Anti-inflammatory T-cell shift in neuropathic pain

Background: The classification of pain into nociceptive and neuropathic pain is based on characteristic symptoms and different pathophysiological mechanisms. In a recent investigation, we found a disrupted TH17/Treg balance in patients suffering from chronic unspecific low back pain (CLBP). These patients did not show any signs of neuropathy. There is evidence for a considerable impact of the immune system also in neuropathic pain. However, the role of the adaptive immune system is still unclear. In the present study, we investigated systemic T-cell subset responses and T-cell related cytokine profiles in patients with chronic neuropathic pain. Methods: We analyzed T-cell subsets, mRNA expression and T-cell-related cytokine profiles in 26 patients suffering from neuropathic pain in comparison to 26 healthy controls. Using multicolor flow cytometry (FACS), we quantified the number of T helper cells 1 (TH1), TH2, TH17 and regulatory T-cells (Tregs). Forkhead-Box-Protein 3 (FoxP3), Transforming growth factor-beta (TGF-beta) and RAR-related orphan receptor-gamma T (ROR-gamma T) mRNA expression was determined by quantitative real-time PCR (qPCR) and levels of pain-related cytokines were measured by Human Cytokine Multiplex Immunoassay (Macrophage inflammatory protein-1 alpha (MIP-1 alpha), Tumor necrosis factor-alpha (TNF-alpha), Interferon-gamma (IFN-gamma), Interleukin (IL) -4, IL-6, IL-10, IL-17, and IL-23). Results: We found a TH17/Treg imbalance with significantly increased anti-inflammatory Tregs and decreased pro-inflammatory TH17 cells in patients with neuropathic pain as compared to healthy controls. These results were confirmed on mRNA level: Treg-related FoxP3 and TGF-beta mRNA expression was elevated, whereas expression of TH17-related ROR gamma T was reduced. Cytokine analyses revealed only marginal changes. Conclusions: Our investigation revealed a clear shift of T-cell subsets towards anti-inflammation in patients with neuropathic pain. Interestingly, this is quite similar to our previous findings in CLBP patients, but even more pronounced. Therefore, it remains to be elucidated in future investigations whether the immune changes represent an underlying pathophysiological mechanism or an epiphenomenon induced by ongoing pain and stress

Differential expression of P2X7 receptor and IL-1 beta in nociceptive and neuropathic pain

Background: Despite substantial progress, pathogenesis and therapy of chronic pain are still the focus of many investigations. The ATP-gated P2X7 receptor (P2X7R) has previously been shown to play a central role in animal models of nociceptive inflammatory and neuropathic pain. Recently, we found that the adaptive immune system is involved in the pathophysiology of chronic nociceptive and neuropathic pain in humans. So far, data regarding P2X7R expression patterns on cells of the adaptive immune system of pain patients are scarce. We therefore analyzed the P2X7R expression on peripheral blood lymphocytes and monocytes, as well as serum levels of IL-1 beta in patients suffering from chronic nociceptive and neuropathic pain in comparison to healthy volunteers in order to identify individuals who might benefit from a P2X7R modulating therapy. Methods: P2X7R messenger RNA (mRNA) and protein expression were determined in patients with either chronic nociceptive low back pain (CLBP) or neuropathic pain (NeP), and in healthy volunteers by quantitative real-time PCR (qPCR) and by fluorescence-assisted cell-sorting (FACS), respectively. IL-1 beta serum levels were measured with a multiplex cytokine assay. Results: Compared to healthy volunteers, P2X7R mRNA (1.6-fold, p = 0.038) and protein levels were significantly increased on monocytes (NeP: 24.6 +/- 6.2, healthy volunteers: 17.0 +/- 5.4;p = 0.002) and lymphocytes (NeP: 21.8 +/- 6.5, healthy volunteers: 15.6 +/- 5.2;p = 0.009) of patients with NeP, but not in patients with CLBP. Similarly, IL-1 beta serum concentrations were significantly elevated only in NeP patients (1.4-fold, p = 0.04). Conclusions: A significant upregulation of P2X7R and increased IL-1 beta release seems to be a particular phenomenon in patients with NeP. P2X7R inhibitors may therefore represent a potential option for the treatment of this frequently intractable type of pain

Disrupted TH17/Treg balance in patients with chronic low back pain.

Chronic low back pain (CLBP) is a leading cause of disability and costs in health care systems worldwide. Despite extensive research, the exact pathogenesis of CLBP, particularly the individual risk of chronification remains unclear. To investigate a possible role of the adaptive immune system in the pathophysiology of CLBP, we analyzed T cell related cytokine profiles, T cell related mRNA expression patterns and the distribution of T cell subsets in 37 patients suffering from nonspecific CLBP before and after multimodal therapy in comparison to 25 healthy controls. Serum patterns of marker cytokines were analyzed by Luminex technology, mRNA expression of cytokines and specific transcription factors was measured by real-time PCR, and distribution of TH1-, TH2-, TH17- and regulatory T cell (Tregs) subsets was determined by multicolor flow cytometry. We found that CLBP patients exhibit an increased number of anti-inflammatory Tregs, while pro-inflammatory TH17 cells are decreased, resulting in an altered TH17/Treg ratio. Accordingly, FoxP3 and TGF-β-mRNA expression was elevated, while expression of IL-23 was reduced. Serum cytokine analyses proved to be unsuitable to monitor the adaptive immune response in CLBP patients. We further show that even after successful therapy with lasting reduction of pain, T cell subset patterns remained altered after a follow-up period of 6 months. These findings suggest an involvement of TH17/Treg cells in the pathogenesis of CLBP and emphasize the importance of these cells in the crosstalk of pain and immune response.German Clinical Trial Register: Registration Trial DRKS00005954

Soluble intercellular adhesion molecule-1: a potential biomarker for pain intensity in chronic pain patients

Aim: Pain therapy is strongly guided by patients’ self-reporting. However, when self-reporting is not an option, pain assessment becomes a challenge and may lead to undertreatment of painful conditions. Pain is a complex and multifactorial phenomenon. Recent work has connected pain pathophysiology also with the inflammatory system. We therefore hypothesized that pain intensity could be predicted by cytokine-levels. Patients &amp; methods: In this observational, single-center study, we investigated 30 serum cytokines to predict pain intensity in a screening/follow-up set of 95 chronic pain patients and controls. We then prospectively validated soluble intercellular adhesion molecule-1 (sICAM-1)'s discriminatory capability (n = 21). Results &amp; conclusion: sICAM-1 was significantly associated with patient-reported pain intensity and yielded differential serum levels in patients of varying degrees of pain intensity. Changes in pain ratings over time correlated with changes in sICAM-1 levels. Our findings suggest the possibility of a clinical use of sICAM-1 as a potential biomarker for pain intensity. </jats:p

Expression levels of T cell related cytokine mRNA measured by qPCR.

<p>TNF-α, IL-4 and IL-10 were neither detectable in CD4⁺ T cells of CLBP patients nor in healthy controls. The expression of IFN-γ did not exhibit significant differences in CLBP patients as compared to healthy controls (IFN-γ: 4.19±3.54 in CLBP patients vs. 3.60±2.20 in healthy controls, n.s.; Fig. 2A) whereas IL-23 expression of in CD4⁺ T cell samples of CLBP patients was found to be significantly decreased (4.88±2.44 in CLBP patients vs. 7.73±3.77 in healthy controls, p = 0.006; Fig. 2B).</p

Gating strategy for the detection of Tregs.

<p>PBMCs extracellular stained with PerCP labeled anti-human CD4-antibody, PE labeled anti CD25-antibody, Brilliant Violet (BV570) labeled anti CD127-antibody and intracellular stained with Alexa Fluor (AF488) labeled anti-human FoxP3-antibody. Lymphocyte population was gated from PBMCs according to forward scatter (FSC) characteristics and side scatter (SSC) characteristics (left). Gated lymphocytes were then separated in CD4⁺CD25^high cells/T cells (middle) and CD4⁺CD25^highCD127^lowFOXP3⁺ cells/CD4⁺T cells (right, named Treg). Upper row represents the result of a healthy control with less CD4⁺CD25^high T cells (3.28%) and less CD4⁺CD25^highCD127^lowFoxP3⁺ T cells (1.94%) compared to a patient with CLBP (lower row, 5.74% and 3.11%).</p

Expression levels of T cell subset related mRNA measured by qPCR.

<p>TGF-β and FoxP3 mRNA expression, specific for Tregs, was significantly higher in patients with CLBP than in healthy controls (TGF-β: 0.21±0.07 in CLBP patients vs. 0.14±0.05 in healthy controls, p = 0.014; Fig. 3A), (FoxP3: 0.21±0.14 in CLBP patients vs. 0.14±0.06 in healthy controls, p = 0.009; Fig. 3B). TH17 specific expression of IL-17 was neither detectable in CD4⁺ samples of CLBP patients nor in healthy controls. Expression levels of RORγT did not differ in CLBP patients and healthy controls (RORγT: 0.028±0.02 in CLBP patients vs. 0.025±0.01 in healthy controls, p = n.s.; Fig. 3C).</p

RT-PCR Assay Characteristics and Primer Sequences.

<p>RT-PCR Assay Characteristics and Primer Sequences.</p