Matthias Ebbinghaus

A de novo gain-of-function mutation in SCN11A causes loss of pain perception

The sensation of pain protects the body from serious injury. Using exome sequencing, we identified a specific de novo missense mutation in SCN11A in individuals with the congenital inability to experience pain who suffer from recurrent tissue damage and severe mutilations. Heterozygous knock-in mice carrying the orthologous mutation showed reduced sensitivity to pain and self-inflicted tissue lesions, recapitulating aspects of the human phenotype. SCN11A encodes Nav1.9, a voltage-gated sodium ion channel that is primarily expressed in nociceptors, which function as key relay stations for the electrical transmission of pain signals from the periphery to the central nervous system. Mutant Nav1.9 channels displayed excessive activity at resting voltages, causing sustained depolarization of nociceptors, impaired generation of action potentials and aberrant synaptic transmission. The gain-of-function mechanism that underlies this channelopathy suggests an alternative way to modulate pain perception.

Interleukin-17 sensitizes joint nociceptors to mechanical stimuli and contributes to arthritic pain through neuronal interleukin-17 receptors in rodents

OBJECTIVE Interleukin-17 (IL-17) is considered a proinflammatory cytokine, but whether neuronal IL-17 receptors contribute to the generation of arthritic pain is unknown. This study was undertaken to explore whether IL-17A acts on neurons, whether it sensitizes joint nociceptors, and whether neutralization of IL-17 is antinociceptive. METHODS We recorded action potentials from rat joint nociceptors after intraarticular injection of IL-17A. We studied the expression of the IL-17A receptor in the rat dorsal root ganglia (DRG), explored the effect of IL-17A on signaling pathways in cultured rat DRG neurons, and using patch clamp recordings, monitored changes of excitability by IL-17A. We tested whether an antibody to IL-17 influences pain behaviors in mice with antigen-induced arthritis (AIA). RESULTS A single injection of IL-17A into the rat knee joint elicited a slowly developing and long-lasting sensitization of nociceptive C fibers of the joint to mechanical stimuli, which was not attenuated by neutralizing tumor necrosis factor α or IL-6. The IL-17A receptor was visualized in most rat DRG neurons, the cell bodies of primary sensory neurons. In isolated and cultured rat DRG neurons, IL-17A caused rapid phosphorylation of protein kinase B and ERK, and it rapidly enhanced excitability. In mice with unilateral AIA in the knee, an antibody against IL-17 improved the guarding score and reduced secondary mechanical hyperalgesia at the ipsilateral paw. CONCLUSION Our findings indicate that IL-17A has the potential to act as a pain mediator by targeting IL-17 receptors in nociceptive neurons, and these receptors are particularly involved in inflammation-evoked mechanical hyperalgesia.

The anti-inflammatory effects of sympathectomy in murine antigen-induced arthritis are associated with a reduction of Th1 and Th17 responses

Background Both facilitatory and inhibitory effects of the sympathetic nervous system (SNS) on experimental arthritis have been reported. It is unknown whether such bidirectional effects are inherent to all experimental arthritis models and/or whether critical time windows exist for influences of the SNS on inflammation. Objectives To assess the effect of sympathectomy at different time points on the course and severity of murine antigen-induced arthritis (AIA). Methods AIA was induced in mice. Chemical sympathectomy with 6-hydroxydopamine was carried out either neonatally, in the immunisation phase, or immediately before AIA elicitation, or during the chronic phase. In sympathectomised and non-sympathectomised AIA mice the inflammatory process (joint swelling, histopathology of inflammation and joint destruction), pain-related behaviour and cellular and humoral immune responses were analysed. Results Sympathectomy during AIA induction or neonatal sympathectomy significantly reduced the severity of acute AIA. Neither sympathectomy in the immunisation phase nor in the chronic phase influenced AIA. Flare-up reactions were reduced by sympathectomy just before flare-up or during the initial acute AIA stage. Sympathectomised AIA mice showed less hyperalgesia. Sympathectomy significantly reduced interleukin (IL) 2, IL-17 and transforming growth factor β in supernatants from lymph nodes and/or spleen cells and antigen-specific Th1-associated IgG2a in serum; IgG1 titres were unaffected. The ß blocker, propranolol, and the norepinephrine reuptake inhibitor bupropion produced similar anti-inflammatory effects, whereas the ß-adrenergic agonist isoproterenol increased AIA severity in neonatally sympathectomised mice. Conclusions Sympathetic activity mainly increases the severity of acute episodes of immune-mediated arthritis. Therapeutic reduction of sympathetic activity at acute stages attenuates inflammation, hyperalgesia and proinflammatory immune parameters.

The role of interleukin‐1β in arthritic pain: Main involvement in thermal, but not mechanical, hyperalgesia in rat antigen‐induced arthritis

OBJECTIVE Interleukin-1β (IL-1β) is considered a pronociceptive cytokine, but its role in the generation of arthritic pain is unknown. The aim of this study was to investigate the role of IL-1β in arthritic pain and to explore the antinociceptive potential of the IL-1 receptor type I (IL-1RI) antagonist anakinra. METHODS Antigen-induced arthritis (AIA) was induced in rats. Expression of IL-1RI in the dorsal root ganglia (DRGs) was determined, and the effects of anakinra on inflammation, pain-related behavior, and receptor expression were assessed. In cultured DRG neurons, the effect of IL-1β on the expression of the transient receptor potential vanilloid 1 (TRPV-1) ion channel was examined. Recordings of action potentials from joint nociceptors were made after intraarticular injection of IL-1β into the rat knee joints. RESULTS AIA generated pronounced and persistent mechanical and thermal hyperalgesia, and IL-1RI expression in the lumbar DRGs was significantly up-regulated. Treatment with anakinra did not significantly reduce the severity of arthritis or mechanical hyperalgesia, but did result in a pronounced reduction in thermal hyperalgesia. In cultured DRG neurons, IL-1β up-regulated the expression of TRPV-1, a major transduction molecule involved in thermal hyperalgesia. During AIA, anakinra treatment down-regulated the expression of TRPV-1, consistent with the pronounced reduction in thermal hyperalgesia. IL-1β increased the mechanosensitivity of C-fibers of the joint, but reduced the mechanosensitivity of Aδ-fibers, thus having opposite effects on these mechanonociceptive nerve fibers. CONCLUSION In the context of arthritic knee pain, IL-1β and IL-1 receptors appear to be involved in thermal, rather than mechanical, hyperalgesia. Therefore, neutralization of IL-1β may be mainly antinociceptive in disease states characterized by thermal hyperalgesia, but not in disease states mainly characterized by mechanical hyperalgesia.

Interleukin-6-dependent influence of nociceptive sensory neurons on antigen-induced arthritis

IntroductionInterleukin-6 (IL-6) is an important mediator of inflammation. In addition to cells involved in inflammation, sensory nociceptive neurons express the IL-6 signal-transducer glycoprotein 130 (gp130). These neurons are not only involved in pain generation but also produce neurogenic inflammation by release of neuropeptides such as calcitonin gene-related peptide (CGRP). Whether IL-6 activation of sensory neurons contributes to the induction of inflammation is unknown. This study explored whether the action of IL-6 on sensory neurons plays a role in the generation of neurogenic inflammation and arthritis induction.MethodsIn SNS-gp130−/− mice lacking gp130 selectively in sensory neurons and appropriate control littermates (SNS-gp130flox/flox), we induced antigen-induced arthritis (AIA), and assessed swelling, histopathological arthritis scores, pain scores, expression of CGRP in sensory neurons, serum concentrations of CGRP and cytokines, and the cytokine release from single cell suspensions from lymph nodes and spleens. In wild-type mice CGRP release was determined during development of AIA and, in cultured sensory neurons, upon IL-6 stimulation.ResultsCompared to SNS-gp130flox/flox mice SNS-gp130−/− mice showed significantly weaker initial swelling, reduced serum concentrations of CGRP, IL-6, and IL-2, no inflammation-evoked upregulation of CGRP in sensory neurons, but similar histopathological arthritis scores during AIA. During the initial swelling phase of AIA, CGRP was significantly increased in the serum, knee and spleen. In vitro, IL-6 augmented the release of CGRP from cultured sensory neurons. Upon antigen-specific restimulation lymphocytes from SNS-gp130−/− mice released more interleukin-17 and interferon-γ than lymphocytes from SNS-gp130flox/flox mice. In naive lymphocytes from SNS-gp130flox/flox and SNS-gp130−/− mice CGRP reduced the release of IL-2 (a cytokine which inhibits the release of interleukin-17 and interferon-γ).ConclusionsIL-6 signaling in sensory neurons plays a role in the expression of arthritis. Selective deletion of gp130 signaling in sensory neurons reduces the swelling of the joint (most likely by reducing neurogenic inflammation) but increases some proinflammatory systemic cellular responses such as the release of interleukin-17 and interferon-γ from lymphocytes upon antigen-specific restimulation. Thus IL-6 signaling in sensory neurons is not only involved in pain generation but also in the coordination of the inflammatory response.

Interleukin-17A is involved in mechanical hyperalgesia but not in the severity of murine antigen-induced arthritis

Interleukin-17A (IL-17A) is considered an important pro-inflammatory cytokine but its importance in joint diseases such as rheumatoid arthritis (RA) is unclear. It has also been reported that IL-17A may induce pain but it is unclear whether pro-inflammatory and pro-nociceptive effects are linked. Here we studied in wild type (WT) and IL-17A knockout (IL-17AKO) mice inflammation and hyperalgesia in antigen-induced arthritis (AIA). We found that the severity and time course of AIA were indistinguishable in WT and IL-17AKO mice. Furthermore, the reduction of inflammation by sympathectomy, usually observed in WT mice, was preserved in IL-17AKO mice. Both findings suggest that IL-17A is redundant in AIA pathology. However, in the course of AIA IL-17AKO mice showed less mechanical hyperalgesia than WT mice indicating that IL-17A contributes to pain even if it is not crucial for arthritis pathology. In support for a role of IL-17A and other members of the IL-17 family in the generation of pain we found that sensory neurones in the dorsal root ganglia (DRG) express all IL-17 receptor subtypes. Furthermore, in isolated DRG neurones most IL-17 isoforms increased tetrodotoxin- (TTX-) resistant sodium currents which indicate a role of IL-17 members in inflammation-evoked sensitization of sensory nociceptive neurones.

Long-Lasting Activation of the Transcription Factor CREB in Sensory Neurons by Interleukin-1β During Antigen-Induced Arthritis in Rats: A Mechanism of Persistent Arthritis Pain?

In spite of successful treatment of immune‐mediated arthritis, many patients still experience pain. We undertook this study to investigate whether antigen‐induced arthritis (AIA) in rats triggers neuronal changes in sensory neurons that outlast the inflammatory process.

Does chloride channel accessory 3 have a role in arthritis pain? A study on murine antigen-induced arthritis.

Calcium-activated chloride channels (CaCCs) are thought to regulate neuronal excitability, and recently chloride (Cl(-)) regulation in DRG neurons has attracted much attention in pain research. Furthermore, the activity of CaCCs is modified by a family of CLCA proteins. In acute antigen-induced arthritis (AIA), a remarkable up-regulation of the murine chloride channel accessory 3 (mClca3) was shown in dorsal root ganglion (DRG) neurons. Therefore we tested the hypothesis that mClca3 is involved in arthritic pain perception. In mClca3 knock-out mice and wild-type control mice, AIA was induced and measures of inflammation and pain were assessed. In the very acute phase of AIA, joint swelling was reduced in mClca3 knock-out mice. This effect disappeared during the course of AIA. We could not show significant differences in mechanical hyperalgesia between both groups of mice, neither at the acute nor at the chronic stage (21 days of AIA). Additional experiments on thermal hyperalgesia in wild-type and mClca3 knock-out mice in the first 3 days of AIA did not show a difference either. In addition, niflumic acid, an antagonist at CaCCs, did not significantly influence hyperalgesia during AIA. Thus, we were not able to provide evidence for a role of CaCCs, and in particular of mClca3, on the expression of arthritis or inflammation-evoked hyperalgesia.

The differential impact of TH+ neuronal cell therapy in models of experimental arthritis

Background A long-lasting anti-inflammatory effect of generated catecholamine-producing tyrosine hydroxylase-positive (TH+) cells was shown in collagen type II-induced arthritis (CIA). Here, we investigated the importance of TH+ cells in another model of rheumatoid arthritis. Methods TH+ cells were generated from murine bone -derived mesenchymal stem cells by specific catecholaminergic factors. Antigen-induced arthritis (AIA) was induced in C57BL/6J mice and one group was treated by TH+ cell-transfer. The appearance of natural TH+ cells, clinical signs of arthritis, pain and immune parameters were observed during AIA. Results In contrast to CIA, here, only a small number of natural occurring TH+ cells were detectable in spleen in the acute phase of arthritis. After TH+ cell transfer, joint swelling was only moderately reduced in acute AIA, but mice showed significantly less guarding and less reduction of withdrawal threshold for mechanical stimulation on the inflamed hind limb. These effects were attenuated towards later phases of the disease. Furthermore both, in TH+ treated mice cytokines in knee joint extracts and immunoglobulins and cytokines in sera were not reduced in acute AIA. Moreover, significantly enhanced values of IL-17, IgG1 and IgG3 were detected. Conclusion In acute AIA, TH+ cells have rather an anti-nociceptive than an anti-inflammatory effect. The increase of inflammatory parameters corresponds to the effect of the sympathetic system on the acute phase of the AIA.

Is IL-17 causally involved in the proinflammatory role of the sympathetic nervous system?

The proinflammatory cytokine Interleukin 17 (IL-17) is considered an important mediator of inflammation in several autoimmune diseases. It is secreted by immune cells such as Th17 cells, and its receptors are ubiquitously expressed. Recently we observed in the model of murine antigen-induced arthritis (AIA) that the anti-inflammatory and the anti-nociceptive effect of sympathectomy was accompanied by a significant reduction of Th17 responses (Ebbinghaus et al., Ann Rheum Dis 2012;71:253–261). This raised the question whether IL-17 is a major player in neuro-immune interactions in murine AIA. In order to further elucidate the role of IL-17 in AIA and in the proinflammatory effect of the sympathetic nervous system, we performed several experiments in AIA using IL-17 knockout mice, a neutralizing anti-IL-17-antibody and adrenoceptor agonists. We found that destruction of sympathetic neurons (by chemical sympathectomy) in the absence of IL-17 still acts anti-inflammatory in AIA. Furthermore neutralization of IL-17 in wild-type C57Bl/6 mice did not effectively reduce joint swelling but significantly reduced secondary mechanical hyperalgesia and attenuated gait abnormalities of the ipsilateral hind limb in the acute stage of inflammation. In addition treatment of AIA-derived lymphocytes ex vivo with α- or β-adrenoceptor agonists did not change their ability to produce IL-17 following re-stimulation with the antigen. Taken together we found that IL-17 alone is not the major player in AIA. Furthermore we did not obtain evidence that IL-17 is causally involved in the proinflammatory effect of the sympathetic nervous system. Nonetheless we found a robust involvement of IL-17 in inflammation-evoked mechanical hyperalgesia.