STAT1 Inhibitor Reduced Bone Destruction in the Periodontitis Lesion Area of the Periodontitis-Nos3-/- Group

To investigate the influence of STAT1 in the development of periodontitis with Nos3-/--related hypertension, we analyzed the bone resorption in the periodontal area by micro–computed tomography. Bacteria-induced periodontitis increased bone resorption (red dotted area), which demonstrated successful model establishment (Fig. 1). The periodontitis lesion area was exacerbated in the periodontitis-Nos3-/- group as expected. Interestingly, the inhibitor group showed a significant bone protection effect as compared with the periodontitis-Nos3-/- group (Fig. 1A). More severe bone destruction was found in the periodontitis-Nos3-/- group, whereas the STAT1 inhibitor could ameliorate the bone erosion (Fig. 1B, C).

Figure 1.

Figure 1.

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Inhibition of STAT1 (signal transducer and activator of transcription 1) by specific inhibitor reduced bone resorption in the periodontitis lesion area of the periodontitis-Nos3-/- group. (A) Three-dimensional reconstruction of the maxilla of different groups. Vertical bone resorption is indicated by red dotted area. (B) Quantification of alveolar bone resorption area and CEJ-ABC length: distance from the cement-enamel junction (CEJ) to the alveolar bone crest (ABC). (C) Quantification of bone volume/total volume, Tb.N (trabecular number), and Tb.Sp (trabecular separation). (D) Hematoxylin and eosin staining of periodontal tissue of different groups. The width of periodontal ligament is indicated by black dotted areas. (E) Quantification of periodontal ligament width. (F) The systolic and diastolic blood pressure was measured at 16 wk. (G) Relative mRNA expression levels of Nos3 were detected by quantitative reverse transcription polymerase chain reaction. Gapdh was used as the endogenous control. C, nonnephritic phosphate-buffered saline–treated group; I, inhibitor-treated group; KO, Nos3 knockout group; P, periodontitis group; P KO, periodontitis with Nos3 knockout group. *P < 0.05. **P < 0.01. ***P < 0.001. Repeated 3 times. Data are presented as the mean ± SD (n = 10 per group) and compared with the control.

We then used hematoxylin and eosin staining to determine the alveolar bone resorption and quantitatively analyzed the width of the periodontal ligament of different groups (Fig. 1D, E). The results verified that alveolar bone resorption was increased in the periodontitis-Nos3-/- group as compared with the periodontitis-only group, which was reversed by the STAT1 inhibitor (periodontitis-Nos3-/--inhibitor group). The data showed that Nos3-/- mice have higher blood, systolic, and diastolic pressure as compared with the control group (Fig. 1F). The expression of Nos3 was also detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR) to confirm the knockout level (Fig. 1G). Collectively, these data suggested that Nos3 knockout exacerbates periodontal disease and that inhibition of STAT1 can exert a protective effect on periodontal bone resorption, indicating that STAT1 may be involved in the link between periodontitis and Nos3-/--related hypertension.

Inhibition of STAT1 Decreased Downstream SOCS3 Expression in the Periodontitis Lesion Area

To explore the underlying mechanism, we then analyzed the expression of STAT1 and its related cytokines (Fig. 2). Immunohistochemistry staining indicated that phosphorylated STAT1 (pSTAT1)–positive cells were significantly increased in the periodontitis area of the periodontitis-Nos3-/- group than in the periodontitis-only group (Fig. 2A, B). Western blot analysis showed a similar result that pSTAT1 expression was significantly increased in the periodontitis-Nos3-/- group (Fig. 2C). In addition, the protein expression levels of SOCS3 were higher in the Nos3-/- mouse model and the coexistence model of 2 diseases (Fig. 2D). Results of the qRT-PCR assay also revealed that the expression of STAT1 downstream factors Cxcl9 and Socs3 was increased significantly, indicating that Nos3 knockout can activate the functional status of STAT1 in the periodontal lesion region (Fig. 2E). The inhibitor fludarabine was found to significantly prevent STAT1 phosphorylation (Fig. 2A, C) and decrease the expression of SOCS3 (Fig. 2D, E). These data suggested that Nos3 knockout–related hypertension may significantly activate STAT1 and upregulate the expression of SOCS3 in vivo.

Figure 2.

Figure 2.

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Inhibition of STAT1 (signal transducer and activator of transcription 1) decreased the STAT1 downstream SOCS3 (suppressor of cytokine signaling 3) expression in the periodontitis lesion area. (A) Immunohistochemistry analysis of pSTAT1 (phosphorylated STAT1) in the periodontitis lesion areas of different groups; the red arrow refers to cells expressing pSTAT1. (B) Quantification of pSTAT1-positive cells numbers in the periodontal lesion area. Western blot analysis of (C) pSTAT1 and STAT1 expression and (D) SOCS3 expression in the periodontal lesion area of different groups; representative images and the summary of the normalized quantification are shown. (E) Quantitative reverse transcription polymerase chain reaction of Stat1, Cxcl9, and Socs3 in different groups. C, nonnephritic phosphate-buffered saline–treated group; I, inhibitor-treated group; KO, Nos3 knockout group; P, periodontitis group; P KO, periodontitis with Nos3 knockout group. *P < 0.05. **P < 0.01. ***P < 0.001. Repeated 3 times. Data are presented as mean ± SD (n = 10 per group) and compared with the control.

Inhibition of STAT1 Can Decrease Macrophage Infiltration and Inflammatory Cytokines in the Periodontitis-Nos3-/- Group

Using the immunofluorescence assay, we demonstrated that macrophages infiltrated in the lesion area of periodontitis. Macrophages were significantly increased in the periodontitis-Nos3-/- group as compared with the periodontitis-only group, and the infiltration of macrophages was obviously decreased in the periodontitis-Nos3-/- group with the inhibitor (Fig. 3A–C). Inhibitor treatment also decreased the proinflammatory gene expression of Il6, Tnfα, Il1β, and Ifnγ in the periodontitis-Nos3-/- group (Fig. 3D). This result, as confirmed by ELISA, showed that IL6 and TNFα were increased in the periodontitis-Nos3-/- group as compared with the inhibitor-treated group (Fig. 3E). We also explored whether infiltration of the other types of immunocytes changed in the 2 disease models and whether they were affected by the STAT1 inhibitor. The results showed that B- and T-lymphocyte infiltration increased significantly in the periodontitis-Nos3-/- group and that inhibition of STAT1 could reduce this infiltration (Appendix Figs. 1A, B and 2A, B). However, there was no significant difference in the neutrophil infiltration (Appendix Fig. 3A, B), and qRT-PCR assay of phenotypic markers of these immunocytes confirmed the result (Appendix Figs. 1C, 2C, 3C). In summary, the data suggested that STAT1 plays a critical role in upregulating the inflammation response, especially under the premise of periodontitis-Nos3-/-, and that application of a STAT1 inhibitor can decrease the inflammatory status.

Figure 3.

Figure 3.

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Inhibition of the STAT1 (signal transducer and activator of transcription 1) decreased macrophage infiltration and inflammatory cytokine expression in the periodontitis-Nos3-/- group. (A) Immunofluorescence staining of F4/80-positive (green) macrophages in the periodontitis lesion area in different groups. The positive stained cells are showed in white inset boxes of enlarged images. (B) Quantification of F4/80-positive macrophage numbers in the periodontal area. The mRNA expression of (C) F4/80 and (D) Il6, Tnfα, Il1β, and Ifnγ in mandibular bone was detected via quantitative reverse transcription polymerase chain reaction, and data are normalized to Gapdh. (E) ELISA analysis of IL6 and TNFα in serum of different groups. C, nonnephritic phosphate-buffered saline–treated group; I, inhibitor-treated group; KO, Nos3 knockout group; P, periodontitis group; P KO, periodontitis with Nos3 knockout group. *P < 0.05. **P < 0.01. ***P < 0.001. Repeated 3 times. Data are presented as the mean ± SD (n = 10 per group) and compared with the control.

Inhibition of STAT1 Increased STAT3 and IL10 Expression in the Periodontitis Lesion Area in the Periodontitis-Nos3-/- Group

To determine whether Nos3-/--related hypertension affects the STAT3 anti-inflammatory pathway, we examined the expression of STAT3 in the periodontal lesions of each group. The number of pSTAT3-positive cells in the periodontitis-only group was significantly higher than in the control group. Knockout of Nos3 decreased the pSTAT3 expression. Quantitative analysis showed that the difference was statistically significant (Fig. 4A, B). Western blot and quantitative analysis also confirmed the immunohistochemistry results (Fig. 4C). Interestingly, when the inflammatory cytokine IL6 was highly expressed in the periodontitis group (Fig. 3D, E), the IL6/STAT3 anti-inflammatory pathway was activated simultaneously, and the anti-inflammatory cytokine IL10 was increased (Fig. 4D, E). Although the expression of IL6 was increased, the STAT3 pathway was not significantly activated in the Nos3-/--periodontitis group (Fig. 4C); moreover, the expression level of IL10 was significantly decreased in the Nos3-/--periodontitis group as compared with the periodontitis group (Fig. 4D, E). To investigate whether highly activated STAT1 in the periodontitis-Nos3-/- group affects STAT3 expression and function, we used fludarabine to inhibit STAT1. As expected, inhibition of STAT1 in the periodontitis-Nos3-/- group increased STAT3 phosphorylation (Fig. 4A, C) and its downstream anti-inflammatory factor expression as compared with the noninhibitor group (Fig. 4D, E). This result indicated that Nos3-/--related hypertension can further exacerbate inflammation during the development of periodontitis. These in vivo data verified the evident inhibitory role of STAT1 on the STAT3 anti-inflammatory function and chemokine expression in the coexistence model of 2 diseases.

Figure 4.

Figure 4.

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Inhibition of STAT1 (signal transducer and activator of transcription 1) increased the STAT3 and IL10 expression in the periodontitis lesion area in the periodontitis-Nos3-/- group. (A) Immunohistochemistry analysis of pSTAT3 (phosphorylated STAT3) in the periodontitis lesion areas of different groups; the red arrow refers to cells expressing pSTAT3. (B) Quantification of pSTAT3-positive cells numbers in the periodontal area. Western blot analysis of (C) pSTAT3 and STAT3 expression and (D) IL10 expression in the periodontal lesion area. Representative images and the summary of the normalized quantification are shown. (E) Quantitative reverse transcription polymerase chain reaction of Stat3 and Il10 in different groups. C, nonnephritic phosphate-buffered saline–treated group; I, inhibitor-treated group; KO, Nos3 knockout group; P, periodontitis group; P KO, periodontitis with Nos3 knockout group. *P < 0.05. **P < 0.01. ***P < 0.001. Repeated 3 times. Data are presented as the mean ± SD (n = 10 per group) and compared with the control.

Ang II with Lipopolysaccharide Stimulation Increased STAT1 Phosphorylation and Decreased STAT3 Phosphorylation and Related Chemokine Expression In Vitro

To test whether the Ang II–induced STAT1 signaling pathway plays a crucial role in regulating the immune response, we performed an in vitro experiment. Macrophage cell lines (RAW 264.7) and fibroblast cell lines (L929) were treated with Ang II (100 nM) or lipopolysaccharide (LPS; 200 ng/ml) for 12 h. After Ang II stimulation, STAT1 phosphorylation and SOCS3 expression in macrophage cell lines were significantly increased as compared with the control group and the LPS stimulation group (Fig. 5A). Furthermore, the mRNA expression level of the STAT1 downstream factors Cxl9 and Socs3 in the AngII and LPS–costimulated group was significantly higher (Fig. 5B). Similar to the in vivo results, STAT3 and its downstream anti-inflammatory cytokine IL-10 were increased in the LPS-stimulated group. In the Ang II and LPS–costimulation group, the STAT3 phosphorylation level and the gene expression level of Il10 were significantly lower than that in the LPS stimulation group (Fig. 5C, D). Western blot assay of IL10 verified these results (Fig. 5E). The mRNA expression of Il6, Tnfα, Il1β, and Ifnγ were also upregulated with the Ang II and LPS cotreatment (Fig. 5F). ELISA showed similar results and confirmed the qRT-PCR result (Fig. 5G).

Figure 5.

Figure 5.

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Effect of angiotensin II (Ang II) and lipopolysaccharide (LPS) on STAT1 (signal transducer and activator of transcription 1) and STAT3 phosphorylation and related chemokine expression in vitro. (A) Western blot analysis of pSTAT1 (phosphorylated STAT1), STAT1, and suppressor of cytokine signaling 3 (SOCS3) expression in different groups; representative images and the summary of the normalized quantification are shown. (B) The mRNA expressions of Cxcl9 and Socs3 in different groups were detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR). (C) Western blot analysis of pSTAT3 and STAT3 expression in different groups; representative images and the summary of the normalized quantification are shown. (D) The mRNA expressions of Stat3 and Il10 in different groups were detected by qRT-PCR. (E) Western blot analysis of IL10 expression in different groups, representative images and the summary of the normalized quantification are shown. (F) qRT-PCR of proinflammatory chemokines in the different groups. (G) ELISA analysis of IL6 and TNFα in the different groups. (H) Proposed mechanism diagram shows that hypertension promotes periodontitis by activating the JAK/STAT1 proinflammatory pathway and inhibiting the IL6/STAT3 anti-inflammatory pathway response to AngII. RAW cells treated with or without Ang II (100 nM) and LPS (200 ng/mL) for 12 h. *P < 0.05. **P < 0.01. ***P < 0.001. NS, no significance. Repeated 3 times. Data are presented as the mean ± SD (n = 5 per group) and compared with the control.

As the major nonimmune cells in the periodontal environment, fibroblasts exhibited similar results as macrophages when treated with Ang II and LPS. Ang II promoted STAT1 phosphorylation and SOCS3 protein levels (Appendix Fig. 4A, B), whereas stimulation of LPS caused STAT3 to initiate the anti-inflammatory mechanism and increased IL10 expression (Appendix Fig. 4C, D). In the presence of costimulation, STAT3 phosphorylation and IL10 protein level were significantly lower than in the LPS stimulation group, also similar to the macrophage (Appendix Fig. 4). The mRNA expression of Il6, Tnfα, Il1β, and Ifnγ and the protein level of IL6 and TNFα were also upregulated with the Ang II–LPS cotreatment (Appendix Fig. 4E, F). We confirmed these findings by stimulating the monocytes isolated from Nos3-/- mice. As expected, the results showed that knockout of Nos3 can increase STAT1 phosphorylation and proinflammatory signaling and decrease STAT3 phosphorylation and anti-inflammatory signaling (Appendix Fig. 5). Our results showed that the Ang II–induced STAT1 signaling pathway dramatically increased the inflammatory cytokine expression with LPS stimulation and inhibited STAT3 and its downstream pathway activation.

Discussion

Hypertension is a multifactorial disease, and NOS3 has been shown to be associated with it (Stauss et al. 1999). Epidemiologic surveys have revealed that NOS3 is a genetic susceptibility factor for essential hypertension in the population (Wang et al. 2015; Gamil et al. 2017). Previous reports showed that Nos3-/- mice have higher blood, systolic, and diastolic pressure as compared with a control group (Shesely et al. 1996), and the current study showed a similar result. There is a significant association between hypertension and periodontitis (Paizan and Vilela-Martin 2014), and the potential mechanisms of hypertension affecting periodontitis are still unclear. Similarly, bacteria-induced inflammation and aberrant host inflammatory responses contribute significantly to periodontitis pathogenesis (Darveau 2010). Given our current result, we proposed that the Ang II–induced STAT1 pathway may regulate inflammation in periodontitis and Nos3-/--related hypertension.

STAT1 has been proven to regulate inflammation (dePrati et al. 2005). Ang II can activate the STAT pathway (Marrero et al. 1995) and regulate immune cells through STAT1 pathway activation (Kodama et al. 1998). To investigate the role of the Ang II–induced STAT1 pathway in regulating inflammation in periodontitis and Nos3-/--related hypertension, we established a mouse periodontitis model and applied a Nos3-/- mouse model with significantly high blood pressure. The in vivo study revealed that pSTAT1 expression was increased in the lesion area of the periodontitis-Nos3-/- group and that macrophage infiltration, expression of proinflammatory cytokines, and expression of SOCS3 (the suppressor of STAT3) protein were increased. Interestingly, even though pSTAT1 increased in the periodontitis group as compared with the control group, when periodontitis was accompanied by Nos3-/--related hypertension, the increase was higher (Fig. 2C). In the presence of STAT1 inhibitor, STAT1 was overall inhibited in the group. However, the inhibitor dramatically affects the expression of STAT1 in the coexistence of the 2 diseases. These results together indicated that STAT1 may play a leading role only in the coexistence of these 2 diseases.

STAT3, is usually considered an anti-inflammatory factor activated by IL6. LPS augments IL6 and STAT3 expression, leading to further anti-inflammatory responses in plasma and in local tissues (Greenhill et al. 2011). IL6/STAT3 serve to block apoptosis in cells during the inflammatory process and reversely modulate the LPS-driven proinflammatory responses (Greenhill et al. 2011; Scheller et al. 2011). Literature has indicated that the functional status of the STAT3 protein is upregulated in periodontitis and plays an important role in its pathogenesis (Ambili et al. 2017). In our in vivo study, the pSTAT3 protein was upregulated in the periodontitis group, while it was downregulated in the periodontitis-Nos3-/--related hypertension group (Fig. 4C). The inhibition of STAT1 could increase pSTAT3 expression in the periodontitis-Nos3-/--related hypertension group, which demonstrates the inhibition function of STAT1 toward STAT3. Interestingly, we found that inhibition of STAT1 did not recover STAT3 in the periodontitis-Nos3-/--related hypertension group to a similar level as compared with the periodontitis-only group, which may indicate that there are other undiscovered mechanisms participating in the regulation of STAT3. Our in vitro study showed that Ang II induced STAT1 phosphorylation and SOCS3 expression in macrophages and fibroblasts and that the IL6/STAT3 pathway could be induced by LPS. When both stimuli were present, the inflammatory response was aggravated, and the anti-inflammatory pathway was inhibited. These results together showed that STAT1 has an inhibitory effect on STAT3 in the coexistence of these 2 diseases.

The antagonistic effects of STAT1 and STAT3 are supported by many physiologic and pathologic studies. In common, STAT1 and STAT3 counteract each other in regulating immune and inflammatory responses, and the balance between them is important (Avalle et al. 2012). Activation of the STAT1 pathway induces SOCS3 (the suppressor of STAT3) expression in ventricular myocytes, tubular epithelial cells, and mesangial cells (Villarino et al. 2015). Meanwhile, the IL6/STAT3 pathway could be suppressed by SOCS3 protein (Yoshimura et al. 2007). Our in vivo and in vitro results clearly showed that with the coexistence of Nos3-/--related hypertension and periodontitis, pSTAT1 and downstream SOCS3 were elevated, whereas pSTAT3 was decreased. After inhibition of pSTAT1, SOCS3 was decreased, whereas pSTAT3 was increased. Our current data indicated that Nos3-/--related hypertension disrupts the state of STAT1/STAT3 in periodontitis and that STAT1 predominance tends to aggravate inflammation.

In this study, different cell types were explored to examine their critical role in STAT1-related functions. Monocytes are precursor cells of macrophages. It is essential for the following inflammatory responses, since it travels via blood to peripheral lesion areas (Shi and Pamer 2011). After conditioning, monocytes can differentiate into macrophages. As antigen-presenting cells and secretory cells, they are crucial in regulating immune responses and processing inflammation (Chen and Zhang 2017). Our in vivo findings showed extensively exacerbated macrophage infiltration in the lesion area in the periodontitis-Nos3-/- group, which drew our attention to macrophages. More interestingly, immunofluorescence staining of STAT1 and STAT3 revealed their expression in lesion areas of the periodontitis-Nos3-/- group and their colocalization with macrophages (Appendix Fig. 6). Fibroblasts, as main representative cells of the periodontal environment, may also act as an immune cell (Jönsson et al. 2011). The results of the current study showed that fibroblasts may also play an important function during the STAT1 and STAT3 antagonistic effects in the periodontitis-Nos3-/- group. As for other immune cells, the 2 diseases together have a certain effect on their infiltration, and with STAT1 inhibition, the results showed that the infiltration of B- and T-lymphocytes decreased in the periondontitis-Nos3-/- group, which certified that STAT1 plays a critical role in upregulating the inflammatory response, especially under the premise of periodontitis-Nos3-/-.

The stimulation factor and related receptors involved in the STAT pathway are also considered in our current study. Our previous and current results showed that Ang II increased in lesion area (Appendix Fig. 7A) and augmented the inflammatory response both in vivo and in vitro (Li et al. 2019). We also analyzed the LPS receptor (TLR4) and Ang II receptor (AT1R) in vivo. The results showed that the mRNA expression of Agtrap was increased in the Nos3-/- model and that the mRNA expression of Tlr4 was elevated in the periodontitis model. However, the expression of receptors affected by the inhibitor was not modulated significantly (Appendix Fig. 7B, C).

In conclusion, as shown in the schematic representation (Fig. 5G), this study preliminarily verified that STAT1 plays an important role in the occurrence of Nos3-/--related hypertension promoting periodontitis and that increased STAT1 may impair the anti-inflammatory function of STAT3 when the 2 diseases occur simultaneously. Essentially, this study provides novel and interesting mechanistic insights into the impact of Nos3-/--related hypertension during the development of periodontitis. These observations underscored the crucial pathogenic effect of the Ang II–STAT1 signaling pathway.

Author Contributions

W. Wei, X. Xiao, contributed to conception, design, data acquisition, analysis, and interpretation, drafted and critically revised the manuscript; J. Li, contributed to design and data acquisition, critically revised the manuscript; H. Ding, W. Pan, W. Yin, L. Xue, contributed to conception, critically revised the manuscript; S. Deng, contributed to conception, design, and data analysis, drafted and critically revised the manuscript; Q. Lu, Y. Yue, contributed to conception, data analysis, critically revised the manuscript; Y. Tian, contributed to design, critically revised the manuscript; M. Wang, contributed to conception, design, and data analysis, critically revised the manuscript; L. Hao, contributed to conception, design, data analysis, and interpretation, drafted and critically revised the manuscript. All authors gave final approval and agree to be accountable for all aspects of the work.

Supplemental Material

DS_10.1177_0022034519858063 – Supplemental material for Activation of the STAT1 Pathway Accelerates Periodontitis in Nos3-/- Mice
Click here for additional data file. (2.2MB, pdf)

Supplemental material, DS_10.1177_0022034519858063 for Activation of the STAT1 Pathway Accelerates Periodontitis in Nos3-/- Mice by W. Wei, X. Xiao, J. Li, H. Ding, W. Pan, S. Deng, W. Yin, L. Xue, Q. Lu, Y. Yue, Y. Tian, M. Wang and L. Hao in Journal of Dental Research

Acknowledgments

We appreciate Dr. L. Chen from the Analytical and Testing Center at Sichuan University for her generous help in micro–computed tomography scanning for 3-dimensional reconstruction of the samples.

Footnotes

A supplemental appendix to this article is available online.

This work was supported by the National Natural Science Foundation of China (81570987 and 81601909) and the Sichuan Provincial Science and Technology Foundation (2019YFS0359, 2018FZ0027, and 2018FZ0042).

The authors declare no potential conflicts of interest with respect to the authorship and/or publication of this article.

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Supplementary Materials

DS_10.1177_0022034519858063 – Supplemental material for Activation of the STAT1 Pathway Accelerates Periodontitis in Nos3-/- Mice
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Supplemental material, DS_10.1177_0022034519858063 for Activation of the STAT1 Pathway Accelerates Periodontitis in Nos3-/- Mice by W. Wei, X. Xiao, J. Li, H. Ding, W. Pan, S. Deng, W. Yin, L. Xue, Q. Lu, Y. Yue, Y. Tian, M. Wang and L. Hao in Journal of Dental Research

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