METTL3-dependent N6-methyladenosine RNA modification promotes diabetes-associated periodontitis via mediating macrophage pyroptosis

doi:10.21203/rs.3.rs-3981894/v1

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METTL3-dependent N6-methyladenosine RNA modification promotes diabetes-associated periodontitis via mediating macrophage pyroptosis

https://doi.org/10.21203/rs.3.rs-3981894/v1

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Patients with diabetes are twice as likely to suffer from periodontitis (PD) than those without diabetes, and diabetes-associated PD is distinguished by increased inflammation and aggravated tissue damage. However, the underlying mechanisms remain largely unknown, leading to undetermined effective treatment for diabetes-associated PD. One of the most significant epigenetic alterations, N⁶-methyladenosine (m⁶A) modifications are implicated in a number of developmental and pathological processes. Here, we found that Methyltransferase-like 3 (METTL3) expression was upregulated in diabetes-associated PD mice and macrophages under Porphyromonas gingivalis and high glucose stimulation. Functionally, m⁶A RNA hypermethylation and pyroptosis were eliminated by METTL3 knockdown, but more significant alterations were brought about by METTL3 overexpression. Moreover, using methylation RNA immunoprecipitation, nod-like receptor family pyrin domain-containing 3 (NLRP3) was found to be a target of METTL3. Mechanistically, METTL3 increased m⁶A methylation level of NLRP3 and further affects macrophage pyroptosis. Binding of insulin-like growth factor 2 binding protein 3 (IGF2BP3) to the m⁶A-modified domains of NLRP3 was necessary for its stability. Lastly, the silencing of METTL3 mediated by adeno-associated virus 9 (AAV9) ameliorated periodental inflammation and alveolar bone loss in diabetes-associated PD mice. Collectively, we have shown that METTL3 induced m⁶A alterations of NLRP3 and enhanced its stability through pathways depended on IGF2BP3, thereby inducing macrophage pyroptosis in diabetes-associated PD progression, providing evidence that the METTL3/NLRP3 axis is a new and promising target for treating diabetes-associated PD.

Biological sciences/Cell biology/Mechanisms of disease

Biological sciences/Cell biology/Cell death

Biological sciences/Drug discovery/Target identification

Diabetes mellitus is one of the most prevalent chronic illnesses worldwide, with its incidence rising in recent years ¹. Characterized by hyperglycemia for the reason of insulin resistance or inadequate insulin production, diabetes and its complications have aroused the public’s attention globally for the increased morbidity and mortality ². Periodontitis (PD) is twice as prevalent in diabetics compared to nondiabetics ³, characterized by refractory inflammation, aggravated alveolar bone loss and tooth loss ^4,5, and less favorable responses to conventional treatment ^6,7. Meanwhile, poorly controlled periodontitis also adversely worsens diabetic complications associated with hyperglycaemia ^8,9. Individuals with diabetes and periodontitis may have better long-term glucose control following long-term periodontal therapy ¹⁰. Taking these into consideration, it would be imperative and important to investigate the underlying mechanisms and develop the effective treatment of diabetes-associated PD, which may contribute not only to an improvement in oral health, but also to a reduction of diabetes and its complications.

Macrophages are the main cells participated in innate immune response to the invasion of periodontal pathogens (i.e., Porphyromonas gingivalis) during the initiation of periodontitis ¹¹. Subsequently, macrophages undergo the unique programmed cell death process of pyroptosis ¹². Hyperglycaemia in diabetes has been reported to cause dysfunction of the immune responses, including altering immune cell functions and activation states ¹¹, which might be closely related to pyroptosis ¹². In contrast to apoptosis, which preserves membrane integrity, pyroptosis is triggered by inflammasomes and causes pore formation and membrane rupture, releasing inflammatory factors¹³. The most well-studied inflammasome, the Nod-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome, contributes to the start of pyroptosis by encouraging the activation of cleaved-caspase-1, which cleaves GSDMD and produces IL-1β and IL-18 ¹⁴. While it has been found that periodontitis and the NLRP3 inflammasome have a close association ¹⁵, the physiological functions of pyroptosis in diabetes-associated PD remain largely unknown.

In eukaryotic mRNA, the most prevalent internal modification is N⁶-methyladenosine (m⁶A), which methylates adenosine on the nitrogen-6 position by various regulators ¹⁶. The coordinated actions of methyltransferases, demethylases, and binding proteins—also known as “Writers”, “Erasers”, and “Readers”—mediate the process of RNA m⁶A methylation ¹⁷. The first methyltransferase to be discovered, methyltransferase-like 3 (METTL3), is an essential enzyme in the traditional METTL3-METTL14-WTAP methyltransferase complex ¹⁸. According to earlier research, METTL3 was involved in cellular functions and multiple diseases, such as tooth root formation ¹⁹ or osteogenic differentiation²⁰. However, very little study has been conducted on the function of METTL3-dependent m⁶A modification in pyroptosis and diabetes-related periodontitis.

With a focus on METTL3-mediated control of macrophage pyroptosis, the current work aims to clarify the processes underlying periodontal degradation in diabetes-associated PD. Our fingdings uncovered METTL3-mediated m⁶A modification was implicated in the development of diabetes-associated PD via regulating the macrophage pyroptosis in an NLRP3-dependent manner. Therapeutically, local inhibition of METTL3 in diabetes-associated PD models contributed to reduced macrophage pyroptosis, diminished inflammatory response, and decreased alveolar bone loss. Altogether, our results indicate that METTL3 promotes m⁶A modifications of NLRP3 and enhances its stability, thereby inducing macrophage pyroptosis in diabetes-associated PD.

High glucose aggravates periodontal tissue damage in diabetes-associated PD mice via regulating P. gingivalis-induced macrophage pyroptosis.

To investigate the impact of hyperglycaemia on inflammatory destruction and alveolar bone damage along the progression of periodontitis, we employed ligature-induced periodontitis, a classic model of periodontitis. Diabetic mice were effectively made hyperglycemic after injections of STZ (streptozotocin) according to the detection of fasting blood glucose (Fig. S1a). In comparison to control mice, maxillae of PD mice showed a considerable loss of alveolar bone as demonstrated by micro-CT studies. Importantly, the diabetic mice exhibited a significant increase in the distance from the cemento-enamel junction (CEJ) to the alveolar bone crest (ABC), whereas a decreased bone volume (BV/TV) in periodontitis model (Fig. 1a). Moreover, measures of the histomorphometric analysis of tissue slices stained with hematoxylin and eosin (H&E) indicated the similar results as micro-CT showed (Fig. 1b).

To investigate the factors in charge of the aggravated periodontal damage in diabetes-associated PD mice compared with control PD mice, we examined the expression of pyroptosis-related molecules using slices of maxillae fixed in paraffin by immunofluorescence (IF) staining. Concretely, in sections of PD mice, we found a large number of NLRP3⁺F4/80⁺, GSDMD⁺F4/80⁺, Caspase-1⁺F4/80⁺, and IL-1β⁺F4/80⁺ macrophages. Notably, the parameters above were increased in diabetes-associated PD mice compared with the control PD mice (Fig. 1c-f). Additionally, most of the NLRP3⁺, GSDMD⁺, Caspase-1⁺or IL-1β⁺ cells might co-localize with F4/80⁺ macrophages, indicating that the majority of pyroptotic cells in the gingiva were macrophages (Fig. 1c-f). Quantitative polymerase chain reaction (qPCR) and Western blot investigation of mice gingival tissues further confirmed it (Fig. 1g, h).

Next, RAW264.7 cells and bone marrow-derived macrophages (BMDMs) were cultivated with P. gingivalis (multiplicity of infection [MOI] = 100) or high glucose (25mM). We found high glucose increased cell viability before 24h and dropped at 48h with analysis utilizing the Cell Counting Kit-8 (CCK-8) (Fig. 1I and Fig. S1b). Lactate dehydrogenase (LDH) release assay showed a similar trend with CCK8 (Fig. 1j and Fig. S1c). Considering these results, we determined that 24 hours was a suitable time interval for coculturing with P. gingivalis and high glucose. Molecules associated to pyroptosis had higher expression levels in P. gingivalis or hyperglycemia-treated BMDMs (Fig. 1k, l) and RAWs (Fig. S1d, e). In addition, high glucose culture remarkably enhanced pyroptosis in P. gingivalis-stimulated cells (Fig. 1k, l and Fig. S1d, e). The secretion level of IL-1β detected by ELISA futher confirmed it (Fig. S1f).

Macrophage pyroptosis inhibits osteoblast activity both in vivo and in vitro.

To evaluate the impact of macrophage pyroptosis on osteoclastic bone resorption and osteoblastic bone formation in diabetes-associated PD mice, ALP and TRAP activity were detected histochemically in paraffin slices of the maxillae. Histomorphometric analysis revealed that osteoclast and osteoblast activity were stimulated in PD mice (Fig. 2a, b). However, hyperglycemia promoted osteoclast differentiation, whereas inhibited osteogenic differentiation in diabetes-associated PD mice than in control PD mice (Fig. 2a, b). Further, IF staining was performed and OCN was used as a marker for osteoblast. The trend of OCN⁺ cells was observed consistent with that of ALP-positive osteoblasts (Fig. 2c). Moreover, fewer OCN⁺ osteoblasts were seen in the GSDMD⁺ cell distribution (Fig. 2c). Therefore, we speculate that macrophage pyroptosis may inhibit osteoblast activity.

The effects of macrophage pyroptosis on osteogenic differentiation were investigated using the conditional medium (CM) culture system (Fig. 2d) and the transwell coculture method (Fig. 2g). Western blotting (Fig. 2e) and qPCR (Fig. 2f) results showed decreased expression levels of Runx2, Osterix, Bsp, and Ocn in pyroptotic macrophages CM compared with normal macrophages CM. Unsurprisingly, the parameters decreased more in P. gingivalis plus high glucose (HP)-induced pyroptotic macrophages CM than in P. gingivalis (NP) or high glucose (HG) individually induced pyroptotic macrophages CM (Fig. 2e and Fig. 2f). Besides, after two days of co-culture, similar results were obtained (Fig. 2g and Fig. 2h). We then repeated the above experiments using RAWs and obtained consistent results (Fig. S2).

METTL3 mediates macrophage pyroptosis in response to P. gingivalis and high glucose stimulation.

Since increasing evidence showed that m⁶A modifications play important roles in various physiological processes, we next investigated m⁶A RNA modifications in mice gingival tissues. In PD mice, global RNA m⁶A levels were elevated, and in diabetes-associated PD animals, these levels were significantly higher than in control PD mice (Fig. 3a). The mRNA levels of methyltransferases (Mettl3, Mettl14, Mettl16, and Wtap) were then analyzed. Interestingly, Mettl3 and Mettl14 levels in PD mice were much greater than those in control groups, according to qPCR analysis (Fig. 3b). In particular, diabetes-associated PD mice showed the highest Mettl3 and Mettl14 expressions (Fig. 3b). We further validated the results with additional in vitro experiments using BMDMs and RAWs. Dot blot and qPCR analysis showed a similar variation tendency of METTL3-mediated m⁶A modifications with that in mice both in BMDMs (Fig. 3c, d) and RAWs (Fig. S3a, b). However, the expression of Mettl14 in RAWs underwent different stimulations, but no discernible alterations occurred (Fig. S3b). These indicated that METTL3 was the most promising candidate for further investigation. Then, western blot and IF staining were used to determine the protein level of METTL3, and the results were in line with qPCR findings in both BMDMs (Fig. 3e, f) and RAWs (Fig. 3e and Fig. S3c). Moreover, we confirmed the key role of METTL3 by testing tissue proteins (Fig. 3g). In addition, double IF staining was performed and we observed a large number of METTL3⁺F4/80⁺ macrophages in PD mice (Fig. 3h). More importantly, the parameter was increased in diabetes-associated PD mice in contrast to the control PD mice (Fig. 3h).

In order to assess the precise role of METTL3 in macrophages in response to inflammatory stimuli, qPCR was used to confirm METTL3 knockdown in BMDMs using siRNA transfection (Fig. S3d). After the elimination of METTL3, the total m⁶A modified mRNA levels were significantly reduced (Fig. 3i). In comparison to the negative control groups, the METTL3 knockdown groups exhibited a decrease in both LDH and IL-1β release (Fig. 3j, k). Although the production of LDH and IL-1β in HP group were more than other groups, the indicators in HP group were reduced to the same level as NP group showed after METTL3 knockdown (Fig. 3j, k). These results suggested a potential role for METTL3 in macrophage pyroptosis. Furthermore, considering the outcomes of a 2-day CM culture (Fig. 3l, m) and transwell coculture system (Fig. 3n, o), we concluded that METTL3 knockdown showed an obvious rescuing effect on pyroptosis-induced inhibition of osteogenic differentiation.

NLRP3 serves as a target of METTL3 via an IGF2BP3-dependent mechanism.

In order to confirm the close relationship and determine the underlying mechanism, we transfected siRNA or overexpression plasmid, respectively, to knockdown and overexpress METTL3 in BMDMs. METTL3 knockdown dramatically reduced NLRP3, GSDMD, Caspase-1, and IL-1β mRNA levels (Fig. 4a) and protein levels (Fig. 4b). Additionally, compared to any of the groups, the combination group had a more noticeable drop, and the expression levels of these indicators in combination-primed BMDMs were largely consistent with those in P. gingivalis-primed BMDMs following METTL3 knockdown (Fig. 4a, b). Correspondingly, METTL3 overexpression increased the expression levels of above indicators (Fig. 4c, d) in HP group. Most importantly, except for NLRP3, other indicators remained to be constant after METTL3 overexpression in the groups without stimulation (Fig. 4c, d), suggesting NLRP3 transcripts is an important functional substrate of METTL3.

Using the online SRAMP database (http://www.cuilab.cn/sramp) to analyze the putative m⁶A binding sites, we confirmed the presence of 25 m⁶A residues spread across the NLRP3 sequence, 10 of which were classified as as high/very high confidence (Fig. 4e). We divided the 10 potential functional sites into 5 regions and designed specific primers, respectively. Methylated RNA immunoprecipitation (MeRIP)-qPCR revealed that stimuli induced a substantial hypermethylation on the third region of NLRP3 mRNA (Fig. 4f). In particular, the HP group exhibited the most enrichment of NLRP3 mRNA, whereas the hypermethylation was abolished when METTL3 was silenced. (Fig. 4f). Subsequently, we observed a decreased NLRP3 mRNA half-life in BMDMs lacking METTL3, indicating that NLRP3's m⁶A alteration enhanced its mRNA stability (Fig. 4g).

Given that members of the insulin-like growth factor 2 binding protein (IGF2BP) family are essential for identifying m⁶A alterations and maintaining mRNA stability, we evaluated the contribution of IGF2BP1/2/3 to the stabilization of NLRP3 mRNA. Each of them was targeted by two distinct siRNAs, and the effectiveness of these constructs was verified (Fig. 4h). We found that suppression of IGF2BP1 or IGF2BP2 had a minimal impact, whereas silencing IGF2BP3 significantly decreased NLRP3 mRNA expression (Fig. 4h). The upregulated gene expression levels of pyroptosis-related molecules in response to different stimulations were completely blocked by IGF2BP3 knockdown (Fig. 4i, j). The relationship between IGF2BP3 and NLRP3 mRNA was further validated using RIP-qPCR in RAWs (Fig. 4k). Finally, we observed a decrease in the stability of NLRP3 mRNA while IGF2BP3 knockdown, demonstrating that IGF2BP3 is essential for maintaining the stability of NLRP3 mRNA (Fig. 4l).

Local delivery of shMettl3 attenuates diabetes-associated PD via modulating pyroptosis.

To ascertain the functional implications of the METTL3-mediated NLRP3 m⁶A hypermethylation observed in vitro and its relevance in vivo, We administered an adeno-associated virus (AAV) that encoded either a scrambled control shRNA (shCtrl) or a shRNA targeting Mettl3 (shMettl3) into the periodontium of mice immediately on day 2, 4, 6, and 8 after silk ligature (Fig. 5a). The injection of shMettl3 significantly ameliorated periodontal damage both in PD and diabetes-associated PD mice (Fig. 5b, c). Intriguingly, no significant difference was observed between PD and diabetes-associated PD mice after treatment with shMettl3, whereas the disease phenotype of diabetes-associated PD mice was more severe than PD mice in shCtrl groups (Fig. 5b, c). In addition, the administration of shMettl3 supported the proliferation of osteoblasts and reduced the osteoclasts activity in PD and diabetes-associated PD mice compared with shCtrl groups (Fig. 5d, e). Similarly, there were not any discernible variations between PD and diabetes-associated PD mice after shMettl3 injection, suggesting that shMettl3 significantly reversed the additive effects of high glucose (Fig. 5d, e).

Next, qPCR was used to detect Mettl3 knockdown using mice gingival tissues (Fig. S4). In response to downregulated Mettl3, Nlrp3 expression was dramatically downregulated, thereby effectively abrogating pyroptosis-related gene expression levels (Fig. S4). Using IF staining, the number of METTL3⁺, NLPR3⁺, GSDMD⁺, Caspase-1⁺, and IL-1β⁺ macrophages significantly diminished in maxillae of mice receiving shMettl3 in contrast to those receiving shCtrl (Fig. 5f-j). Importantly, diabetes-associated PD mice achieved a better efficacy and the data were indistinguishable from shMettl3-injected PD mice (Fig. 5f-j). Collectively, these findings showed that shMettl3 downregulated NLRP3 expression, thereby inhibiting macrophage pyroptosis, attenuating periodontal destruction in diabetes-associated PD mice.

Given that the sixth complication of diabetes is periodontitis, accumulating evidence imply the bidirectional correlation between diabetes and periodontitis ^21,22. Persistent inflammatory responses and dysregulated repair in diabetic complications, result in the unfavorable therapeutic efficacy for diabetes-associated periodontitis ²³. In turn, the mortality was observed higher in diabetic cardiovascular disease or diabetic nephropathy patients with comorbid periodontitis compared to individuals with minimal or no periodontitis ²⁴. Thus, an efficient strategy for improving the prognosis of diabetes-associated periodontitis should be established. In the current study, we found that high levels of pyroptosis caused significant periodontal tissue damage in diabetes-associated periodontitis, which was attributed to the upregulation of METTL3-mediated m⁶A modification in macrophages. Mechanistically, METTL3 enhanced NLRP3 m⁶A RNA methylation and boosted its RNA stability through IGF2BP3-dependent pathways, hence promoting macrophage pyroptosis (Fig. 6). Importantly, we performed in vivo experiments by locally delivering shMettl3 in diabetes-associated PD models and found METTL3 silencing protected against inflammation and alveolar bone loss in PD mice with diabetes.

Pyroptosis, a recently identified proinflammatory process of programmed cell death, has demonstrated criticality in the regulation of microbial infections ²⁵. There is growing evidence that suggests pyroptosis may be involved in the pathophysiology of a number of diseases, including rheumatoid arthritis ²⁶, diabetes mellitus ²⁷, and cardiovascular diseases ²⁸. Fewer studies, however, demonstrated a strong correlation between pyroptosis and the advancement of diabetes-associated PD. Considering that P. gingivalis may induce pyroptosis in macrophages ²⁹, which was also confirmed in this study, we examined the relationship between diabetes-related periodontitis and pyroptosis. Our findings demonstrated that hyperglycemia exacerbated P. gingivalis-induced macrophage pyroptosis, which might contribute to the dysregulated immune response in diabetes-associated PD. Of note, a latest report indicated that fibroblasts around the pyroptotic macrophages exacerbated cell senescence in the periodontal tissues ¹². Nevertheless, we focused on alveolar bone remodeling and found that macrophage pyroptosis might lead to alveolar bone loss by inhibiting osteoblast activity. Therefore, we speculate that macrophage pyroptosis exert complicated effects on diabetes-associated PD progression and need further study.

Recently, the m⁶A modification in mRNA, engaged in a variety of biological processes, including mRNA maturation or degradation ³⁰, is closely related to diabetic complications. METTL3 expression was determined elevated in cells from individuals with diabetic nephropathy and METTL3 knockout resulted in alleviation of inflammation and apoptosis ³¹. Interestingly, another study demonstrated that METTL3 expression in podocytes was downregulated in response to elevated glucose, resulting in exacerbated inflammation ³². As for diabetes-associated periodontitis, a recent study revealed that the stimulation of advanced glycation end products (AGEs) increased fat mass and obesity-associated protein (FTO), an RNA m⁶A demethylase and FTO knockdown protected the osteogenic development of mesenchymal stem cells from bone marrow exposed to AGEs ³³. However, single stimulation of high glucose may not represent the appropriate immune microenvironment in diabetes-associated periodontitis. Moreover, the key role of immune cells can not be ignored in diabetes-associated PD and it has not been studied yet whether m⁶A modification in macrophages affects diabetes-associated periodontitis. Therefore, in our study, the combined stimulation of P. gingivalis and high glucose was utilized to mimic the microenvironment, and the findings showed that METTL3 was the predominant methyltransferase mediating macrophage pyroptosis, which revealed METTL3's unique functional involvement in pyroptosis and pyroptosis-induced diabetes-associated PD. Unlike the studies showed indirect functional regulation of pyroptosis by m⁶A modifications ^34–36, our results identified NLRP3 as the direct target gene of METTL3 in macrophage pyroptosis under high glucose condition. Target transcript fate is frequently determined by m⁶A selective binding proteins, such as IGF2BP family members, including IGF2BP1/2/3, which assist in keeping these mRNAs stable and translating ³⁷. According to our results, we verified that IGF2BP3, not IGF2BP1/2, was essential for maintaining the stability of NLRP3 mRNA under METTL3 regulation.

While it was discovered that a METTL3 inhibitor might treat myeloid leukemia, demonstrating that METTL3 might be a therapeutic target for diseases, unfortunately, METTL3 inhibitors are still in the preclinical phase of development ³⁸. Consequently, it is essential to create novel therapy approaches that prioritize METTL3 in order to prevent diabetes-associated PD. We evaluated the efficacy of METTL3-targeted therapy with AAV encoding shMettl3 and illustrated shMettl3 inhibited pyroptosis in a NLRP3-dependent manner, thereby ameliorating diabetes-associated PD. The detailed analysis of effects on diabetes and its complications after effective management of diabetes-associated PD requires additional study. Notably, while periodontal damage was decreased by locally injected shMettl3, the benefits were not complete, indicating that either the local concentration of shMettl3 was inadequate or that other variables likely had a role in the inflammation. Another limitation of our study is that the detailed function of METTL3 has not been verified in METTL3-floxed with LysM-cre or CX3CR1-cre mice. Moreover, we will give careful consideration to the intricate mechanism that underlies the enhanced expression of METTL3 produced by P. gingivalis and high glucose in our upcoming research.

In conclusion, using ligature-induced periodontitis in diabetic murine models, we demonstrated that METTL3 inhibited macrophage pyroptosis via m⁶A methylation of NLRP3 mRNA in a way depended on IGF2BP3. In addition, inhibition of METTL3 protected against periodontal inflammation and alveolar bone loss in diabetes-associated PD mice. Thus, our research reveals the poorly understood molecular pathways by which m⁶A results in diabetes-associated PD and suggests the potential therapies targeting METTL3/NLRP3 axis for the management of diabetes-associated PD.

Animals

The Hubei Provincial Center for Disease Control and Prevention Laboratory Animal Research Center provided five-week-old C57BL/6 mice for our acquisition. To create diabetes models, after 1 week of environment acclimation, mice received 55 mg/kg of streptozotocin (STZ; Sigma-Aldrich, China) intraperitoneally every day for five days ¹². At 8 weeks of age, diabetes in animals was verified using the blood glycemia test. Then, a 5 − 0 silk ligature was placed around the maxillary second molar to generate experimental PD. Ten days following the ligation, mice were sacrificed ¹⁵. All animal experiments were approved by the Ethics Committee of the School and Hospital of Stomatology, Wuhan University (S07923080I).

Micro-CT, histology, and histomorphometric analyses

Murine maxillary tissues were dissected without soft tissue, preserved in 4% paraformaldehyde for an entire night, and then scanned using a Quantum GX microCT scanner µCT (PerkinElmer, Inc, Waltham, MA, USA). The CT was operated at 100 kV, 200 µA, at a pixel size of 10 µm. Separated maxillae were decalcified in 14% EDTA and preserved in 4% paraformaldehyde for histology and histomorphometric studies. Following dehydration, the tissues were fixed in paraffin and sectioned into 4-µm-thick pieces. For regular histology, the paraffin sections were stained with H&E. Additionally, TRAP activity was used to identify osteoclasts, while ALP activity was used to identify osteoblasts. Using an Olympus fluorescent microscope, representative pictures were taken.

Immunofluorescence staining

The sections that had been deparaffinized underwent heat-mediated antigen retrieval, were blocked in H₂O₂ for half an hour, and then spent an additional half an hour at room temperature in PBS containing 5% BSA and 0.2% Triton X-100. Afterward, the sections were stained overnight with primary antibody against F4/80 (Abcam, ab16911), NLRPP3 (Sigma-Aldrich, SAB5700723), Caspase-1 (Abmart, MU167952), GSDMD (Abclonal, A18281), IL-1β (R&D, AF-401-NA), METTL3 (Abclonal, A8370) or OCN (Proteintech, 23418-1-AP) at 4℃. Tissues were soaked in PBS for fifteen minutes, and then they were incubated with goat anti-rat FITC (Abclonal, AS019) or goat anti-mouse Cy3 (Abclonal, AS008) or goat anti-rabbit Cy3 (Abclonal, AS007) or goat anti-mouse FITC (Abclonal, AS001) at room temperature. DAPI-containing mounting medium was used to mount the slides (ZhongShan Jinqiao Biotechnology, China). Using an Olympus fluorescent microscope, representative pictures were taken.

The methods for cell staining with IF were comparable. 4% paraformaldehyde was used to fix the cells for 15 minutes. 0.1% Triton X-100 was used to permeabilize the cells for 15 minutes. Finally, 10% goat serum albumin was used to block the cells for 1 hour at 37°C. Then, the cells were then exposed to the designated primary antibody against METTL3 (Abclonal, A8370) overnight at 4°C. Tissues were soaked in PBS for fifteen minutes, and then they were incubated with goat anti-rabbit Cy3 (Abclonal, AS007) at room temperature. Finally, samples were mounted with anti-fade fluorescence mounting media containing DAPI. Using an Olympus fluorescent microscope, representative pictures were taken.

Cell culture and Bacteria Culture

RAW264.7 and MC3T3-E1 (MC3T3) cell lines served as storage cells in laboratories, and were tested for mycoplasma contamination before the experiment. DMEM with 10% fetal bovine serum (FBS) supplement was used to maintain the RAW264.7 cells. and The culture medium used for MC3T3 cells was α-MEM with 10% FBS. Osteogenic medium prepared with DMEM, 10% FBS, 10 mM sodium β-glycerophosphate, 10 nM dexamethasone, and 50 µg/mL ascorbic acid were used to stimulate the formation of osteogenic differentiation. WT mice were used to isolate and produce bone marrow-derived macrophages (BMDMs), as previously mentioned ³⁹. In short, bone marrow (BM) cells were removed by flushing with α-MEM/2% FBS after tibiae and femora were harvested. Following lysing of red blood cells, BM cells were cultivated in α-MEM/10% FBS for three days with M-CSF (abclonal, China) to produce BMDMs. To simulate the high glucose environment in vitro, the cells were cultured with 25 mM glucose.

The ATCC 33277 strain of P. gingivalis was cultivated in trypticase soy broth (TSB) supplemented with 1µg/mL vitamin K1, 0.1% yeast extract, and 5µg/mL hemin (Sigma). The culture was conducted under anaerobic conditions (80% N₂, 10% H₂, and 10% CO₂) at 37°C. The concentration of bacterial resuspension was determined by measuring the optical density (OD) at 600 nm (1 OD is equal to a concentration of 109 P. gingivalis/mL) ⁴⁰. To simulate the in vitro microenvironment, P. gingivalis (MOI = 100) was treated with the cells.

Western Blotting

By centrifuging at 12000 rpm for 15 minutes at 4 ˚C, total cellular proteins were extracted using M-PER mammalian protein extraction reagent (Thermo Scientific). Proteins (20–30 µg) from every sample were separated using 12% SDS-PAGE and subsequently placed onto polyvinylidene difluoride (PVDF) membranes (Millipore). The membranes were blocked for two hours at room temperature using 5% skim milk, and then each primary antibody was incubated for an additional night at 4°C: NLRPP3 (Sigma-Aldrich, SAB5700723), Caspase-1 (Abmart, MU167952), GSDMD (Abclonal, A18281), IL-1β (Abclonal, A16288), METTL3 (Abclonal, A8370), IGF2BP3 (Abclonal, A4444), Runx2 (Abcam, ab236639), Osterix (Abcam, ab22552), BSP (Affinity, DF7738), OCN (Proteintech, 23418-1-AP), β-actin (Proteintech, 66009-1-AP), GAPDH (Abclonal, AC001). Subsequently, the membranes were incubated with HRP-conjugated secondary antibody for one hour on the next day following three rounds of PBST washing. Bands were visualized using the Enhanced Chemiluminescence Detection Reagents (Advansta) by the Odyssey LI-COR scanner.

Quantitative real-time PCR

TRIzol (Invitrogen) Reagent was used to extract total RNA. PrimeScript RT Master Mix (Takara, RR036A) was used to reverse transcribe cDNAs, and particular primers were used for RT-qPCR. Using the 2-^ΔΔCt technique, the relative expression of mRNA was assessed and normalized to GAPDH expression. The sequences of primer sets are shown in Supplementary Table S1 and Table S2 on line.

Lactate dehydrogenase (LDH) release assay and Cell viability assay

Placed in 96-well plates, 1×10⁴ cells were stimulated with either high glucose or P. gingivalis. The Cell Counting Kit-8 (CCK-8) was used to determine the viability of the cells (Beyotime, China). Following the manufacturer's instructions, the LDH test (Beyotime, China) was performed.

ELISA

Following cell culture, the conditioned media was removed and analyzed using ELISA.

The mouse IL-1β ELISA kit (Abclonal, RK00006) was acquired and utilized in compliance with the guidelines provided by the manufacturer.

Conditional medium (CM) based coculture and transwell coculture

In 6-well plates, macrophages were seeded the day before pyroptosis induction. Following a 24-hour induction of macrophage pyroptosis using P. gingivalis or high glucose, the medium was disposed of and cleaned using PBS. After giving the macrophages another 24 hours in the serum-free media (2 mL/well), the supernatants were removed. After passing through sterile filters with particle sizes of 0.22 µm, the supernatants were combined with an equivalent amount of osteogenic media. In 6-well plates, MC3T3 cells were planted at a density of 3 × 10⁵/well. To determine the effects of the CM on MC3T3 osteogenic differentiation, CM replaced the medium the next day, and cultured for 2 days. Through the use of western blotting and RT-qPCR, the expressions of markers associated to osteogenesis were found.

MC3T3 cells and macrophages were cocultured in a 6-well-transwell coculture system to assess the effects of macrophage pyroptosis on osteogenic differentiation (0.4 µm pore). In short, MC3T3 cells were sown in 6-well plates and macrophages were seeded in the up inserts. After macrophage pyroptosis induction with P. gingivalis or high glucose for 24 h, the medium was was disposed of and cleaned with PBS. After that, the upper inserts were placed on 6-well plates and cultured for two days using osteogenic media. In MC3T3 cells, the expression of markers associated to osteogenesis was quantified.

Overexpression and knockdown of genes

Following the manufacturer's instructions, Lipofectamine 2000 (Invitrogen) was used to transfect plasmids or siRNA. 48 to 72 hours later, the samples were analyzed. The chosen sequences are mentioned in table S3.

Dot blot assay

The amount of m⁶A in the poly-A tailing of total RNA was assessed by RNA m⁶A dot blot analysis as described ⁴¹. TRIzol (Invitrogen) was used to isolate total RNA in accordance with the manufacturer's instructions. The RNAs (200 and 400 ng respectively) were double diluted and spotted onto a Hybond-N⁺ membrane (GE Healthcare). Following ultraviolet crosslinking and blocking in 5% nonfat milk, the membranes were treated with the m⁶A antibody (Abclonal, A17924) overnight. The secondary antibody was then applied to the membranes and incubated for an hour at room temperature. Bands were visualized using the Enhanced Chemiluminescence Detection Reagents (Advansta) by the Odyssey LI-COR scanner. Afterward, the quantity of total RNA was shown using 0.02% methylene blue in 0.3 M sodium acetate (pH 5.2).

qPCR of Methylated RNA immunoprecipitation (MeRIP)

The MeRIP-qPCR procedure was followed as previously mentioned ²⁰. Essentially, mRNA was purified from total RNA using the GenEluteTM mRNA Miniprep Kit (Sigma). RNA was randomly fragmented using reagents for RNA fragmentation. m⁶A immunoprecipitation was performed using a particular anti-m⁶A antibody (Abclonal). Protein A magnetic beads were pre-bound with anti-m⁶A antibody and left in reaction buffer for half an hour. After being treated for an hour at 4°C with protein A magnetic beads attached to the m⁶A antibody, the fragmented mRNA was rinsed with low- and high-salt reaction buffers. M⁶A-antibody-bound RNA was extracted from the Dynabeads using Buffer RLT (Qiagen) and further incubated with Dynabeads MyOne Silane (Life Technologies). The combination of RNA and Dynabeads was precipitated using 100% ethanol, then rinsed with 70% ethanol before being re-suspended in water free of nucleases. Once the beads were drawn to the tube's side by a magnetic field, the supernatant was carefully collected. After the binding of m⁶A-IP, reverse transcription and qRT-PCR were performed to confirm the enrichment. For m⁶A-meRIP-qPCR, the effect of m⁶A methylation can be analyzed by quantifying changes in specific target genes.

RNA immunoprecipitation (RIP)

Following the manufacturer's instructions, RIP was performed using the Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Bersinbio). To summarize, RIP lysis buffer was used to lyse macrophages (1 × 10⁷cells per 150 µl). Prepared cell lysates were treated with magnetic beads coated with 5 µg of particular antibodies against mouse IgG (Bersinbio) or IGF2BP3 (Abclonal) for 4 hours at room temperature. Next, in order to separate the immunoprecipitated RNA, the RNA-protein complexes underwent six rounds of washing and were then treated with proteinase K digestion buffer. qPCR was used to assess the relative interaction between NLRP3 and IGF2BP3.

RNA stability

In order to evaluate the stability of TAB3, actinomycin (5 µg/ml) was treated with the cells to stop transcription. At 0, 3, and 6 hours following termination, samples were collected. NLRP3 expression was assessed by real-time PCR after total RNA was extracted.

Statistical analysis

All results are given as mean ± SD. The statistical analysis was carried out with GraphPad Prism 9 (GraphPad Software Inc., San Diego, CA, USA). The 2-tailed unpaired Student's t test was used to assess differences between two groups. One way ANOVA followed by Dunnett’s post-hoc multiple comparisons was used to compare three or more groups. p values < 0.05 were considered statistically significant.

DATA AVAILABILITY

The data supporting the findings are available from the corresponding author upon reasonable request.

AUTHOR CONTRIBUTIONS

Q.Y. contributed to conception, design, data acquisition, analysis and interpretation, drafted and critically revised the manuscript; J.X., Y.L., Z.Y., C.W., J.S. contributed to data acquisition, analysis and interpretation, and critically revised the manuscript; H.W., H.L., X.W., L.M., X.H. contributed to data analysis, critically revised the manuscript; Z.C. contributed to conception, design, data analysis, critically revised the manuscript. All authors gave final approval and agree to be accountable for all aspects of the work.

ACKNOWLEDGEMENTS

This research was supported by the National Natural Science Foundation of China to Zhengguo Cao (No. 82370967 and No. 82170963), Li Ma (No. 82301092) and Xiaoxuan Wang (No. 82101022).

CONFLICT OF INTEREST STATEMENT

The authors declare no conflict of interests.

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METTL3-dependent N6-methyladenosine RNA modification promotes diabetes-associated periodontitis via mediating macrophage pyroptosis

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INTRODUCTION

RESULTS

DISCUSSION

MATERIALS AND METHODS

Animals

Micro-CT, histology, and histomorphometric analyses

Immunofluorescence staining

Cell culture and Bacteria Culture

Western Blotting

Quantitative real-time PCR

Lactate dehydrogenase (LDH) release assay and Cell viability assay

ELISA

Conditional medium (CM) based coculture and transwell coculture

Overexpression and knockdown of genes

Dot blot assay

qPCR of Methylated RNA immunoprecipitation (MeRIP)

RNA immunoprecipitation (RIP)

RNA stability

Statistical analysis

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References

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