Background
Neuropathic pain represents a major problem in clinical practice. In contrast to nociceptive pain, which is caused by damage or potential damage to tissue, neuropathic pain occurs due to a lesion or disease of the peripheral or central nervous system. It is characterized by burning and lancinating pain sensations and further somatosensory disturbances like hypo- and hypersensitivity. Very often, neuropathic pain is more difficult to treat and more refractory to common analgesics, including non-steroidal anti-inflammatory drugs and opioids, than nociceptive pain [
1,
2]. Despite extensive research, the underlying pathophysiological mechanisms of neuropathic pain are still not fully understood. In recent years, increasing evidence indicates a pivotal role of the immune system in neuropathic pain [
3,
4]. The majority of previously published data link pain syndromes with higher levels of pro-inflammatory cytokines. Due to these findings, attempts were made in numerous studies to reduce neuropathic pain by blocking pro-inflammatory or enhancing anti-inflammatory immune cells and cytokines [
3]. For example, in animal models of neuropathy, tumor necrosis factor-α (TNF-α), Interleukin (IL) -6, IL-17 and Makrophage inflammatory protein1-α (MIP1-α) blockers reduced pain hypersensitivity [
5-
9]. Comparable results were obtained by increasing the anti-inflammatory cytokines IL-4, IL-10 or Transforming growth factor-β (TGF-β) [
10-
12]. Nevertheless, despite those promising experimental findings, there are no pharmacological agents available for the specific immunological therapy of neuropathic pain until now.
Cytokines and neutrophils are important during the early stages of acute pain, whereas T-lymphocytes seem to play a central role in chronic neuropathic pain [
13]. Regarding T-cells as key players of the adaptive immune system, a TH1/TH2 imbalance has already been shown in patients with complex regional pain syndrome (CRPS) and chronic pelvic pain [
14,
15]. In recent years, TH1/TH2 dichotomy has been extended by the identification of two other CD4
+ T-cell lineages: TH17 and regulatory T-cells (Tregs) [
16]. TH17 cells appear to be the key effector T-cells in a variety of human autoimmune diseases and Tregs play a vital role in controlling adaptive immune responses. In neuropathy, TH17 has been linked to increased pain sensitivity and destructive effects promoting persistent pain [
16], while Tregs were found to be mainly involved in the endogenous recovery [
17]. Recently published data showed an increased proportion of Tregs in patients with postherpetic neuralgia [
18]. Assuming a beneficial role for an anti-inflammatory T-cell shift, a phase one trial was carried out with an anti-CD28 antibody, preferentially expanding TH2 cells and Tregs in human volunteers. Despite promising results in several animal models, the clinical trial had to be cancelled because of severe side effects [
19]. In patients with neuropathic pain, the role of T-cell subsets has not yet been investigated.
In a recent study, we found that patients suffering from nociceptive, non-specific chronic low back pain (CLBP), without any signs of neuropathic pain components, display a clear disruption of the TH17/Treg balance as compared to healthy volunteers [
20]. Since clinical and pathophysiological mechanisms differ considerably between nociceptive and neuropathic pain, we aimed to detail changes of pro- and anti-inflammatory T-cell subsets and the respective relative mRNA expression, as well as pain-related cytokine levels in patients with chronic neuropathic pain in comparison to pain free controls. While the cytokine measurement did not reveal any relevant results, we found a distinct anti-inflammatory shift of the T-cell subsets and their respective mRNA expression.
Discussion
Neuropathic pain is a severe and frequent condition which affects up to 18% of the population [
29]. The pathophysiological mechanisms leading to chronification of neuropathic pain are a major focus of interest, but are not yet completely elucidated. Recent data indicate a critical involvement of the innate and adaptive immune system in the pathophysiology of chronification. Several types of immune cells have been implicated in the pathogenesis of neuropathic pain [
3]. The innate immune system has been shown to be important during the early stages of acute pain, represented particularly by neutrophils [
13]. Regarding chronification, T-lymphocytes, as key players of the adaptive immune system, seem to be of major importance [
30,
31]. Traditionally, it has been suggested that neuropathic pain is associated with a pro-inflammatory immune response. Therefore, mainly anti-inflammatory treatments targeting cytokines and immune cells have been evaluated in several animal models of neuropathic pain [
3]. In a recent study, neuropathic pain induced by experimental autoimmune neuritis was successfully attenuated by expanding Tregs [
17]. In humans, the first Treg-expanding tests were stopped because of life-threatening side effects [
19]. Nevertheless, the modulation of T-cells is still the focus of intense research [
32].
In the present study, we analyzed the pain-related cytokines MIP-1α, TNF-α, IFN-γ, IL-4, IL-6, IL-10, IL-17 and IL-23 in the peripheral blood of 26 patients with neuropathic pain and compared the results with those of 26 healthy controls. We found that the serum levels of IL-4, TNF-α and IFN-γ were below the detectable limit and no differences were found regarding IL-6, IL-10 and IL-17. Only the pro-inflammatory cytokines MIP-1α and IL-23 were significantly higher in neuropathic pain. It has to be noted though, that except for IL-23, values of all cytokines measured were below the lowest concentration of an analyte in a sample that can be reliably detected. This shortcoming of serum cytokine measurements in pain syndromes has also been described by other authors [
33]. Taken together, our results indicated that serum cytokine levels alone are not sufficient to monitor the adaptive immune response in neuropathic pain and led us to analyze the cellular compartment.
By routine laboratory studies regarding the differential leucocyte count, we found only an unchanged number of neutrophil granulocytes and a slightly reduced number of lymphocytes in the peripheral blood of patients with neuropathic pain. Using multicolor flow cytometry, we subsequently quantified the numbers of TH1, TH2, TH17 and Treg cells in the peripheral blood of our patients. Contrary to our initial assumption, we found clear indications for an anti-inflammatory T-cell shift: Pro-inflammatory TH17 cells were significantly decreased, whereas anti-inflammatory Tregs were significantly increased. Consequently, the corresponding TH17/Treg ratio was distinctly shifted towards an anti-inflammatory immune response. To confirm the quantitative observations of the TH17 and Treg frequency, we also determined the relative mRNA expression of the TH17 cell-specific transcription factor RORγT, as well as TGF-β and the Treg-specific transcription factor FoxP3 by quantitative real-time PCR. A diminished RORγT mRNA expression was in line with the reduced number of TH17 cells, while a notably elevated FoxP3 and TGF-β mRNA expression confirmed the increased Treg frequency. Regarding patient subgroups, we found no differences in respect of the anti-inflammatory T-cell shift and mRNA expressions between patients suffering from orofacial pain, postherpetic neuralgia and other types of peripheral neuropathies (Table
3). The question arises whether the observed changes are of clinical relevance, particularly in view of the overall low portion of the specific T-cell subsets. Furthermore, it would be interesting and relevant to investigate whether the immune changes can also be found in the affected tissue.
Our results are, at first glance, unexpected since the majority of previously published data describes the association between pain and ‘immune activation’ based on investigations of TH1 and TH2 cells, as well as cytokines. This previous TH1/TH2 paradigm has, however, been revised and updated with the discovery of TH17 cells and the more specific detection of Tregs. Our findings, together with recently published data regarding various T-cell subsets, point to a strong association between pain and ‘immune suppression’. Interestingly, the T-cell response in the present study is comparable with our recent findings in patients with CLBP, who also presented with high pain and stress levels, but had no signs of neuropathic pain [
20]. There is a general consensus that neuropathic and nociceptive pain are distinct entities, although basic research clearly reveals a huge overlap of underlying pathophysiological mechanisms, including neurotransmitters and cytokines [
2]. Our results show for the first time that in both neuropathic and nociceptive pain the adaptive immune system is altered in the same anti-inflammatory way. The context of chronic stress and immune suppression has been described for many years, although not extensively with regard to TH17 cells and Tregs [
34]. An anti-inflammatory T-cell shift has been found in patients with chronic mild depression or chronic fatigue syndrome [
35-
37], and both disorders are frequently associated with all types of chronic pain.
Concerning the cellular mechanisms, T-cell differentiation mainly depends on the cytokine milieu of the microenvironment, but other pathways have also been shown to be involved. For example, the hypothalamic-pituitary-adrenal axis mediates immune regulation through binding of stress hormones like adrenocorticotropic hormone or cortisol to their cognate receptors at the surface of T-cells. Furthermore, the sympathetic nervous system is known to induce immune dysregulation via adrenaline and noradrenaline [
38]. These processes in turn play an important role in negative emotional states, such as stress and depression. Our patients with neuropathic pain also suffered from stress and psychological burden as revealed by the enhanced KAB values. We therefore hypothesize that the altered immune responses in both of our studies might reflect a particular chronic pain-related stress reaction
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
All authors read and approved the final manuscript. BL performed all experiments and wrote the manuscript. BL and BRA recruited the patients and prepared the blood samples. BL, BRA, JH, SK and SCA analyzed the data. BL, BRA and SCA designed the experiments.