Kombinierte anti-inflammatorische Effekte des Endocannabinoid-Systems und Cortisol in arthritischem Gewebe - eine nicht neuronale Rolle der Endocannabinoide
Zusammenfassung der Projektergebnisse
In this project, we aimed to clarify the non-neuronal role of endocannabinoids together with cortisol to combat chronic inflammation in rheumatoid arthritis (RA). Our first goal was to identify and quantify endocannabinoids produced by RA synovial fibroblasts (RASF) under basal and stimulated conditions. In a next step, we determined endocannabinoid receptor levels under basal and stimulated conditions and found RASF to express CB1, CB2, TRPV1, TRPA1, GPR55 and GRPR18 protein. Of note, CB1, CB2, and TRPV1 were up-regulated by TNF treatment while GPR18 was up-regulated only by interferon gamma. The most pronounced effect was detected with TRPA1 which was strongly increased by TNF. Next, we investigated the functional impact of GPR18 and GPR55 activation under TNF-stimulated conditions. We found that N-arachidonyl glycine (GPR18 agonist) and lysophosphatidylinositiol (GPR55 agonist) reduced IL-6, IL-8 and MMP3 production by RASF. Interestingly, this was not mediated by GPR18 or GPR55 since the antagonist O-1918 was not able to inhibit the effect. In addition, we assessed the anti-inflammatory effect of anandamide and found that this endocannabinoid mediates its effects by activating/desensitizing TRPA1 and TRPV1 ion channels. We next aimed to elucidate the mechanism by which cannabinoids exert their antiinflammatory effect. We postulated that CB1 switches its g-protein coupling from the inhibitory Gαi subunit to the calcium mobilizing Gq subunit. We found that changes in gprotein coupling are not responsible for the anti-inflammatory effect of the synthetic cannabinoid WIN55,212 on cytokine production by RASF. We rather found that WIN55,212 mediates its effect mainly by activating/desensitizing TRPV1 and TRPA1 ion channels although CB2 was also involved in its anti-inflammatory effect. Since cannabinoids are known modulators of intracellular calcium levels, we investigated the impact of several (endo)cannabinoids on the mobilization of intracellular calcium. We found that neither anandamide, WIN55,212 or JWH015 modulated calcium levels in concentrations below 1µM. However, when increasing the concentration of WIN55,212 (and also the synthetic cannabinoid CP47,497) to 10µM we found robust calcium mobilization. Interestingly, the effects on calcium were recapitualted by specific agonists for TRPA1 suggesting that this ion channel is the target of cannabinioids in higher concentrations. In a next step, we planned on investigating the impact of cannabinoids but also of cortisol on cellular energy supply. Unfortunately, reporter mice did not show a specific fluorescence signal in mitochondria which prevented further analysis of endocannabinoid/cortisol effects. We then assessed the effects of activation of AMP-activated kinase (AMPK) on the antiinflammatory effects of anandamide and found that AMPK activation inhibits the effects of anandamide. Since AMPK regulates autophagy, we postulated that endocannabinoids are anti-inflammatory due to a change in autophagic flux. Autophagy is regulated by intracellular calcium and we therefore conducted calcium assays with the TRPA1 agonist polygodial, since TRPA1 was previously identified to be the "effector" receptor of anandamide in RASF. We found that TRPA1 activation increases intracellular calcium and this effect was strongly enhanced by TNF pre-stimulation. Additionally, this increase in calcium was accompanied by extensive cell death. TRPA1 might therefore be an attractive therapeutic target to selectively delete pro-inflammatory RASF. We then determined the cellular localization of TRPA1 and found this ion channel located in lysosomes, since depletion of lysosomal calcium stores abrogated TRPA1-induced currents. Other TRP family members like TRPML1 play an important role in lysosome function and autophagy and therefore, we will delineate the role of TRPA1 in this respect in future studies. To investigate the impact of endocannabinoids on arthritis, we treated mice with established collagen-induced arthritis with the FAAH inhibitor JNJ1661010, which prevents the in vivo degradation of ethanolamide-based endocannabinoids. FAAH inhibition slightly reduced arthritic score in mice, but this effect was not enhanced by low-dose glucocorticoids.
Projektbezogene Publikationen (Auswahl)
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2015. A sustained high fat diet for two years decreases IgM and IL-1 beta in ageing Wistar rats. Immun. Ageing 12: 12
Pongratz, G., T. Lowin, R. Kob, R. Buettner, T. Bertsch, and L. C. Bollheimer
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2015. Anti-inflammatory effects of N-acylethanolamines in rheumatoid arthritis synovial cells are mediated by TRPV1 and TRPA1 in a COX-2 dependent manner. Arthritis Res. Ther. 17: 321
Lowin, T., M. Apitz, S. Anders, and R. H. Straub
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2015. Cannabinoid-based drugs targeting CB1 and TRPV1, the sympathetic nervous system, and arthritis. Arthritis Res. Ther. 17: 226
Lowin, T., and R. H. Straub
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2015. Synovial fibroblasts integrate inflammatory and neuroendocrine stimuli to drive rheumatoid arthritis. Expert. Rev. Clin. Immunol. 11: 1069-1071
Lowin, T., and R. H. Straub
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2016. alpha-MSH modulates cell adhesion and inflammatory responses of synovial fibroblasts from osteoarthritis patients. Biochem. Pharmacol. 116: 89-99
Bohm, M., M. Apel, T. Lowin, J. Lorenz, Z. Jenei-Lanzl, S. Capellino, H. Dosoki, T. A. Luger, R. H. Straub, and S. Grassel
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2016. The synthetic cannabinoid WIN55,212-2 mesylate decreases the production of inflammatory mediators in rheumatoid arthritis synovial fibroblasts by activating CB2, TRPV1, TRPA1 and yet unidentified receptor targets. J. Inflamm. (Lond) 13: 15
Lowin, T., G. Pongratz, and R. H. Straub
