Background Activation of the NLRP3 inflammasome is a major inflammatory pathway in monocytes in response to various exogenous and endogenous stimuli. However, negative regulation of inflammasome activity is not well understood. Glucocorticoids (GC) are drugs of choice for the treatment of many inflammatory diseases. Recently, we could show that treatment of monocytes with GC leads to re-programming towards a specific population involved in resolution of inflammation. Gene analysis has shown up-regulated expression of 12/15-lipoxygenase (12/15-LOX) in GC-and LPS/GC-treated monocytes. 12/15-LOX reacts with polyunsaturated-fatty-acids to generate anti-inflammatory lipid-mediators, which contribute to resolution of inflammation.
Objectives The aim of our study was to determine the contribution of 12/15-LOX 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 and/or LPS as well as various inhibitors or stimulants. Gene expression was analyzed using qRT-PCR. Protein expression was examined by Western-Blot, Flow-Cytometry and ELISA. T-cell response was analyzed by co-culture of stimulated monocytes with allogenic T-cells.
Results 12/15-LOX-/- monocytes showed slightly higher secretion of IL-1β as compared to wt cells after LPS stimulation. The differences between wt and 12/15-LOX-/- were much more pronounced when monocytes were additionally exposed to ATP. LPS treatment markedly enhanced expression of pro-IL-1β in 12/15-LOX-/- monocytes. No differences could be observed between wt and 12/15-LOX-/- monocytes in secretion of other proinflammatory mediators as well as the expression of inflammasome components. However, expression of cleaved caspase-11 was up-regulated in 12/15-LOX-/- monocytes exposed to LPS. Additionally, inhibition of caspase-11, caspase-1 and 5-LOX significantly reduced the high secretion of IL-1β in 12/15 LOX-/- monocytes. Interestingly, 12/15-LOX-/- rather than wt monocytes stimulated with LPS led to enhanced T-cell proliferation.
Conclusions Our results demonstrate that 12/15-LOX plays a regulatory role during inflammatory immune response by counteracting the NLRP3 inflammasome activity through down-regulation of caspase-11 and 5-LOX activity. Thus, we identified a novel negative regulatory pathway of inflammasome activity.
Barczyk, K. et al. Glucocorticoids promote survival of anti-inflammatory macrophages via stimulation of adenosine receptor A3. Cancer 116, 1–3 (2010).
Conrad, D.J. The arachidonate 12/15 lipoxygenases. A review of tissue expression and biologic function. Clinical reviews in allergy&immunology17, 71–89 (1999).
Disclosure of Interest None declared