Role Of Mac-1-NOX2 In Sepsis Associated Neuroinflammation

ObjectiveThe purpose of this study is to investigate the effect and the underlying mechanism of Mac-1-NOX2 signaling pathwayin sepsis induced-neuroinflammation mediated by microglial cells,the intrinsic innate immune cells in the central nervous system（CNS）.Here we used the genetic abalation of Mac-1 or NOX2 mice to detect the profiles of pro-inflammatory factors and using immunostaining to detect the changes of microglia morphology in sepsis condition.The results of this study could provide the insight in the treatment of sepsis-induced neuroinflammation or neuronal damage.ContentIn in vivo studies,we used wild-type and Mac-1 KO mice injected with lipopolysaccharide（LPS,5mg/kg,i.p.）to induce sepsis and the subsequent neuroinflammation.We performed immunostaining to determine the morphological changes of microglia,and real-time PCR to detect the level of pro-inflammatory factors m RNA in mouse brains.In in vitro studies,we prepared different primary cultures including enriched-microglia,neuron-glial cultures from wild-type,Mac-1KO and NOX2 KO mice to study the molecule mechanism of Mac-1-NOX2 signaling pathway in activatedmicroglia.Methods1.Build up sepsis-induced neuroinflammatorion model.Both C57BL/6J（wild-type）and Mac-1 KO mice-were intraperitoneally injected with LPS（5 mg/kg）.We used saline-injected mice as the control group.2.At 1 and 7 days after LPS injection,anti-ionized calcium binding adapter molecule 1（Iba-1）antibody was used for immunohistochemial stainingto observe morphology of microglia.3.Saline-or LPS-injected mouse brains were collected at 1 hour and 7 days after injection for real-time RT-PCR analysis to detect the pro-inflammatory m RNA expressions,such as TNF-α,IL-1β,COX2 and MCP-1 m RNA in the brain.4.Primary neuron-glial cultures prepared from C57BL/6J mice and Mac-1KO mouse embryos（gestation day E14-E15）were treated with saline or LPS.Cell pellets were collected at different time points after LPS treatment and analyzed the microglia activation marker,Iba-1,expression by Western blot.5.Prepare enriched-microglia from C57BL/6J mice neuron-glial cell primary culture which were treated with LPS and set up the control groups.The microglia marker protein expression level of Iba-1 were detected at 0,1,2,3,5 days after LPS treatment by Western Blot,respectively.Primary neuron-glial cultures prepared from C57BL/6J mice and NOX2 KO mouse embryos（gestation day E14-E15）were treated with saline or LPS.Cell pellets were collected at different time points after LPS treatment and analyzed the microglia activation marker,Iba-1,expression by Western blot.6.Prepared enriched-microglia cultures from C57BL/6J and Mac-1KO pups（postnatal day 1 to day 3）,and treated with saline or LPS for 1 hour.Cells were fixed and performed immunofluorescence staining to observe the translocation of P47^phox.Results1.The microglia in C57BL/6J and Mac-1 KO mouse brains injected saline were maintained in resting condition,the cells showed with lots of dendrites,small cell body,which have no apparent activation phenomenon.The microglia in bothmouse brains injected LPS became activate at one day after LPS injection,the cell morphology showedwith thick branchesand cell body size was enlarged,and the number of microglia was increased.There is no obvious difference between these two groupsincell morphology.The microglia morphology showed obvious different between C57BL/6J and Mac-1 KO mice at 7 days after LPS injection.The C57BL/6J mice microglia cells were still maintainedin activation stage,but microglia in Mac-1 KO mice returned to the resting stage.2.The level of pro-inflammatory gene m RNA expression in C57BL/6J and Mac-1 KO mouse brainswere significantly increased at 1 hour after LPS injection compared to thesaline control group.There is no obvious difference between these two strains of mice.At 7 days after LPS injection,the pro-inflammatory m RNA in the mouse brains was significantly reduced compared to 1 hour after LPS injection.The pro-inflammatory m RNA in C57BL/6J mouse brains were still maintained in low grade expression,but in Mac-1 KO mouse brains these gene expressions have returned to basal level.3.Neuron-glial cultures from C57BL/6J and Mac-1 KO mouse embryos were treated with LPS and collected cell ppellets at 0,1,2,3,5 days after treatment.The Iba-1 protein expressed in microglia was graduallyincreased and peaked at 5 days after treatment.The Iba-1 expression in C57BL/6J and Mac-1 KO neuron-glial cultures has no difference at day 1 and day 2 after LPS treatment,but from day 3 after LPS treatment,the Iba-1 expression in Mac-1 KO cultures was significantly less than that of in C57BL/6J cultures（P<0.05）.4.Neuron-glial cultures from C57BL/6J and NOX2 KO mouse embryos were treated with LPS and collected cell ppellets at 0,1,2,3,5 days after treatment.The Iba-1 protein expressed in microglia was gradually increased and peaked at 5 days after treatment.The Iba-1 expression in C57BL/6J and NOX2 KO neuron-glial cultures has no difference at day 1 and day 2 after LPS treatment,but from day 3 after LPS treatment,the Iba-1 expression in NOX2 KO cultures was significantly less than that of in C57BL/6J cultures（P<0.05）.5.In resting stage,microglial NOX2 P47^phoxsubunit was located in the cytoplasm.After stimulating with LPS for 1 hour,it showed that P47^phox in C57BL/6J microglia translocated to the cell membrane,while P47^phox in Mac-1 KO microglia had no obvious translocation.Conclusion1.Sepsis can cause microglia activation,increased pro-inflammatory factor expression,and elicited neuroinflammation in the CNS.2.Mac-1 plays a key role in microglia activation and sustains pro-inflammatory factor expression in chronic inflammation caused by sepsis.3.Microglial NOX2 activation is important in neuroinflammation caused by sepsis.4.Mac-1-NOX2 signal axis is essential in microglia activation,which could be the potential target for the treatment of CNS inflammation.