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

Abstract

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.

Fig 1. Influence of 15-epi-LXA₄ on bioluminescence signals in dermal inflammation.

Schematic division of the shaved back of mice as the experimental setup (A) and with corresponding bioluminescence images (B). Results of the bioluminescence imaging show a time course of signal intensity (C) and the signal intensity at 24 h after OXA-challenge (D).

Fig 2. Calibration curves for LXA₄, RvD1, PDx and 7-MaR1 in skin (n = 3) and PBS.

Calibration curves for each analyte were prepared in skin of 3 different mice as well as PBS and compared. Exemplary curves of LXA₄ (A), RvD1 (B), 7-MaR1 (C) and PDX (D) are shown.

Fig 3. Levels of prostanoids, LTB₄ and HETE at 24 h after application of 15-epi-LXA₄ and NaCl.

Levels of different prostanoids, products of the COX-pathway, like PGD₂ (C), PGE₂ (D), PGF_2α (E) and TXB₂(F), as well as products of the LOX-pathway, such as LTB₄ (B), its precursor 5-HETE (A) and 15-HETE (G), were determined at 24 h after application of 15-epi-LXA₄ or NaCl. Concentrations of 15-HETE were also compared 6 and 24 h after application of 15-epi-LXA₄ or NaCl.

Fig 4. In vivo availability of 15-epi-LXA₄, RvD2, PDX and 7-MaR1.

Availability of the SPM was determined by their direct analysis, 30 min and 2 h after their subcutaneous injection into the back skin of the mice. The results for 15-epi-LXA₄ (A), 7-MaR1 (B), RvD2 (C) and PDX (D) are shown as examples.

Fig 5. Distribution behavior of 15-epi-LXA₄, RvD2, PDX and 7-MaR1.

The distribution of the SPM was determined by direct analysis of skin areas, 30 min and 2 h after their subcutaneous injection. The results for 15-epi-LXA₄ (A), 7-MaR1 (B), RvD2 (C) and PDX (D) are shown as examples.