Temporal Patterns of Microglial Activation When Bilirubin Enters into the Brain

Abstract

Event Abstract Back to Event Temporal Patterns of Microglial Activation When Bilirubin Enters into the Brain Sandra L. Silva1, Catarina C. Osório1, Ana R. Vaz1, Andreia Barateiro1, Ana S. Falcão1, Maria A. Brito1, Adelaide Fernandes1, Rui F. Silva1 and Dora Brites1* 1 University of Lisbon, Centro de Patogénese Molecular/iMed.UL, Faculty of Pharmacy, Portugal Microglia turns into an activated state when facing injury and disease entailing two main functions: phagocytosis of cellular debris and propagation of immune response. Once unconjugated bilirubin (UCB) increases proinflammatory cytokine secretion by both astrocytes [1] and microglia [2], we aimed to assess the sequence of events involved in microglia immunophenotype by UCB.Primary cultures of rat microglia were incubated from 15 min to 24 h with 50 μM UCB plus 100 μM HSA, at 37ºC. Phagocytic capacity was evaluated by fluorescent microspheres uptake. NF-κB nuclear translocation was assessed by immunocytochemistry. Activation of MAPKs (p38, ERK1/2), and upregulation of COX-2 and RAGE were determined by Western blot. Apoptosis was estimated by measuring caspase 3, 8 and 9 activities.UCB led to activation of p38 and ERK1/2 peaking between 15-30 min (≈1.3-fold, p<0.05) followed by NF-κB nuclear translocation (≈1.4-fold, p<0.05) at 1 to 2 h of exposure. These events precede the release of proinflammatory cytokines already observed. Interestingly, phagocytic properties of cells exposed to UCB was only observed at 4 h (≈1.5 -fold, p<0.05). From this point on, onset of apoptosis occurs based on the activation of caspases-3, -8 and -9 achieving a maximum increase at 12 h (≈2.3-fold, p<0.05). From 12 to 24 h of microglia exposure to UCB, a COX-2 increased expression was noticed (≈1.15-fold, p<0.01 and ≈1.14-fold, p<0.05, respectively) indicating its involvement in the immunostimulation by UCB. Although at a less extent, RAGE expression is also induced at the same time points (≈1.1-fold, p<0.05 and ≈1.14-fold, p<0.05, respectively) reinforcing the acquirement of macrophage-like properties. Since it is known that amplification of inflammatory response may result from RAGE engagement by ligands such as S100B, usually released by reactive astrocytes, it will be interesting to evaluate, in future studies, whether UCB-induced activation of astrocytes may aggravate microglial response.In conclusion, these results suggest that microglia, when facing UCB, could initially play a repair role by removing dead cells before acquiring a predominantly immunostimulant phenotype. To what extent these data can be transferred into therapy of bilirubin encephalopathy is to be shown in the future.Funded by FCT-PPCDT/SAU-MMO/55955/2004 and FCT-PTDC/SAU-NEU/64385/2006 (to DB)