RESUMO
Periodontal inflammation is largely governed by infiltration of myeloid cells, in particular macrophages. Polarization of Mφ within the gingival tissues is a well-controlled axis and has considerable consequences for how Mφ participate in inflammatory and resolution (tissue repair) phases. We hypothesize that periodontal therapy may instigate a pro-resolution environment favoring M2 Mφ polarization and contribute towards resolution of inflammation post-therapy. We aimed to evaluate the markers of macrophage polarization before and after periodontal therapy. Gingival biopsies were excised from human subjects with generalized severe periodontitis, undergoing routine non-surgical therapy. A second set of biopsies were excised after 4-6 weeks to assess the impact of therapeutic resolution at the molecular level. As controls, gingival biopsies were excised from periodontally healthy subjects, undergoing crown lengthening. Total RNA was isolated from gingival biopsies to evaluate pro- and anti-inflammatory markers associated with macrophage polarization by RT-qPCR. Mean periodontal probing depths, CAL and BOP reduced significantly after therapy and corroborated with the reduced levels of periopathic bacterial transcripts after therapy. Compared to heathy and treated biopsies, higher load of Aa and Pg transcripts were observed in disease. Lower expression of M1Mφ markers (TNF-α, STAT1) were observed after therapy as compared to diseased samples. Conversely, M2Mφ markers (STAT6, IL-10) were highly expressed in post-therapy as opposed to pre-therapy, which correlated with clinical improvement. These findings corroborated with murine ligature-induced periodontitis and resolution model, comparing the respective murine Mφ polarization markers (M1 Mφ: cox2 , iNOS2 and M2 Mφ: tgm2 and arg1 ). Our findings suggest that imbalance in M1 and M2 polarized macrophages by assessment of their markers can provide relevant clinical information on the successful response of periodontal therapy and can be used to target non-responders with exaggerated immune responses.
RESUMO
AIM: To evaluate the potential role of miR-26 family members in periodontal pathogenesis by assessing innate immune responses to periopathic bacteria and regulation of cytoskeletal organization. MATERIALS AND METHODS: Expression of miR-26a-5p and miR-26b-5p was quantified in gingival biopsies derived from healthy and periodontally diseased subjects before and after non-surgical (scaling and root planing) therapy by RT-qPCR. Global pathway analysis and luciferase assays were performed for target identification and validation. Cytokine expression was assessed in miR-26a-5p transfected human oral keratinocytes upon stimulation with either live Porphyromonas gingivalis (Pg), Aggregatibacter actinomycetemcomitans or Pg lipopolysaccharide (LPS). Wound closure assays were performed in cells transfected with miR-26a-5p, while the impact on cytoskeletal organization was assessed by F-actin staining. RESULTS: miR-26a-5p and miR-26b-5p were downregulated in diseased gingiva and restored 4-6 weeks post-therapy to levels comparable with healthy subjects. Target validation assays identified phospholipase C beta 1 as a bona fide novel target exhibiting antagonistic expression pattern in disease and post-therapy cohorts. miR-26a-5p transfected cells secreted higher levels of cytokine/chemokines upon stimulation with periopathogens and demonstrated impaired cell migration and cytoskeletal rearrangement. CONCLUSIONS: Downregulated miR-26a-5p levels in periodontal inflammation may interfere with key cellular functions that may have significant implications for host defence and wound healing.