Aberrant sphingosine-1-phosphate receptor 1 expression on activated naive B cells associated with disease activity and lupus nephritis in systemic lupus erythematosus

Article Figures and data Abstract Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Somatosensory neurons mediate responses to diverse mechanical stimuli, from innocuous touch to noxious pain. While recent studies have identified distinct populations of A mechanonociceptors (AMs) that are required for mechanical pain, the molecular underpinnings of mechanonociception remain unknown. Here, we show that the bioactive lipid sphingosine 1-phosphate (S1P) and S1P Receptor 3 (S1PR3) are critical regulators of acute mechanonociception. Genetic or pharmacological ablation of S1PR3, or blockade of S1P production, significantly impaired the behavioral response to noxious mechanical stimuli, with no effect on responses to innocuous touch or thermal stimuli. These effects are mediated by fast-conducting A mechanonociceptors, which displayed a significant decrease in mechanosensitivity in S1PR3 mutant mice. We show that S1PR3 signaling tunes mechanonociceptor excitability via modulation of KCNQ2/3 channels. Our findings define a new role for S1PR3 in regulating neuronal excitability and establish the importance of S1P/S1PR3 signaling in the setting of mechanical pain thresholds. https://doi.org/10.7554/eLife.33285.001 Introduction Pain is a complex sensation. It serves to protect organisms from harmful stimuli, but can also become chronic and debilitating following tissue injury and disease. Distinct cells and molecules detect noxious thermal and mechanical stimuli. Thermal pain is detected by thermosensitive TRP channels in subsets of nociceptors (Caterina et al., 2000; Vriens et al., 2011), and gentle touch is detected by Piezo2 channels in low-threshold mechanoreceptors (LTMRs) (Ranade et al., 2014; Woo et al., 2014). Aδ high-threshold mechanoreceptors (HTMRs) have been shown to play a key role in responses to painful mechanical stimuli (Arcourt et al., 2017; Ghitani et al., 2017). Recent studies have shown that there are at least two populations of HTMRs that mediate responses to noxious mechanical stimuli. The Npy2r+ subpopulation of HTMRs mediates fast paw withdrawal responses to pinprick stimulation and terminates as free nerve endings in the epidermis (Arcourt et al., 2017). The Calca+ subpopulation of circumferential-HTMRs responds to noxious force and hair pulling, and terminates as circumferential endings wrapped around guard hair follicles (Ghitani et al., 2017). Additionally, somatostatin-expressing interneurons of laminae I-III in the dorsal horn of the spinal cord receive input from nociceptors and are required for behavioral responses to painful mechanical stimuli (Duan et al., 2014). Despite these advances in defining the cells and circuits of mechanical pain, little is known about the molecular signaling pathways in mechanonociceptors. Here, we show that sphingosine 1-phosphate (S1P) is required for mechanical pain sensation. S1P is a bioactive lipid that signals via 5 G-protein coupled S1P Receptors (S1PRs 1–5). S1P signaling, mainly via S1PR1, plays a well-known role in immune cell migration and maturation (Spiegel and Milstien, 2003; Matloubian et al., 2004; Schwab et al., 2005). Additionally, recent studies have shown that S1PRs are expressed throughout the nervous system (Janes et al., 2014; Mair et al., 2011; Camprubí-Robles et al., 2013) and S1P signaling is associated with a variety of neuroinflammatory disorders, including multiple sclerosis (Brinkmann et al., 2010) and Alzheimer's disease (Couttas et al., 2014). S1P has been implicated in spontaneous pain (Camprubí-Robles et al., 2013) and thermal pain hypersensitivity (Mair et al., 2011; Finley et al., 2013; Weth et al., 2015), but due to conflicting accounts of S1P receptor expression in the CNS (Janes et al., 2014; Weth-Malsch et al., 2016) and PNS (Mair et al., 2011; Camprubí-Robles et al., 2013; Usoskin et al., 2015) as well as inconsistent reports on the effects of S1P on neuronal excitability (Camprubí-Robles et al., 2013; Zhang et al., 2006; Li et al., 2015) and pain behaviors (Mair et al., 2011; Camprubí-Robles et al., 2013; Finley et al., 2013; Weth et al., 2015), the role of S1P in somatosensation remains controversial. We found that mice lacking the S1P receptor S1PR3 display striking and selective deficits in behavioral responses to noxious mechanical stimuli. Likewise, peripheral blockade of S1PR3 signaling or S1P production impairs mechanical sensitivity. We show that S1P constitutively enhances the excitability of A mechanonociceptors (AMs) via closure of KCNQ2/3 potassium channels to tune mechanical pain sensitivity. The effects of S1P are completely dependent on S1PR3. While previous studies have shown that elevated S1P triggers acute pain and injury-evoked thermal sensitization (Mair et al., 2011; Camprubí-Robles et al., 2013), we now demonstrate that baseline levels of S1P are necessary and sufficient for setting normal mechanical pain thresholds. By contrast, elevated S1P selectively triggers thermal sensitization via activation of TRPV1+ heat nociceptors, with no effect on mechanical hypersensitivity. Our findings uncover an essential role for constitutive S1P signaling in mechanical pain. Results To identify candidate genes underlying mechanosensation, we previously performed transcriptome analysis of the sensory ganglia innervating the ultra-sensitive tactile organ (the star) of the star-nosed mole (Gerhold et al., 2013). Immunostaining revealed the tactile organ is preferentially innervated by myelinated Aδ fibers (Gerhold et al., 2013), which are primarily mechanosensitive. While our original analysis focused on ion channels enriched in the neurons of the star organ, our dataset also revealed enrichment of several components of the S1P pathway, including S1pr3. Likewise, single-cell RNA seq of mouse dorsal root ganglion (DRG) neurons revealed S1pr3 expression in a subset of myelinated mechanoreceptors (Usoskin et al., 2015) in addition to a subpopulation of peptidergic C nociceptors. S1P promotes excitability in small-diameter, capsaicin-sensitive nociceptors (Mair et al., 2011; Camprubí-Robles et al., 2013; Zhang et al., 2006; Li et al., 2015). In addition, S1PR3 has been shown to mediate spontaneous pain triggered by elevated S1P and thermal sensitization following sterile tissue injury (Camprubí-Robles et al., 2013). However, no studies have examined the role of S1PR3 in mechanosensation or in regulating somatosensory behaviors under normal conditions. Given the enrichment of S1pr3 in mechanosensory neurons of the star-nosed mole and mouse, we hypothesized that S1P signaling via S1PR3 may also play a role in mechanosensation. Thus, we set out to define the role of S1P signaling and S1PR3 in somatosensory mechanoreceptors. S1PR3 mediates acute mechanical pain We first examined a variety of somatosensory behaviors in mice lacking S1PR3 (Kono et al., 2004) (S1pr3tm1Rlp/Mmnc; referred to herein as S1PR3 KO). We initially investigated baseline responses to mechanical stimuli. S1PR3 KO mice displayed a dramatic loss of mechanical sensitivity (Figure 1A; see Figure 1—source data 1), as von Frey paw withdrawal thresholds were significantly elevated in S1PR3 KO mice relative to WT and S1PR3 HET littermates (mean thresholds: 1.737 g vs. 0.736 and 0.610 g, respectively). Moreover, S1PR3 KO mice demonstrated decreased responses to a range of noxious tactile stimuli (2–6 g; Figure 1B) and to noxious pinprick stimulation (Figure 1C), but normal responsiveness to innocuous tactile stimuli (0.6–1.4 g; Figure 1B). S1PR3 KO mice exhibited normal tape removal attempts (Ranade et al., 2014) (Figure 1D), righting reflexes (Figure 1—figure supplement 1A), radiant heat withdrawal latencies (Figure 1E), and itch-evoked scratching (Figure 1—figure supplement 1B). These results demonstrate a selective role for S1PR3 in acute mechanical pain. Figure 1 with 1 supplement see all Download asset Open asset S1PR3 mediates acute mechanical pain. (A) von Frey 50% withdrawal threshold measurements for S1pr3+/+ (WT, N = 8), S1pr3+/- (HET, N = 7) and S1pr3-/- (KO, N = 12) mice. p<0.0001 (one-way ANOVA). Tukey-Kramer post hoc comparisons for KO and HET to WT indicated on graph. (B) von Frey force-response graph for WT (N = 8) versus KO (N = 12) animals; pgenotype <0.0001 (two-way ANOVA). Tukey HSD comparisons between genotypes are indicated for given forces. (C) % withdrawal to pinprick stimulation of hindpaw for HET versus KO animals; p<0.0001 (unpaired t-test; N = 5–7 mice per group). (D) Number of attempted removal bouts in tape assay for WT (N = 2), HET (N = 2), and KO (N = 5) mice; p=0.172 (one-way ANOVA). (E) Baseline radiant heat measurements for WT (N = 8), HET (N = 3), and KO (N = 5) mice; p=0.444 (one-way ANOVA). (F) von Frey 50% withdrawal threshold measurements for mice pre- and post-injection of 500 µM TY 52156 (N = 10), 10 µM W146 (N = 6), or 1% DMSO-PBS vehicle (N = 17); p=0.016, 0.650 (two-tailed paired t-test comparing vehicle- vs. drug-injected paw). (G) von Frey force-response graph for mice injected with either 1% DMSO-PBS (N = 4) or 500 µM TY 52156 (N = 4); ptreatment <0.0001 (two-way ANOVA). Tukey HSD comparisons were made between treatment groups and significant differences at a given force are indicated on graph. Error bars represent mean ± SD. https://doi.org/10.7554/eLife.33285.002 Figure 1—source data 1 S1PR3 mediates acute mechanical pain. Related to Figure 1. https://doi.org/10.7554/eLife.33285.004 Download elife-33285-fig1-data1-v2.xlsx As a complement to our analysis of somatosensation in S1PR3 KO animals, we employed a pharmacological approach, using the S1PR3-selective antagonist TY 52156 (TY) (Nussbaum et al., 2015). Similar to the phenotype of knockout animals, intradermal injection of 500 µM TY into the mouse hindpaw (the site of testing) triggered a rapid and significant elevation in von Frey paw withdrawal thresholds (Figure 1F) and decreased responsiveness to noxious (2–6 g), but not innocuous (0.6–1.4 g), tactile stimuli (Figure 1G), without affecting noxious heat sensitivity (Figure 1—figure supplement 1C). By contrast, blockade of S1PR1 with the selective antagonist W146 (Finley et al., 2013) had no effect on baseline mechanical or thermal thresholds (Figure 1F; Figure 1—figure supplement 1C). Overall, these data show that S1PR3 signaling sets mechanical pain sensitivity. Endogenous S1P mediates acute mechanical pain We next asked whether peripheral S1P was required for the S1PR3-dependent effects on mechanosensation. We decreased S1P levels via injection of the sphingosine kinase inhibitor SKI II to block local production of S1P (Chiba et al., 2010) or elevated S1P levels via intradermal injection of S1P and measured behaviors 30 min after injection. Decreasing local S1P levels with SKI II significantly reduced mechanical sensitivity (Figure 2A; see Figure 2—source data 1), comparable to the hyposensitivity phenotype observed in S1PR3 KO mice (Figure 1A). Again, similar to what was observed in S1PR3 KO animals (Figure 1E), peripheral blockade of S1P production had no effect on baseline thermal sensitivity (Figure 1—figure supplement 1C). Surprisingly, injecting exogenous S1P (10 µM; maximum solubility in saline vehicle) had no effect on mechanical sensitivity (Figure 2A–B). However, as previously reported (Mair et al., 2011; Camprubí-Robles et al., 2013), S1P injection triggered S1PR3-dependent thermal hypersensitivity and spontaneous pain (Figure 2C–D), demonstrating that the lack of effect on mechanical hypersensitivity is not due to problems with S1P delivery or degradation. Figure 2 Download asset Open asset Endogenous S1P mediates acute mechanical pain. (A) von Frey 50% withdrawal measurements for mice pre- and post-injection of 50 µM SKI II (N = 8) or 10 µM S1P (N = 7); p=0.003, 0.604 (two-tailed paired t-tests). (B) von Frey force-response graph for animals injected with 10 µM S1P or 0.1% MeOH-PBS; pgenotype >0.05 (two-way ANOVA; N = 8 mice per group). No Tukey HSD comparisons at any force between genotypes were significant. (C) Intradermal cheek injection of 10 µM S1P, 2 µM, 0.2 µM, and 20 µL 0.3% methanol PBS (vehicle), with quantification of number of forepaw wipes over the 5 min post-injection interval; p<0.0001 (one-way ANOVA; N = 3 mice per condition). Dunnett's multiple comparisons p-values are represented on graph for comparisons made between treated and vehicle groups. (D) Radiant heat normalized paw withdrawal latencies 20–30 min post injection of 15 µL 10 µM S1P, 0.2 µM S1P. or 0.3% methanol-PBS vehicle (i.d.) into the hind paw of S1PR3 WT or KO mice; p=0.0129 (one-way ANOVA; N = 3–10 mice per condition). Dunnett's multiple comparisons p-values are represented on graph for comparisons made between treated and vehicle groups. (E) von Frey 50% withdrawal measurements for mice pre- (baseline) and post-injection of 50 µM SKI II (N = 14) and 0 (N = 4), 10 (N = 3), 75 (N = 4), or 200 nM S1P (N = 3; one-way ANOVA; p=0.0001). Tukey Kramer comparisons are indicated on graph. Error bars represent mean ± SD. https://doi.org/10.7554/eLife.33285.005 Figure 2—source data 1 Endogenous S1P mediates acute mechanical pain. Related to Figure 2. https://doi.org/10.7554/eLife.33285.006 Download elife-33285-fig2-data1-v2.xlsx These data support a model whereby S1P constitutively activates S1PR3 to set normal mechanical pain thresholds. To further test this model, we asked if the mechanical hyposensitivity elicited after endogenous S1P depletion (via SKI II) could be rescued by local injection of exogenous S1P. Indeed, we found that injection of exogenous S1P reversed SKI II-induced mechanical hyposensitivity in a dose-dependent manner, and observed a maximal effect with 200 nM S1P (Figure 2E). Although quantification of native S1P levels in skin is inaccurate owing to avid lyase activity (Shaner et al., 2009), our data establish that baseline S1P levels are sufficient to maximally exert their effect on S1PR3-dependent mechanical pain, such that increased S1P does not evoke mechanical hypersensitivity, but diminished S1P leads to mechanical hyposensitivity. These data show that constitutive activation of S1PR3 by S1P is required for normal mechanosensitivity. S1PR3 is expressed in A mechanonociceptors and thermal nociceptors Our behavioral data showing distinct roles for S1PR3 in mechanonociception and thermal hypersensitivity suggest that S1PR3 is expressed in distinct subsets of somatosensory neurons. While a previous study suggested that all somatosensory neurons express S1PR3 (Camprubí-Robles et al., 2013), single cell RNA seq data suggests S1pr3 is not expressed by all DRG neurons (Usoskin et al., 2015), and no studies have performed quantitative analysis of S1PR3 staining or co-staining to define subpopulations of S1PR3+ neurons. We thus set out to characterize the somatosensory neuron subtypes expressing S1pr3 using in situ hybridization (ISH) of wild-type somatosensory ganglia and immunohistochemistry (IHC) in an S1pr3mCherry/+ reporter mouse (Sanna et al., 2016). We first used in situ hybridization (ISH) with a specific S1pr3 probe to examine expression patterns of S1pr3 (Figure 3A–B; see Supplementary file 1). In our experiments, 43% of cells from wild-type DRG expressed S1pr3. Co-ISH revealed that one population of S1pr3+ neurons represents Aδ mechanonociceptors (AMs). These cells expressed Scn1a (39.9% of all S1pr3+), a gene that encodes the Nav1.1 sodium channel, which mediates mechanical pain in Aδ fibers (Osteen et al., 2016). S1pr3+ cells also co-expressed Npy2r (20.4% of all S1pr3+), a marker of a subset of mechanonociceptive A fibers (Arcourt et al., 2017). S1pr3 was expressed in 70.6% of Scn1a+ cells and 72% of Npy2r+ cells, comprising a majority of both of these populations. Interestingly, a subset of cells co-expressed S1pr3 and the mechanically sensitive channel Piezo2, which is expressed by Aβ, Aδ, and C fibers (Ranade et al., 2014). The remaining S1pr3+ cells were Trpv1+ and/or Trpa1+ C nociceptors (67.1% of all S1pr3+), which are reported to overlap minimally with the Scn1a+ and Npy2r+ populations (Arcourt et al., 2017; Osteen et al., 2016). Figure 3 with 1 supplement see all Download asset Open asset S1pr3 is expressed in A mechanonociceptors and C thermal nociceptors. (A) (Top) Representative co-ISH of S1pr3 (green; left) with Scn1a, Npy2r, Piezo2, and Trpv1 (magenta; center) in sectioned DRG. Right column: overlay with co-localized regions colored white (10x air objective; scale = 100 µm). (B) Bar chart showing the % of total cells expressing the indicated marker (grey) and the % of total cells co-expressing both marker and S1pr3 (green). See Table S1 for quantification. (C) Representative IHC images of sectioned DRG from S1pr3mCherry/+ animals stained with anti-DsRed (green, S1PR3) and anti-Peripherin (left, magenta) or anti-NF200 (right, magenta). Arrows indicate co-stained cells. Images were acquired using a 10x air objective (scale = 100 µm). (D) Whole-mount skin IHC confocal images with anti-DsRed antibody (S1PR3, green) and anti-NefH antibody (NF200, magenta) in an S1pr3mCherry/+ animal (20x water objective; scale = 50 µm). Arrows indicate co-positive free nerves (left image). Arrowheads indicate NF200- free nerves (left) or S1PR3- circumferential fibers (right image). (E) Sectioned skin IHC with anti-DsRed (S1PR3) and anti-NefH (NF200, left, top right) or anti-DsRed (S1PR3) and anti-beta-tubulin III (BTIII, bottom right) antibody (magenta) in S1pr3mCherry/+ skin (20x air objective; scale = 50 µm). Arrows indicate co-positive free nerve endings (left), S1PR3-negative lanceolate/circumferential hair follicle endings (top right, arrow = circumferential, arrowhead = lanceolate), or S1PR3-negative putative Merkel afferent (bottom right). (F) (Left) Quantification of sectioned DRG IHC experiments showing % of S1PR3+ cells that co-stained with indicated markers (n > 250 cells per marker). (Right) Quantification of sectioned skin IHC experiments showing % of fibers positive for indicated marker that co-stained with S1PR3 (anti-DsRed; n = 10 images per marker from two animals). https://doi.org/10.7554/eLife.33285.007 We next used an S1pr3mCherry/+ reporter mouse, which produces a functional S1PR3-mCherry fusion protein (Sanna et al., 2016), as an independent strategy to explore S1PR3 expression and localization. This strategy was used because we found that anti-S1PR3 antibodies showed broad immunoreactivity in DRG from mice lacking S1PR3, and so we instead used anti-DsRed antibodies to probe expression of the S1PR3 fusion protein (Figure 3—figure supplement 1E). We found that 42.4% of S1PR3+ cells co-stained with anti-Peripherin, demonstrating that S1PR3 is expressed in a subset of small-diameter neurons. We also observed that 69.5% of S1PR3+ cells co-stained with anti-NF200, which marks medium and large-diameter myelinated neurons. Furthermore, we observed that S1PR3+ cells were primarily of small to medium diameter (11.3–35.1 µm), whereas all cells in the DRG ranged from 11.3 to 53.9 µm. Overall, these data support the expression of S1PR3 in subsets of small-diameter thermal nociceptors and medium-diameter mechanonociceptors (Figure 3F). Additionally, no significant differences were observed between WT and S1PR3 KO DRG in number of Trpa1+, Trpv1+, Peripherin+, NF200+, or IB4+ cells (Figure 3—figure supplement 1B–C,F,G). The mean diameters of Trpv1+ neurons (Figure 3—figure supplement 1D, left), NF200+ neurons (Figure 3—figure supplement 1G), or all neurons (Figure 3—figure supplement 1D, right) in WT versus KO DRG were not significantly different, suggesting no loss of major sensory neuronal subtypes in the S1PR3 KO. We then visualized S1PR3 expression in nerve fibers that innervate the skin using anti-DsRed antibodies in whole-mount immunohistochemistry (IHC; Figure 3D). The reporter animals showed no specific antibody staining in epidermal or dermal cells (Figure 3—figure supplement 1I), and single-cell RNA seq of a diverse array of mouse epidermal and dermal cells corroborates this lack of expression (Joost et al., 2016). We observed overlap of S1PR3-expressing free nerve endings with NF200+ myelinated free nerves and NF200- putative C-fiber endings (Figure 3F), but did not observe expression of S1PR3 in NF200+ circumferential or lanceolate hair follicle receptors, or in putative Merkel afferents (Figure 3D–E). β-tubulin III, PGP9.5 (pan-neuronal markers), and NF200 staining in S1PR3 KO skin displayed patterns of epidermal and dermal innervation similar to WT skin, suggesting the phenotypes observed in the S1PR3 KO mice are not due to developmental loss of sensory neuronal innervation (pPGP9.5= 0.443 (n = 93, 38 fibers); pNefH = 0.405 (n = 61, 28 fibers); pBTIII = 0.353 (n = 104, 89 fibers); two-tailed t-tests based on average number of fibers per field of view). These results support expression of S1PR3 in subsets of myelinated A mechanonociceptors and unmyelinated C nociceptors that terminate as free nerve endings. S1P activates thermal nociceptors but not putative AMs Live imaging of cultured DRG neurons from adult reporter animals showed expression of S1PR3-mCherry fusion protein in 48.3% of neurons, mirroring our ISH and IHC results (Figure 4A). To examine the effects of S1P on A mechanonociceptors and C nociceptors, we performed ratiometric calcium imaging and electrophysiology on DRG cultures from reporter mice. Interestingly, only 56.1 ± 22.4% of mCherry-expressing neurons were activated by 1 µM S1P (Representative trace in Figure 4B; representative images in Figure 4C), which our dose-response showed to be the saturating concentration for calcium influx (Figure 4D; EC50 = 155 nM). neurons were also capsaicin-sensitive (n > sensory neurons from S1PR3 KO animals did not to S1P, as (Camprubí-Robles et al., 2013), exhibited responses that were not significantly from WT neurons (Figure 3—figure supplement The mean diameter of neurons was ± whereas the mean diameter of neurons was ± two-tailed We also performed cell experiments with studies (Mair et al., 2011; Zhang et al., 2006; Li et al., 2015), found that S1P in capsaicin-sensitive small diameter cells (Figure This that only the small-diameter, S1PR3+ putative nociceptors are by S1P. We next asked whether the S1PR3+ diameter neurons represent the mechanonociceptors observed by ISH (Figure To this we asked whether the a selective of nociceptors (Osteen et al., 2016), triggers calcium influx in S1PR3-expressing neurons. Indeed, we found that ± of neurons expressed (Figure with our staining showing expression of S1pr3 in nociceptors and the role of neurons in mechanical pain in (Osteen et al., 2016). Figure Download asset Open asset S1P activates thermal nociceptors but not mechanonociceptors. (A) (Left) Representative of in cultured adult DRG neurons from one S1pr3mCherry/+ (Right) Quantification of % of total cells expressing S1pr3 from DRG ISH and from DRG cultures (N = 2 animals (B) Representative from calcium imaging showing two neurons, one which to 1 µM S1P, 1 µM and and one which only to (C) (Left) calcium imaging (left) and after addition of 1 µM S1P in S1pr3 cultured mouse DRG neurons. Bar (Right) % of neurons that are to 1 µM S1P in ratiometric calcium imaging (n > cells from imaging from animals). (D) of mean neuronal calcium to of S1P. and (N = 2 animals). Error bars represent mean ± SD. for all S1P from which EC50 was S1P were also (E) trace of a single wild-type neuron in response to addition of 1 µM S1P and 1 µM with of to S1P and one of one also to Bar = 2 (F) (Left) calcium imaging after addition of 500 nM in S1pr3mCherry/+ neurons, which were used instead of adult DRG neurons because to without (Right) % of neurons that are (N = 1 total S1PR3 KCNQ2/3 channels to excitability We next the molecular by which S1P signaling in nociceptors may mechanical pain. We performed on the medium-diameter S1pr3mCherry/+ DRG neurons = ± which did not display calcium influx (Figure In these cells, 1 µM S1P did not (Figure supplement 1A; see Figure data or in the of injection (Figure supplement 1A; Figure However, S1P the threshold to in an S1PR3-dependent (Figure Figure supplement 1B). Figure 5 with 1 supplement see all Download asset Open asset S1PR3 KCNQ2/3 channels to experiments were performed in S1pr3mCherry/+ or DRG neurons. (A) (Left) of a single in cell and after S1P (Right) % in after S1P for S1pr3mCherry/+ (left, n = 7) and KO (right, n = 12) neurons = two-tailed paired t-tests). (B) % in input after S1P or vehicle two-tailed paired t-test; n = cells per group). (C) The is by The was by of the from the and at n = cells. Data were with a and were measured at the indicated to 20 following a was using the of the after to indicated indicated all bars represent mean ± (D) of a single neuron in cell comparing pre- and using indicated of a single neuron in cell with was in % in after indicated µM S1P, 3 µM or for S1pr3mCherry/+ medium-diameter one-way ANOVA; n = using at % in after indicated = 100 µM for S1pr3mCherry/+ medium-diameter two-tailed paired t-test; n = Figure data 1 S1PR3 KCNQ2/3 channels to Related to Figure Download We then set out to the by which S1PR3 activity neuronal excitability using studies showed that S1P capsaicin-sensitive nociceptors by sodium and potassium et al., 2006; Li et al., 2015). We found that S1P had no such effects on S1PR3+ medium-diameter cells (Figure supplement By contrast, S1P triggered a in input (Figure with the closure of potassium channels. analysis revealed that the by S1P was by potassium (Figure Additionally, S1P significantly reduced (Figure supplement 1F; Figure in an S1PR3-dependent (Figure supplement As in Aδ neurons are primarily mediated by KCNQ2/3 potassium channels et al., et al., we that S1P may modulation of these channels. Furthermore, the of the

FRI0048 Interleukin-6 increases the number of osteoclast precursors in femur bone marrow via up-regulation of sphingosine-1-phosphate receptor 2 in inflammatory arthritic mice

Chronic lymphocytic leukemia (CLL) is characterized by the progressive accumulation of clonal B lymphocytes. Proliferation occurs in lymphoid tissues upon interaction of leukemic cells with a supportive microenvironment. Therefore, the mobilization of tissue-resident CLL cells into the circulation is a useful therapeutic strategy to minimize the reservoir of tumor cells within survival niches. Because the exit of normal lymphocytes from lymphoid tissues depends on the presence of sphingosine-1 phosphate (S1P) and the regulated expression of S1P receptor-1 (S1PR1), we investigated whether the expression and function of S1PR1 can be modulated by key microenvironment signals. We found that activation of CLL cells with CXCL12, fibroblast CD40L(+), BCR cross-linking, or autologous nurse-like cells reduces their S1PR1 expression and the migratory response toward S1P. Moreover, we found that S1PR1 expression was reduced in the proliferative/activated subset of leukemic cells compared with the quiescent subset from the same patient. Similarly, bone marrow-resident CLL cells expressing high levels of the activation marker CD38 showed a lower expression of S1PR1 compared with CD38(low) counterparts. Finally, given that treatment with BCR-associated kinase inhibitors induces a transient redistribution of leukemic cells from lymphoid tissues to circulation, we studied the effect of the Syk inhibitors piceatannol and R406 on S1PR1 expression and function. We found that they enhance S1PR1 expression in CLL cells and their migratory response toward S1P. Based on our results, we suggest that the regulated expression of S1PR1 might modulate the egress of the leukemic clone from lymphoid tissues.

<h3>Background</h3> Proliferative lupus nephritis (LN) is characterized by robust glomerular and tubulo- interstitial inflammation, sub-endothelial deposits of immunoglobulin, and increased endothelial cell permeability. Sphingosine 1- Phosphate Receptor 1 (S1PR1) has...

After induction in secondary lymphoid organs, a subset of antibody-secreting cells (ASCs) homes to the bone marrow (BM) and contributes to long-term antibody production. The factors determining secondary lymphoid organ residence versus BM tropism have been unclear. Here we demonstrate that in mice treated with FTY720 or that lack sphingosine-1-phosphate (S1P) receptor-1 (S1P1) in B cells, IgG ASCs are induced and localize normally in secondary lymphoid organs but they are reduced in numbers in blood and BM. Many IgG ASCs home to BM on day 3 of the secondary response and day 3 splenic ASCs exhibit S1P responsiveness, whereas the cells remaining at day 5 are unable to respond. S1P1 mRNA abundance is higher in ASCs isolated from blood compared to spleen, whereas CXCR4 expression is lower. Blood ASCs also express higher amounts of Kruppel-like factor (KLF)2, a regulator of S1P1 gene expression. These findings establish an essential role for S1P1 in IgG plasma cell homing and they suggest that differential regulation of S1P1 expression in differentiating plasma cells may determine whether they remain in secondary lymphoid organs or home to BM.

Sphingosine‐1‐phosphate (S1P) activation of sphingosine‐1‐phosphate receptor 1 (S1PR1) in endothelial cells (ECs) strengthens the endothelial barrier and prevents lung injury. However mechanisms regulating EC‐S1PR1 expression remains unclear. Recently, we showed that endothelial‐specific deletion of focal adhesion kinase (FAK) in mice severely disrupted lung vascular barrier function. Microarray analysis of FAK depleted cells showed a marked reduction in S1PR1 expression. Thus, we tested the hypothesis that impaired S1PR1 synthesis is responsible for defective endothelial barrier repair in EC‐FAK−/− mice. We confirmed that FAK deletion markedly reduced S1PR1 expression both at the mRNA and protein level in lungs and ECs whereas the expression of S1PR2 and S1PR3 and S1P generating enzymes, sphingosine kinases (SPHK1 and SPHK2) was not reduced. Consistently, FAK depleted ECs and mice conditionally lacking FAK in pulmonary ECs failed to anneal adherens junctions and showed impairment of endothelial barrier function under basal condition and even after S1P application. Rescuing S1PR1 expression in FAK null ECs restored barrier function in the pulmonary vessels of EC‐FAK null mice. Next, we screened for potential transcription factors binding to the S1PR1 promoter which included KLF2, Sox2, FoxP1, GATA1, GATA4 and ERG1. Intriguingly, we observed that rescuing KLF2 expression in FAK depleted ECs restored S1PR1 expression and endothelial barrier function to the levels observed in control cells. In other studies, we observed that KLF2 increased S1PR1 promoter activity while the S1PR1 cDNA lacking KLF2 binding sites failed to increase S1PR1 promoter activity, demonstrating that KLF2 can promote endothelial barrier function in the absence of EC‐FAK by transducing S1PR1. Because FAK maintains intracellular tension by balancing the activities of RhoA and Rac1 GTPases and KLF2 mRNA expression is regulated by methylation of CpG islands, we next address the possible role of increased cellular tension and DNA methyltransferases (DNMT) in suppressing KLF2 gene transcription and thereby S1PR1 expression in EC‐FAK null mice. We found that DNMT activity was elevated by 3‐fold in FAK depleted ECs in a RhoA‐dependent manner. Intriguingly, we found that depletion of FAK increased EC‐stiffness, which was associated with hypermethylation of KLF2 by DNMT3a. Interestingly, inhibition of RhoA in FAK depleted cells restored KLF2 and S1PR1 expression. Thus, our data describes the novel role of FAK in suppressing cellular tension driven‐epigenetic modification of KLF2 and S1PR1 and thereby vascular integrity.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Sphingosine 1-phosphate (S1P)/S1P receptors are involved in human liver fibrosis by action on hepatic myofibroblasts motility

Description SLE is an autoimmune disease characterized by pathogenic autoantibodies (autoAbs) produced by terminally differentiated B lineage antibody-secreting cells (ASCs). The expanded double negative 2 (DN2) B cell subset found in SLE patients gives rise to autoAb-producing ASCs. While many B cell subsets can form ASCs, it’s unclear whether ASCs derived from specific B cell populations, isolated from either SLE or healthy donors (HD), exhibit distinct functional or pathogenic attributes. Using our novel two-step culture system, we generated ASCs in vitro from resting naïve (rNav-ASCs), resting switched memory (rSW-ASCs), and DN2 (DN2-ASCs) primary human B cell subsets isolated from HDs and SLE patients (n = 3-5). Bulk RNA-sequencing revealed differentially expressed genes and enriched pathways between ASCs generated from different B cell subsets. Notably, CXCR3, which directs cells to inflamed tissues, was upregulated in SLE-derived DN2-ASCs relative to rSW-ASCs. Moreover, we identified differences between ASCs generated from phenotypically matched subsets isolated from SLE and HD, with enrichment for inflammatory signatures within SLE rSW-ASCs. Thus, while DN2 cells are thought to be major contributors to SLE pathogenesis, the gene signature of SLE rSW-ASCs suggest rSW memory B cells may also contribute to pathogenesis. We are now performing functional assays to identify key regulators controlling the distinct attributes of ASCs derived from different B subsets in SLE patients. Funding Sources Funding from P01 AI125180 and the Lupus Research Alliance Topic Categories Basic Autoimmunity (BA)

Cryo-EM in combination with biochemistry, flow cytometry and fluorescence microscopy reveals the activation of S1PR1 by CD69, a type II membrane protein, in cis to induce receptor internalization and thereby disrupt cellular responsiveness to S1P gradients.

Lymphocyte egress from lymph nodes requires the G-protein-coupled sphingosine 1-phosphate receptor-1 (S1P1). The activation antigen CD69 associates with and inhibits the function of S1P1, inhibiting egress. Here we undertook biochemical characterization of the requirements for S1P1-CD69 complex formation. Domain swapping experiments between CD69 and the related type II transmembrane protein, NKRp1A, identified a requirement for the transmembrane and membrane proximal domains for specific interaction. Mutagenesis of S1P1 showed a lack of requirement for N-linked glycosylation, tyrosine sulfation, or desensitization motifs but identified a requirement for transmembrane helix 4. Expression of CD69 led to a reduction of S1P1 in cell lysates, likely reflecting degradation. Unexpectedly, the S1P1-CD69 complex exhibited a much longer half-life for binding of S1P than S1P1 alone. In contrast to wild-type CD69, a non-S1P1 binding mutant of CD69 failed to inhibit T cell egress from lymph nodes. These findings identify an integral membrane interaction between CD69 and S1P1 and suggest that CD69 induces an S1P1 conformation that shares some properties of the ligand-bound state, thereby facilitating S1P1 internalization and degradation.

High expression of sphingosine kinase 1 and S1P receptors in chemotherapy-resistant prostate cancer PC3 cells and their camptothecin-induced up-regulation

Recent research has indicated that sphingosine 1-phosphate plays a role in allergy. This study examined the effect of allergen challenge on the expression of sphingosine 1-phosphate receptors on the eosinophils of allergic rhinitis patients, and the effect of steroid treatment on this expression. A prospective, non-randomised study. The study had three parts. Firstly, sphingosine 1-phosphate receptor expression on the eosinophils of allergic rhinitis patients and control patients was determined. Secondly, sphingosine 1-phosphate receptor expression was quantified pre- and post-allergen challenge, before and after a short course of fluticasone propionate; all patients underwent symptom scoring and peak nasal inspiratory flow measurement pre- and post-allergen challenge, both before and after steroid or saline treatment. Thirdly, the effect of sphingosine 1-phosphate on eosinophil migration was examined. The eosinophils of both allergic rhinitis patients and controls expressed sphingosine 1-phosphate1, 3, 4, and 5. Eosinophils from all allergic rhinitis patients demonstrated up-regulation in sphingosine 1-phosphate expression after allergen challenge. These changes were statistically very significant for sphingosine 1-phosphate1, 4, and 5, and moderately significant for sphingosine 1-phosphate3. Sphingosine 1-phosphate receptor expression up-regulation was abolished in the steroid-treated group after allergen challenge; however, the saline-treated group showed no change in sphingosine 1-phosphate receptor expression after allergen challenge. Peak nasal inspiratory flow scores were significantly diminished after allergen challenge prior to treatment, but not after a course of topical nasal steroids. Sphingosine 1-phosphate induced eosinophil chemotaxis was increased following allergen challenge in allergic rhinitis subjects. Local intranasal steroid therapy acts directly to block allergen-induced up-regulation of sphingosine 1-phosphate receptors on the peripheral eosinophils of allergic rhinitis patients, and this is coincident with post-challenge peak nasal inspiratory flow measurement improvements. These observations support the idea that such an increase in sphingosine 1-phosphate receptor expression is clinically relevant in allergic rhinitis, with potential consequences for eosinophil migration and survival.

Aim: Sphingosine 1-phosphate (S1P), sphingolipid derivatives are known anti-inflammatory, anti-apoptotic, and anti-oxidant agent. S1P have been demonstrated to have a role in the cardiovascular system. The purpose of this study was to understand the precise expression and distribution of S1P receptors (S1PRs) in human and rat cardiovascular tissues to know the significance and possible implementation of our experimental studies in rat models.Methods and Results: In this study, we investigated the localization of S1PRs in human heart samples from cardiac surgery department, University of Verona Hospital and rat samples. Immunohistochemical investigation of paraffin-embedded sections illustrated diffused staining of the myocardial samples from human and rat. The signals of the human heart were similar to those of the rat heart in all chambers of the heart. The immunohistochemical expression levels correlated well with the results of RT-PCR-based analysis and western blotting. We confirmed by all techniques that S1PR1 expressed strongly as compared to S1PR3, and are uniformly distributed in all chambers of the heart with no significant difference in human and rat myocardial tissue. S1PR2 expression was significantly weak while S1PR4 and S1PR5 were not detectable in RT-PCR results in both human and rat heart.Conclusion: These results indicate that experimental studies using S1PR agonists on rat models are more likely to have a potential for translation into clinical studies, and second important information revealed by this study is, S1P receptor agonist can be used for cardioprotection in global ischemia-reperfusion injury.

Objectives: Cervical squamous cell carcinoma and cervical adenocarcinoma(CESC) are the second leading cause of deaths from malignant tumors in women, while theirtherapeutic and diagnostic aims are still finited. A growing body of evidence indicatedthat sphingosine-1-phosphate receptor 2 (S1PR2) plays essential roles in the occurrenceand development about several human cancers. Nevertheless, the key mechanism and rolemechanism of S1PR2 in CESC are still unclear.Methods: We first used Tissue Expression (GTEx) and Genotypic Cancer GenomeAtlas (TCGA) data to perform pan-cancer analysis on the expression and prognosis of S1PR2,and found that S1PR2 may have a potential impact on CESC. To generate a protein-proteininteraction (PPI) network using the STRING database. The clusterProfiler package is usedfor feature-rich analysis. The Tumor IMmune Estimation Resource was used to determine theconnection between S1PR2 mRNA expression and immune infiltrates. Results: S1PR2 expression in CESC tissues was down-regulated compared withadjacent normal tissues. Kaplan-Meier analysis indicated that compared with patients withhigh expression of S1PR2, CESC patients with low S1PR2 expression had a worse prognosis.Reduced S1PR2 expression is associated with patients with high clinical stage, morehistological types of squamous cell carcinoma, and poor primary treatment outcomes. Thereceiver operating characteristic curve of S1PR2 was 0.870. Correlation analysis showedthat the mRNA expression of S1PR2 was related to immune infiltrates and tumor purity.Conclusion: Down-regulated S1PR2 expression is related to poor survival andimmune infiltration in CESC. S1PR2 is a potential biomarker for poor prognosis and as apotential target for CESC immune therapy.

Recent studies have shown that activation of liver X receptors (LXRs) attenuates the development of atherosclerosis, not only by regulating lipid metabolism but also by suppressing inflammatory signaling. Sphingosine 1-phosphate receptor 2 (S1PR2), an important inflammatory gene product, plays a role in the development of various inflammatory diseases. It was proposed that S1PR2 might be regulated by LXR-α. In the present study, the effect of LXR-α on tumor necrosis factor-α (TNF-α)-induced S1PR2 expression in human umbilical vein endothelial cells (HUVECs) was investigated and the underlying mechanism was explored. The results demonstrated that TNF-α led to an increase in S1PR2 expression and triggered a downregulation of LXR-α expression in HUVECs as well. Downregulation of LXR-α with specific small interfering RNA (siRNA) remarkably enhanced the primary as well as TNF-α-induced expression of S1PR2 in HUVECs. Activation of LXR-α by agonist GW3965 inhibited both primary and TNF-α-induced S1PR2 expression. GW3965 also attenuated S1PR2-induced endothelial barrier dysfunction. The data further showed that TNF-α induced a significant decrease in miR-130a-3p expression. Overexpression of miR-130a-3p with mimic product reduced S1PR2 protein expression, and inhibition of miR-130a-3p by specific inhibitor resulted in an increase in S1PR2 protein expression. Furthermore, activation of LXRs with agonist enhanced the expression of miR-130a-3p, and knockdown of LXR-α by siRNA suppressed miR-130a-3p expression. These results suggest that LXR-α might downregulate S1PR2 expression via miR-130a-3p in quiescent HUVECs. Stimulation of TNF-α attenuates the activity of LXR-α and results in enhanced S1PR2 expression.