Chronic temporomandibular disorder (TMD), characterized by muscle and/or joint pain of the masticatory system lasting greater than 3 months, affects up to 5% of the population.37,38 Although a number of psychobehavioral (somatic awareness, stress, and sleep13,16,24,32,33,36,39) stressors contribute to TMD, alterations in immune system has been proposed as a key biological mechanism in chronic pain and TMD.7,29,31 However, there is a current gap in the understanding of molecular basis contributing to alterations in immune system in individuals with chronic pain. The “Conserved Transcriptional Response to Adversity” (CTRA) profile, characterized by an upregulation of genes bearing response elements for the proinflammatory transcription factor nuclear factor-kappa B (NF-kB) and a downregulation of innate antiviral response genes bearing response elements for interferon-regulatory factors (IRF), is one molecular mechanism linking psychosocial and behavioral stress to negative health outcomes through alterations in the immune system.9,10
Using a promoter-based bioinformatics analyses of RNA from peripheral blood leukocytes, the CTRA profile can be assessed by quantifying the differential representation of “response elements,” or a sequence of DNA within a promoter permitting binding of transcription factors, in a set of differentially expressed genes empirically associated with an outcome of interest (TMD). Studies have repeatedly found a shift to a more proinflammatory and less Type I interferon-regulated bias within the transcriptome of circulating leukocytes, which is driven by extended periods and high levels of psychosocial, behavioral, and/or environmental stress, and consequent increases in activity of the sympathetic nervous system (cAMP response element-binding protein, CREB). This proinflammatory/anti-interferon transcriptome bias, in turn, can contribute to negative health outcomes related to stress.4,8,21,23,30
For the current study, we investigate whether the activity of 3 specific CTRA-related transcription factors, as indicated by expression of genes bearing response elements for NF-kB, IRF, and CREB, differed as a function of TMD status. This is important for 3 reasons. First, little is known about the relationship of CTRA to chronic pain states because only one study to date has tested whether the CTRA profile is present in patients with irritable bowel syndrome.19 Second, pain conditions such as TMD are highly sensitive to psychosocial stress, and it is possible that the relationship between stress and pain involves changes in proinflammatory gene regulation. Third, the CTRA profile may offer a unique window into the functioning of the cellular components of the immune system, which may not be readily observed with peripheral measurements of cytokines. The current study tested the hypothesis that individuals with TMD would exhibit elevated CTRA expression, which will be characterized by a differential expression of inflammation (upregulated) and antiviral (downregulated) genes bearing response elements for transcription factors, compared with controls. We also explored other aspect of the CTRA profile including differential expression of genes underlying the sympathetic nervous system (SNS)-responsive CREB signaling pathway because altered β-adrenergic signaling has been implicated in TMD.
Detailed information about participants and methods can be found in Supplementary Information (available at http://links.lww.com/PR9/A89).
After informed consent and health history review, all participants underwent a standardized clinical examination (DC-TMD) by a calibrated investigator for TMD.5,34 In participants with TMD (n = 19) and pain-free healthy controls (HCs, n = 17), a blood sample was collected for gene expression profiling (Fig. 1A). Information about sex, age, and history of smoking and alcohol consumption were collected and used as covariates (Table 1).
Table 1 -
|Mean (SD) or %
||TMD* (n = 19)
||HCs (n = 17)
| Age (Years)
| BMI (kg/m2)
| Sex (% female)
| Race (% NHW)‡
| Alcohol consumption (%yes)
*All participants with TMD meet criteria for mixed facial pain diagnosis (myofascial and arthralgia).
†Although the study did not restrict enrollment into the study for smoking, none of the enrolled participants report a history of smoking.
‡Significant group differences: P < 0.05.
TMD, temporomandibular disorder.
2.2. Gene expression profiling and analysis
After RNA extraction (Fig. 1B), genome-wide transcriptional profiling of RNA samples was performed (Fig. 1C). For the gene expression analysis, we first excluded genes with minimal level (or variation) in expression and then identified a set of genes with a >1.2-fold differential expression in TMD vs controls (Supplemental Tables, available at http://links.lww.com/PR9/A89). Due to the limited size of the sample and the hypothesis-testing nature of this study, we did not perform any exploratory/discovery analysis or statistical testing of individual gene transcripts, but rather this gene set served as input to the Transcription Element Listening System (TELiS) promoter-based bioinformatic analyses12 to explore associated transcription factor activity (Fig. 1D) and the CTRA profile (Fig. 1E). As in previous research, point estimates of differential expression served as input because previous research has found such lists to generate more reliable downstream bioinformatics results than gene lists derived from statistical p-/q-values.17,35,47
Sex (χ2 = 0.56, P = 0.46), history of alcohol use (χ2 = 2.5, P = 0.11), BMI (t = 0.3, P = 0.79), and age (t = 1.9, P = 0.07) were similar between the 2 groups. Differences were observed with race (χ2 = 4.5, P = 0.03) with a higher percentage of Whites in the TMD group. None of the participants reported a history of smoking (current or past), so this variable was not included as a covariate in the transcriptome analysis.
3.2. Transcription factor activity
Genome-wide transcriptional profiling by RNA sequencing identified 1173 gene transcripts showing >1.2-fold differential expression in TMD vs HCs (80 upregulated and 1093 downregulated). TELiS bioinformatics analysis of transcription control pathways (Fig. 2) indicated significant upregulation of genes with response elements for the proinflammatory transcription factor NF-kB (2.138 log2 TFBM ratio ± 0.694 standard error, P = 0.002) and significant downregulation of genes with IRF response elements (−1.135 ± 0.541, P = 0.037) in adults with TMD compared with HCs. Results also indicated a nonsignificant trend toward upregulation of the SNS-responsive CREB signaling pathway in patients with TMD (1.092 ± 0.624, P = 0.08).
In this small pilot study, we found preliminary evidence that patients with TMD show a CTRA-characteristic shift in gene regulation within the basal leukocyte transcriptome including greater proinflammatory activity, reduced antiviral activity, and greater adrenergic signaling. These 3 transcription control pathways were targeted a priori based on previous hypotheses linking pain to stress biology and inflammation and were quantified by asymmetrical distribution of binding sites for the targeted transcription factors in upregulated vs downregulated genes.4,8,21,23,30 Consistent with the classic CTRA profile, patients with TMD showed upregulation of genes bearing response elements for the proinflammatory transcription factor NF-kB, suggesting elevated levels of NF-kB activation and a propensity for proinflammatory responses. In addition, patients with TMD showed downregulation of genes bearing response elements for interferon regulatory factors (IRF), the primary inverse component of the CTRA profile.9,10 These results were observed after controlling for demographic and behavioral factors that might otherwise confound relationships between TMD and gene expression.
Evidence for proinflammatory shift in our current sample is supportive of a growing literature about the presence of low-grade inflammation (eg, basal inflammation and exaggerated inflammatory activity)1–3,40,41 in chronic pain. Although some of this literature is conflicting,1 low-grade inflammation can be driven by toll-like receptor-4 (TLR4) signaling,6,18 which induces the transcription factor NF-kB. Our preliminary data support the presence of low-grade inflammation, as reflected by NF-kB, in TMD. Also, limited evidence suggests that interferon signaling pathways can distinguish individuals with pain,27 and infections have been shown to be predictive of widespread pain.22 Finally, these CTRA-characteristic shifts may be related to altered β-adrenergic signaling due to chronic activation of the SNS by psychobehavioral stressors11,14,15,20,39,48. Recent studies have highlighted favorable impacts of β-adrenergic antagonist medication in TMD.42,43 Our preliminary data found directional evidence for CREB activation in TMD, which would be consistent with increased SNS activity in TMD.
Although findings provide initial support for the hypothesis of altered inflammatory and antiviral pathway activity in individuals with TMD, the study is preliminary and has several limitations. First, our current study was not large enough to support any hypothesis-free genome-wide discovery analysis. Consequently, any attempts to interpret the role of specific, differentially expressed genes will require a larger sample size. Second, our gene expression was measured in peripheral blood samples. Although the observed transcriptional shift occurred in total blood leukocytes, future studies should collect information about the leukocyte subset composition of peripheral blood (eg, by flow cytometry) and clarify whether other tissues associated with TMD also show similar transcriptional shifts.
Overall, a shift of transcriptional resources of immune cells in individuals with TMD to a proinflammatory state may contribute to somatic symptoms (pain and fatigue), although it is also possible that chronic pain causes SNS activation (through stressful experience). In addition to limiting healing of damaged tissue of the temporomandibular joint and muscle,26 this shift to a proinflammatory state of peripheral immune cells in individuals with TMD may result in a direct45,48 and/or indirect (eg, synovial fluid of the temporomandibular joint25) enhancement of neuronal sensory processing. Preclinical evidence suggests that elevated levels of peripheral inflammation lead to activation of glial cells in peripheral (eg, trigeminal ganglia) and central nervous systems,46 which can increase release of inflammatory mediators and amplify neuronal excitability, which in turn leads to an increased hypersensitivity to painful stimuli within these neuronal structures.28,44 Future research blocking the pathways identified here and/or manipulating pain or distress may help clarify the causal mechanisms involved. Despite these limitations, our results identify new candidate molecular pathways that can be targeted in future research examining pathogenesis of TMD pain.
G. Schulert has received consulting fees from Novartis and SOBI. The remaining authors have no conflicts of interest to declare.
Supported by the National Institute of Dental and Craniofacial Research of the National Institutes of Health (R00DE022368). The authors would like to acknowledge Duong Do for her technical assistance with RNA extraction and Victor Schneider for recruiting, screening, and collecting outcomes from the study participants.
Appendix A. Supplemental digital content
Supplemental digital content associated with this article can be found online at http://links.lww.com/PR9/A89.
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