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Synthesis of Lipid Mediators during UVB-Induced
Inflammatory Hyperalgesia in Rats and Mice
Marco Sisignano
1
, Carlo Angioni
1
, Nerea Ferreiros
1
, Claus-Dieter Schuh
1
, Jing Suo
1
, Yannick Schreiber
1
,
John M. Dawes
2
, Ana Antunes-Martins
3
, David L. H. Bennett
2
, Stephen B. McMahon
3
, Gerd Geisslinger
1
,
Klaus Scholich
1
*
1Institute of Clinical Pharmacology, pharmazentrum Frankfurt/ZAFES, University Hospital of the Goethe-University, Frankfurt am Main, Germany, 2Nuffield Department of
Clinical Neurosciences, John Radcliffe Hospital, University of Oxford, Oxford, United Kingdom, 3Wolfson CARD, King’s College London, Guy’s Campus, London, United
Kindgom
Abstract
Peripheral sensitization during inflammatory pain is mediated by a variety of endogenous proalgesic mediators including a
number of oxidized lipids, some of which serve endogenous modulators of sensory TRP-channels. These lipids are
eicosanoids of the arachidonic acid and linoleic acid pathway, as well as lysophophatidic acids (LPAs). However, their
regulation pattern during inflammatory pain and their contribution to peripheral sensitization is still unclear. Here, we used
the UVB-model for inflammatory pain to investigate alterations of lipid concentrations at the site of inflammation, the dorsal
root ganglia (DRGs) as well as the spinal dorsal horn and quantified 21 lipid species from five different lipid families at the
peak of inflammation 48 hours post irradiation. We found that known proinflammatory lipids as well as lipids with unknown
roles in inflammatory pain to be strongly increased in the skin, whereas surprisingly little changes of lipid levels were seen in
DRGs or the dorsal horn. Importantly, although there are profound differences between the number of cytochrome (CYP)
genes between mice and rats, CYP-derived lipids were regulated similarly in both species. Since TRPV1 agonists such as LPA
18:1, 9- and 13-HODE, 5- and 12-HETE were elevated in the skin, they may contribute to thermal hyperalgesia and
mechanical allodynia during UVB-induced inflammatory pain. These results may explain why some studies show relatively
weak analgesic effects of cyclooxygenase inhibitors in UVB-induced skin inflammation, as they do not inhibit synthesis of
other proalgesic lipids such as LPA 18:1, 9-and 13-HODE and HETEs.
Citation: Sisignano M, Angioni C, Ferreiros N, Schuh C-D, Suo J, et al. (2013) Synthesis of Lipid Mediators during UVB-Induced Inflammatory Hyperalgesia in Rats
and Mice. PLoS ONE 8(12): e81228. doi:10.1371/journal.pone.0081228
Editor: Andrej A. Romanovsky, St. Joseph’s Hospital and Medical Center, United States of America
Received September 16, 2013; Accepted October 9, 2013; Published December 9, 2013
Copyright: ß2013 Sisignano et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the DFG (German Research Association) grants SCHO817 and SFB1039 TPA08 and 09 and is part of the Europain
Collaboration, which has received support from the Innovative Medicines Initiative Joint Undertaking, under grant agreement no 115007, resources of which are
composed of financial contribution from the European Union’s Seventh Framework Programme (FP7/2007–2013) and EFPIA companies’ in kind contribution,
www.imi.europa.eu. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: Marco.Sisignano@med.uni-frankfurt.de
Introduction
Inflammatory hyperalgesia is mediated through sensory changes
in the inflamed tissue. These include mechanisms of peripheral
sensitization of sensory neurons via release of proalgesic mediators
by immune cells at the site of inflammation. Proalgesic compo-
nents comprise of cytokines, growth factors such as the nerve
growth factor NGF, reactive molecules such as nitric oxide (NO)
and reactive oxygen species (ROS) as well as oxidized lipids [1,2].
Most of these substances specifically bind to receptors at the cell
membrane of sensory neurons which activate signaling cascades,
leading to the activation of protein kinases PKA and PKC [3,4].
The activated protein kinases then phosphorylate ligand gated ion
channels, such as the transient receptor potential (TRP)-channels
or sodium channels, thereby reducing their activation threshold
[5,6].
Apart from already known components that contribute to
inflammatory hyperalgesia, many oxidized lipids have recently
been identified to either directly activate or sensitize nociceptors
by either interacting with transducer ion channels of the transient
receptor potential (TRP)-family or sodium channels, or by
recruiting immune cells to the site of inflammation. Among these
lipids, eicosanoids, oxidized linoleic acid metabolites (OLAMs) and
lysophophatidic acids (LPA) can be found. LPAs can be generated
by the secretory phospholipase D autotaxin through cleavage of
the choline group from lysophosphatidyl choline (LPC) species [7].
Recently, LPA 18:1, the OLAMs 9-, and 13-HODE as well as the
lipoxygenase product 20-HETE were identified as endogenous
activators of the vanilloid receptor TRPV1 [8,9,10]. Moreover,
the epoxylipid and 12-lipoxygenase metabolite Hepoxilin A3
(HXA
3
) has recently been identified as endogenous activators of
TRPV1 and TRPA1 and inflammatory pain [11]. Additionally,
leukotriene B4 acts as a chemoattractant for invading immune
cells during inflammation [12]. Both activation of sensory TRP-
channels and recruitment of immune cells contribute to peripheral
sensitization and inflammatory hyperalgesia and enhanced pain
perception [13,14,15].
The aim of this study was to investigate the extent to which lipid
mediators are regulated in the skin and in the downstream located
nociceptive systems, the DRGs and the spinal cord, during UVB-
induced inflammation and if their production and regulation can
PLOS ONE | www.plosone.org 1 December 2013 | Volume 8 | Issue 12 | e81228
explain the weak analgesic effects of cyclooxygenase inhibitors
during UVB-induced skin inflammation [16,17,18,19]. Therefore,
the levels of prostanoids, leukotrienes, hydroxyeicosatetraenoic
acids (HETEs), expoxylipids, HODEs and lysophophatidic acids
were determined during peripheral UVB-induced inflammatory
hyperalgesia at the time with the strongest nociceptive response.
The well described model of UVB induced skin inflammation
[20,21,22] is thought to have a high translational potential [22].
Moreover, since the number of genes for CYP enzymes differ
strongly between mice on one side and rats and humans on the
other side [23] we put a special focus on the comparison of levels
of CYP-derived lipids.
Materials and Methods
Ethics Statement
All animal experiments were performed according to the
recommendations in the Guide for the Care and Use of
Laboratory Animals of the National Institutes of Health and
approved by the local Ethics Committees for Animal Research
(Darmstadt) with the permit number F95/42. The radiation
procedure was performed under ketamine/xylazin anesthesia, and
all efforts were made to minimize suffering.
Animals and UVB-irradiation
For the irradiation procedure, male C57BL/6 N mice or male
Sprague Dawley rats were purchased from Janvier (Le Geneset-
Saint-Isle, FR) at an age of 6–8 weeks (mice) or 250–300 g (rats)
and anesthesized by intraperitoneal injection of a mixture,
containing ketamine (100 mg?kg
21
) and xylazin (10 mg?kg
21
).
Hair was removed by shaving (AeskulapHIsis GT420) and
additional treatment with a commercial available hair removal
creme (PilcaH) for five minutes. The radiation setup and
calibration device as well as the irradiation procedure were the
same as described by Bishop et al., [16] except using doses of both
1000 mJ/cm
2
and of 1500 mJ/cm
2
for the irradiation of mice and
1000 mJ/cm
2
for Sprague Dawley rats. During the procedure the
eyes of the animals were kept moist using an ointment
(Bepanthen). Shaving and hair removal were also performed in
non-irradiated control animals.
Behavioral Testing
To assess mechanical allodynia, mice were put in test cages on
an elevated grid at least 1 h prior to the measurement to allow
accommodation. Mechanical thresholds of the hind paws were
measured using a Dynamic Plantar Aesthesiometer (Ugo Basile,
Comerio, IT). A steel rod is pushed against the plantar side of the
hindpaw with linear ascending force (0–5 g over 10 s, in 0.5 g/s
intervals) until a fast withdrawal response was observed. Paw
withdrawal latencies were determined in seconds and calculated to
units of Newton (5 g = 0.049 N) and the irradiated and untreated
paws were measured alternately in intervals of 5 minutes.
Determination of Lipids from Tissue Samples by LC-MS/
MS
After dissection, tissue samples were weighted. The weight
ranged from 1 mg (L4–L6-DRGs) to 16 mg (skin) for murine
tissue and from 3 mg (L4–L6-DRGs) to 105 mg (skin) for rat
tissue. Ipsilateral DRGs of the sections L4–L6 out of one animal
were pooled. The procedure was repeated for contralateral L4–
L6-DRGs. Prior to the lipid extraction, tissue samples were
homogenized with 5 zirconium oxide grinding balls for 3 min at
30 s
21
(MM400, Retsch, Haan, Germany).
Lipid extraction and standards. Stock solutions with
2500 ng/ml of the analytes: 5,6 EET, 8,9 EET, 11,12 EET,
14,15 EET, LTB
4
, 5-S-HETE, 12-S-HETE and 15-S-HETE and
the internal standards: 5,6 EET-d11, 8,9 EET-d11, 11,12 EET-d8,
14,15 EET-d8, LTB
4
-d4, 5-S-HETE-d4, 12-S-HETE-d4 and 15-
S-HETE-d4 were prepared in methanol. Working standards were
obtained by further dilution with a concentration range of 0.1–
250 ng/ml for all analytes. For LPAs, Stock solutions with
100,000 ng/ml of all analytes (LPA 16:0, LPA 18:0, LPA 18:1,
LPA 18:2, LPA 18:3 and LPA 20:4) and the internal standard
(LPA 17:0) were prepared in methanol. Working standards were
obtained by further dilution with a concentration range of 0.5–
2500 ng/ml for all the analytes. For prostanoids, Stock solutions
with 50,000 ng/ml of all analytes (PGE
2
, PGD
2
, 6-keto-PGF
1a
,
TXB
2
and PGF
2a
) and the internal standards (PGE
2
-d4, PGD
2
-
d4, 6-keto-PGF
1a
-d4, TXB
2
-d4 and PGF
2a
-d4) were prepared in
methanol. Working standards were obtained by further dilution
with a concentration range of 0.1–1,250 ng/ml for PGE
2
, PGD
2
,
6-keto-PGF
1a
and TXB
2
and 0.4–5,000 ng/ml for PGF
2a
.
Sample pretreatment was performed using liquid–liquid extrac-
tion. Therefore, homogenated tissue was extracted twice with
600 ml of ethyl acetate (EETs, leukotrienes and prostanoids) or
500 ml of 1-butanol saturated with water (LPAs). The combined
organic phases were removed at a temperature of 45uC under a
gentle stream of nitrogen. The residues were reconstituted with
50 ml of methanol/water/butylated hydroxytoluene (BHT)
(50:50:10
23
, v/v/v) (EETs and leukotrienes), 50 ml of methanol
(LPAs) or 50 ml of acetonitrile/water/formic acid (20:80:0.0025,
v/v/v) (prostanoids) and then centrifuged for 2 min at 10,000 g,
and transferred to glass vials waiting for analysis.
Instrumentation for lipid measurement. The LC-MS/
MS system consisted of a QTrap 5500 (AB Sciex, Darmstadt,
Germany) equipped with a Turbo-V source operating in negative
electrospray ionization mode, an Agilent 1200 binary HPLC
pump and degasser (Agilent, Waldbronn, Germany), and an HTC
Pal autosampler (CTC analytics, Zwingen, Switzerland). High-
purity nitrogen for the mass spectrometer was produced by a
NGM 22-LC-MS nitrogen generator (cmc Instruments, Eschborn,
Germany).
For the chromatographic separation of EETs and leukotrienes,
a Gemini NX C18 column and precolumn were used (15062mm
inner diameter, 5 mm particle size, and 110 A
˚pore size;
Phenomenex, Aschaffenburg, Germany). A linear gradient was
used at a flow rate of 0.5 ml/min with a total run time of
17.5 min. Mobile phase A consist of water:ammonia (100:0.05, v/
v), and mobile phase B of acetonitrile:ammonia (100:0.05, v/v).
The gradient changed from 85% A to 10% within 12 min. These
conditions were held for 1 min. Then, the mobile phase shifted
back to 85% A within 0.5 min and it was maintained for 4 min to
reequilibrate the column.
The chromatographic separation of LPAs was achieved using a
Luna C18 column (2062 mm inner diameter, 3 mm) and a
precolumn of same material, (Phenomenex, Aschaffenburg,
Germany) and a linear gradient at a flow rate of 0.4 ml/min
were used. Total chromatographic time was 7 min. Mobile phase
A consisted of 50 mM ammonium acetate/formic acid (100:0.2,
v/v), and mobile phase B of acetonitrile/formic acid (100:0.2, v/
v). The gradient started with 60% A changing to 5% within 1 min
and maintained for 2.5 min. Within 0.5 min, the mobile phase
shifted back to 60% A and was held for 3 min to re-equilibrate the
column.
For the chromatographic separation of prostanoids, a Synergi
4 u Hydro-RP column (15062 mm inner diameter, 4 mm,
Phenomenex, Aschaffenburg, Germany) and a precolumn of same
Lipids in UVB-Induced Inflammatory Pain
PLOS ONE | www.plosone.org 2 December 2013 | Volume 8 | Issue 12 | e81228
material were used. Chromatographic separation was carried out
in gradient elution mode at a flow rate of 0.3 ml/min. Total run
time was 16 min. Mobile phase A consisted of water/formic acid
(100:0.0025, v/v), and mobile phase B of acetonitrile/formic acid
(100:0.0025, v/v). The linear gradient started with 90% A for
1 min and then changed to 60% A within 1 min. It was held for
1 min at 60% in phase A. Within 1 min, the mobile phase shifted
to 50% in phase A and was held for 2 min. Within 2 min, the
mobile phase shifted to 10% A and was held for 1 min.
Composition of the gradient shifted back to 90% A in one min
and it was maintained for 6 min to re-equilibrate the column.
20 ml (EETs, leukotrienes, and LPAs) or 45 ml (prostanoids) of
the extracted samples were injected into the LC-MS/MS system.
Quantification was performed with Analyst software version 1.5
(Applied Biosystems) using the internal standard method (isotope-
dilution mass spectrometry). Ratios of analyte peak area and
internal standard area (y-axis) were plotted against concentration
(x-axis), and calibration curves were calculated by least-squares
regression with 1/square concentration weighting.
Data Analysis and Statistics
All data are presented as mean 6SEM. To determine
statistically significant differences in all behavioral experiments,
ANOVA for repeated measures was used, followed by Bonferro-
ni’s post hoc correction using GraphPad Prism. For lipid
measurements comparing only two groups, Student’s t-test was
carried out. A confidence interval of 95% and a corresponding p-
value of ,0.05 were considered statistically significant.
Results
To investigate alterations in concentrations of lipid levels during
inflammatory hyperalgesia, we chose a UVB model of skin
inflammation first described for rats by Bishop et al. [16]. First we
tested two irradiation doses for BL/6 mice (1000 mJ/cm
2
and
1500 mJ/cm
2
) because of the stronger pigmentation in mice as
described previously [24]. Mice were irradiated on the plantar site
of the left hind paw, while the right hind paw was not irradiated
and was used as contralateral control. To evaluate mechanical
hyperalgesia in mice, the paw withdrawal latency was monitored
6 h –7 d after irradiation. We observed significantly decreased
mechanical thresholds of the treated mice 24 h after irradiation
with a dose of 1500 mJ/cm
2
but not with the lower dose of
1000 mJ/cm
2
. After three days the mice seemed to recover as the
mechanical thresholds slowly increased and reached baseline level
at day seven post irradiation (Fig. 1). Notably, the strongest and
most stable decrease of mechanical thresholds is within 48 h post
irradiation consistent with behavioral data from rats [16].
Therefore, at this time point skin, L4–L6-DRGs and the
corresponding section of the ipsilateral dorsal horn were dissected
and lipid concentrations were determined by LC-MS/MS. In
mice LPAs, epoxylipids and metabolites, leukotrienes and
prostanoids were measured in skin tissue, L4–L6-DRGs and the
corresponding section of the dorsal horn.
Since COX-metabolites are reliable markers for inflammatory
responses and contribute to peripheral inflammatory hyperalgesia
[1], we first quantified the concentrations of prostaglandin (PG)
D
2
, PGE
2
, PGF
2
aas well as thromboxane B
2
(TXB
2
) and 6-Keto-
PGF
1a
, the stable metabolites of TXA
2
and PGI
2
respectively, in
the tissue samples. Consistent with previous findings from human
skin [25], the concentrations of PGE
2
, were strongly elevated in
murine skin. Moreover the concentrations of TXB
2
and PGF
2
a
were significantly increased at the site of irradiation in skin samples
from irradiated mice (Fig. 2A). Surprisingly, none of the
prostanoids increased in the lumbar DRGs L4–L6 (Fig. 2B) or
in the corresponding dorsal horn sections. In the spinal tissue, 6-
Keto-PGF
1a
levels even decreased in irradiated mice as compared
to untreated mice (Fig. 2C). The concentration changes of
prostanoids in rat tissue were very similar to the murine tissue
(Figure S1).
In the group of LOX-metabolites, LTB
4
, 5-, 12-, 15- and 20-
HETE were measured. Interestingly, the concentrations of two 5-
LOX metabolites LTB
4
and 5-HETE were increased in the skin of
irradiated mice compared with the controls (Fig. 3A). Both, LTB
4
and 5-HETE, are known to be chemoattractant to neutrophils
promoting their migration to the site of inflammation [26,27,28].
In addition, in vitro 5-HETE has been shown to activate directly
TRPV1 [29]. In irradiated rat skin 5- and 15-HETE were
increased compared to the control tissue (Figure S1). In contrast to
the results in the skin, in L4–L6-DRGs of both irradiated mice and
rats only the concentrations of 12-HETE, an endogenous TRPV1
agonist [30], were increased in both tissues (Fig. 3B, Figure S1). In
corresponding sections of the dorsal horn no changes in the levels
of LOX-metabolites could be observed in mice or rats (Fig. 3C,
Figure S1).
Similar to prostanoids and leukotrienes, LPAs with different
chain lengths and saturation states 16:0, 18:0, 18:1, 18:2 were
significantly elevated in the inflamed skin as compared to the
control tissue (Figs. 3D). In contrast, LPA levels were not altered in
lumbar DRGs and the dorsal horn (Figs. 3E, 3F). The oxidized
linoleic metabolites 9- and 13-hydroxyoctadecadienoic acids (9-
and 13-HODE) are endogenous TRPV1-agonists, which are
elevated in heated rat skin [8,31]. Both 9–and 13-HODE were
elevated in the irradiated murine skin (Fig. 3G), while their levels
did not change in the DRGs or spinal dorsal horn tissue as
compared to the untreated mice (Figs. 3H, 3I). Taken together the
data show so far that there is a strong upregulation of the synthesis
of several lipid species in the skin, while there is, with the exception
of 12-HETE, no increased lipid synthesis seen in DRGs or the
spinal cord. These data suggest that lipid signaling is mainly
involved in peripheral responses to UVB irradiation and seems not
to play a major role in potential central mechanisms of UV-B-
induced hyperalgesia.
Next we addressed the question, whether or not the lipids,
which were found to be increased in irradiated skin, can evoke
mechanical allodynia. Since prostanoids, 9- and 13-HODE, as
Figure 1. Mechanical allodynia after UV-B-irradiation in BL/6N-
mice. Mice were irradiated at the plantar side of the hind paw with the
indicated UV-B-doses. Mechanical thresholds were measured up to
seven days post irradiation with the dynamic plantar test; bl: baseline.
Data represents the average 6SEM from 10 animals per group;
**p,0.01, ***p,0.001, two way repeated-measures ANOVA with
Bonferroni post-hoc test.
doi:10.1371/journal.pone.0081228.g001
Lipids in UVB-Induced Inflammatory Pain
PLOS ONE | www.plosone.org 3 December 2013 | Volume 8 | Issue 12 | e81228
well as LPA 18:1 have already been shown to cause mechanical
allodynia upon intraplantar injection in mice [1,8,9], we injected
LTB
4
, 5-HETE (both 10 mlofa6mM solution), LPA 16:0 and
LPA 18:0 (both 10 mlofa10mM solution) in hind paws of mice
and determined mechanical thresholds. Indeed, injection of LTB4
and 5-HETE caused a significant reduction of mechanical
thresholds for 5–6 hours (Figs. 4A, 4B). Moreover, LPA 18:0 but
not LPA 16:0 caused significant and long lasting reduction of
Figure 2. Prostanoid concentrations in skin, DRG and spinal dorsal horn samples from irradiated mice 48 h post irradiation.
Concentrations of prostanoids from isolated skin (A), L4–L6-DRGs (B) and spinal dorsal horn tissue (C) of mice comparing untreated (black bars) versus
irradiated skin (grey bars); 6-k-PGF1a: 6-keto-Prostaglandin F1a. Data represent mean 6SEM from six mice per group; *p,0.05, **p,0.01,
***p,0.001, student’s t-test.
doi:10.1371/journal.pone.0081228.g002
Figure 3. Concentrations of HETEs, LPAs and HODEs in skin, L4–L6-DRGs and spinal dorsal horn samples from irradiated mice.
Shown are the concentrations of LTB
4
, 5-, 12-, 15- and 20-HETE from isolated skin (A), L4–L6-DRGs (B) and the corresponding section of the dorsal
horn (C)., n.d, not detectable. (D–F) Levels of LPAs 16:0, 18:0, 18:1 and 18:2 in skin (D), L4–L6-DRGs (E) and the spinal dorsal horn (F) of irradiated
versus untreated mice. (G–I) Shown are the concentrations of 9- and 13-HODE from skin (G), L4–L6-DRGs (H) and the dorsal horn of the spinal cord (I).
Data represent mean 6SEM from six mice per group, *p,0.05, **p,0.01, student’s t-test.
doi:10.1371/journal.pone.0081228.g003
Lipids in UVB-Induced Inflammatory Pain
PLOS ONE | www.plosone.org 4 December 2013 | Volume 8 | Issue 12 | e81228
mechanical thresholds four to 24 hours after intraplantar injection
in mice (Figs. 4C). Thus, with the exception of LPA 16:0, all lipids,
which are upregulated in the skin after UVB irridation, are able to
induce mechanical allodynia in mice. These data strongly suggest
that the nociceptive response to UV-irradiation is based on several
mediators, which origin from different COX-independent meta-
bolic pathways.
The well described model of UVB induced skin inflammation
[20,21,22] is thought to have a high translational potential [22].
However, since the number of genes for CYP enzymes differ
strongly between mice on one side and rats and humans on the
other side [23], we put a special focus on the comparison of levels
of CYP-derived lipids. To compare lipid alterations in mice and
rats, we irradiated rats as previously described with a UVB-dose of
1000 mJ/cm
2
[16]. Both arachidonic acid and linoleic acid can be
converted to epoxy-lipids by CYP-epoxygenases [32,33,34].
Members of the epoxyeicosatrienoic acids (EETs), epoxy-metab-
olites of arachidonic acid, are endogenous modulators of the
transient receptor potential (TRP) ion channels TRPV4 and
TRPA1 [35,36]. Moreover, DiHOMEs, hydroxy-metabolites of
linoleic acid, formerly called ‘‘leukotoxin-diols’’ have been shown
to be produced in inflammatory leukocytes and display cytotoxic
effects by causing respiratory burst [37,38]. We determined the
concentrations of EpOMEs (epoxy-metabolites of linoleic acid)
and their dihydro-metabolites DiHOMEs, as well as EETs in skin
samples of both irradiated rats and mice. Among the group of
EETs, only 14,15-EET was detectable in these tissues. In
irradiated murine skin samples all measured lipids from this
group except 14,15-EET were significantly elevated (Fig. 5A).
Similarly, in irradiated rat skin samples the concentrations of all
detectable lipids from this group were significantly increased
(Fig. 5B). In summary, although there is a profound difference in
the number of CYP genes between rats and mice, the synthesis of
lipids metabolized through CYP enzymes in response to UVB
irradiation is very similar in both species.
Discussion
Oxidized metabolites of arachidonic acid and linoleic acid as
well as lysophosphatidic acid (LPA) 18:1 have already been shown
to activate TRP-channels in sensory neurons, leading to enhanced
thermal or mechanical hyperalgesia during inflammation [8,9,30].
Here we combined a UVB-model of local skin inflammation and
an analytical approach to investigate whether or not the
concentrations of prostanoids, leukotrienes, lysophosphatidic acids
and CYP-generated lipids are altered at the site of inflammation,
in the DRGs and in the dorsal horn of the spinal cord.
Interestingly, among all investigated lipid groups, the strongest
concentration increases were observed at the site of inflammation
(Table 1).
PGE
2
is capable of sensitizing primary afferent neurons by
binding one of its four G-protein coupled receptors (EP1-EP4)
leading to TRPV1-sensitization through the PLC-PKC pathway
(EP1) or the cAMP-PKA-pathway (EP2 and EP4) and contributing
to thermal and mechanical hyperalgesia [39,40]. In the group of
measured prostanoids, both PGE
2
and TXB
2
were found to be
elevated in irradiated skin tissue of mice, showing that cycloox-
ygenase-2 and the terminal PGE-synthases and TXA-synthase are
upregulated during peripheral inflammation. Additionally, PGF2a
was found to be elevated after irradiation, indicating that PGI-
synthase is specifically activated in mice during peripheral UVB-
induced skin inflammation. Moreover, the concentrations of LPA
18:1, 9-HODE were significantly elevated in the inflamed skin
tissue of mice. These lipids have been reported to be either direct
or indirect activators of TRPV1 [8,9] and may thus contribute to
thermal hyperalgesia and mechanical allodynia during UVB-
Figure 4. Mechanical thresholds of C57/Bl6 mice after injection
of LTB4, 5-HETE, LPA 16:0 or LPA 18:0. Shown are paw withdrawal
thresholds of wild type BL6 mice after intraplantar injection of LTB
4
(10 mlofa6mM solution, A), 5-HETE (10 mlofa6mM solution B), LPA
16:0 and LPA 18:0 (both 10 mlofa10mM solution, C) and the
corresponding vehicle (0.4% Ethanol (v/v) for LTB
4
and 5-HETE, 1%
DMSO (v/v) for LPA 16:0 and LPA 18:0). Mechanical thresholds were
monitored until 6 h post injection (LTB4 and 5-HETE) or until 30 h post
injection (LPA 16:0 and LPA 18:0). Data represent mean 6SEM from 6–
8 mice per group (LTB
4
and 5-HETE) or 7–11 mice per group (LPA 16:0
and LPA 18:0); *p,0.05, **p,0.01, ***p,0.001 two way repeated-
measures ANOVA with Bonferroni post-hoc test. Structures were
obtained from lipidmaps.org.
doi:10.1371/journal.pone.0081228.g004
Lipids in UVB-Induced Inflammatory Pain
PLOS ONE | www.plosone.org 5 December 2013 | Volume 8 | Issue 12 | e81228
induced inflammation. Fittingly, 9-HODE has been shown to be
generated in heated skin and recently HODEs were shown to be
markedly upregulated in murine skin biopsies in a post-burn pain
model of partial-thickness Injury [8,41].
Moreover, we show that LTB4, 5-HETE, LPA 16:0 and LPA
18:0 increased in inflamed skin tissue. LTB
4
, and 5-HETE caused
a significant reduction of the mechanical thresholds lasting up to
six hours post injection. Additionally, injection of LPA 18:0 but
not LPA 16:0 resulted in long lasting decreased mechanical
thresholds four to 24 hours after injection, indicating an indirect
and possibly secondary sensitizing function for LPA 18:0 in
peripheral inflammatory hyperalgesia. Several explanations for the
pronociceptive effects of the 5-LO products 5S-HETE and LTB4
have been described that may explain their effects on pain
thresholds. For example, LTB4 receptors which are expressed on
peripheral sensory neurons are thought to be involved in the
sensitization of nociceptors [42], while 5S-HETE can directly
activate TRPV1 [43]. Also, both lipids are able to activate
cytosolic phospholipase A
2
(PLA
2
) and, therefore, to stimulate the
synthesis of pronociceptive prostaglandins (i.e. PGE
2
) [44]. Finally,
LTB4 and 5S-HETE have a strong chemoattractive potential
causing neutrophil recruitment [27,28] or monocyte migration
[45].
These results may explain, why COX-inhibitors administered
even at high doses have relatively weak antinociceptive effects in
reversing thermal hyperalgesia or mechanical allodynia in
irradiated rats [16,17,18,19]. The upregulation of COX-indepen-
dent TRPV1 agonists LPA 18:1, 9-HODE and 5-HETE and
other proalgesic acting lipids, such as LTB
4
and LPA 18:0 may still
cause activation and/or sensitization of TRPV1 and subsequently
thermal hyperalgesia and mechanical allodynia even if prostanoid
synthesis is inhibited. According to these results, a selective
TRPV1-antagonist may be more effective for treating UVB-
induced inflammatory pain than cyclooxygenase inhibitors.
Epoxylipids are generated by CYP-epoxygenases of the
subfamilies 2C and 2J [34]. Interestingly almost all measured
epoxylipids and metabolites were found to be elevated in
irradiated skin of both rats and mice, leading to the conclusion
that upregulation of CYP epoxygenases 2C and 2J and possibly of
phospholipase A
2
, delivering arachidonic acid and linoleic acid as
substrate, occurs in both species during UVB-induced skin
inflammation. Notably, mice and rats are equipped with a
different number and isoform-constellation of CYP-epoxygenases
[23]. However, given the concentration differences of epoxylipids
in irradiated skin of both species, these different isoforms do not
differ in generation or preference of epoxylipids between the two
investigated species, and seem to be regulated similarly during
peripheral inflammation. This is consistent with previous findings
investigating the synthesis and regulation of epoxylipids in rats and
mice under pathophysiological conditions in the cardiovascular
context [46]. Ruparel et al. showed upregulation of CYP2J4 in
trigeminal ganglia (TGs) of rats during CFA-induced inflamma-
tory pain, and showed CYP2J4 expression in TG-neurons, thus
pointing toward a role of CYPs and CYP-derived lipids in
inflammatory pain [47]. We also found CYP-lipids in increased
concentrations during UVB-induced inflammatory pain, however
not in DRGs, but in the dorsal horn and most predominantly in
the skin. These different regulatory locations of CYP-epoxygenases
may be due to differences of the inflammatory models. It is still
unclear which cells produce CYP-derived lipids in the skin, but it is
Figure 5. Comparison of epoxylipid-levels in skin tissue from irradiated mice and rats. Shown are the concentrations of 9,10- and 12,13-
EpOME, and their metabolites 9,10–12,13-DiHOME, as well as 14,15-EET in skin from irradiated mice (A) and rats (B). Data represent mean 6SEM from
five rats and six mice per group; *p,0.05, **p,0.01, ***p,0.001, student’s t-test.
doi:10.1371/journal.pone.0081228.g005
Table 1. Overview of lipid synthesis during UVB-induced inflammatory pain in skin, DRG and dorsal horn tissue from C57Bl6/N
mice and Sprague Dawley rats.
skin DRGs dorsal horn
Prost HETE LPA CYP HODE Prost HETE LPAs CYP HODE Prost HETE LPAs CYP HODE
mouse qqqqq,q12-
HETE
,,,,,,q14,15-
EET
,
rat qqqqq,q12-
HETE
,,, qPGD
2
,, , ,
The tissue was dissected from six mice and six rats per group 48 hours post irradiation (1500 mJ/cm
2
for mice and 1000 mJ/cm
2
for rats) and lipids were extracted and
quantified with LC-MS/MS. Prost: prostanoides, arrow indicates upregulation;
,indicates no significant difference.
doi:10.1371/journal.pone.0081228.t001
Lipids in UVB-Induced Inflammatory Pain
PLOS ONE | www.plosone.org 6 December 2013 | Volume 8 | Issue 12 | e81228
possible that they are released by resident immune cells as part of
oxidative stress response during UVB-induced skin inflammation.
Recently, TRPV4 expressed in keratinocytes has been exposed
to be involved in generating UV-dependent inflammatory
hyperalgesia. However, the involvement of endogenous TRPV4
modulators has not been investigated [48]. Perhaps synthesis of
oxidized lipids as endogenous TRPV4 activators and the
subsequent neuronal and immune cell responses is a necessary
regulatory step in generating UVB-dependent inflammatory pain.
It is unclear why the observed concentration changes are most
exclusively located in the periphery and at the site of inflamma-
tion. These findings are consistent with the observation that
hyperalgesia following UVB is thought to principally be mediated
by peripheral sensitization [20].
One may speculate that the area of skin inflammation is small
and that only a part of the nociceptors in the skin are activated,
leading to comparably low nociceptive input to the dorsal horn as
compared to an invasive model of inflammation such as
carrageenan or complete freud’s adjuvant (CFA) This also leads
to minor changes in lipid concentrations in the central nervous
system.
In summary, members of LPAs, Epoxylipids, HODEs, leuko-
trienes and prostaglandins were found to be significantly increased
in skin samples from mice in a UVB-model of peripheral
inflammation. In particular, we found already known endogenous
TRPV1-agonists, such as HODEs, LPA 18:1, 5- and 12-HETE to
be increased in irradiated skin. Moreover, LPA 18:0, a lipid that
was not formerly related to inflammatory pain, was found in
increased concentrations in irradiated skin and caused long lasting
mechanical allodynia in mice when injected intraplantarly. The
high abundance of these proalgesic COX-independently generat-
ed lipids may explain, why COX-inhibitors such as ibuprofen only
show weak antinociceptive effects in UVB-induced mechanical
allodynia in rodents [16,17,18,19] and indicate that TRPV1-
antagonists may be more promising in treating UVB-induced
inflammatory pain.
Supporting Information
Figure S1 Prostanoid- and HETE-levels in skin, DRG
and spinal dosal horn tissue from irradiated SD-rats.
Concentrations of prostanoids from isolated skin (A) L4–L6-DRGs
(B) and the corresponding section of the spinal dorsal horn (C)
from irradiated (1000 mJ/cm
2
, grey bars) versus untreated rats
(black bars). (D–F) Levels of HETEs in skin (D), DRG (E) and
dorsal horn tissue (F) from rats. Data represent mean 6SEM from
five rats per group; *p,0.05, **p,0.01, student’s t-test, n.d, not
detectable.
(TIF)
Author Contributions
Conceived and designed the experiments: KS GG AAM JD DB SM.
Performed the experiments: MS JS CDS. Analyzed the data: MS CA NF
YS JS CDS. Wrote the paper: MS KS.
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