Jing Suo

Synthesis of lipid mediators during UVB-induced inflammatory hyperalgesia in rats and mice.

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.

Prostacyclin mediates neuropathic pain through interleukin 1β-expressing resident macrophages.

Summary Prostacyclin synthesized at the sites of nerve injuries regulates the accumulation of resident macrophages. These cells express IL1&bgr;, which supports the development of neuropathic pain. ABSTRACT Prostacyclin is an important mediator of peripheral pain sensation. Here, we investigated its potential participation in mediating neuropathic pain and found that prostacyclin receptor (IP) knockout mice exhibited markedly decreased pain behavior. Application of an IP antagonist to the injury site or selective IP deficiency in myeloid cells mimicked the antinociceptive effect observed in IP knockout mice. At the site of nerve injury, IP was expressed in interleukin (IL) 1&bgr;‐containing resident macrophages, which were less common in IP knockout mice. Local administration of the IP agonist cicaprost inhibited macrophage migration in vitro and promoted accumulation of IP‐ and IL1&bgr;‐expressing cells as well as an increase of IL1&bgr; concentrations at the application site in vivo. Fittingly, the IL1‐receptor antagonist anakinra (IL‐1ra) decreased neuropathic pain behavior in wild‐type mice but not in IP knockout mice. Finally, continuous, but not single administration, of the cyclooxygenase inhibitor meloxicam early after nerve injury decreased pain behavior and the number of resident macrophages. Thus, early synthesis of prostacyclin at the site of injury causes accumulation of IL1&bgr;‐expressing macrophages as a key step in neuropathic pain after traumatic injury.

Analysis of sphingolipid and prostaglandin synthesis during zymosan-induced inflammation

Sphingosine-1-phosphate (S1P) is generated through phosphorylation of sphingosine by two sphingosine kinases (SPHK-1 and -2). As extra- and intracellular messenger S1P fulfils multiple roles in inflammation such as mediating proinflammatory inputs or acting as chemoattractant. In addition, S1P induces cyclooxygenase-2 (COX-2) expression and the synthesis of proinflammatory prostanoids in several cell types. Here, we analysed in vivo the regulation of S1P level as well as potential interactions between S1P and COX-dependent prostaglandin synthesis during zymosan-induced inflammation. S1P and prostanoid levels were determined in the blood and at the site of inflammation under basal conditions and during zymosan-induced inflammation using wild type and SPHK-1 and -2 knockout mice. We found that alterations in S1P levels did not correlate with changes in plasma- or tissue-concentrations of the prostanoids as well as COX-2 expression. In the inflamed tissue S1P and prostanoid concentrations were reciprocally regulated. Prostaglandin levels increased over 6h, while S1P and sphingosine level decreased during the same time, which makes an induction of prostanoid synthesis by S1P in zymosan-induced inflammation unlikely. Additionally, despite altered S1P levels wild type and SPHK knockout mice showed similar behavioural nociceptive responses and oedema sizes suggesting minor functions of S1P in this inflammatory model.

GPVI and thromboxane receptor on platelets promote proinflammatory macrophage phenotypes during cutaneous inflammation.

Platelets are well known for their role in hemostasis but are also increasingly recognized for their supporting role in innate immune responses. Here, we studied the role of platelets in the development of peripheral inflammation and found that platelets colocalize with macrophages in the inflamed tissue outside of blood vessels in different animal models for cutaneous inflammation. Collagen-treatment of macrophages isolated from paws during zymosan-induced inflammation induced thromboxane synthesis through the platelet-expressed collagen receptor glycoprotein VI. Deletion of glycoprotein VI or its downstream effector thromboxane A2 receptor (TP) reduced zymosan-induced mechanical allodynia without altering macrophage recruitment or formation of macrophage/platelet complexes. Instead, macrophages in inflamed paws of glycoprotein VI- and TP-deficient mice exhibited an increased expression of anti-inflammatory markers and synthesized less proinflammatory mediators (prostaglandin E2 and IL6). TP expression on platelets was necessary to mediate increased prostaglandin E2 and IL6 synthesis, whereas TP expression on macrophages was sufficient to decrease the expression of the anti-inflammatory macrophage marker CD206, showing that TP activation on platelets and macrophages regulates different aspects of macrophage activation.

Neutrophils mediate edema formation but not mechanical allodynia during zymosan‐induced inflammation

Inflammatory pain is based on stimulation and sensitization of peripheral endings of sensory neurons (nociceptors) by pronociceptive mediators. These mediators can be released by resident cells, as well as invading immune cells. Although neutrophils are known to release various mediators, which can stimulate or sensitize nociceptors, the extent of their contribution to nociceptive responses is unclear. Here, we studied the contribution of neutrophils to zymosan‐induced inflammatory pain, which is characterized by an early recruitment of high numbers of neutrophils. Surprisingly, antibody‐mediated neutrophil depletion caused a complete loss of edema formation but had no effect on mechanical pain thresholds. Blockage of the interaction between neutrophils and platelets or endothelial cells using antibodies directed against CD11b and CD162 reduced neutrophil recruitment to the site of inflammation. Again, the treatment decreased zymosan‐induced edemas without altering mechanical pain thresholds. Also, HLB‐219 mice, which have five to 10 times less platelets than WT mice, showed reduced neutrophil recruitment to the site of inflammation and decreased edema sizes, whereas, again, mechanical thresholds were unaltered. The effects observed in HLB‐219 mice were relatively small and not reproduced in vWF‐deficient mice or after antibody‐mediated blockage of GPIbα. Flow chamber and transmigration assays showed that platelets were not necessary for neutrophil adhesion to endothelial cells but increased their transmigration. Taken together, zymosan‐induced mechanical allodynia is, in contrast to edema formation, independent of neutrophils, and recruitment of neutrophils is only partly influenced by interactions with platelets.

Targeting CYP2J to reduce paclitaxel-induced peripheral neuropathic pain.

Significance Chemotherapy-induced peripheral neuropathic pain (CIPNP) is a severe side effect that affects up to 80% of patients during cancer treatment. Pharmacological treatment options are poor, as CIPNP differs mechanistically from other chronic pain states. Here, we describe a mechanism by which pronociceptive oxidized lipid mediators are generated by the cytochrome P450-epoxygenase CYP2J2 in sensory neurons during CIPNP. Blocking the synthesis of these oxidized lipids with telmisartan, which we identified as a potent CYP2J2 inhibitor in a large drug repurposing screen, caused a robust reduction of paclitaxel-induced pain in mice in vivo. We therefore consider targeting CYP2J2 with telmisartan as a treatment option for chemotherapy-induced peripheral neuropathic pain in patients. Chemotherapy-induced peripheral neuropathic pain (CIPNP) is a severe dose- and therapy-limiting side effect of widely used cytostatics that is particularly difficult to treat. Here, we report increased expression of the cytochrome-P450-epoxygenase CYP2J6 and increased concentrations of its linoleic acid metabolite 9,10-EpOME (9,10-epoxy-12Z-octadecenoic acid) in dorsal root ganglia (DRGs) of paclitaxel-treated mice as a model of CIPNP. The lipid sensitizes TRPV1 ion channels in primary sensory neurons and causes increased frequency of spontaneous excitatory postsynaptic currents in spinal cord nociceptive neurons, increased CGRP release from sciatic nerves and DRGs, and a reduction in mechanical and thermal pain hypersensitivity. In a drug repurposing screen targeting CYP2J2, the human ortholog of murine CYP2J6, we identified telmisartan, a widely used angiotensin II receptor antagonist, as a potent inhibitor. In a translational approach, administration of telmisartan reduces EpOME concentrations in DRGs and in plasma and reverses mechanical hypersensitivity in paclitaxel-treated mice. We therefore suggest inhibition of CYP2J isoforms with telmisartan as a treatment option for paclitaxel-induced neuropathic pain.

Antinociceptive effects of FTY720 during trauma-induced neuropathic pain are mediated by spinal S1P receptors.

Abstract FTY720 (fingolimod) is, after its phosphorylation by sphingosine kinase (SPHK) 2, a potent, non-selective sphingosine-1-phosphate (S1P) receptor agonist. FTY720 has been shown to reduce the nociceptive behavior in the paclitaxel model for chemotherapy-induced neuropathic pain through downregulation of S1P receptor 1 (S1P1) in microglia of the spinal cord. Here, we investigated the mechanisms underlying the antinociceptive effects of FTY720 in a model for trauma-induced neuropathic pain. We found that intrathecal administration of phosphorylated FTY720 (FTY720-P) decreased trauma-induced pain behavior in mice, while intraplantar administered FTY720-P had no effect. FTY720-P, but not FTY720, reduced the nociceptive behavior in SPHK2-deficient mice, suggesting the involvement of S1P receptors. Fittingly, intrathecal administration of antagonists for S1P1 or S1P3, W146 and Cay10444 respectively, abolished the antinociceptive effects of systemically administered FTY720, demonstrating that activation of both receptors in the spinal cord is necessary to induce antinociceptive effects by FTY720. Accordingly, intrathecal administration of S1P1 receptor agonists was not sufficient to evoke an antinociceptive effect. Taken together, the data show that, in contrast to its effects on chemotherapy-induced neuropathy, FTY720 reduces trauma-induced neuropathic pain by simultaneous activation of spinal S1P1 and S1P3 receptor subtypes.

The leukotriene B4 receptors BLT1 and BLT2 form an antagonistic sensitizing system in peripheral sensory neurons

Sensitization of the heat-activated ion channel transient receptor potential vanilloid 1 (TRPV1) through lipids is a fundamental mechanism during inflammation-induced peripheral sensitization. Leukotriene B4 is a proinflammatory lipid mediator whose role in peripheral nociceptive sensitization is not well understood to date. Two major G-protein-coupled receptors for leukotriene B4 have been identified: the high-affinity receptor BLT1 and the low-affinity receptor BLT2. Transcriptional screening for the expression G-protein-coupled receptors in murine dorsal root ganglia showed that both receptors were among the highest expressed in dorsal root ganglia. Calcium imaging revealed a sensitization of TRPV1-mediated calcium increases in a relative narrow concentration range for leukotriene B4 (100–200 nm). Selective antagonists and neurons from knock-out mice demonstrated a BLT1-dependent sensitization of TRPV1-mediated calcium increases. Accordingly, leukotriene B4-induced thermal hyperalgesia was mediated through BLT1 and TRPV1 as shown using the respective knock-out mice. Importantly, higher leukotriene B4 concentrations (>0.5 μm) and BLT2 agonists abolished sensitization of the TRPV1-mediated calcium increases. Also, BLT2 activation inhibited protein kinase C- and protein kinase A-mediated sensitization processes through the phosphatase calcineurin. Consequently, a selective BLT2-receptor agonist increased thermal and mechanical withdrawal thresholds during zymosan-induced inflammation. In accordance with these data, immunohistochemical analysis showed that both leukotriene B4 receptors were expressed in peripheral sensory neurons. Thus, the data show that the two leukotriene B4 receptors have opposing roles in the sensitization of peripheral sensory neurons forming a self-restricting system.

In Vivo Availability of Pro-Resolving Lipid Mediators in Oxazolone Induced Dermal Inflammation in the Mouse

The activation and infiltration of polymorphonuclear neutrophils (PMN) are critical key steps in inflammation. PMN-mediated inflammation is limited by anti-inflammatory and pro-resolving mechanisms, including specialized pro-resolving lipid mediators (SPM). We examined the effects of 15-epi-LXA4 on inflammation and the biosynthesis of pro-inflammatory mediators, such as prostaglandins, leukotriene B4 and various hydroxyeicosatetraenoic acids and SPM, in an oxazolone (OXA)-induced hypersensitivity model for dermal inflammation. 15-epi-LXA4 (100 μM, 5 μL subcutaneously injected) significantly (P < 0.05) reduced inflammation in skin, 24 hours after the OXA challenge, as compared to skin treated with vehicle. No significant influence on the biosynthesis of prostaglandins or leukotriene B4 was observed, whereas the level of 15S-hydroxy-eicosatetraenoic acid was significantly (P < 0.05) lower in the skin areas treated with 15-epi-LXA4. In spite of the use of a fully validated analytical procedure, no SPM were detected in the biological samples. To investigate the reason for the lack of analytical signal, we tried to mimic the production of SPM (lipoxins, resolvins, maresin and protectin) by injecting them subcutaneously into the skin of mice and studying the in vivo availability and distribution of the compounds. All analytes showed very little lateral distribution in skin tissue and their levels were markedly decreased (> 95%) 2 hours after injection. However, docosahexaenoic acid derivatives were biologically more stable than SPM derived from arachidonic acid or eicosapentaenoic acid.

Myc binding protein 2 suppresses M2‐like phenotypes in macrophages during zymosan‐induced inflammation in mice

MYCBP2 is an E3 ubiquitin ligase, which is well characterized as a key element in the inhibition of neuronal growth, synapse formation and synaptic strength by regulating several signaling pathways. Although MYCBP2 was suspected to be expressed also in immune cells, to date nothing is known about its role in inflammation. We used Multi‐epitope ligand cartography (MELC), a method for multiple sequential immunohistology, to show that MYCBP2 is strongly expressed in monocyte‐derived macrophages during zymosan‐induced inflammation. We generated a myeloid‐specific knockout mouse and found that loss of MYCBP2 in myeloid cells reduced nociceptive (painful) behavior during the resolution phase (1–3 days after zymosan injection). Quantitative MELC analyses and flow cytometric analysis showed an increased number of CD206‐expressing macrophages in the inflamed paw tissue. Fittingly, CD206 and arginase 1 expression was upregulated in MYCBP2‐deficient bone marrow‐derived macrophages after polarization with IL10 or IL4. The regulation of protein expression in these macrophages by MYCBP2 varied depending on the polarization signal. The increased IL10‐induced CD206 expression in MYCBP2‐deficient macrophages was mediated by p38 MAPK, while IL4‐induced CD206 expression in MYCBP2‐deficient macrophages was mediated by protein kinase A.