Inflammatory modulators of central sensitization in fibromyalgia and temporomandibular disorders: a pilot study

Abstract

Background: This thesis is expanding on a previous pilot study that was entitled G protein-coupled Receptor Kinase 2 (GRK2) and Toll-like Receptors as Regulators of Central Sensitization in Fibromyalgia and Chronic Temporomandibular Disorders, by Drangsholt et. al. 2018 (designated here as “pilot study-A”). Fibromyalgia (FM) and chronic temporomandibular disorders (TMD) are both chronic pain syndromes that are challenging to manage, especially since they are not explained by tissue lesions. They both feature an increased activity of pain pathways in the central nervous system, i.e. central sensitization (CS). However, the molecular mechanisms underlying CS are unclear, which hampers the development of targeted treatments. Because of the strong evidence that implicates the immune system in modulating central pain pathways, pilot study-A examined the expression levels of GRK2, a negative modulator of the immune response, and the toll-like receptors’ (TLR) downstream cytokine interleukin 1-beta (IL1β), a positive modulator of the immune response. Importantly, both inflammatory modulators are found centrally in glial cells and in peripheral blood mononuclear cells (PBMCs). Our ability to measure these molecules in PBMCs is clinically important because we can’t measure them in glial cells of living human subjects. In pilot study-A, the researcher successfully enrolled 11 TMD patients, 9 fibromyalgia (FM) patients and 18 controls to examine the levels of GRK2 and IL1β after TLR stimulation in blood samples from these three groups. The hypotheses of that study were: 1) PBMCs from FM and chronic TMD patients have lower basal GRK2 levels. 2) These levels are negatively correlated with indices of CS. 3) the blood of FM and TMD patients displays exaggerated IL1β production after TLRs stimulation. 4) IL1β levels correlate positively with indices of CS. In a preliminary analysis of the recruited patients, pilot study-A exhibited a negative correlation between Nociceptive Flexion Reflex Threshold (NFRT) and IL1β level following TLR stimulation, which was in line with one of the hypotheses of the study. Aims: With this thesis I worked to accomplish two aims: 1) Complete the goal of pilot study-A of recruiting patients to reach 18 recruits for the TMD and FM groups and to analyze both the levels of IL1β after TLR stimulation and basal GRK2 levels in all groups at n = 18 and test the hypotheses of the previous study. 2) Make use of the full complement of blood samples at n = 18 for each of the three study groups to expand on pilot study-A by measuring other molecular candidates of the immune system that I hypothesize to modulate CS. Specifically, I wished to look at the protein levels of tumor necrosis factor- alpha (TNFα) which is downstream of TLR activation in both glial cells and PBMC. Hypotheses: The pro-inflammatory cytokines (IL1β and TNFα) are expected to be elevated in FM and TMD samples relative to controls post-TLR stimulation, while the anti-inflammatory GRK2 is expected to be lower in FM and TMD samples relative to controls. With respect to indices of CS, I expected to find a positive correlation with pro-inflammatory cytokine expression and a negative correlation with GRK2 levels. Methods: I successfully added 2 subjects to the TMD research group (total n = 13) and 8 subjects to the FM research group (total n = 17) to the cohort from pilot study-A. I assessed CS and production of TNFα and IL1β post-TLR stimulation in vitro. I also isolated PBMCs to acquire protein lysates which underwent a Western Blot assay to quantify GRK2 levels. Results: Basal GRK 2 levels are pending to be analyzed with a working antibody to GRK2. Statistically significant differences were identified between the study groups regarding IL1β production post-TLR2 stimulation. Namely, the FM group had a lower production of IL1β compared to controls (CON). TNFα measurements showed similar trends to IL1β in relation to study group designation but did not reach statistical significance. Both NFRT and pain area were negatively correlated with IL1β level post-TLR2 stimulation. Both TNFα and IL1β were positively correlated with the wind up ratio (WUR) measurement of CS at the dorsum of the foot. Conclusion: In line with the hypotheses of the study, post-TLR2 stimulation in PBMCs there is a negative correlation between IL1β expression and NFRT. There is also a positive correlation of both TNFα and IL1β expression post-TLR2 stimulation on one hand, with Dorsum of foot WUR on the other. These correlations suggest that cytokines can be used as biomarkers for CS. However, and counter to the proposed hypotheses, Both FM and TMD study groups have higher NFRT values. In addition, IL1β expression post-TLR2 stimulation in PBMCs is significantly lower in the FM group compared to the CON group, while the TMD group was intermediate. This suggests that there might be an impaired TLR2 responsiveness associated with chronic pain which could possibly be part of the mechanisms underlying CS in the spinal cord, considering the mechanistic similarities between PBMCs and glial cells in the contexts of pain and immunity.