TET1 Knockdown Inhibits Porphyromonas gingivalis LPS/IFN-γ-Induced M1 Macrophage Polarization through the NF-κB Pathway in THP-1 Cells

Tet-eleven translocation 1 (TET1) is a dioxygenase that plays an important role in decreasing the abundance of DNA methylation and changing the expression levels of specific genes related to inflammation. Porphyromonas gingivalis (Pg.) lipopolysaccharide (LPS) can induce periodontal diseases that present with severe bone loss and collagen fiber destruction accompanied by a high number of M1 macrophages. M1-polarized macrophages are pivotal immune cells that promote the progression of the periodontal inflammatory response, but the function of TET1 during M1 macrophage activation is still unknown. Our results showed that the mRNA and protein expression levels of TET1 decreased in THP-1 cells during M1 macrophage differentiation. TET1 knockdown resulted in a significant decrease in the production of proinflammatory markers such as IL-6, TNF-α, CCL2, and HLA-DR in Pg. LPS/IFN-γ- and Escherichia coli (E. coli) LPS/IFN-γ-induced M1 macrophages. Mechanistically, TET1 knockdown downregulated the activity of the NF-κB signaling pathway. After treatment with the NF-κB inhibitor BAY 11-7082, M1 marker expression showed no significant difference between the TET1 knockdown group and the control group. Taken together, these results suggest that TET1 depletion inhibited Pg. LPS/IFN-γ-induced M1 macrophage polarization through the NF-κB pathway in THP-1 cells.

Periodontal diseases, which include gingivitis and periodontitis, are progressive inflammatory diseases caused by bacterial infection accompanied by the host immune response, periodontal tissue destruction, and alveolar bone loss [11]. The gram-negative bacteria Porphyromonas gingivalis (Pg.) is one of the most important periodontal pathogens that is frequently observed in the subgingival biofilm [12,13]. Pg. infection increases the necroptosis rate among defense cells and promotes severe bone destruction in the periodontal tissue [14]. Murine teeth injected with Pg. LPS show histological lesions that many vacuolated macrophages infiltrate and alveolar bone resorption around the periodontal ligament lesion [15]. A higher proportion of M1 macrophages correlates positively with periodontitis [16]. The M1-related cytokines IL-1β and IL-6, which are secreted by macrophages, induce the expression of matrix metalloproteinases (MMPs) in human gingival fibroblasts (HGFs). MMPs then cause the destruction of gingival collagen fibers in inflamed periodontal tissue under high-glucose conditions [17]. These findings demonstrate that M1 macrophages might play an important role in the occurrence and development of periodontitis.
DNA methylation is an important epigenetic modification that serves as a critical switch for gene expression and widely impacts cellular physiological functions. DNA methylation is dynamically balanced by methylation and demethylation. While DNA methyltransferases (DNMTs) serve as methylation writers and maintainers to generate 5-methylcytosine (5mC), ten-eleven translocation proteins (TETs) act as methylation erasers to oxidize 5mC into 5-hydroxymethylcytosine (5hmC) [18,19]. TETs are Fe 2+ -and α-ketoglutarate (α-KG)-dependent dioxygenases that are classified as TET1, TET2, and TET3. Increasing evidence has demonstrated that TETs play important roles in tumor progression and embryonic development. Recently, TETs have gained attention in the inflammatory process. Upon Escherichia coli (E. coli) LPS stimulation, TET1 downregulates pro-IL-1β and IL-1β transcription in the THP-1 cell line [20]. In regulatory T cells (Tregs), transforming growth factor-β (TGF-β)-activated Smad3 and interleukin-2 (IL-2)-activated signal transducers and activators of transcription 5 (Stat5) induce Tet1 and Tet2 binding to the fork-head-family transcription factor (Foxp3) promoter. Tet1 and Tet2 then enhanced the Foxp3 promoter hypomethylation, which promoted Treg differentiation to further regulate the autoimmune response [21]. TET2 participates in the LPS-induced inflammatory response in human dental pulp cells by epigenetically regulating the transcription of the NF-κB signaling pathway transduction molecule MyD88 [22]. Thus, TET proteins can impact the expression of particular genes and change the process of inflammation by different mechanisms. Our previous study found that the level of TET1 markedly decreased while the levels of TET2 and TET3 showed no significant changes during Pg. LPS/IFN-γ-induced M1 macrophage polarization in THP-1 cells. However, whether TET1 is involved in the regulation of macrophage activation during the periodontal inflammatory process is still unknown.
In this study, we examined the expression of TET1 and the consequences of TET1 knockdown during M1 macrophage polarization in THP-1 cells. Furthermore, we investigated whether TET1 inhibits the NF-κB signaling pathway during M1 macrophage activation. As a result, we found that TET1 modulates M1 macrophage differentiation by the NF-κB pathway.

Pg. LPS/IFN-γ Polarized M0 Macrophages into M1 Proinflammatory Macrophages
To verify that monocytes differentiated into naive macrophages, the expression of the human cluster of differentiation 68 (CD68) was detected by fluorescence-activated cell sorting analysis (FACS) [23]. After PMA treatment for 24 h, the expression of CD68 was significantly increased ( Figure 1A). Morphological evidence showed that THP-1 cells were round and small, while M0 macrophages were clearly larger and contained many granules when viewed under an inverted microscope ( Figure 1B). Thus, the THP-1 cells were differentiated into macrophages (M0). To transform M0 macrophages into M1 proinflammatory macrophages, the cells were treated with LPS/IFN-γ, as previously described [24,25]. For cell viability, no significant difference was shown for 0, 1, and 10 ng/mL IFN-γ at 24 and 48 h. A higher concentration of IFN-γ treatments suppressed the cell proliferation rates. No significant difference was shown for 0-1000 ng/mL Pg. LPS and 10 ng/mL IFN-γ within 72 h ( Figure 2B). Cell viability was inhibited in the groups above 0.1 ng/mL E. coli LPS and 10 ng/mL IFN-γ at 48 h and 72 h ( Figure 2C). The treatments of 100 ng/mL Pg. LPS/10 ng/mL IFN-γ and 0.1 ng/mL E. coli LPS/10 ng/mL IFN-γ were chosen to establish the M1 macrophage model.
To verify the efficiency of M1 macrophage activation, the expression levels of classic M1 markers, such as IL-6, TNF-α, CCR7, CCL2, and HLA-DR, were assessed by a real-time quantitative polymerase chain reaction (RT-qPCR) and FACS. Following Pg. LPS/IFN-γ or E. coli LPS/IFN-γ stimulation, the expression levels of M1 markers significantly increased ( Figures 2D-2G). Both Pg. LPS/IFN-γ and E. coli LPS/IFN-γ promoted round M0 macrophages to develop a flat morphology with spindle pseudopodia that could be viewed with an inverted microscope, which further confirms the LPS-IFN-γ-induced M1 macrophage polarization ( Figure 1B). To transform M0 macrophages into M1 proinflammatory macrophages, the cells were treated with LPS/IFN-γ, as previously described [24,25]. For cell viability, no significant difference was shown for 0, 1, and 10 ng/mL IFN-γ at 24 and 48 h. A higher concentration of IFN-γ treatments suppressed the cell proliferation rates. No significant difference was shown for 0-1000 ng/mL Pg. LPS and 10 ng/mL IFN-γ within 72 h ( Figure 2B). Cell viability was inhibited in the groups above 0.1 ng/mL E. coli LPS and 10 ng/mL IFN-γ at 48 h and 72 h ( Figure 2C). The treatments of 100 ng/mL Pg. LPS/10 ng/mL IFN-γ and 0.1 ng/mL E. coli LPS/10 ng/mL IFN-γ were chosen to establish the M1 macrophage model.
To verify the efficiency of M1 macrophage activation, the expression levels of classic M1 markers, such as IL-6, TNF-α, CCR7, CCL2, and HLA-DR, were assessed by a real-time quantitative polymerase chain reaction (RT-qPCR) and FACS. Following Pg. LPS/IFN-γ or E. coli LPS/IFN-γ stimulation, the expression levels of M1 markers significantly increased ( Figure 2D-G). Both Pg. LPS/IFN-γ and E. coli LPS/IFN-γ promoted round M0 macrophages to develop a flat morphology with spindle pseudopodia that could be viewed with an inverted microscope, which further confirms the LPS-IFN-γ-induced M1 macrophage polarization ( Figure 1B). The expression of HLA-DR in macrophages was analyzed by flow cytometry. M0 macrophages were used as a control. All of the results represent the mean ± standard deviation of three independent experiments (n = 3). * p < 0.05, ** p < 0.01, and *** p < 0.0001 represent significant differences compared with the control.

TET1 Expression Decreased in LPS/IFN-γ-Induced M1 Macrophages
To study the TET1 expression pattern during the generation of M1 macrophages, we detected the TET1 mRNA and protein levels in Pg. LPS/IFN-γ or E. coli LPS/IFN-γ-induced macrophages. The mRNA expression of TET1 was repressed after LPS/IFN-γ treatment at 6 h, 12 h, and 24 h. The suppression of TET1 mRNA expression was more clear with E. coli LPS/IFN-γ treatment than with The expression of HLA-DR in macrophages was analyzed by flow cytometry. M0 macrophages were used as a control. All of the results represent the mean ± standard deviation of three independent experiments (n = 3). * p < 0.05, ** p < 0.01, and *** p < 0.0001 represent significant differences compared with the control.

TET1 Expression Decreased in LPS/IFN-γ-Induced M1 Macrophages
To study the TET1 expression pattern during the generation of M1 macrophages, we detected the TET1 mRNA and protein levels in Pg. LPS/IFN-γ or E. coli LPS/IFN-γ-induced macrophages. The mRNA expression of TET1 was repressed after LPS/IFN-γ treatment at 6 h, 12 h, and 24 h. The suppression of TET1 mRNA expression was more clear with E. coli LPS/IFN-γ treatment than with Pg. LPS/IFN-γ stimulation ( Figure 3A,C). The TET1 protein level also decreased in Pg. LPS/IFN-γ-and E. coli LPS/IFN-γ-treated cells ( Figure 3B,D).

TET1 Knockdown Inhibited the Expression of Proinflammatory Cytokines and Chemokines during LPS/IFN-γ-Induced M1 Macrophage Activation
To evaluate the function of TET1 in M1 macrophage activation, TET1 siRNAs or a control siRNA were transfected into M0 cells. Compared to the control group, the #3 TET1 siRNA group exhibited a decrease in TET1 expression of up to 70% to 80% at both the mRNA and protein levels ( Figure 4A-C). Therefore, the #3 TET1 siRNA sequence resulted in the best knockdown effect and was further utilized in the following experiments.
To explore the effect of TET1 knockdown on M1 macrophage activation, cells were treated with Pg. LPS/IFN-γ or E. coli LPS/IFN-γ for 6, 12, and 24 h, and M1 markers were analyzed after TET1 knockdown. Following LPS/IFN-γ treatment, the mRNA levels of IL-6, TNF-α, and CCL2 showed different degrees of decrease in the TET1 knockdown group ( Figure 5A). The IL-6 protein level significantly decreased in the TET1 knockdown group compared with the control group, and TNF-α expression was decreased at 24 h after LPS/IFN-γ stimulation ( Figure 5B). TET1 knockdown slightly inhibited the induction of HLA-DR protein in E. coli LPS/IFN-γ-induced macrophages, but did not affect HLA-DR expression in Pg. LPS/IFN-γ-induced M1 macrophages at 24 h ( Figure 5C).

TET1 Knockdown Attenuated M1 Macrophage Polarization through the NF-κB Signaling Pathway
The NF-κB signaling pathway plays an indispensable role in M1 macrophage activation. To investigate the role of TET1 in the regulation of the NF-κB signaling pathway during M1 macrophage activation, the expression of the phosphorylated forms of the key factors IKKα/β, IκBα, and p65 was examined by Western blot analysis. The results revealed that TET1 knockdown decreased the levels of phosphorylated IKKα/β, IκBα, and p65 in LPS/IFN-γ-induced M1 macrophages at the indicated time points ( Figure 6A-D). IκB kinase (IKK) consists of two subunits including IKKα and IKKβ. Its activation facilitates the phosphorylation, ubiquitination, and proteasomal degradation of IκBα [26]. IkBα is a molecular inhibitor of NF-κB and its phosphorylation drives its ubiquitination and proteasome degradation [27]. As an effector member of NF-κB pathway, p65 can be liberated from IκBα and then phosphorylates and translocates into the nucleus, which indicates the activation of NF-κB signaling [28]. The levels of phosphorylated IKKα/β, IκBα, and p65 decreased in the TET1 knockdown group during M1 macrophage activation, which indicates that TET1 depletion inhibited the NF-kB signaling pathway.

TET1 Knockdown Inhibited the Expression of Proinflammatory Cytokines and Chemokines during LPS/IFN-γ-Induced M1 Macrophage Activation
To evaluate the function of TET1 in M1 macrophage activation, TET1 siRNAs or a control siRNA were transfected into M0 cells. Compared to the control group, the #3 TET1 siRNA group exhibited a decrease in TET1 expression of up to 70% to 80% at both the mRNA and protein levels ( Figure 4A-C). Therefore, the #3 TET1 siRNA sequence resulted in the best knockdown effect and was further utilized in the following experiments.   VINCULIN was used as an internal control. All of the results represent the mean ± standard deviation of three independent experiments (n = 3). * p < 0.05, ** p < 0.01, and *** p < 0.0001 represent significant differences compared with the control.

TET1 Knockdown Inhibited the Expression of Proinflammatory Cytokines and Chemokines during LPS/IFN-γ-Induced M1 Macrophage Activation
To evaluate the function of TET1 in M1 macrophage activation, TET1 siRNAs or a control siRNA were transfected into M0 cells. Compared to the control group, the #3 TET1 siRNA group exhibited a decrease in TET1 expression of up to 70% to 80% at both the mRNA and protein levels ( Figure 4A-C). Therefore, the #3 TET1 siRNA sequence resulted in the best knockdown effect and was further utilized in the following experiments.  All of the results represent the mean ± standard deviation of three independent experiments (n = 3). * p < 0.05, ** p < 0.01, and *** p < 0.0001 represent significant differences compared with the control. knockdown. Following LPS/IFN-γ treatment, the mRNA levels of IL-6, TNF-α, and CCL2 showed different degrees of decrease in the TET1 knockdown group ( Figure 5A). The IL-6 protein level significantly decreased in the TET1 knockdown group compared with the control group, and TNF-α expression was decreased at 24 h after LPS/IFN-γ stimulation ( Figure 5B). TET1 knockdown slightly inhibited the induction of HLA-DR protein in E. coli LPS/IFN-γ-induced macrophages, but did not affect HLA-DR expression in Pg. LPS/IFN-γ-induced M1 macrophages at 24 h ( Figure 5C).  All of the results represent the mean ± standard deviation of three independent experiments (n = 3). * p < 0.05 and ** p < 0.01 represent significant differences compared with the control.

Discussion
Periodontitis is an inflammatory disease caused by infection with periodontal pathogens such as Pg., which results in tissue destruction in the periodontal tissue accompanied by extensive macrophage infiltration [29][30][31]. Macrophages are heterogeneous and plastic cells that can be polarized into different phenotypes and exhibit various functions in response to surrounding stimuli [32]. The cell walls of gram-negative bacteria contain LPS, which can trigger the inflammatory response and generate M1 macrophages with or without IFN-γ [33,34]. In response to Pg. infection, M1 macrophages widely infiltrate and cause severe bone destruction in the periodontium [35]. An interesting phenomenon was discovered, and it showed that M1 macrophage numbers are significantly increased and there is a small change in the M2 phenotype in periodontitis in nonhuman primates [36]. Pg. LPS can induce the NF-κB signaling pathway activation to generate M1-related cytokines such as IL-6, TNF-α, IL-1β, and nitric oxide (NO) in macrophages during the progression of periodontitis [30,37]. In a mouse periodontitis model established by exposure to Pg., human β-defensin 3 (hBD3) reduces M1 macrophage polarization by inhibiting the phosphorylation of the NF-κB p65 subunit, which results in less alveolar bone loss [38]. To verify the function of Pg. LPS/IFN-γ in M1 macrophage differentiation, we examined the expression levels of M1 markers such as IL-6, TNF-α, CCR7, CCL2, and HLA-DR in THP-1-derived macrophages. Since E. coli LPS/IFN-γ treatment is a classic protocol to induce M1 macrophages, this treatment was chosen as a positive control for the induction of M1 macrophages in the present study. We demonstrated that the mRNA and protein levels of the M1 markers were reinforced after Pg. LPS/IFN-γ treatment, which suggests that Pg. LPS/IFN-γ induced M1 polarization in macrophages. Compared to E. coli LPS, which has well-defined activity, Pg. LPS showed less biological reactivity.
As one of the most important epigenetic modifications, DNA methylation is considered to be an important factor for the inflammatory response and immune cell differentiation [39]. A previous study showed that DNMTs promoted the expression of genes involved in inflammation, such as IL-6, TNF-α, IL-1β, NO, and CCL2, in mouse macrophages via the methylation of CpG-enriched sites in the liver X receptor α (LXRα) and peroxisome proliferator-activated receptor γ1 (PPARγ1) promoters. LXRα and PPARγ1 could inhibit the ability of NF-κB to drive inflammatory gene expression [40]. The combination of the DNMT inhibitor 5-Aza-2-deoxycytidine (Aza) and the histone deacetylase (HDAC) inhibitor Trichostatin A (TSA) decreased the number of LPS-induced M1 macrophages and increased the number of M2 macrophages by inhibiting HuR and activating the STAT3-Bcl2 pathways [41]. Compared to DNA methylation mediated by DNMTs, the role of DNA demethylation during macrophage activation and inflammation has been investigated in only a few studies. To evaluate the role of the DNA demethylase TET1 in M1 macrophage activation, we detected the expression level of TET1 in THP-1 cells after Pg. LPS/IFN-γ treatment. The trend of TET1 expression level was contrary to that of the M1 markers IL-6, TNF-α, CCR7, and CCL2 in THP-1-derived macrophages induced with Pg. LPS/IFN-γ treatment. This result indicated that TET1 might participate in the regulation of Pg. LPS/IFN-γ-stimulated macrophage M1 activation.
Several lines of evidence support the conclusion that TET1 is relevant for activating immune cells [42,43]. High TET1 expression is related to lower infiltration by immune cells, such as T and B cells, in basal-like breast cancer [44]. In an inflammatory rat burn injury model, elevated Tet1 expression helps to demethylate the M1-related gene iNOS, which results in the upregulation of expression and the inflammatory response [45]. Melatonin alleviates inflammation and increases the ratio of M2 to M1 macrophages by transporting exosomes from adipose cells. Macrophages absorb exosomes enriched in α-KG and promote TET-mediated DNA demethylation in adipose tissue [46]. To explore the effect of TET1 on the inflammatory response in Pg. LPS/IFN-γ-induced M1 macrophages, we silenced TET1 expression in THP-1 cells and detected the mRNA and protein levels of M1-related cytokines. The data showed that the expression levels of IL-6, TNF-α, and CCL2 decreased after TET1 knockdown, which indicates that TET1 could positively regulate M1 macrophage polarization.
The NF-κB signaling pathway plays an important role in the inflammatory response and macrophage polarization. A growing body of evidence showed that the NF-κB signaling pathway can be regulated by DNA methylation [47,48]. In germinal center B cells, elevated DNMT1 expression causes hypomethylation of the promoter of the nuclear factor kappa B kinase subunit epsilon (IKBKE) inhibitor, which is one of the NF-κB pathway signaling components that induces IKB phosphorylation [47]. The DNA methyltransferase inhibitor Aza facilitates the nuclear translocation of NF-κB and promotes the TLR4-NF-κB signaling pathway [49]. Aza treatment can promote the activation of IKKa/β and IκBα and facilitates, which results in high cell survival and proliferation in gastric cancer cells [50]. To determine whether TET1 influences the activation of the NF-κB pathway during Pg. LPS/IFN-γ-induced macrophage M1 polarization, we detected the phosphorylation levels of several key signaling molecules in the NF-κB signaling pathway by Western blot analysis. The results showed that TET1 depletion reduced the phosphorylation levels of IKKα/β, p65, and IκBα under LPS/IFN-γ treatment conditions. To further clarify the function of the NF-κB signaling pathway in M1 macrophage activation, an NF-κB inhibitor was used to block this signaling pathway. After treatment with the NF-κB inhibitor, the mRNA expression levels of the proinflammatory markers IL-6, TNF-α, and CCL2 showed no difference between the TET1-deficient group and the control group. These data demonstrated that TET1 depletion reduced M1 macrophage polarization by inhibiting the activation of the NF-κB signaling pathway.
In conclusion, this study indicated that TET1 expression decreased during Pg. LPS/IFN-γ-induced M1 polarization in THP-1 macrophages. Under Pg. LPS/IFN-γ stimulation conditions, TET1 knockdown inhibited M1 macrophage polarization through the NF-κB signaling pathway. These findings may offer a new strategy to research macrophage polarization to regulate the inflammatory response.

Cell Counting Kit-8 (CCK8) Assay
A CCK8 assay tested the proliferation of cells (Dojindo, Kumamoto, Japan) according to the manufacturer's instructions. After 24, 48, or 72 h of incubation, the supernatant was refreshed with RPMI-1640 medium containing CCK-8 for another 2 h. An automated microplate reader (Sunrise, Tecan, Hombrechtikon, Switzerland) was used to read the optical density (OD) at 450 nm.

TET1 Small Interfering RNA (siRNA) Transfection
When M0 macrophages reached 60% confluence, they were transfected with a TET1 siRNA (100 nM) or negative control siRNA with Lipofectamine RNAi MAX (Invitrogen, Carlsbad, CA, USA), according to the manufacturer's instructions. The siRNA sequences are listed in Table 1. Cells were lysed with the TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and RNA was reverse transcribed into complementary DNA (cDNA) by PrimeScript™ RT Master Mix (Takara, Tokyo, Japan) following the manufacturer's instructions. Then, the cDNA was used as a template for PCR with a LightCycler 480 thermocycler and SYBR green I Master Mix (Roche, Basel, Switzerland). Primers were designed by using Primer Express v3.0 software (Life Technologies Corp., Carlsbad, CA, USA) and are shown in Table 2.

Western Blot Analysis
Total protein was extracted by using the RIPA buffer (Beyotime, Haimen, China) and measured by using a BCA protein assay kit (ComWin Biotech, Beijing, China). A total of 30 µg of protein was subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). The membranes were blocked with 5% bovine serum albumin for 1 h and incubated with the following primary antibodies against TET1 (GeneTex, Irvine, CA, USA), IKKα/β, phospho-IKKα/β, p65, phospho-p65, IκBα, phospho-IκBα, and VINCULIN (Cell Signaling Technology, CST, Danvers, MA, USA). After washing, the membranes were incubated with secondary antibodies (CST, Danvers, MA, USA) for 1 h. The proteins were visualized using an enhanced chemiluminescence system (Millipore, Billerica, MA, USA) and analyzed by ImageJ software (National Institutes of Health, Baltimore, MD, USA).

Enzyme-Linked Immunosorbent Assay (ELISA)
ELISA kits were used to test the IL-6 and TNF-α protein concentrations in culture supernatants (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions. A microplate reader (Tecan, Hombrechtikon, Switzerland) was used to detect the optical density at 450 nm.

Fluorescence-Activated Cell Sorting Analysis (FACS)
Cells were washed and stained for 20 min at room temperature with monoclonal antibodies specific for CD68 and HLA-DR (eBioscience, Carlsbad, CA, USA). Then, the samples were analyzed by flow cytometry (Beckman, San Francisco, CA, USA) and FlowJo software.

Statistical Analysis
Each experiment was repeated at least three times. All data were statistically analyzed by using the SPSS 20.0 software package (SPSS Inc., Chicago, IL, USA). The differences between groups were evaluated by a Student's t-test. p < 0.05 was considered statistically significant.

Conflicts of Interest:
The authors declare no conflict of interest.