No abstract

It has been suggested that microRNAs (miRs) are involved in the immune regulation of periodontitis. However, it is unclear whether and how miRs regulate the function of B cells in the context of periodontitis. This study is to explore the role of miR-146a on the inflammatory cytokine production of B cells challenged by Porphyromonas gingivalis (P. gingivalis) lipopolysaccharide (LPS). Primary B cells were harvested from mouse spleen. Quantitative real-time polymerase chain reaction (qPCR), enzyme-linked immunosorbent assay (ELISA) were used to detect the expression of inflammatory cytokines in B cells in the presence or absence of P. gingivalis LPS and/or miR-146a. Bioinformatics, luciferase reporter assay and overexpression assay were used to explore the binding target of miR-146a. Our results showed that miR-146a level in B cells was elevated by P. gingivalis LPS stimulation, and the mRNA expressions of interleukin (IL)-1β, 6 and 10, and IL-1 receptor associated kinase-1 (IRAK1), but not TNF receptor associated factor 6 (TRAF6), were also upregulated. The expression levels of IL-1β, 6, 10 and IRAK1 were reduced in the presence of miR-146a mimic, but were elevated by the addition of miR-146a inhibitor. MiR-146a could bind with IRAK1 3' untranslated region (UTR) but not TRAF6 3'-UTR. Overexpression of IRAK1 reversed the inhibitory effects of miR-146a on IL-1β, 6 and 10. In summary, miR-146a inhibits inflammatory cytokine production in B cells through directly targeting IRAK1, suggesting a regulatory role of miR-146a in B cell-mediated periodontal inflammation.

Background:The present study was to determine the role of Toll-like receptor 4 (TLR4) signaling in inflammation and alveolar bone resorption using a murine model of Porphyromonas gingivalis-associated ligature-induced peri-implantitis. Methods:Smooth surface titanium implants were placed in the left maxilla alveolar bone 6 weeks after extraction of first and second molars in Wild-type (WT) and TLR4 −/− (TLR4 KO) mice. Silk ligatures immersed with P. gingivalis were tied around the implants 4 weeks after the implant placement and confirmation of osteointegration. Two weeks after the ligation, bone resorption, osteoclastogenesis, cellular inflammatory responses, and gingival mRNA expression levels of cytokines were assessed by micro-computed tomography, tartrate-resistant acid phosphatase (TRAP) staining, immunobiological examination and Real-time quantitative polymerase chain reaction, respectively. Results:In both WT and TLR4 KO mice, the bone resorption around implants was significantly increased in the P. gingivalis/ligation group compared with control group. In P. gingivalis/ligation group, the levels of bone resorption, TRAP+ cell formation, and gingival CD3+ and CD45+ cell infiltration were significantly decreased in TLR4 KO mice compared with that in WT mice. Receptor activator of nuclear factor-kappa B ligand /osteoprotegerin (RANKL/OPG) ratio was significantly increased after P. gingivalis/ligation treatment in WT mice not in TLR4 KO mice. When comparing the P. gingivalis/ligation group with the respective control group, gingival mRNA expressions of IL-1 , IFN-, and 1L-17 were significantly increased in TLR4 KO mice. Conclusion:This study suggests that TLR4 mediates alveolar bone resorption in P. gingivalis associated ligature-induced peri-implantitis through regulation of immune B cell infiltration, RANKL/OPG expression ratio, and differential inflammatory cytokine production. K E Y W O R D Sbone resorption, peri-implantitis, TLR4 J Periodontol. 2020;91:671-682.

Human lymphocytes exposed to Aggregatibacter actinomycetemcomitans (Aa) cytolethal distending toxin (Cdt) undergo cell cycle arrest and apoptosis. In previous studies, we demonstrated that the active Cdt subunit, CdtB, is a potent phosphatidylinositol (PI) 3,4,5-triphosphate phosphatase. Moreover, AaCdt-treated cells exhibit evidence of PI-3-kinase (PI-3K) signaling blockade characterized by reduced levels of PIP3, pAkt, and pGSK3β. We have also demonstrated that PI-3K blockade is a requisite of AaCdt-induced toxicity in lymphocytes. In this study, we extended our observations to include assessment of Cdts from Haemophilus ducreyi (HdCdt) and Campylobacter jejuni (CjCdt). We now report that the CdtB subunit from HdCdt and CjCdt, similar to that of AaCdt, exhibit potent PIP3 phosphatase activity and that Jurkat cells treated with these Cdts exhibit PI-3K signaling blockade: reduced levels of pAkt and pGSK3β. Since non-phosphorylated GSK3β is the active form of this kinase, we compared Cdts for dependence on GSK3β activity. Two GSK3β inhibitors were employed, LY2090314 and CHIR99021; both inhibitors blocked the ability of Cdts to induce cell cycle arrest. We have previously demonstrated that AaCdt induces increases in the CDK inhibitor, p21CIP1/WAF1, and, further, that this was a requisite for toxin-induced cell death via apoptosis. We now demonstrate that HdCdt and CjCdt also share this requirement. It is also noteworthy that p21CIP1/WAF1 was not involved in the ability of the three Cdts to induce cell cycle arrest. Finally, we demonstrate that, like AaCdt, HdCdt is dependent upon the host cell protein, cellugyrin, for its toxicity (and presumably internalization of CdtB); CjCdt was not dependent upon this protein. The implications of these findings as they relate to Cdt’s molecular mode of action are discussed.