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. 2015 Aug 4;10(8):e0134608.
doi: 10.1371/journal.pone.0134608. eCollection 2015.

Increased Eotaxin and MCP-1 Levels in Serum From Individuals With Periodontitis and in Human Gingival Fibroblasts Exposed to Pro-Inflammatory Cytokines

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Free PMC article

Increased Eotaxin and MCP-1 Levels in Serum From Individuals With Periodontitis and in Human Gingival Fibroblasts Exposed to Pro-Inflammatory Cytokines

Elisabeth A Boström et al. PLoS One. .
Free PMC article

Abstract

Periodontitis is a chronic inflammatory disease of tooth supporting tissues resulting in periodontal tissue destruction, which may ultimately lead to tooth loss. The disease is characterized by continuous leukocyte infiltration, likely mediated by local chemokine production but the pathogenic mechanisms are not fully elucidated. There are no reliable serologic biomarkers for the diagnosis of periodontitis, which is today based solely on the degree of local tissue destruction, and there is no available biological treatment tool. Prompted by the increasing interest in periodontitis and systemic inflammatory mediators we mapped serum cytokine and chemokine levels from periodontitis subjects and healthy controls. We used multivariate partial least squares (PLS) modeling and identified monocyte chemoattractant protein-1 (MCP-1) and eotaxin as clearly associated with periodontitis along with C-reactive protein (CRP), years of smoking and age, whereas the number of remaining teeth was associated with being healthy. Moreover, body mass index correlated significantly with serum MCP-1 and CRP, but not with eotaxin. We detected higher MCP-1 protein levels in inflamed gingival connective tissue compared to healthy but the eotaxin levels were undetectable. Primary human gingival fibroblasts displayed strongly increased expression of MCP-1 and eotaxin mRNA and protein when challenged with tumor necrosis factor-α (TNF-α and interleukin-1β (IL-1β), key mediators of periodontal inflammation. We also demonstrated that the upregulated chemokine expression was dependent on the NF-κΒ pathway. In summary, we identify higher levels of CRP, eotaxin and MCP-1 in serum of periodontitis patients. This, together with our finding that both CRP and MCP-1 correlates with BMI points towards an increased systemic inflammatory load in patients with periodontitis and high BMI. Targeting eotaxin and MCP-1 in periodontitis may result in reduced leukocyte infiltration and inflammation in periodontitis and maybe prevent tooth loss.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Multivariate PLS modeling results in a model with clustered periodontitis cases from periodontally healthy and significantly enhanced levels of inflammatory mediators in serum from periodontally diseased.
(A) PLS scatter plot illustrating separation of periodontally diseased from healthy subjects by their serum concentrations of inflammatory markers and subject characteristics (see labels in B). The scores t1 and t2 are the new PCS created variables summarizing the x variables; (B) PLS column loading plot showing PLS correlation coefficients with 95% CI for the variables in the model, i.e. the model behind the separation in A. Bars for which the 95% whisker does not pass zero are statistically significant. Serum levels of eotaxin (pg/ml) (C), MCP-1 (pg/ml) (D), and CRP (mg/l) (E) in individuals with periodontitis and in periodontally healthy. Data are presented as arithmetic means (eotaxin and MCP-1) or geometric mean (CRP) with 95% confidence intervals. To circumvent confounding by BMI, data for MCP-1 and CRP are presented in BMI strata, i.e. BMI<25, BMI 25–30, BMI>30.
Fig 2
Fig 2. Presence of MCP-1 in inflamed gingival connective tissue.
MCP-1 protein concentration measured by ELISA in gingival tissue homogenates of periodontitis (n = 3) and periodontally healthy (n = 3) subjects. Data expressed as means ± SEM.
Fig 3
Fig 3. Time- and dose-dependent increase of eotaxin and MCP-1 protein expression in gingival fibroblasts stimulated by TNF-α and IL-1β.
(A and C) TNF-α (50 ng/ml) stimulates eotaxin and MCP-1 protein expression, and (B and D) IL-1β (100 pg/ml) stimulates eotaxin and MCP-1 protein expression a time-dependent manner. (E and G) TNF-α stimulates eotaxin and MCP-1 protein expression, and (F and H) IL-1β stimulates eotaxin and MCP-1 protein expression in a dose-dependent manner.
Fig 4
Fig 4. Time- and dose-dependent increase of eotaxin and MCP-1 mRNA expression in gingival fibroblasts by TNF-α and IL-1β.
(A and C) TNF-α (50 ng/ml) increases eotaxin and MCP-1 mRNA expression, and (B and D) IL-1β (100 pg/ml) increases eotaxin and MCP-1 mRNA expression in a time-dependent manner. Analysis performed at 1h and 3h. (E and G) TNF-α increases eotaxin and MCP-1 mRNA expression, and (F and H) IL-1β increases eotaxin and MCP-1 mRNA expression in a dose-dependent manner. Analysis performed at 24h. Data expressed as means ± SEM. Data represent three individual experiments.
Fig 5
Fig 5. The canonical pathway of NF-κB activation is required for TNF-α and IL-1β stimulated eotaxin and MCP-1 gene expression in gingival fibroblasts.
(A) Time-dependent decrease of IκBα protein in gingival fibroblasts exposed to TNF-α (50 ng/ml) and IL-1β (100 pg/ml) cultured for 5, 10, 15 and 30 min. The pharmacological inhibitors (B) BMS-345541 (6 μM) and (C) IKK-2 inhibitor V (10 μM) (D) Celastrol (0.1 μM) inhibit the expression of eotaxin and MCP-1 mRNA in gingival fibroblasts exposed to TNF-α (10 ng/ml) and IL-1β (30 pg/ml). Analysis performed at 48 h. Data expressed as means ± SEM.
Fig 6
Fig 6. Schematic figure of the canonical versus non-canonical NF-κB signaling pathways, and downstream targets of inhibitors.

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Grant support

This study was supported by funds from the Swedish Research Council (http://www.vr.se/) (EAB, UL), Stockholm County Council/ ALF Project (http://www.sll.se/) (EAB), Karolinska Institutet Funds (http://www.ki.se) (EAB), the Swedish Patent Revenue Fund (EAB), Ollie and Elof Ericssons Stiftelse (http://www.oestiftelse.se/) (EAB), Tore Nilsson Stiftelse (http://www.torenilsonsstiftelse.nu/) (EAB), Faculty of Medicine, Umeå University (http://www.medfak.umu.se/) (PL), County Council of Västerbotten (https://www.vll.se) (PL, UL), the Swedish Rheumatism Association (https://reumatikerforbundet.org/) (PL, UL), Swedish Dental Society (http://www.tandlakarforbundet.se/Om-oss/Svenska-Tandlakare-Sallskapet/) (PL, EK, MM), COMBINE, the ALF/LUA Research Grant in Gothenburg, the Lundberg Foundation, and the Royal 80 Year Fund of King Gustav V (UL). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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