Background 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 cells1. 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 inflammation2
Objectives 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.
Methods Bone marrow-derived monocytes were isolated from wild type (wt) C57BL/6 and 12/15-LOX–/– 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.
Results 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–/– 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–/– 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–/– cells. No differences could be observed between wt and 12/15-LOX–/– 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–/– monocytes. However, Western blot analysis of expression and activation of caspase-1 showed increased processing of caspase-1 in lysates of 12/15-LOX–/– 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–/– mice exposed to LPS.
Conclusions 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.
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 & Immunology 17, 71-89 (1999).
Disclosure of Interest : None declared
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