PGESubscript2/Subscript Inhibits IL10 Product

Authors: ChunHsien Chu ShihHeng Chen Qingshan Wang Robert Langenbach Hong Li Darryl Zeldin ShiouLan Chen Shijun Wang Huiming Gao RuBand Lu JauShyong Hong

Publish Date: 2014/09/14

Volume: 52, Issue: 1, Pages: 587-600

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Abstract

Regulatory mechanisms of the expression of interleukin10 IL10 in brain inflammatory conditions remain elusive To address this issue we used multiple primary brain cell cultures to study the expression of IL10 in lipopolysaccharide LPSelicited inflammatory conditions In neuron–glia cultures LPS triggered wellorchestrated expression of various immune factors in the following order tumor necrosis factorα TNFα cyclooxygenase2 COX2 prostaglandin E2 PGE2 and lastly IL10 and these inflammatory mediators were mainly produced from microglia While exogenous application of individual earlierreleased proinflammatory factors eg TNFα IL1β or PGE2 failed to induce IL10 expression removal of LPS from the cultures showed the requirement of continuing presence of LPS for IL10 expression Interestingly genetic disruption of tnfα its receptors tnfr1/r2 and cox2 and pharmacological inhibition of COX2 activity enhanced LPSinduced IL10 production in microglia which suggests negative regulation of IL10 induction by the earlierreleased TNFα and PGE2 Further studies showed that negative regulation of IL10 production by TNFα is mediated by PGE2 Mechanistic studies indicated that PGE2elicited suppression of IL10 induction was eliminated by genetic disruption of the PGE2 receptor EP2 and was mimicked by the specific agonist for the EP2 butaprost but not agonists for the other three EP receptors Inhibition of cAMPdependent signal transduction failed to affect PGE2mediated inhibition of IL10 production suggesting that a G proteinindependent pathway was involved Indeed deficiency in βarrestin1 or βarrestin2 abolished PGE2elicited suppression of IL10 production In conclusion we have demonstrated that COX2derived PGE2 inhibits IL10 expression in brain microglia through a novel EP2 and βarrestindependent signaling pathwayThis work was supported in part by the Intramural Research Program of the NIH/NIEHS ES090082 ES025043 the National Natural Science Foundation of China and the award to highlevel innovative and entrepreneurial talents of Jiangsu Province of China We thank Anthony Lockhart for the assistance with animal colony management and maintenance of the timed pregnant miceEffects of polymyxin B on neuronglia cultures treated with LPS A Immunostaining of neuronglia cells treated with LPS 15 ng/ml and polymyxin B 10 μg/ml at 48 hours using antiIba1 antibody Iba1 served as a microglia marker B 3H dopamine uptake of neuronglia cells treated with LPS 15 ng/ml and polymyxin B 10 μg/ml at 7 days Results of dopamine uptake were represented as percentage of vehicletreated control cultures Data were expressed as means ± SEM from three independent experiments run in triplicate p 005 compared with corresponding vehicletreated control cultures p 005 compared with corresponding LPStreated cultures Magnification 200X GIF 71 kbBoth TLR4 and Mac1 receptors participate in induction of IL10 by LPS Mixedglia cultures from wildtype FeJ and TLR4deficient HeJ mice A or from wildtype and Mac1deficient mice B were treated with LPS 15 ng/ml IL10 production in the supernatant of these cells was measured at 48 hours following LPS treatment by ELISA Results are shown as the mean ± SEM from 3 independent experiments p 005 compared with vehicletreated control cultures p 005 compared with corresponding LPStreated wildtype cultures C LPS induces IL10 production through MAPK and NFκB signaling pathways Neuronglia cultures were pretreated with a variety of protein kinase inhibitors including SP6000125 5 μM for JNK SB203580 1 μM for p38 U0126 10 μM for ERK Bay 117821 10 μM for NFκB RpcAMPs 10 μM for PKA and Calphostin C 1 μM for PKC for 1 hour and then exposure to LPS 15 ng/ml IL10 production in the supernatant of these cells was measured at 48 hours following LPS treatment by ELISA Results are represented as percentage of LPStreated group and shown as the mean ± SEM from 3 independent experiments p 005 compared with corresponding LPStreated wildtype cultures GIF 24 kbDeficiency in tnfα or its receptor genes reduces LPSinduced proinflammatory factors in microglia A Mixedglia cultures were prepared from wildtype TNFαdeficient or TNFR1/R2deficient mice After 2 weeks of cells seeding immunocytochemical analysis of microglia in these mixedglia cultures was performed by using antiIba1 antibody B After 3 hours of LPS treatment 15 ng/ml TNFα secretion into the supernatant of mixedglia cultures was detected by ELISA Results are shown as the mean ± SEM from 3 independent experiments p 005 compared with vehicletreated control cultures p 005 compared with corresponding LPStreated wildtype cultures C Expression of COX2 and iNOS mRNA in mixedglia cultures at 6 hours after LPS treatment was measured by RTPCR Results are represented as percentage of LPStreated group and shown as the mean ± SEM from 3 independent experiments p 005 compared with corresponding LPStreated wildtype cultures GIF 108 kbLPS induces similar amount of TNFα production in wildtype and COX2deficient mixedglia cultures Three hours after these cultures were treated with LPS 15 ng/ml TNFα secretion into the supernatant of these cultures was detected by ELISA Results are shown as the mean ± SEM from 3 independent experiments p 005 compared with vehicletreated control cultures GIF 6 kbProtein kinase inhibition fails to restore reduction of IL10 release by PGE2 PGE2 and protein kinase inhibitors including wortmannin 50 nM for PI3K A SB216763 1 μM for GSK3β B U0126 10 μM for ERK C SP600125 5 μM for JNK D and PD98059 50 μM for MEK1/2 E were added into neuronglia cultures after these cultures were treated with LPS for 24 hours IL10 release into the supernatants was detected at 24 hours following PGE2 treatment 48 hours after LPS treatment by ELISA The experiment has been performed three times Results are shown as the mean ± SEM p 005 compared with corresponding vehicletreated control cultures p 005 relative to corresponding LPStreated cultures NS nonsignificant GIF 73 kbLPS induces similar amount of TNFα production in wildtype βarrestin1 deficient and βarrestin2 deficient mixedglia cultures Three hours after these cultures were treated with LPS 15 ng/ml TNFα secretion into the supernatant of these cells were detected by ELISA Results are shown as the mean ± SEM from 3 independent experiments p 005 compared with vehicletreated control cultures GIF 9 kb

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