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Genetic evidence for involvement of neuronally expressed S1P₁ receptor in nociceptor sensitization and inflammatory pain.

Mair N, Benetti C, Andratsch M, Leitner MG, Constantin CE, Camprubí-Robles M, Quarta S, Biasio W, Kuner R, Gibbins IL, Kress M, Haberberger RV - PLoS ONE (2011)

Bottom Line: Immune cells, epithelia and blood cells generate high levels of S1P in inflamed tissue.We found that the S1P₁ receptor for S1P is expressed in subpopulations of sensory neurons including nociceptors.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Physiology, Department of Physiology and Medical Physics, Innsbruck Medical University, Innsbruck, Austria. norbert.mair@i-med.ac.at

ABSTRACT
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.

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SNS-S1P1−/− mice are largely protected from S1P-induced hypersensitivity.(A) Deletion of exon 2 in nociceptive neurons with the SNS-Cre recombination methods in SNS-Cre:S1P1fl/fl (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 S1P1fl/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 S1P1fl/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 S1P1fl/fl mice.
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pone-0017268-g005: SNS-S1P1−/− mice are largely protected from S1P-induced hypersensitivity.(A) Deletion of exon 2 in nociceptive neurons with the SNS-Cre recombination methods in SNS-Cre:S1P1fl/fl (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 S1P1fl/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 S1P1fl/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 S1P1fl/fl mice.

Mentions: Mice deficient of the S1P1 receptor die 12.5 days after conception or birth, depending on the genetic background [41]. Therefore, conditional knockout mice lacking S1P1 selectively in nociceptive neurons of the dorsal root ganglion (DRG) were generated using the Cre recombinase loxP strategy and conditional removal of exon 2 (Fig. 5A) which encodes the entire coding region of S1P1 [42]. This was achieved by mating homozygous mice carrying the loxP-flanked (floxed) S1P1 (S1P1fl/fl) [42] with a mouse line expressing Cre recombinase under the transcriptional control of the nociceptor-specific Nav1.8 gene (SNS-Cre) [43]. In SNS-Cre mice, gene recombination reportedly occurs in around 90% of small diameter (≤28 µm) nociceptive sensory neurons, commences at birth and does not affect gene expression in the spinal cord, brain or any other organs in the body [43]–[45]. Sequence comparisons of PCRs using primers P1 and P3 of cDNA from S1P1+/+, S1P1fl/fl and SNS-S1P1−/− DRG confirmed that DRG neurons from SNS-S1P1−/− mice lacked mRNA for S1P1 receptors. Moreover, antibodies directed against the S1P1 receptor [46] showed immunoreactivity to S1P1 receptor protein in DRG from S1P1fl/fl but not SNS-S1P1−/− mice (Fig. 5B, C). For quantitative analysis, we recorded capsaicin-induced calcium transients in DRG neurons before and after conditioning stimulation with S1P which regularly induced brief calcium transients itself. After S1P the capsaicin responses were strongly facilitated (Fig. 5D). The number of neurons sensitized to capsaicin after conditioning stimulation with S1P was significantly lower in SNS-S1P1−/− mice compared with S1P1fl/fl mice (p<0.01; Student's unpaired t-test; Fig. 5E) but the magnitude of the increased response in sensitized neurons was similar for both strains (Fig. 5F).


Genetic evidence for involvement of neuronally expressed S1P₁ receptor in nociceptor sensitization and inflammatory pain.

Mair N, Benetti C, Andratsch M, Leitner MG, Constantin CE, Camprubí-Robles M, Quarta S, Biasio W, Kuner R, Gibbins IL, Kress M, Haberberger RV - PLoS ONE (2011)

SNS-S1P1−/− mice are largely protected from S1P-induced hypersensitivity.(A) Deletion of exon 2 in nociceptive neurons with the SNS-Cre recombination methods in SNS-Cre:S1P1fl/fl (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 S1P1fl/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 S1P1fl/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 S1P1fl/fl mice.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3040773&req=5

pone-0017268-g005: SNS-S1P1−/− mice are largely protected from S1P-induced hypersensitivity.(A) Deletion of exon 2 in nociceptive neurons with the SNS-Cre recombination methods in SNS-Cre:S1P1fl/fl (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 S1P1fl/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 S1P1fl/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 S1P1fl/fl mice.
Mentions: Mice deficient of the S1P1 receptor die 12.5 days after conception or birth, depending on the genetic background [41]. Therefore, conditional knockout mice lacking S1P1 selectively in nociceptive neurons of the dorsal root ganglion (DRG) were generated using the Cre recombinase loxP strategy and conditional removal of exon 2 (Fig. 5A) which encodes the entire coding region of S1P1 [42]. This was achieved by mating homozygous mice carrying the loxP-flanked (floxed) S1P1 (S1P1fl/fl) [42] with a mouse line expressing Cre recombinase under the transcriptional control of the nociceptor-specific Nav1.8 gene (SNS-Cre) [43]. In SNS-Cre mice, gene recombination reportedly occurs in around 90% of small diameter (≤28 µm) nociceptive sensory neurons, commences at birth and does not affect gene expression in the spinal cord, brain or any other organs in the body [43]–[45]. Sequence comparisons of PCRs using primers P1 and P3 of cDNA from S1P1+/+, S1P1fl/fl and SNS-S1P1−/− DRG confirmed that DRG neurons from SNS-S1P1−/− mice lacked mRNA for S1P1 receptors. Moreover, antibodies directed against the S1P1 receptor [46] showed immunoreactivity to S1P1 receptor protein in DRG from S1P1fl/fl but not SNS-S1P1−/− mice (Fig. 5B, C). For quantitative analysis, we recorded capsaicin-induced calcium transients in DRG neurons before and after conditioning stimulation with S1P which regularly induced brief calcium transients itself. After S1P the capsaicin responses were strongly facilitated (Fig. 5D). The number of neurons sensitized to capsaicin after conditioning stimulation with S1P was significantly lower in SNS-S1P1−/− mice compared with S1P1fl/fl mice (p<0.01; Student's unpaired t-test; Fig. 5E) but the magnitude of the increased response in sensitized neurons was similar for both strains (Fig. 5F).

Bottom Line: Immune cells, epithelia and blood cells generate high levels of S1P in inflamed tissue.We found that the S1P₁ receptor for S1P is expressed in subpopulations of sensory neurons including nociceptors.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Physiology, Department of Physiology and Medical Physics, Innsbruck Medical University, Innsbruck, Austria. norbert.mair@i-med.ac.at

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus