Porphyromonas gingivalis inhibits M2 activation of macrophages by suppressing α‐ketoglutarate production in mice

Reprograming of metabolic pathways is critical in governing the polarization of macrophages into classical proinflammatory M1 or alternative anti‐inflammatory M2 phenotypes in metabolic diseases, such as diabetes. Porphyromonas gingivalis, a keystone pathogen of periodontitis, causes an imbalance in M1/M2 activation, resulting in a hyperinflammatory environment that promotes the pathogenesis of periodontitis. However, whether P. gingivalis infection modulates metabolic pathways to alter macrophage polarization remains unclear. Bone‐marrow‐derived macrophages (BMDMs) were collected from 6‐week‐old female C57BL/6 mice and stimulated with P. gingivalis, P. gingivalis‐derived LPS or IL‐4. Relative gene expression and protein production were measured by quantitative real‐time PCR, RNA sequencing and western blotting. Colorimetric assays were also performed to assess the amounts of α‐ketoglutarate (α‐KG) and succinate. P. gingivalis or P. gingivalis‐derived LPS‐induced inflammatory responses enhanced M1 macrophages and suppressed M2 macrophages, even in the presence of IL‐4. P. gingivalis inhibited Idh1/2 and Gpt1/2 mRNA expression, and increased Akgdh mRNA expression, thus decreasing the ratio of α‐KG/succinate. Supplementation of cell‐permeable dimethyl‐α‐KG dramatically restored M2 activation during P. gingivalis infection. Our study suggests that P. gingivalis maintains a hyperinflammatory state by suppressing the production of α‐KG by M2 macrophages.     

Interleukin-1 beta (IL-1 beta) levels in gingival crevicular fluid from adults with previous evidence of destructive periodontitis. A cross sectional study.

We have estimated the levels of Interleukin-1 beta (IL-1 beta) by ELISA in gingival crevicular fluid (GCF) at 58 sites from 37 patients with adult periodontitis. GCF was collected for 5 s on filter papers and a 2nd sample was collected for 30 s 1 min later. 68/116 strips yielded detectable levels of IL-1 beta. IL-1 beta was present in both the 1st and 2nd samples at 28 sites, in the 1st only at 4 sites and in the 2nd only at 8 sites; 18 sites were below the level of detection for the assay. When the concentrations of IL-1 beta were calculated in the original volume of GCF on each strip, the mean value for positive strips was 34.16 +/- 29.45 (SD) pg/microliters with a range from 1.75 to 97.13 pg/microliters. There were no statistically significant correlations with the plaque index, bleeding index or probable crevice depth (pocket depth). The results indicate that IL-1 is present in the GCF from a proportion of sites with evidence of previous periodontal destruction.     

Activation of NLRP3 inflammasome in macrophages by mycoplasmal lipoproteins and lipopeptides

The NLRP3 inflammasome, an intracellular sensor consisting of the nucleotide‐binding oligomerization domain‐like receptor family, pyrin domain containing 3 (NLRP3), the adaptor protein apoptosis‐associated speck‐like protein containing a caspase‐recruitment domain (ASC), and procaspase‐1, plays critical roles in host defense against microbial pathogens by inducing production of interleukin‐1β (IL‐1β) and IL‐18. Mycoplasma salivarium and Mycoplasma pneumoniae cells activated murine bone marrow‐derived macrophages (BMMs) to induce production of IL‐1α, IL‐1β, and IL‐18. The IL‐1β production‐inducing activities of these mycoplasmas toward BMMs from Toll‐like receptor 2 (TLR2)‐deficient mice were significantly attenuated compared with those from C57BL/6 mice (B6BMMs). This result suggests the possibility that their lipoproteins as TLR2 agonists are involved in the activity. Lipoproteins of M. salivarium and M. pneumoniae (MsLP and MpLP), and the M. salivarium‐derived lipopeptide FSL‐1 induced IL‐1β production by B6BMMs, but not by BMMs from caspase‐1‐, NLRP3‐ or ASC‐deficient mice. The activities of MsLP and MpLP were not downregulated by the proteinase K treatment, suggesting that the active sites are their N‐terminal lipopeptide moieties. B6BMMs internalized the mycoplasmal N‐terminal lipopeptide FSL‐1 at least 30 min after incubation, FSL‐1‐containing endosomes started to fuse with the lysosomes around 2 hours, and then FSL‐1 translocated into the cytosol from LAMP‐1⁺ endosomes. The artificial delivery of FSL‐1 into the cytosol of B6BMMs drastically enhanced the IL‐1β production‐inducing activity. FSL‐1 as well as the representative NLRP3 inflammasome activator nigericin induced the NLRP3/ASC speck, but FSL‐1 located in a compartment different from the NLRP3/ASC speck.     

Activation of interleukin 1β gene transcription by human cytomegalovirus: molecular mechanisms and relevance to periodontitis

In recent years, studies have demonstrated an association between human cytomegalovirus (HCMV) and destructive periodontal disease. It has been shown that reactivation of HCMV in periodontitis lesions may be related to progressing periodontal disease. Several possible mechanisms by which HCMV exerts periodontopathic potential have been previously proposed. These are reviewed and include the upregulation of bone resorptive cytokines such as interleukin‐1beta (IL‐1β) and tumor necrosis factor‐alpha (TNF‐α) by active HCMV infection at the periodontitis site. This review focuses on the molecular basis of IL‐1β gene activation by HCMV immediate early (IE) gene products. A novel hypothesis is also described whereby HCMV plays a significant role in the pathogenesis of periodontal disease by the ability of its IE proteins to strongly transactivate IL‐1β gene expression. More studies are needed to further explore this hypothesis and clarify the association between HCMV and periodontitis.     

Involvement of P2X(7) purinergic receptor and MEK1/2 in interleukin-8 up-regulation by LL-37 in human gingival fibroblasts

Montreekachon P, Chotjumlong P, Bolscher JGM, Nazmi K, Reutrakul V, Krisanaprakornkit S. Involvement of P2X ₇ purinergic receptor and MEK1/2 in interleukin‐8 up‐regulation by LL‐37 in human gingival fibroblasts. J Periodont Res 2011; 46: 327–337.© 2011 John Wiley & Sons A/S Background and Objective: The antimicrobial peptide LL‐37, derived from human neutrophils, can directly chemoattract leukocytes and up‐regulate the expression of several immune‐related genes in various cell types. In this study, we wanted to determine the immunoregulatory effect of LL‐37 on interleukin‐8 (IL‐8) expression in human gingival fibroblasts (HGFs) and to characterize intracellular signaling pathway(s) and receptor(s) involved in IL‐8 induction. Material and Methods: Cultured fibroblasts were treated with different concentrations of LL‐37 or interleukin‐1β (IL‐1β), as a positive control, for specific periods of time in the presence or absence of various inhibitors. RT‐PCR and real‐time PCR were conducted to analyze the expression of IL‐8 mRNA, and the IL‐8 levels in cell‐free culture media were measured using ELISAs. The MTT assay was performed to determine the cytotoxicity of LL‐37. Results: Nontoxic concentrations of LL‐37 (up to 10 μm ) and IL‐1β significantly up‐regulated the expression of IL‐8 mRNA in a dose‐dependent manner (p < 0.05). The IL‐8 protein levels were consistently significantly elevated in conditioned media of LL‐37‐treated HGFs (p < 0.05). IL‐8 up‐regulation by LL‐37 was completely abrogated by 20 μm U0126, consistent with transient phosphorylation of p44/42 MAP kinases. Moreover, pretreatment with Brilliant Blue G (a selective antagonist of the P2X₇ receptor) and the neutralizing antibody against P2X₇ blocked IL‐8 up‐regulation in a dose‐dependent manner, consistent with expression of the P2X₇ receptor in HGFs. Conclusion: These findings indicate that LL‐37 induces IL‐8 expression via the P2X₇ receptor and the MEK1/2‐dependent p44/42 MAP kinases in HGFs, suggesting both direct and indirect involvement of LL‐37 in neutrophil recruitment into an inflammatory site within diseased periodontal tissues.     

Sema3A/Nrp1信号轴在慢性牙周炎牙周组织中的表达及在牙周骨质破坏中的作用

目的:研究轴突导向蛋白3A(semaphorins3A,Sema3A)及其受体神经纤毛蛋白-1(neuropilin-1,Nrp1)在慢性牙周炎发生发展中的作用.方法:20只SD大鼠随机分为2组(n=10),实验组建立慢性牙周炎模型,对照组常规饲养,8周后处死.micro-CT测量大鼠釉牙骨质界到牙槽嵴顶距离(CEJ-ABC),免疫组化染色计算Sema3A/Nrp1阳性染色积分光密度,并进行相关性分析.门诊收集慢性牙周炎样本及正常样本各10例,进行免疫荧光和qRT-PCR检测.结果:实验组釉CEJ-ABC明显高于对照组(P＜0.05),Sema3A/Nrp表达强度明显低于对照组(P＜0.05),CEJ-ABC与Sema3A/NrP呈负相关(P＜0.05).人牙周炎样本Sema3A/Nrp1荧光强度及mRNA相对表达量均低于对照组(P＜0.05).结论:Sema3A/Nrp1表达量的减少参与了慢性牙周炎骨破坏进程.     

慢性牙周炎患者唾液中miR-146a的表达及其与龈沟炎症、MMP-8/TIMP-1水平的关系

目的： 探讨慢性牙周炎患者唾液中miR-146a的表达及其与龈沟炎症、基质金属蛋白酶8（MMP-8）、基质金属蛋白酶抑制剂1（TIMP-1）水平的关系。方法： 选择2015年3月—2017年1月间收治的慢性牙周炎患者68例作为慢性牙周炎组,同期在本院进行体检的健康志愿者50例作为正常对照组。检测2组研究对象唾液中miR-146a的表达量,龈沟液中炎症因子、MMP-8/TIMP-1的水平及牙周临床症状指标。采用SPSS24.软件中的Pearson检验评估慢性牙周炎患者唾液中miR-146a的表达量与病情严重程度的相关关系。结果： 慢性牙周炎患者唾液中miR-146a的表达量显著高于正常对照组（P<0.05）,龈沟液中炎症因子（IL-1β、IL-6、IL-35、TNF-α）水平、牙周临床症状指标（PD、AL、PLI、BI）以及龈沟液中MMP-8、TIMP-1的水平显著高于正常对照组（P<0.05）。Pearson检验发现,慢性牙周炎患者唾液中miR-146a表达量与龈沟炎症程度、牙周临床症状严重程度及MMP-8/TIMP-1水平呈正相关。结论： 慢性牙周炎患者唾液中miR-146a表达量异常增高,且与龈沟炎症程度、牙周损伤程度一致。     

The role of LFA-1 in osteoclast development induced by co-cultures of mouse bone marrow cells and MC3T3-G2/PA6 cells

Interactions between leukocyte function-associated antigen-1 (LFA-1) and intercellular adhesion molecule-1 (ICAM-1) influence the development of osteoclasts. However, little is known about how these adhesion molecules are involved in the process of osteoclast development. This study evaluated the role of LFA-1 and its ligands in osteoclast development and bone resorption. Co-cultures of bone marrow cells from LFA-1-deficient mice and MC3T3-G2/PA6 (PA6) cells were cultured in the presence of 1alpha,25(OH)2D3 and dexamethasone for 7 days. The number of TRAP-positive cells that were generated by bone marrow cells from LFA-1-deficient mice was smaller than that generated by bone marrow cells from wild-type mice. In addition, the bone-resorbing activity of osteoclast-like cells that were generated from LFA-1-deficient mice was lower than that generated by osteoclast-like cells from wild-type mice. Immunofluorescence flow cytometry showed that osteoclast stromal PA6 cells expressed the cell adhesion molecules, ICAM-1 and VCAM-1. When monoclonal antibodies to mice VCAM-1, CD11b or CD18 were added separately to the co-culture system, the number of TRAP-positive cells that were generated from LFA-1-deficient mice was 20-30% smaller than that generated from wild-type mice. The formation of TRAP-positive cells from both LFA-1 deficient and wild-type mice was especially inhibited by anti-CD18 antibody, in comparison to the addition of normal IgG serum. These results suggest that LFA-1 adhesion molecules play a role in osteoclast development by affecting adhesion between stromal cells and osteoclast progenitors before the occurrence of ODF-ODF receptor signaling. CD18 appears to be a key adhesion molecule in cell-to-cell contacts during the early stage of osteoclast development.     

Variational expression of functionally different macrophage markers (27E10, 25F9, RM3/1) in normal gingiva and inflammatory periodontal disease

Abstract Keratinocytes and macrophages share under immunologically activated conditions several surface proteins. We investigated immunohistochemically with monoclonal antibodies and the APAAP technique the expression pattern of 27E10 antigen (inflammatory macrophages), 25F9 antigen (resident macrophages) and RM3/1 antigen (intermediate macrophages in healing tissue) in 29 specimen biopsies of different stages of gingivitis and periodontitis. Macrophages of each subtype exhibited a different localization pattern depending on the stage of inflammation. Furthermore, suprabasal oral gingival epithelia showed a constant 27E10 expression, independent of the stage of inflammation. In contrast, all layers of the sulcus and pocket epithelia in gingivitis and periodontitis were strongly 27E10-positive, indicating immunological activation. 25F9 antigen was expressed on basal keratinocytes independent of the stage of inflammation, whereas RM3/1 was constantly negative on keratinocytes. The expression pattern of these functionally different macrophage markers on lesional macrophages and keratinocytes indicates varying differentiation and activation and suggests a participation of these cells in the local immune response in periodontal infection.     

Prostaglandin E2 potentiates interleukin-1β induced interleukin-6 production by human gingival fibroblasts

Abstract Increased levels of cytokines and prostanoids have been detected in inflamed gingival tissue and may play an important role in periodontal pathogenesis. Recent studies suggest that monocytic products, such as interleukin (IL)-1β, could stimulate IL-6 production by human gingival fibroblasts (HGF). In this context, the production of local cytokines and inflammatory mediators could regulate the secretory capacity of resident gingival fibroblasts. Therefore, the purpose of this study was to determine if PGE₂ induced by IL-1β could potentiate the IL-6 response by HGF. Utilizing an ELISA, it was determined that maximal IL-6 occurred when HGF were stimulated with 0.10–10 nM IL-1β. These concentrations of IL-1β also induced a small, but significant increase in PGE₂ production by HGF. Interestingly, the combination of ILγβ and PGE₂ induced a synergistic rise in IL-6 production by HGF. Moreover, inclusion of indomethacin caused a 20% reduction in IL-6 production and totally eliminated PGE₂ production. These findings provide additional rationale for the clinical use of NSAIDs in the management of periodontal disease due to their ability to attenuate production of both PGE₂, and IL-6. These results suggest the endogenous PGE₂ induced by IL-1β plays an important regulatory role in IL 6 production by HGF. Moreover, they support the concept that elevated PGE₂ induced during inflammation can regulate HGF secretory function.