SIRT6-regulated macrophage efferocytosis epigenetically controls inflammation resolution of diabetic periodontitis

Rationale: Diabetes exacerbates the prevalence and severity of periodontitis, leading to severe periodontal destruction and ultimately tooth loss. Delayed resolution of inflammation is a major contributor to diabetic periodontitis (DP) pathogenesis, but the underlying mechanisms of this imbalanced immune homeostasis remain unclear. Methods: We collected periodontium from periodontitis with or without diabetes to confirm the dysfunctional neutrophils and macrophages in aggravated inflammatory damage and impaired inflammation resolution. Our in vitro experiments confirmed that SIRT6 inhibited macrophage efferocytosis by restraining miR-216a-5p-216b-5p-217 cluster maturation through ''non-canonical'' microprocessor complex (RNA pulldown, RIP, immunostaining, CHIP, Luciferase assays, and FISH). Moreover, we constructed m6SKO mice that underwent LIP-induced periodontitis to explore the in vitro and in vivo effect of SIRT6 on macrophage efferocytosis. Finally, antagomiR-217, a miRNA antagonism, was delivered into the periodontium to treat LIP-induced diabetic periodontitis. Results: We discovered that insufficient SIRT6 as a histone deacetylase in macrophages led to unresolved inflammation and aggravated periodontitis in both human and mouse DP with accumulated apoptotic neutrophil (AN) and higher generation of neutrophil extracellular traps. Mechanistically, we validated that macrophage underwent high glucose stimulation resulting in disturbance of the SIRT6-miR-216/217 axis that triggered impeded efferocytosis of AN through targeting the DEL-1/CD36 axis directly. Furthermore, we demonstrated the inhibitory role of SIRT6 for MIR217HG transcription and identified a non-canonical action of microprocessor that SIRT6 epigenetically hindered the splicing of the primary miR-216/217 via the complex of hnRNPA2B1, DGCR8, and Drosha. Notably, by constructing myeloid-specific deletion of SIRT6 mice and locally delivering antagomir-217 in DP models, we strengthened the in vivo effect of this axis in regulating macrophage efferocytosis and inflammation resolution in DP. Conclusions: Our findings delineated the emerging role of SIRT6 in mediating metabolic dysfunction-associated inflammation, and therapeutically targeting this regulatory axis might be a promising strategy for treating diabetes-associated inflammatory diseases.

infiltration and phenotypic polarization of macrophages.
(A) Neutrophils were isolated by erythrocyte sedimentation and centrifugation. (B) Flow cytometry showed that the spontaneous apoptosis of neutrophils was not increased after 6 hours of high glucose culture in vitro. (C) Transwell migration in macrophages after efferocytosis, and apoptotic stimulation. (D) Wound healing assays were used to investigate the migratory ability of macrophages stimulated by efferocytosis, and apoptosis. (E) Efferocytosis promotes macrophages to secrete antiinflammatory mediators such as IL-10 and TGF-β. (F) Macrophages co-cultured with apoptotic neutrophils secrete a large number of inflammatory mediators, such as IL-6 and TNF-α. (G-H) WB and immunofluorescence analysis of macrophage differentiation stimulated by efferocytosis, and apoptosis. The results were presented as means ± S.D. *p < 0.05; **p < 0.01; ***p > 0.001 by ANOVA or 2-tailed, unpaired Student's t test.   The results were presented as means ± S.D. *p < 0.05; **p < 0.01 by ANOVA or 2tailed, unpaired Student's t test. The white dotted line indicates the boundary between the root and the alveolar bone and the gingiva.

Supplementary materials and methods miRNA microarray
Total RNA was extracted from macrophages and macrophages after SIRT6 inhibition and met the RNA quality control requirements of the array. The Personalbio Company (Personalbio, Nanjing) performed the miRNA microarray assay. Hiseq single end mode is adopted for sequencing and genome alignment analysis using miRDeep2 (Mackowiak SD, 2011) software. Analyze the data obtained by Affymetrix Expression Console software according to the MAS5 method. The spots with a | log2 ratio| ≥ 0.263 and a p <0.05 were selected for analysis.

Wound Healing and Cellular Transwell assay
Cell migration was determined by scratch wound healing and Cellular transwell assay. Macrophages were inoculated into six-well plates, subjected to serum starvation in a serum-free medium for 24 h, and then an artificial wound was created using a 200 mL pipette tip. Images were taken at 0 h, 24 h, and 48 h using an inverted microscope.
For in vitro invasion assay, approximately 1 × 10 5 cells of differentiated macrophages were plated in serum-free media in the top chamber (Millipore, USA). Medium supplemented with 10% FBS was added to the lower chamber. The cells were then incubated at 37 • C in 5% CO2 for 48 h. The chambers were then fixed with 4% paraformaldehyde, and stained with crystal violet, and the number of macrophages was counted under a microscope.

RNA extraction and quantitative RT-PCR (qRT-PCR)
Total RNA was extracted using TRIzol according to the guideline and reversely transcribed into complementary DNA (cDNA) by a HiScript II Q RT SuperMix for qPCR kit (Vazyme, China). The RT of miRNA was conducted using the tailing method.
For Real-time qPCR, the StepOne Plus RT PCR System (Thermo, USA) was used by SYBR Green (Vazyme, China). The relative expression normalized to GAPDH or U6 was analyzed with the formula 2 -ΔΔCt .
Membranes were blocked by 5% fat-free milk for 2 h and incubated with primary antibodies overnight. After washing with TBST three times, the membranes were then incubated with the appropriate HRP-conjugated secondary antibody (1:10,000). Protein bands were detected with an ECL detection kit. ImageJ software was performed to analyze the Western blot.

High glucose stimulation of macrophages
In this experiment, 1640 medium with three Glucose concentrations was used: normal glucose concentration (NG): 5.5mmol/L, ordinary 1640 medium: 11.11 mmol/L, and high glucose 1640 medium (HG): 25mmol/L. NG and HG are prepared from glucose-free 1640 medium RPMI culture medium (Gibco, Waltham, MA, USA) and glucose, respectively. After the glucose is dissolved, filter sterilization. Conventional culture of THP-1 and induction of macrophages were performed using an ordinary 1640 medium, and subsequent stimulation with different sugar concentrations was performed using a 1640 medium with corresponding sugar concentrations.

Analysis of single-cell RNA-seq and GO analysis.
Single-cell RNA-seq raw data generated in skin specimens from DFU-Nonhealers (DFU, diabetic foot ulceration) and foot skin of healthy non-diabetic subjects were retrieved from NCBI Gene Expression Omnibus (GEO). DFU-Non-healers labeling GSM5050530, GSM5050533, GSM5050557, GSM5050558, and GSM5050563, foot skin of healthy non-diabetic labeling GSM5050534, GSM5050538, GSM5050534, GSM5050534, GSM5050552, GSM5050552, GSM5050556, GSM5050556, GSM5050556, GSM5050568, and GSM5050574. Download the Sequence Read Archive (SRA) raw data from the NCBI server. Single-cell RNA-seq analysis and GO analysis based on previous studies.

antagomir-217
antagomir-217 and antagomir-NC were designed and synthesized by RiboBio (Guangzhou, China). A microsyringe (5μL Hamilton, Switzerland) was used for injection. From the first day of ligation, the injection was once every three days, with a total of 4 injections. The corresponding drugs were injected into the mesial and distal gingiva of the mouse's buccal and palatal sides of the maxillary second molar using a micro-syringe, respectively. The injection dose of antagomir-217 and antagomir-NC was 10 nmol per mouse.

Supplementary Material 1 Abbreviations
Teeth were numbered according to the International Federation of Dentists (FDI). BI: bleeding index, PD: probing depth.