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THU0554 12/15-Lipoxygenase Counteracts the Activity of NLRP3 Inflammasome in Monocytes
  1. Y. Kusche,
  2. K. Barczyk-Kahlert,
  3. J. Roth
  1. Institut der Immunologie, Münster, Germany

Abstract

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.

References

  1. 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).

  2. 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

DOI 10.1136/annrheumdis-2014-eular.5356

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