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, 96 (3), 285-291

Estrogen-Induced Monocytic Response Correlates With TMD Pain: A Case Control Study

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Estrogen-Induced Monocytic Response Correlates With TMD Pain: A Case Control Study

M C Ribeiro-Dasilva et al. J Dent Res.

Abstract

Temporomandibular disorders (TMD) are a set of conditions characterized by pain and dysfunction in the temporomandibular joint and muscles of mastication. These pain conditions are associated with considerable morbidity, societal costs, and reduced quality of life. The prevalence varies between 4% and 10%, with females at higher risk, and a higher prevalence occurs during reproductive years. The increased prevalence of TMD in females and low prevalence in childhood reinforce that sex hormones, like estrogen, play an important, complex role in the pathophysiology of these disorders. The goal of this study was to determine whether women with TMD exhibit a monocytic hyperinflammatory response compared with control women, and to examine associations of monocytic inflammatory responses with clinical pain. Eighteen women, aged 18 to 35 y, were seen during their follicular menstrual phase. A blood sample was collected, a clinical questionnaire about pain history was administered, and a Research Diagnostic Criteria (RDC) exam was performed. Extracted monocytes were stimulated with the toll-like receptor (TLR)-4 ligand, lipopolysaccharide (LPS), in the presence and absence of estrogen, and the levels of IL6 expression evaluated. Women with TMD showed a systemic hyperinflammatory phenotype, manifested by an increased monocytic release of cytokines after an inflammatory insult, and this was further increased by estrogen. In addition, monocytes from participants who self-reported more pain on the VAS scale produced higher levels of IL6 compared with those from participants who self-reported lower pain sensitivity. These data suggest that an estrogen-induced hyperinflammatory phenotype in women with TMD may at least in part contribute to heightened clinical pain, perhaps via central sensitization.

Keywords: cytokines; estrogen receptors; inflammation; lipopolysaccharide; monocytes; toll-like receptors.

Conflict of interest statement

The authors declare no potential conflicts of interest with respect to the authorship and/or publication of this article.

Figures

Figure 1.
Figure 1.
Elevated IL6 in temporomandibular disorder (TMD)-associated monocytes at rest. Monocytes (1 × 104) isolated from the peripheral blood of participants with (n = 9) and without (n = 9) TMD were (A, B) unstimulated or (C–E) stimulated with 500 ng/mL of ultra-pure LPS from Escherichia coli. The 24-h conditioned supernatants were assessed for IL6 expression by ELISA. (A, C) Data are presented as pg/ml. Bars represent mean and SEM. Black, TMD; white, control (CON). *P = 0.0286; ^P = 0.002; independent Student’s t test. (B, D) Data are presented as fold change over control: (B) 8.2 (1.03) and (D) 12.6 (3.38). (E) Data are presented as fold change of mRNA over unstimulated control. Bars represent mean and SEM. Black, TMD; gray, control. *P = 0.0241; Student’s t test.
Figure 2.
Figure 2.
Estrogen enhances TMD-associated monocytic TLR4-hyperresponsiveness. Monocytes (1 × 104) isolated from the peripheral blood of participants with (n = 9) and without (n = 9) TMD, were stimulated with 500 ng/mL of ultra-pure LPS from Escherichia coli in the presence or absence of 10−9 M 17β-estradiol (E2). Conditioned supernatants were then examined for IL6 expression by ELISA and qPCR after 24 h and 6 h, respectively. (A) Data are presented as pg/mL. Bars represent mean and SEM. Black, TMD; gray, control; cross-hatched, E2. *P = 0.04 (TMD); P = 0.02 (CON) LPS vs. E2; Student’s t test. (B) Data are presented as fold change over control. Mean fold change is 14.2 (4.31). (A, B) Bars represent mean and SEM. Black, TMD; gray, control; cross-hatched, E2. (C) Data are presented as fold change of mRNA for treated over unstimulated using box-and-whisker plot. Black, TMD; gray, control; cross-hatched, E2. *P < 0.0001, LPS vs. E2; ^P < 0.0001 TMD vs. CON. One-way ANOVA with Bonferroni’s multiple corrections.
Figure 3.
Figure 3.
Estrogen receptor (ER) use in TMD-associated monocytic TLR4-hyperresponsiveness. Monocytes (1×104) isolated from the peripheral blood of participants with (n = 9) and without (n = 9) TMD were (A) unstimulated or (B) stimulated with 500 ng/mL of ultra-pure LPS from Escherichia coli in the presence and absence of 10−9 M 17β-estradiol (E2) or 10 nM of inhibitors to ERa (MMP), ERβ (PTHP) and ERa+ ERβ inhibitor, GPR30 agonist (ICI) (see Methods for inhibitors). (A) The relative surface expression of ERa and ERβ was determined by flow cytometry. Data are presented as the mean fluorescence intensity, which is directly proportional to the number of receptors expressed on the cell surface of a given cell. Data are presented as box-and-whisker plots. Black, TMD; gray, control; *P < 0.05. TMD vs. CON for ERβ. One-way ANOVA with Bonferroni’s multiple corrections. (B) Data are presented as fold change over expression following TLR4-stimulation using box-and-whisker plots. Black, TMD; gray, control. *P < 0.05. TMD vs. CON E2. ^P < 0.05 E2 vs. all other stimulations within an experimental group. One-way ANOVA with Bonferroni’s multiple corrections.
Figure 4.
Figure 4.
TMD-associated monocytic TLR4-hyper-responsiveness correlates with self-reported pain. IL6 levels from monocytic cultures stimulated with 500 ng/mL of ultra-pure LPS from Escherichia coli in the presence and absence of 10−9 M 17β-estradiol (E2) were log transformed to determine relative expression levels. (A) VAS scores from TMD participants were stratified as low self-reported pain (VAS <4.5; black circles) and high self-reported pain (VAS >4.5; white circles). Data are presented as mean and SEM of relative expression of IL6 as well as relative expression for each participant (each dot represents a single participant). *P < 0.0137. Student’s t test. (B) Within the TMD cohort, VAS scores and relative expression levels of IL6 following TLR4 stimulation were correlated using Pearson’s correlation (P = 0.0328). (C) Within the TMD cohort, VAS scores and relative expression levels of IL6 following TLR4 and E2 stimulation were correlated using Pearson’s correlation (P = 0.0338).

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