THU0554 12/15-Lipoxygenase Counteracts the Activity of NLRP3 Inflammasome in Monocytes
Authors: Y. Kusche, K. Barczyk-Kahlert, J. Roth
Journal: Annals of the Rheumatic Diseases (2014)

<h3>Background</h3> Glucocorticoids (GC) are drugs of choice for the treatment of many autoimmune diseases. Our previous studies have shown that GC treatment does not generally suppress monocyte functions but induces a distinct anti-inflammatory phenotype in these cells<sup>1</sup>. Similarly the treatment of inflammatory monocytes with GC leads to re-programming of the cells towards a specific population involved in resolution of inflammation. Gene expression analysis has shown up-regulated expression of 12/15-lipoxygenase (12/15-LOX) in GC- and LPS/GC-treated monocytes. Lipid mediators play a critical role during the inflammatory response and contribute not only to the initiation but also to the resolution of inflammation. 12/15-lipoxygenase reacts with polyunsaturated fatty acids to generate anti-inflammatory lipid mediators which contribute to the resolution of inflammation<sup>2</sup> <h3>Objectives</h3> The aim of our study was to determine the contribution of 12/15-LOX and its product Lipoxin A4 on the inflammatory response on murine monocytes. <h3>Methods</h3> Bone marrow-derived monocytes were isolated from wild type (wt) C57BL/6 and 12/15-LOX<sup>–/–</sup> mice and stimulated with GC, LPS and GC and LPS as well as various inhibitors or stimulants for different time periods. Gene expression profiles were analyzed using quantitative RT-PCR. Protein expression was examined by Western Blot, Flow Cytometry and ELISA. <h3>Results</h3> Monocytes deficient in 12/15-LOX showed slightly but already significantly higher secretion of IL-1b as compared to their wt counterparts after stimulation with only LPS. The differences between wt and 12/15-LOX<sup>–/–</sup> were much more pronounced, when monocytes were additionally exposed to ATP. qRT-PCR and Western blot analysis of monocytes lysates revealed a slightly enhanced expression of pro- IL-1b in 12/15-LOX<sup>–/–</sup> monocytes stimulated with LPS alone as compared to wt monocytes. Additional treatment with ATP enhanced the secretion and processing of mature IL-1b in the 12/15-LOX<sup>–/–</sup> cells. No differences could be observed between wt and 12/15-LOX<sup>–/–</sup> monocytes in secretion of other proinflammatory mediators like TNFa, MCP-1, IL-6 as well as the anti-inflammatory cytokine IL-10. Analysis of expression of inflammasome components like NLRP3 and ASC revealed no alterations between wt and 12/15-LOX<sup>–/–</sup> monocytes. However, Western blot analysis of expression and activation of caspase-1 showed increased processing of caspase-1 in lysates of 12/15-LOX<sup>–/–</sup> monocytes pre-treated with LPS and GC/LPS as compared to control wt monocytes. Similarly the expression of caspase-11 seemed to be up-regulated in monocytes isolated from 12/15-LOX<sup>–/–</sup> mice exposed to LPS. <h3>Conclusions</h3> Our results demonstrate that 12/15-LOX play a regulatory role during inflammatory immune response by counteracting/modifying the (NLRP3) inflammasome activity. Further analysis is needed to decipher the exact pathway and molecular mechanism underlying the regulatory role of 12/15-LOX and its metabolites in dampening the inflammatory response of monocytes. <h3>References</h3> Barczyk K, Ehrchen J, Tenbrock K, Ahlmann M, Kneidl J, Viemann D, Roth J. Glucocorticoids promote survival of anti-inflammatory macrophages via stimulation of adenosine receptor A3. Blood 116:446-55 (2010). Conrad, D.J. The arachidonate 12/15 lipoxygenases. A review of tissue expression and biologic function. Clinical Reviews in Allergy &amp; Immunology 17, 71-89 (1999). <h3>Disclosure of Interest</h3> : None declared <h3>DOI</h3> 10.1136/annrheumdis-2014-eular.5356