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Department of Biomedical Sciences, College of Veterinary Medicine, NC State University, Raleigh, NC, 27695, USAComparative Medicine Institute, NC State University, Raleigh, NC, 27695, USA
Lipid Peroxidation Unit, Laboratory of Clinical Investigation, National Institute on Aging and Intramural Program of the National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Baltimore, MD, 21224, USA
Lipid Peroxidation Unit, Laboratory of Clinical Investigation, National Institute on Aging and Intramural Program of the National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Baltimore, MD, 21224, USANeurosciences Graduate Program, University of California San Diego, La Jolla, California 92093, USA
Lipid Peroxidation Unit, Laboratory of Clinical Investigation, National Institute on Aging and Intramural Program of the National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Baltimore, MD, 21224, USA
Lipid Peroxidation Unit, Laboratory of Clinical Investigation, National Institute on Aging and Intramural Program of the National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Baltimore, MD, 21224, USASchool of Medicine, Virginia Commonwealth University, Richmond, VA 23298, USA
Lipid Peroxidation Unit, Laboratory of Clinical Investigation, National Institute on Aging and Intramural Program of the National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Baltimore, MD, 21224, USA
Department of Biomedical Sciences, College of Veterinary Medicine, NC State University, Raleigh, NC, 27695, USAComparative Medicine Institute, NC State University, Raleigh, NC, 27695, USA
Psoriasis is characterized by intense pruritus, with a subset of psoriatic individuals experiencing thermal hypersensitivity. However, the pathophysiology of thermal hypersensitivity in psoriasis and other skin conditions remains enigmatic. Linoleic acid (LA) is an omega-6 fatty acid that is concentrated in the skin, and oxidation of LA into metabolites with multiple hydroxyl and epoxide functional groups has been demonstrated to play a role in skin barrier function. Previously, we identified several LA-derived mediators that were more concentrated in psoriatic lesions, but the role of these lipids in psoriasis remains unknown. Here, we report that two such compounds 9,10-epoxy-13-hydroxy-octadecenoate (9,13-EHL) and 9,10,13-trihydroxy-octadecenoate (9,10,13-THL) are present as free fatty acids and induce nociceptive behavior in mice but not rats. Chemically stabilizing 9,13-EHL and 9,10,13-THL, through the addition of methyl groups, we observed pain and hypersensitization in mice. The nociceptive responses suggest an involvement of the TRPA1 channel, while hypersensitive responses induced by these mediators may require both TRPA1 and TRPV1 channels. Furthermore, we showed that 9,10,13-THL-induced calcium transients in sensory neurons are mediated through the Gβγ subunit of an unidentified GPCR. Overall, mechanistic insights from this study will guide the development of potential therapeutic targets for the treatment of pain and hypersensitivity.
The Precise Structures and Stereochemistry of Trihydroxy-linoleates Esterified in Human and Porcine Epidermis and Their Significance in Skin Barrier Function: IMPLICATION OF AN EPOXIDE HYDROLASE IN THE TRANSFORMATIONS OF LINOLEATE.
Lipoxygenases mediate the effect of essential fatty acid in skin barrier formation: a proposed role in releasing omega-hydroxyceramide for construction of the corneocyte lipid envelope.
). Free (unesterified) oxylipins, which tend to be more labile and bioactive than their esterified (structural) counterparts, play key roles as signaling molecules that mediate immune activation following injury (
Beyond the classic eicosanoids: Peripherally-acting oxygenated metabolites of polyunsaturated fatty acids mediate pain associated with tissue injury and inflammation.
). Classically, oxylipins derived from omega-6 (n-6) arachidonic acid (e.g., prostaglandins, leukotrienes) have been well established for their involvement in mediating inflammation and pain (
Beyond the classic eicosanoids: Peripherally-acting oxygenated metabolites of polyunsaturated fatty acids mediate pain associated with tissue injury and inflammation.
The contribution of the endogenous TRPV1 ligands 9-HODE and 13-HODE to nociceptive processing and their role in peripheral inflammatory pain mechanisms.
Two oxidized linoleic acid metabolites, 9- and 13-hydroxyoctadecenoate (HODE), have been demonstrated to contribute to nociceptive responses in preclinical models (
The contribution of the endogenous TRPV1 ligands 9-HODE and 13-HODE to nociceptive processing and their role in peripheral inflammatory pain mechanisms.
, Tyrrell et al., 2021a). Brash and colleagues identified hydroxy-epoxide and keto-epoxide derivatives of LA as esterified lipids in skin and proposed a structural role for these oxylipins maintaining integrity of the skin water barrier (
). Loss of function mutations to the enzymes that synthesize hydroxy-epoxide and keto-epoxide derivatives of LA, Arachidonate Lipoxygenases (ALOX12B and ALOXE3) and dehydrogenases such as Short Chain Dehydrogenase/Reductase Family 9C Member 7 (SDR9C7) has been shown to cause severe impairments to skin barrier formation (
). Brash and colleagues also recently demonstrated that several LA derived oxylipins were present, esterified to ceramides, in epidermis and more abundant in skin of patients with psoriasis compared to healthy controls (
). We observed that hydroxy-epoxide and keto-epoxide derivatives of LA were elevated in psoriatic lesions and suggested that the free acid forms of these oxylipins could potentially contribute to pain or itch (
). However, the mechanisms and pathways linking these LA derivatives to pain through cutaneous-nerve axis remains unknown.
To address these gaps, we first quantified the amount of 13-hydroxy-9,10-epoxy octadecenoate (9,13-EHL) and the trihydroxy-linoleate derivative, 9,10,13-trihydroxy-octadecenoate (9,10,13-THL), in free and total pools of skin from humans and rats using liquid chromatography-tandem mass spectrometry (LC-MS/MS). We further modified these endogenous compounds via the addition of a methyl group to specific carbons to prevent degradation. Using multiple approaches, such as calcium imaging, mouse genetics, behavior, and pharmacology, we investigated the nociceptive response of these derivatives and the receptors that transduce pain response. In summary, our study suggests that free 9,10,13-THL and their analogs are potential pain mediators in mice, which advances our understanding of pain and hypersensitivity that could be associated with inflammatory skin diseases.
RESULTS
Linoleic acid derived oxylipins are present in human skin
Oxylipins are family of oxygenated derivatives that are formed from polyunsaturated fatty acids. LA-derived oxylipins have been reported to be esterified into complex lipid species in skin as part of formation of the water barrier (Figure 1a) and released by hydrolysis (Figure 1b) where they may act as signaling molecules (
Lipoxygenases mediate the effect of essential fatty acid in skin barrier formation: a proposed role in releasing omega-hydroxyceramide for construction of the corneocyte lipid envelope.
). Using LC-MS/MS, we identified several oxylipins as free acids in skin. Additionally, we used alkaline hydrolysis to liberate oxylipins esterified into complex lipids, and then using LC-MS/MS, we estimated the total (free plus esterified) pool concentrations for each oxylipin (Figure 1h-k), 9,13-EHL (Figure 1d) was approximately 20-40-fold (Figure 1h and i) more abundant than 9,10,13-THL (Figure 1e), suggesting that 9,13-EHL could serve as a substrate for 9,10,13-THL synthesis (Figure 1c). Oxylipins were up to 10-fold more abundant in the total pool as compared to the free pool, indicating skin has the potential to store and release large amounts of oxylipins as free acids for signaling (Figure 1h-j). Notably, in the free fatty acid pool, oxylipins derived from LA were at least 2-fold more abundant than the classic pain mediator, PGE2 (Figure 1k). These LC-MS/MS measurements in human skin suggest that LA derived oxylipins are well situated to play a role in signaling in skin.
Figure 1Linoleic acid (LA) metabolites are found in both the free and total pools of fatty acids in human skin. A box represents the schematic depiction of a) LA or oxylipins (i.e., 9,13-EHL and 9,10,13-THL) derived from LA are esterified into lipid membranes. b) Hydrolysis by enzymes such as phospholipases, can release esterified oxylipins, as free acids, from the membrane where they can bind to receptors or participate in molecular processes. c) Epoxide hydrolysis in a low pH environment or by epoxide hydrolases can convert 9,13-EHL to 9,10,13-THL. d and e) Free acid oxylipins can be esterified into the phospholipid bilayer. f) Proposed active sites (i.e., pharmacophores) of oxylipins are highlighted. g) Stable analogs of 9,13-EHL and 9,10,13-THL can be synthesized to prevent esterification, and/or prevent degradation of the pharmacophore. Concentrations of oxylipins measured h) 9,13-EHL, i) 9,10,13-THL, j) 9-HODE, and k) PGE2 in human skin. Data in panels h – k are presented as Mean ± Standard Deviation (SD); no statistical tests were run on these data. Each data point corresponds to one biological replicate, n=3.
Concentrations of 9,13-EHL and 9,10,13-THL are modified by inflammation
To assess the impact of inflammatory insult on oxylipin concentrations in skin, rats were injected intradermal with Complete Freund’s Adjuvant (CFA). Consistent with previous reports, pronounced swelling was observed (
); however, we did not observe changes in nociceptive behavior that were statistically significant from control (Figure 2a-c). CFA injection evoked decreases in concentrations of LA-derived oxylipins and increases in PGE2 concentration (Figure 2d-g), consistent with gene expression studies that demonstrated genes encoding enzymes capable of PGE2 synthesis were induced by inflammation, while Alox12B and Aloxe3 were not (
). Previously, it was reported that the requisite enzymes for synthesizing LA-derived oxylipins were unaffected by inflammation, however, expression of several phospholipases increased in response to inflammation (
). Since inflammation evoked significant edema in tissues, and inflammation has been demonstrated to induce expression of phospholipases, which release esterified oxylipins into the free fatty acid pool, we calculated the proportion of each LA-derived oxylipin in the free fatty acid pool, thus correcting for increased tissue volume of edema. With this correction, we found that the proportion of 9,10,13-THL and 9-HODE, in the free lipid pool, were increased by CFA injection (Figure 2h). These results are consistent with previous reports in rats where inflammation evoked increased gene expression of phospholipase and prostaglandin synthase encoding transcripts (
Figure 2Effect of inflammation on oxylipin concentrations in skin. Rats were injected (n=24 per group) with Complete Freund’s Adjuvant (CFA) a common, experimental inflammatory stimulus or PBS, intradermal. Behavioral response as indicated were measured a) ipsilateral wiping, b) bi-lateral wiping and c) scratching of the injection site (n = 24, 18, 10 per group at 4 hours, 1 day and 4 days post injection, respectively). Abundance of mediators as indicated in CFA-induced inflammation were compared to PBS injection, d) 9-hydroxyoctadecenoate (9-HODE) (**p < 0.0001 for 4 hours post-CFA and 1 day post-CFA) e) Prostaglandin E2 (PGE2) (**p = 0.0006 for 1 day post-CFA and **p < 0.0001 for 4 days post-CFA). f) 13-hydroxy-9,10-epoxy octadecenoate (9,13-EHL) (***p < 0.0001 for 4 hours post-CFA and 1-day post-CFA) and (g) 9,10,13-trihydroxyoctadecenoate (9,10,13-THL) (*p = 0.048 for 4 hours post-CFA). h) The proportion of 9-HODE (*p = 0.0220 for 1-day post-CFA), 9,13-EHL, and 9,10,13-THL (*p = 0.0184 for 4-days post-CFA) present in skin as free acid was measured at 4- and 1-day(s) after CFA injection compared to PBS. Data are presented as Mean ± SD. In Panels A – C, no significant differences were found as determined by 1-way ANOVA with a Holm-Šídák correction for multiple comparisons. Significance in Panels D – H were determined using a 2-way ANOVA with a Holm-Šídák correction for multiple comparisons. Each data point refers to one biological replicate, n≥6.
In vitro activation of sensory neurons by 9,13-EHL, 9,10,13-THL and their stable analogs.
To investigate whether 9,13-EHL, 9,10,13-THL (natural compounds) their stable analogs and putative pharmacophores evoke calcium influx in sensory neurons, we used Fura-2AM based calcium imaging to primary cultured dorsal root ganglia (DRG) neurons. The quantification of neurons responding to oxylipins were shown in Figure 3a. We used 1 μM of both natural and stable analogues based on 9,10,13 THL dose response curve (Figure 3b). We found approximately 7.0 ± 3.1% of neurons were activated in response to 9,13-EHL and around 8.0 ± 6.2 % of neurons responded to 9,10,13-THL. In addition, we tested the methylated analogs (Table 1 for specific modifications and preparations) of these natural compounds on DRG sensory neurons (
Stable analogs of 13hydroxy-9,10-trans-epoxy-(11E)-octadecenoate (13,9-HEL), an oxidized derivative of linoleic acid implicated in the epidermal skin barrier.
). These analogs were developed to provide stability by preventing oxylipins from further esterification and dehydrogenation, which in turn induce signal efficiently and effectively. As predicted, we found 8.0 ± 6.2 % of neurons responded to methylated stable analogues of 9,13-EHL: 2,2, methyl-13-hydroxy-9,10-epoxy octadecenoate (2,2M-9,13-EHL), 13 ± 3.8% of neurons responded to 13-methyl, 9,10-epoxy octadecenoate (13M-9,13-EHL), 23.8 ± 6.8% of neurons activated to 2,2,13-methyl-9,10-epoxy octadecenoate (2,2,13M-9,13-EHL). Additionally, 8.5 ± 6.3% of neurons reacted to 2-hydroxy-5,6-epoxy-hept-3(E)-ene, the proposed active pharmacophore of 9,13-EHL. Similarly, we found that neurons responded to methylated stable analogues of 9,10,13-THL: 5.4 ± 5.6% of neurons responded to 2,2-methyl-9,10,13-hydroxy octadecenoate (2,2M-9,10,13-THL) and 6.6 ± 4.5% of neurons exposed to 2,2,13-methyl-9,10,13-hydroxy octadecenoate (2,2,13M-9,10,13-THL). Finally, 7.7 ± 0.7% of neurons exposed to 2,5,6-trihdroxy-hept-3(E)-ene, the proposed active pharmacophore of 9,10,13-THL. The percentages of neurons responding to these oxylipins and oxylipin analogues are in line with known inflammatory mediators, such as 9-HODE (12.7 ± 4.2 % of DRG neurons) and PGE2 (13.1 ± 3.8% of neurons) as shown (Figure 3a). Collectively, these data indicate that both natural compounds and their stable analogues activate sensory neurons.
Figure 3Sensory neurons activation/inhibition using calcium imaging. a) Quantification of calcium imaging responses in primary culture DRG sensory neurons to vehicle (1% ethanol in 1X PBS {v/v}), 9HODE, PGE2, 9,13EHL, 9,10,13THL, stable analogues of 9,13EHL and 9,10,13THL, and their small molecule pharmacophores. All compounds were tested at a concentration of 1 μM and normalized to 100 mM KCl responses unless noted. b) Dose response curve of the endogenous mediator 9,10,13-THL, each dot represents a single coverslip, n=2. c) DRG neurons pre-incubated with vehicle or Gallein (100 μM) exposed to 9,10,13-THL and capsaicin (*p = 0.0339) as determined by a 2-tailed paired Student’s t-test. d) DRG neurons pre-incubated with vehicle or pertussis toxin (PTX, 1 ng/mL) and exposed to 9,10,13-THL and capsaicin, ns = not significant based on a 2-tailed paired Student’s t-test. e) Neurons responding to 9,10,13-THL, AITC (100 μM), and capsaicin in WT, TRPA1 KO, and TRPV1 KO mice by measuring calcium transients. Significance was determined using 2-way ANOVA with a Holm-Šídák correction for multiple comparisons. For 9,10,13-THL: **p = 0.0311 (WT vs TRPA1 KO) and **p = 0.002 (WT vs TRPV1 KO). For AITC, ***p = 0.001 (WT vs TRPA1 KO). For Capsaicin, ****p < 0.0001 (WT vs TRPV1 KO). All data is presented as Mean ± SD. Each data point represents an average of coverslips/mouse, n≥3.
In an effort to understand which type of receptor either ion channels or G-protein coupled receptor to which 9,10,13-THL binds, we used Gallein, which inhibits dissociation of the Gβγ subunits of G-protein Coupled Receptors (GPCRs), and pertussis toxin (PTX), which inhibits the functions of the Gα subunits of GPCRs, to determine which pathway 9,10,13-THL uses to evoke calcium transients. We used GPCR subunit inhibitors because other nociceptive fatty acid metabolites have been reported to work through GPCRs (
Discovery of {4-[4,9-bis(ethyloxy)-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl]-2-fluorophenyl} acetic acid (GSK726701A), a novel EP4 receptor partial agonist for the treatment of pain.
Pharmacology of grapiprant, a novel EP4 antagonist: receptor binding, efficacy in a rodent postoperative pain model, and a dose estimation for controlling pain in dogs.
). Our results indicate that 9,10,13-THL binds to a yet unknown GPCR that causes nociceptive responses through intracellular Gβγ pathways, but not through PTX mediated Gα pathways (Figure 3c and 3d, p = 0.0339 determined by a 2-tailed paired Student’s t-test). Next, we examined if these lipid mediators activate TRP channels acting downstream of these GPCR as reported earlier for other lipid mediators. Similar to other lipid mediators, we found a significant decrease in the percentage of neurons responding to 9,10,13-THL in TRPA1 (p = 0.0009) and TRPV1 KO mice (p = 0.0026). We found the expected decreases in percentage of neurons responding to AITC in TRPA1 KO mice and capsaicin in TRPV1 KO mice (Figure 3e), a 2-way ANOVA was used to determine the significant difference. Overall, our result suggests that 9,10,13-THL mediates it affects by first binding to a GPCR, leading to activation of its Gβγ subunit, which then leads to subsequent activation of TRPA1 and/or TRPV1.
Bioactive lipids and their analogues induced acute nociceptive responses in mice.
Since these compounds are found in higher abundance in patients with psoriasis and can induce calcium influx into DRG sensory neurons (
), we next examined if injecting these compounds in mice [intradermally (i.d.)] could induce pain or itch behavior. Here, we used the cheek model that could differentiate between pain and itch behavior (
) for an initial 5 minutes and over a total of 30 minutes. During the first 5 minutes, we found a significant increase in the number of wiping bouts for 9,10,13-THL (p = 0.0008), 2,2M-9,13-EHL (p = 0.0155), 13M-9,13-EHL (p = 0.0007), 2.2,13M-9,13-EHL (p ≤ 0.0001), and 2,5,6-trihydroxy-hept-3(E)-ene (p = 0.0037) compared to the vehicle injection (Figure 4a), the Kruskal-Wallis test with Dunn’s test for multiple comparisons was used to determine significance. The remaining compounds did not induce significantly different number of wiping bouts as compared to vehicle. In contrast, we found significant increase in the number of wiping bouts, compared to control, was observed for all compounds, except 9,13-EHL, (Figure 4b) for 30 minutes duration. There was no significant difference seen when comparing the wiping bouts between 9,10,13-THL and PGE2, 9-HODE, and the stabilized analogs of 9,13-EHL and 9,10,13-THL. We further compared 9,13-EHL and 9,10,13-THL to 9HODE, PGE2, and their respective stable analogues to see if there are significant differences (Table 2). Interestingly, we found most of the 13-EHL synthetic analogues are still causing pain in mice. Scratching bouts of ipsilateral directed cheek behavior was not significantly different when compared to vehicle injection (Figure 4c), the Kruskal-Wallis test with Dunn’s test for multiple comparisons was used to determine significance.
Figure 4Nociceptive responses to oxylipins in control and knockout mice. a) The cheek wipes were measured in the first 5 minutes, b) and 30 minutes. Significance was determined using the Kruskal-Wallis test with Dunn’s test for multiple comparisons; 9,13-EHL vs Vehicle, 9-HODE, and 2-hydroxy-5,6-epoxy-hept-3-(E)-ene is not significant; other p-values when compared against 9,13-EHL are as follows: vs PGE2 **p = 0.059, vs 2,2M-9,13-EHL *p = 0.0230, vs 13M-9,13-EHL **p = 0.0023, vs 2,2,13M-9,13-EHL *p = 0.023, vs 9,10,13-THL *p =0.0282. The same statistical tests were used to compare 9,10,13-THL vs Vehicle **p = 0.0033, vs 9-HODE was not significant, vs PGE2 was not significant, vs 2,2M-9,10,13-THL *p = 0.0282, vs 2,2,13M-9,10,13-THL was not significant, and vs 2,5,6-trihydroxy-hept-3-(E)-ene was not significant. c) quantification of ipsilateral directed cheek scratching bouts following injection of vehicle (1% ethanol in 1X PBS {v/v}) and various mediators (100 μg/ 20 μL i.d.). No significant differences were found in the number ipsilateral cheek directed scratching bouts using the same Kruskal-Wallis test with Dunn’s correction for multiple comparisons. d) Total time spent on paw directed nociceptive behavior response following intraplantar injection of over 5 minutes. The significance was determined using a Kruskal-Wallis test with a Dunn’s test for multiple comparisons: vehicle vs 9-HODE **p = 0.0090, vehicle vs PGE2 **p = 0.0013, vehicle vs 9,10,13-THL **p = 0.0029. e) Total number of wipes quantified following vehicle or 100 μg/ 20 μL 9,10,13-THL (i.d.) in WT, TRPA1 KO, and TRPV1 KO mice. Significance was determined using a 1-way ANOVA with the Holm-Šídák correction for multiple comparisons: vehicle vs TRPA1 KO was not significant, vehicle vs TRPV1 KO ***p = 0.0004, vehicle vs WT ****p < 0.0001, TRPA1 KO vs TRPV1 KO ****p < 0.0001, TRPA1 KO vs WT ***p = 0.0007, TRPV1 KO vs WT ***p = 0.0007. f) Time kinetics of behavior of 9,10,13-THL post injection in 5-minute intervals. Significance was determined using the same parameters as panel e: for the 5-minute bin, no significant differences were found between the number of ipsilateral cheek directed wipes in the vehicle treated mice and the TRPA1 KO mice treated with 9,10,13-THL. Similarly, no significant differences were found between the 9,10,13-THL treated TRPA1 KO and 9,10,13-THL treated TRPV1 KO mice. The other significance values are as follows: Vehicle vs WT **p = 0.0040, Vehicle vs TRPV1 KO *p = 0.0104, WT vs TRPA1 KO **p = 0.0035, WT vs TRPV1 KO *p = 0.0104. Data is presented as Mean ± SD and each data point in panels a – e correspond to one biological replicate, n≥5.
Table 2Calculated p – values for specific comparisons related to figure 4b. Significance was determined using the Kruskal-Wallis test with an uncorrected Dunn’s test for multiple comparisons. The Kruskal-Wallis test was selected because of the non-parametric nature of the behavioral results.
We next performed intraplantar injections of vehicle, PGE2, 9-HODE, and 9,10,13-THL to examine if these compounds evoke pain responses mediated through the DRG. From this point on, we chose to focus on 9,10,13-THL because of the two natural ligands (the other being 9,13-EHL), 9,10,13-THL produced significantly more wiping responses in the cheek assay. We found that all three oxylipins induced significantly higher nociceptive (biting/licking, flinching, guarding, and lifting) response compared to the vehicle (Figure 4d): PGE2 (p = 0.0011), 9-HODE (p = 0.0006), and 9,10,13-THL (p = 0.0002). Surprisingly, in rats none of the oxylipins evoked a nociceptive response (Figure 5 a-d). To identify the TRP channels by which 9,10,13-THL causes pain, we performed the cheek assay in TRPV1 and TRPA1 KO mice and scored their behavior over 30 minutes (Figure 4e). We found a significant decrease in the number of ipsilateral cheek wipes in TRPA1 (p < 0.0001) and TRPV1 KO (p = 0.0015) mice as compared to the controls. The number of ipsilateral directed cheek wipes in the TRPA1 KO mice was not significantly different from vehicle-injected mice. There was also a significant increase in the number of ipsilateral cheek wipes in the TRPV1 KO mice as compared to the TRPA1 KO mice (p = 0.0005), the Kruskal-Wallis test with Dunn’s test for multiple comparisons was used to determine significance (Figure 4d-f). We found that a majority of the cheek wipes occurred during the first 5-minutes post-injection (Figure 4f, time kinetics), and that loss of TRPA1 and TRPV1 resulted in a significant decrease in the number of cheek wipes during this first 5-minute period.
Figure 5Nocifensive response in rats following injection of Vehicle, 9,10,13-THL, and PGE2. The number of a) ipsilateral wipes, b) ipsilateral and contralateral wipes, c) combined total number of wipes from panels A and B, and d) number of scratching bouts following injection of vehicle, 9,10,13-THL, or PGE2. No significant differences were found using a 1-way ANOVA with a Holm-Šídák correction for multiple comparisons. Data is presented as Mean ± SD and each data point corresponds to one biological replicate, n=8.
TRPA1 and TRPV1 are required for 9,10,13-THL-induced thermal sensitization
Since 9,10,13-THL is capable of inducing acute nociceptive behaviors, we further examined, if 9,10,13-THL can induce thermal (hot and cold) or mechanical hypersensitivity. For heat hypersensitivity, we found that following intraplantar injection of 9,10,13-THL, wild type mice demonstrated significantly faster withdrawal times (p = 0.0007) when placed on a +50°C hot plate as compared to vehicle injected mice (Figure 6a). This response was lost when comparing vehicle and 9,10,13-THL injected TRPA1 KO mice, further validating our acute behavior wherein wiping responses are diminished in TRPA1 KO mice. As expected, we found no difference in behavior response between vehicle and 9,10,13-THL injected TRPV1 KO mice. Further, we did not find any significant differences in withdrawal latency in TRPA1/V1 double KO mice.
Figure 6Peripheral sensitization induced by 9,10,13-THL in normal and knockout mice. a) Changes in withdrawal latency on hot plate after 5 minutes incubation of intraplantar injection of 9,10,13-THL (327 μM in 10 μL). For WT vehicle vs WT 9,10,13-THL **p = 0.0047, for WT 9,10,13-THL vs everything else ****p < 0.0001. b) Changes in withdrawal latency on cold plate after 5 minutes incubation of intraplantar injection of 9,10,13-THL (327 μM in 10 μL). For WT vehicle vs WT 9,10,13-THL *p = 0.0498, for WT 9,10,13-THL vs everything else ****p < 0.0001. c) Changes in withdrawal force in the mechanical assay after 5 minutes incubation of intraplantar injection of 9,10,13-THL (327 μM in 10 μL). No significant differences were found within groups when comparing treatment (vehicle vs 9,10,13-THL) or between strains. d) Changes in withdrawal latency in the dry ice assay after 5 minutes of incubation of intraplantar injection of 9,10,13-THL (327 μM in 10 μL). No significant differences were found within groups when comparing treatment (vehicle vs 9,10,13-THL) or between strains. Data is presented as mean ± SD and each data point corresponds with a single biological replicate, n≥6. Significance was determined by comparing vehicle injected versus 9,10,13-THL injected behavior responses within each genotype. Significance was determined using a Mann-Whitney U-test when comparing within strains. A 2-way ANOVA with the Holm-Šídák correction for multiple comparisons was used when testing for significance between strains and treatments; ns = not significant.
) (Figure 6c). For dry ice test, we did not find a significant difference between vehicle injected and 9,10,13-THL injected wild type and their KO littermates (TRPA1 KO, TRPV1 KO or TRPA1/V1 DKO mice). However, we did see a significant difference in withdrawal latency in wild type mice injected with 9,10,13-THL versus vehicle on the 5 °C cold plate (p = 0.0357). This difference in withdrawal latency was not seen when comparing the vehicle and 9,10,13-THL injected TRPA1 KO, TRPV1 KO, or TRPA1/V1 DKO mice (Figure 6b). We found no significant differences in any groups either injected with vehicle and 9,10,13-THL (Figure 6d) for mechanical sensitivity. A Mann-Whitney U-test when comparing within strains. A 2-way ANOVA with the Holm-Šídák correction for multiple comparisons was used when testing for significance between strains and treatments.
DISCUSSION
Thermal and mechanical sensitivity have been reported in psoriatic skin lesions of individuals to which specific endogenous mediators and mechanisms are unknown. Here, we: 1) characterized the abundance and regional localizations of 9,13-EHL and 9,10,13-THL in human and rat skin, 2) investigated the activities of these endogenous lipids, as well as methylated stable analogs, and their putative pharmacophores in sensitizing DRG neurons and eliciting pain-related behaviors, 3) we found that the 9,10,13-THL mediated effect is GPCRβγ-dependent manner. The reduction in thermal sensitivity using TRPA1/TRPV1 knockout mice suggests that these channels are acting downstream of this GPCR. Collective findings confirm 9,13-EHL and 9,10,13-THL are present as free and esterified skin lipids and introduce free 9,10,13-THL as an endogenous lipid autacoid that could play a role in mediating acute pain responses in mice.
Structure-function relationships and metabolism of 9,13-EHL and 9,10,13-THL
The effects of 9,10,13-THL and related compounds in mice had a rapid onset and short duration, with >80% effects observed in first 5 min. These acute/hyper-acute pain-related responses observed in mice suggest free acid oxylipins are labile and are rapidly inactivated in in vivo conditions. Two possible mechanisms for rapid inactivation include acylation and esterification back into lipid membranes and dehydrogenase-induced conversion of the hydroxyl to ketone moieties. These hypothetical mechanisms could also explain why 9,13-EHL produces little-to-no behavior response despite activating a similar number of neurons as compared to 9,10,13-THL in the calcium imaging assay. These inactivation pathways are not present ex vivo in the calcium imaging set up but are present in vivo in the cheek assay.
To determine why 9,10,13-THL induced significantly more cheek wipes than 9,13-EHL, we used a targeted synthetic chemistry approach using free 9,13-EHL and 9,10,13-THL as bio templates for designing three classes of stable analogs: (1) Addition of 2,2-dimethyl moiety to prevent re-acylation/esterification, effectively trapping lipids in the free acid pool; 2) methyl addition to block dehydrogenation of the hydroxyl moiety, or 3) both (
Stable analogs of 13hydroxy-9,10-trans-epoxy-(11E)-octadecenoate (13,9-HEL), an oxidized derivative of linoleic acid implicated in the epidermal skin barrier.
). Our in vitro calcium imaging and behavior assay results demonstrate that analogs generally maintained activities of free acids and that several of the stable analogs induced more robust behavior responses than the unmodified free acid counterpart. There were no observed differences in bioactivity between 9,10,13-THL and its stable analogs. We proposed two different possibilities to account for these findings. Firstly, free 9,13-EHL is rapidly inactivated via either dehydrogenation and/or re-esterification into a lipid membrane. This hypothesis is supported by the significant increase in behavior response of the methylated derivates. A second possibility is that 9,13-EHL may be converted into 9,10,13-THL which then mediates a nociceptive response.
These LA derivates have similar functional group placements as two arachidonic acid derivates: hepoxilin B3, which also contains an epoxy and hydroxy moiety (
), and its trihydroxy derivative, referred to as trioxilin B. Similar to LA derivatives in the present study, these compounds are produced in the epidermis (
). Further, these compounds are also incorporated into the phospholipid membrane in cells within psoriatic lesions. Unlike these LA derivatives, hepoxilin B3 has only been shown to potentiate algesic responses when produced in the spinal cord (
). Since hepoxilin B3 is unstable and quickly transformed into its trihydroxy derivative, it seems likely that 9,13-EHL is hydrolyzed into 9,10,13-THL, its trihydroxy derivative through a similar pathway. More research is needed to determine if 9,10,13-THL is produced via the same pathway as trioxilin B3 due to their similarity in functional groups.
Our in vitro calcium imaging and behavioral findings indicate that 9,10,13-THL is the more active metabolite in this family as compared to 9,13-EHL. Mammalian epoxide hydrolase is an enzyme in the skin that is capable of converting 9,13-EHL into 9,10,13-THL, which may generate sufficient amounts of 9,10,13-THL to induce the biological activity in conferring pain (
). Our observations are further supported by results from 2,2M-9,13-EHL, 13M-9,13-EHL, and 2,2,13M-9,13-EHL, which indicate that 9,13-EHL alone is quickly taken out of the free pool, as modifying 9,13-EHL was required to induce behavior responses similar to 9,10,13-THL following injection. Future experiments are needed to identify the expression and functional activities of epoxide hydrolase in normal and psoriatic skin.
Free 9,10,13-THL as an autacoid mediator of pain and hypersensitivity responses
We observed that LA-derived oxylipins were most concentrated as esterified lipids, consistent with their proposed structural role in skin (
The Precise Structures and Stereochemistry of Trihydroxy-linoleates Esterified in Human and Porcine Epidermis and Their Significance in Skin Barrier Function: IMPLICATION OF AN EPOXIDE HYDROLASE IN THE TRANSFORMATIONS OF LINOLEATE.
). However, bioactive oxylipins are classically ascribed to free acids and we observed an increased proportion of 9, 10,13-THL as free acid in skin, after CFA injection. Previously, we observed that 9,13-EHL is elevated in the free pool of human psoriatic skin lesions, and that free 9,13-EHL evokes CGRP release only in a low pH environment (
). Therefore, we reasoned that an acid-derived trihydroxy derivative of linoleic acid, rather than 9,13-EHL itself, may be responsible for the observed sensitization to CGRP release. Consistent with this hypothesis, cheek injection of free 9,10,13-THL, but not 9,13-EHL, in mice evoked acute nociceptive, but not pruritic, behaviors. Interestingly, the effect of 9,10,13-THL on spontaneous nociceptive behavior was not significantly different than PGE2, a classic oxidized lipid mediator of pain (
The contribution of the endogenous TRPV1 ligands 9-HODE and 13-HODE to nociceptive processing and their role in peripheral inflammatory pain mechanisms.
). Furthermore, injection of 9,10,13-THL into the mouse hind paw evoked a similar number of spontaneous nociceptive behaviors compared to PGE2 and 9-HODE. Together, these findings suggest that preformed 9,13-EHL and 9,10,13-THL act as structural lipids in membranes but may be released by lipases, forming free acids that contribute to acute pain responses by activating sensory neurons following injury or inflammation. However, caution should be taken in interpreting these cheek assay results, as we were unable to find the nociceptive behavioral effects in rats as compared our mice data, even using concentrations of PGE2 roughly three times that has been reported to show a change in behavior sensitization (
). Additionally, we found that injection of complete Freund’s Adjuvant (CFA) did not induce a significant number of ipsilateral cheek directed wipes in rats as compared to vehicle. The report from Klein et al (
Facial injections of pruritogens or algogens elicit distinct behavior responses in rats and excite overlapping populations of primary sensory and trigeminal subnucleus caudalis neurons.
). All these results suggest there is a species-specific difference for oxylipin-induced facial nociception.
Identifying 9,10,13-THL, ex vivo, as a potential oxylipin involved in pain allowed us to focus on in vivo efforts interrogating the molecular mechanisms through which oxylipins impact nociceptive responses. We demonstrated that 9,10,13-THL induced spontaneous nociceptive behavior via activation of TRPA1 channel. Interestingly, TRPA1 has been demonstrated to mediate their responses to other lipid autocoids including prostaglandins and cysteine modifying agent (
Cruz-Orengo L, Dhaka A, Heuermann RJ, Young TJ, Montana MC, Cavanaugh EJ, et al. Cutaneous nociception evoked by 15-delta PGJ2 via activation of ion channel TRPA1. Mol Pain 2008;4:30. del Camino D, Murphy S, Heiry M, Barrett LB, Earley TJ, Cook CA, et al. TRPA1 contributes to cold hypersensitivity. J Neurosci 2010;30(45):15165-15174.
). Moreover, 9,10,13-THL appeared to evoke hypersensitivity to noxious heat and noxious cold via a TRPA1-dependent mechanism. However, it is unlikely that 9,10,13-THL signals directly through TRPA1 as its effect was blocked by the Gβγ inhibitor, Gallein, making it is likely that 9,10,13-THL affecting pain via GPCR activation, which intracellularly activates TRPA1.
We found that 9,10,13-THL induces hypersensitive responses to noxious heat and cold temperatures. TRPA1 mediates noxious cold (del Camino et al., 2010,
). Interestingly, our results indicate that 9,10,13-THL requires both TRPA1 and TRPV1 for sensitization possibly through the formation of heteromeric complexes that have been shown previously to play a role in pain hypersensitivity (
). This hypothesis is supported by the following evidence: 1) 9,10,13-THL behavior and calcium imaging responses are lost in TRPA1, but not in TRPV1 KO mice and 2) 9,10,13-THL hypersensitivity to noxious heat is lost in TRPA1 KO mice, even though these mice have intact TRPV1. Further, we found sensitivity to noxious cold temperatures that was lost in both TRPA1 and TRPV1 KO mice, despite TRPV1 KO mice having functional TRPA1. This result was unexpected and adds to our hypothesis that 9,10,13-THL sensitizes noxious thermal responses using both TRPA1 and TRPV1 acting downstream of GPCR activation. The overall pattern seen in our results are similar to what has been demonstrated as the mechanism behind bradykinin and PGE2’s ability to cause hypersensitivity to noxious heat (
). Several questions arise from our study that needs to be determined in the future; 1) identification of GPCR acting upstream of TRP channels, 2) intracellular mechanism that regulates the TRP channels activity that helps facilitate these acute pain behavior and hypersensitization.
Approximately 73% of psoriasis patients experience burning/heat discomfort in their psoriasis plaques (
). In both instances, these sensations are reported to be independent of an external stimulus. Based on our results here, we hypothesize that these hot and cold sensations could potentially be due to release of 9,10,13-THL from lipid membranes. These hot/burning/cold sensations were not rated highly using the pain qualities assessment scale (
). While these sensations might not be noxiously painful, they are known to contribute to significant changes in sleep, mood, work ability, and interpersonal relationships experienced by psoriasis patients (
). Therefore, understanding the underlying mechanisms leading to these sensations is worthwhile for improving quality of life in people with psoriasis and should be addressed in future work.
Our results with Gallein and pertussis toxin indicate that 9,10,13-THL activates TRPA1 intracellularly through a GPCR’s Gβγ subunits, but not the Gα subunit. Biased activation of downstream effects from GPCR-ligand interactions has been reported (
); therefore, 9,10,13-THL preferentially activating sensory neurons through Gβγ mediated processes is not improbable. DRG neurons do express relatively high levels of the Gzα GPCR subunit (
) including DRG sensory neurons expressing TRPA1. Overall, our results indicate that 9,10,13-THL requires dissociation of the Gβγ subunits and does not use PTX sensitive Gα subunits. Future work is needed to identify the GPCR to which 9,10,13-THL binds causing neuronal activation and the specific stereoisomers of 9,10,13-THL that are involved.
Overall, our results demonstrate an attenuation of calcium influx and acute pain behavior in mice lacking TRPA1 channels in response to 9,10,13 THL. Furthermore, 9,10,13-THL sensitizes responses to noxious heat and cold using both TRPA1 and TRPV1 channels. Finally, we identified an active region of these compounds, which could be used to generate new antagonists for the receptor of these endogenous oxylipins. Future work should be aimed at identifying the receptor acting upstream of TRP channels, providing novel therapeutic leads for treating pain.
MATERIALS & METHODS
Oxylipin Measurements in Human Skin
Full thickness skin was collected from axilla, plantar aspect of the foot, and lumbar aspect of the dorsum of human tissue donors acutely after death and immediately frozen at -80 °C until processing using the methods described earlier by Ramsden et al (
). Aliquots were then purified using solid phase extraction and free and total (esterified + free) oxylipins were measured using liquid chromatography tandem mass spectrometry as reported (
We obtained approval from the NIH Office of Human Subjects Research and Protection (OHSRP) to obtain and analyze fully de-identified postmortem donor skin specimens provided the Washington Regional Transplant Community (WRTC, Falls Church, Virginia). All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) at North Carolina State University (IACUC # 19-167-B) and National Institutes of Health (IACUC # 1402-17). Mice and rats were housed in standard conditions with a 12/12-hour light dark cycle with ad libitum access to food and water. TRPV1 knockout (KO) (Stock No. 003770), TRPA1 KO (Stock No. 006401) and C57Bl6/J (Stock No. 000664) mice were ordered from the Jackson Lab. TRPV1 and TRPA1 knockout mice were maintained on the C57Bl6/J background.
Chemicals
All compounds were stored in ethanol at -80 °C. Ethanol was evaporated under a steady stream of nitrogen and the compounds were re-dissolved into 1X PBS containing 1% ethanol (v/v) immediately before injection. PGE2 (cat. # 14010); 9-HODE (cat. # 34800); 9,13-EHL and 9,10,13-THL were purchased from Cayman Chemical. Stable analogues of racemic 9,13-EHL and 9,10,13-THL were synthesized as reported (
Stable analogs of 13hydroxy-9,10-trans-epoxy-(11E)-octadecenoate (13,9-HEL), an oxidized derivative of linoleic acid implicated in the epidermal skin barrier.
All experimenters were blind to the structure of the compounds when performing behavior assays. Each compound was injected into the right cheek (100 μg in 20 μL) using sterile insulin needles. Nociceptive behavior responses in mice were assed using the cheek model after being recorded for 30 minutes (
). In this model, painful nociceptive inputs elicit a wiping response with the forepaw(s) directed at the ipsilateral cheek while nocifensive scratching inputs elicit a scratching behavior performed by the hindpaw directed at the ipsilateral cheek.
Mouse Hindpaw Behavior:
Mice were placed in a 4 in x 4 in Plexiglas chamber on a clear Plexiglas pane above a camera. Mice were recorded for 5 minutes. Following recording, mice were used for one of the following behavior tests: +50 °C hot plate, crushed dry ice (
Sprague Dawley rats (Charles River) weighing 250-300g were placed in a 30cm x 30cm plastic testing box (with a camera mounted above) for 1 hour per day for 3 days for habituation to testing conditions (n = 24). Rats were then anesthetized using sevoflurane and injected with 50μl of Complete Freund’s Adjuvant (CFA) or PBS intradermally into the cheeks. Rats were returned to their home cage and observed for up to 4 days post injection. At 4 hours, 1 day, and 4 days post-injection rats were placed in the testing box and recorded for 30 min. Wiping and scratching were quantified as reported (
). Six, eight and 10 rats from each group were euthanized at 4 hours, 1 day and 4-days, respectively. Cheek skin from the injection site was weighed and stored at -80C. Free and total pool oxylipins concentrations in skin were measured as described above for human skin.
Rat Fatty Acid Metabolite Mediator Injections
To broaden our understanding of the nociceptive behavior evoked by oxylipins, we repeated cheek injection experiments in male, Sprague Dawley rats (Charles River). Rats were habituated to testing boxes as described above. Rats were restrained by swaddling with a towel and injected, intradermally with 20 μL of either 9,10,13-THL (100 μg, n=8), PGE2 (100 μg, n=8) or vehicle (1% ethanol in saline v/v, n=8). After injection, rats were placed in testing boxes and recorded for 30 min. Wiping and scratching were quantified as reported (
). All experimenters were blind to the structure of the compounds when performing calcium imaging. In the figures, each data point corresponds to a single biological replicate.
Statistical analysis
Statistical analysis was performed using GraphPad Prism (version 9.0 and higher). Data was tested for normality using the Shaprio-Wilk test. Any data set that fell on a normal distribution was tested for significance using Student’s t-tests or a 1-way ANOVA with a Holm-Šídák correction for multiple comparisons. Data sets falling on a non-normal distribution were tested for significance using the Mann-Whitney U-test or the Kruskal-Wallis test with a Dunn’s test for multiple comparisons. Two-way ANOVA with a Holm-Šídák correction for multiple comparisons was used for the rat time course studies found in Figure 2. Samples were assumed to have similar standard deviations.
The contribution of the endogenous TRPV1 ligands 9-HODE and 13-HODE to nociceptive processing and their role in peripheral inflammatory pain mechanisms.
The Precise Structures and Stereochemistry of Trihydroxy-linoleates Esterified in Human and Porcine Epidermis and Their Significance in Skin Barrier Function: IMPLICATION OF AN EPOXIDE HYDROLASE IN THE TRANSFORMATIONS OF LINOLEATE.
Cruz-Orengo L, Dhaka A, Heuermann RJ, Young TJ, Montana MC, Cavanaugh EJ, et al. Cutaneous nociception evoked by 15-delta PGJ2 via activation of ion channel TRPA1. Mol Pain 2008;4:30. del Camino D, Murphy S, Heiry M, Barrett LB, Earley TJ, Cook CA, et al. TRPA1 contributes to cold hypersensitivity. J Neurosci 2010;30(45):15165-15174.
Discovery of {4-[4,9-bis(ethyloxy)-1-oxo-1,3-dihydro-2H-benzo[f]isoindol-2-yl]-2-fluorophenyl} acetic acid (GSK726701A), a novel EP4 receptor partial agonist for the treatment of pain.
Stable analogs of 13hydroxy-9,10-trans-epoxy-(11E)-octadecenoate (13,9-HEL), an oxidized derivative of linoleic acid implicated in the epidermal skin barrier.
Facial injections of pruritogens or algogens elicit distinct behavior responses in rats and excite overlapping populations of primary sensory and trigeminal subnucleus caudalis neurons.
Pharmacology of grapiprant, a novel EP4 antagonist: receptor binding, efficacy in a rodent postoperative pain model, and a dose estimation for controlling pain in dogs.
Beyond the classic eicosanoids: Peripherally-acting oxygenated metabolites of polyunsaturated fatty acids mediate pain associated with tissue injury and inflammation.
Lipoxygenases mediate the effect of essential fatty acid in skin barrier formation: a proposed role in releasing omega-hydroxyceramide for construction of the corneocyte lipid envelope.
This work was funded by startup from the NC State, NIAMS/NIH (AR077692) and gifts provided by JMD to SKM. CER, AFD, JRJ, GSK, and KMM were supported by the intramural research program of the National Institute on Aging and the National Institute of Alcohol Abuse and Alcoholism, National Institutes of Health.
Disclosures: The NIA (NIH) has claimed intellectual property related to stable analogs of oxylipins (PCT/US2018/041086), with C.E.R. and G.S.K. named as inventors. All other authors declare that they have no competing interests.
AUTHOR CONTRIBUTIONS
Conceptualization: SKM, CER, and JMD; Experimental Design: SKM, CER, JJW, & AFD; Supervision & Funding: SKM & CER; Experiment & Data Analysis Performed: JJW, AFD, JRJ, GSK, KMM, CER, and SKM; Manuscript draft and Review: JJW, AFD & SKM.
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