Deletion of Interleukin-6 Signal Transducer gp130 in Small Sensory Neurons Attenuates Mechanonociception and Down-Regulates TRPA1 Expression

Creative Commons Attribution-Noncommercial-Share Alike 3.0 License Unported (http://creativecommons.org/licenses/by-nc-sa/3.0). agreement This allows data and text mining, use of figures in presentations, and posting the article online, as long as the original article is attributed.Glycoprotein 130 (gp130) is the signal transducing receptor subunit for cytokines of the interleukin-6 (IL-6) family, and it is expressed in a multitude of cell types of the immune and nervous system. IL-6-like cytokines are not only key regulators of innate immunity and inflammation but are also essential factors for the differentiation and development of the somatosensory system. Mice with a null mutation of gp130 in primary nociceptive afferents (SNS-gp130−/−) are largely protected from hypersensitivity to mechanical stimuli in mouse models of pathological pain. Therefore, we set out to investigate how neuronal gp130 regulates mechanonociception. SNS-gp130−/− mice revealed reduced mechanosensitivity to high mechanical forces in the von Frey assay in vivo, and this was associated with a reduced sensitivity of nociceptive primary afferents in vitro. Together with these findings, transient receptor potential ankyrin 1 (TRPA1) mRNA expression was significantly reduced in DRG from SNS-gp130−/− mice. This was also reflected by a reduced number of neurons responding with calcium transients to TRPA1 agonists in primary DRG cultures. Downregulation of Trpa1 expression was predominantly discovered in nonpeptidergic neurons, with the deficit becoming evident during stages of early postnatal development. Regulation of Trpa1 mRNA expression levels downstream of gp130 involved the classical Janus kinase family-signal transducer and activator of transcription pathway. Our results closely link proinflammatory cytokines to the expression of TRPA1, both of which have been shown to contribute to hypersensitive pain states. We suggest that gp130 has an essential role in mechanonociception and in the regulation of TRPA1 expression

Oncostatin M induces heat hypersensitivity by gp130-dependent sensitization of TRPV1 in sensory neurons

Oncostatin M (OSM) is a member of the interleukin-6 cytokine family and regulates eg. gene activation, cell survival, proliferation and differentiation. OSM binds to a receptor complex consisting of the ubiquitously expressed signal transducer gp130 and the ligand binding OSM receptor subunit, which is expressed on a specific subset of primary afferent neurons. In the present study, the effect of OSM on heat nociception was investigated in nociceptor-specific gp130 knock-out (SNS-gp130-/-) and gp130 floxed (gp130fl/fl) mice

Sphingosine-1-phosphate-induced nociceptor excitation and ongoing pain behavior in mice and humans is largely mediated by S1P3 receptor

The biolipid sphingosine-1-phosphate (S1P) is an essential modulator of innate immunity, cell migration, and wound healing. It is released locally upon acute tissue injury from endothelial cells and activated thrombocytes and, therefore, may give rise to acute post-traumatic pain sensation via a yet elusive molecular mechanism. We have used an interdisciplinary approach to address this question, and we find that intradermal injection of S1P induced significant licking and flinching behavior in wild-type mice and a dose-dependent flare reaction in human skin as a sign of acute activation of nociceptive nerve terminals. Notably, S1P evoked a small excitatory ionic current that resulted in nociceptor depolarization and action potential firing. This ionic current was preserved in “cation-free” solution and blocked by the nonspecific Cl− channel inhibitor niflumic acid and by preincubation with the G-protein inhibitor GDP-β-S. Notably, S1P3 receptor was detected in virtually all neurons in human and mouse DRG. In line with this finding, S1P-induced neuronal responses and spontaneous pain behavior in vivo were substantially reduced in S1P3−/− mice, whereas in control S1P1 floxed (S1P1fl/fl) mice and mice with a nociceptor-specific deletion of S1P1−/− receptor (SNS-S1P1−/−), neither the S1P-induced responses in vitro nor the S1P-evoked pain-like behavior was altered. Therefore, these findings indicate that S1P evokes significant nociception via G-protein-dependent activation of an excitatory Cl− conductance that is largely mediated by S1P3 receptors present in nociceptors, and point to these receptors as valuable therapeutic targets for post-traumatic pain.The authors thank K. Braun, T. Martha, and M. Doblander for expert technical assistance. This work was supported by la Generalitat Valenciana and the Ministerio de Economia y Competitividad (A.V.F.M.), the Australian National Health and Medical Research Council Project Grant 535055 to R.V.H., the Intramural Research Programs of the National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases to R.L.P., and the Austrian Research Funding Agency FWF Project Grants P20562, P25345, and SPIN to M.K

Genetic Evidence for Involvement of Neuronally Expressed S1P1 Receptor in Nociceptor Sensitization and Inflammatory Pain

Sphingosine-1-phosphate (S1P) is a key regulator of immune response. Immune cells, epithelia and blood cells generate high levels of S1P in inflamed tissue. However, it is not known if S1P acts on the endings of nociceptive neurons, thereby contributing to the generation of inflammatory pain. We found that the S1P1 receptor for S1P is expressed in subpopulations of sensory neurons including nociceptors. Both S1P and agonists at the S1P1 receptor induced hypersensitivity to noxious thermal stimulation in vitro and in vivo. S1P-induced hypersensitivity was strongly attenuated in mice lacking TRPV1 channels. S1P and inflammation-induced hypersensitivity was significantly reduced in mice with a conditional nociceptor-specific deletion of the S1P1 receptor. Our data show that neuronally expressed S1P1 receptors play a significant role in regulating nociceptor function and that S1P/S1P1 signaling may be a key player in the onset of thermal hypersensitivity and hyperalgesia associated with inflammation

Expression of S1P₁ receptors in sensory neurons.

<p>(A) S1P receptor mRNA expression was detected with reverse transcription PCR in DRG explants. (B) Quantitative real-time PCR revealed expression of S1P₁, S1P₂ and S1P₃ mRNA in DRG explants (total), acutely isolated neurons (acute) and 1-day-old cultures (1 d) (n = 5 experiments). In contrast, S1P₄ and S1P₅ mRNA levels were lower in DRG explants and absent in isolated neurons. (C) Immunoreactivity for S1P₁ was present in neurons and intraganglionic capillaries (arrowhead). S1P₁-IR was colocalized with immunoreactivity for peripherin, whereas S1P₁-IR was absent in NF200-positive neurons. Scale bars  = 50 µm. (D) S1P₁ receptor colocalized with the small neuron marker I-B4 in the vast majority of cultured neurons but usually not with CGRP or Nf200, a marker for myelinated neurons (n = 4 experiments, scale bars  = 20 µm). (E) Size distribution of S1P₁-IR positive neurons revealed that S1P₁-IR expressing cells are amongst the small diameter neurons (n = 6 experiments, 304 neurons). Only 2% of S1P₁-IR+ neurons had diameters >20 µm. (F) Expression of S1P₁ immunoreactivity was absent after preabsorption of the antibodies with the corresponding peptide.</p

SNS-S1P₁^−/− mice are largely protected from S1P-induced hypersensitivity.

<p>(A) Deletion of exon 2 in nociceptive neurons with the SNS-Cre recombination methods in <i>SNS-Cre:S1P₁^fl/fl</i> (SNS-S1P₁^−/−) mice. (B) Taqman®-PCR analysis of DRG explants revealed an almost complete absence of S1P₁ mRNA (n = 10) in SNS-S1P₁^−/− mice in comparison to control S1P₁^fl/fl mice (n = 9, **p<0.01; Mann-Whitney U-test). (C) S1P₁ receptor immunoreactivity is expressed in a subpopulation of small size sensory neurons in DRG sections obtained from S1P₁^fl/fl but not in SNS-S1P₁^−/− mice. There is no difference in the expression profile of CGRP immunoreactivity. (D) Example of a neuron that responded to capsaicin (arrows) with calcium transients. S1P itself induced a brief transient which recovered immediately and the following response to capsaicin was strongly increased. (E, F) The percentage of neurons responding to S1P with an increase in capsaicin-induced calcium transients was significantly reduced in SNS-S1P₁^−/− mice compared to S1P₁^fl/fl mice.</p

Reduced thermal hypersensitivity in S1P₁^−/−mice.

<p>(A) Injection of the S1P₁ agonist SEW2871 induced a significant transient decrease in paw withdrawal latencies in S1P₁^fl/fl (n = 9) which was significantly less pronounced than in SNS-S1P₁^−/− mice (n = 10, *p<0.05; ANOVA). (B, C) While only a minor reduction of paw withdrawal latencies was observed in both mouse strains with local low dose S1P injection, we observed a significant decrease in paw withdrawal latencies in S1P₁^fl/fl mice (n = 7) which was similar to wt. In SNS-S1P₁^−/− mice the degree of hypersensitivity was significantly ameliorated in comparison to S1P₁^fl/fl mice (n = 9, *p<0.05, ** p<0.01; ANOVA). (D) CFA (20 µl) injection into the plantar hindpaw induced a pronounced decrease of PWL which was significantly attenuated in S1P₁-Cre mice (p<0.05, n = 4; ANOVA). (E) Paw swelling was similar in SNS-S1P₁^−/− and S1P₁^fl/fl mice (n = 4).</p