Figure 6.

LCA stimulates NLRP3 activation in KC and provokes a pro-inflammatory phenotype. Stimulation of KC with LCA (+ Nigericin) activates the NLRP3 inflammasome as validated by increase of mRNA of Nlrp3, Il-1b and Il-18 (B) and elevated levels of intra- and extracellular IL-1β (A,C). After exposure to LCA + Nigericin more Caspase-1/PI double positive cells were noted in FACS (D), which indicates pyroptotic cell death. Assessing possible pathways of NLRP3 activation, measurement of DCF fluorescence in KC showed an increase of ROS production after LCA (+ Nigericin) (E). Markers of proinflammatory macrophages (Tnf, iNos, Ccr8, Ccl22 and ActivinA) increased after LCA stimulation, whereas expression of ArginaseA as marker for anti-inflammatory macrophages was decreased (F). More Galectin 3-positive KC were found after stimulation with LCA + Nigericin compared to medium (G). * p < 0.05; ** p < 0.01; *** p < 0.001.