Study On Microglia Activation And Associated Production Of Inflammatory Factors Induced By PHEV Infection

To explore the response of microglia during a neurotrophic coronavirus-procinehemagglutinating encephalomyelitis virus (PHEV) infection, experiments in vivoand vitro were tested on PHEV-injected mice and microglia (BV2cells) respectivelyto determine microglia activation and associated production of inflammatory factorsinduced by PHEV infection, laying a foundation for overall exposure ofimmunologic function played by microglia and its intrinsic mechanism duringPHEV infection.BALB/c mice that were easily infected with PHEV were chosen as experimentalobjects for experiments in vivo. Microglia proliferation and activation in the brainsof mice at different time post PHEV infection were detected from the aspects ofgene transcription, protein expression and histology by detecting the specific markerof microglia Iba1protein using real-time PCR, western blotting, immuno-histochemistry (IHC) and indirect immunofluorescence staining, respectively. Inaddition, expression of some inflammatory factors in mice cerebral cortex postPHEV infection was detected by gene chip technology. The results showed that themodels of PHEV-infected mice brain tissues were established by vaccinating PHEVthrough method of nasal drops, providing a solid foundation for investigatingwhether microglia could activate during PHEV infection. Compared with the emptycontrol group, transcription of Iba1gene and expression of Iba1protein in infectiongroups increased as infection time extended. The mice brain tissues were made intoparafin section for IHC and IFA, showing that PHEV infection stimulated microgliaactivation, the amount of microglia increased and the cells gathered closer. Cellularmorphology changed much, the cell bodies became bigger and turned amebiformshaped. The experiments above proved that obvious proliferation and activation ofmicroglia had been induced by PHEV infection, and microglia activation might bepersistent during the entire process of PHEV infection. Results of gene chip showed that expression of IL-1β, IL-6, TNF-α, IFN-α, IFN-β, CXCL10and CCL2in micecerebral cortex post PHEV infection increased at different levels, especially that ofCXCL10and CCL2.BV2cells that had similar characteristics as primary mice microglia were chosenas experimental object for experiment in vitro. Experiments were performed todetect whether PHEV could infect BV2cells and expression of Iba1, variouscytokines, cell signal transduction pathway related proteins in BV2cells post PHEVstimulation, and their internal connecting links were analyzed. IFA proved thatobvious PHEV virus particles could not be seen in BV2cells, but the redfluorescence that labeled Iba1increased to some extent post PHEV stimulation.Real-time PCR proved expression of PHEV gene decreased post activated andinactivated PHEV virus stimulation, which might be related to degradation of virusparticles and PHEV being unable to copy in BV2cells. Therefore, we think PHEVcould not infect BV2cells. Real-time PCR proved that activated and inactivatedPHEV virus both induced the increase of Iba1gene expression in BV2cells.Western Blot proved expression of Iba1protein by BV2cells increased post PHEVstimulation. The experiments above proved that PHEV could not infect BV2cells,but the virus could stimulate BV2cells to activate. Real-time PCR provedexpression of IL-1β, IL-6, TNF-α, IFN-α and IFN-β by BV2cells post PHEVstimulation increased at different levels, but expression of CXCL10and CCL2changed little. ELISA proved that release of IL-1β, IL-6, TNF-α and IFN-α by BV2cells also increased at different levels. In addition, expression of some cell signaltransduction pathway related proteins were detected by real-time PCR, whichshowed that expression of TLR4, TLR9and RIG-I made little change, butexpression of TLR7, MyD88, MDA5, NF-κB, ASC and Caspase-1in BV2cells postPHEV stimulation increased significantly, laying a foundation for exposure ofintrinsic mechanism of mediating expression of related inflammatory factors in BV2cells.The trend of expression of IL-1β, IL-6, TNF-α, IFN-α and IFN-β by BV2cellspost PHEV stimulation was similar to the results of the mice cerebral cortex postPHEV infection, which increased significantly. Expression of CXCL10and CCL2 which was at a low level in BV2cells increased significantly in mice cerebral cortex,which suggested that microglia was a main source of inflammatory factors in CNS,playing a important role in viral encephalitis caused by PHEV. However, microgliaonly takes a small proportion of CNS cells, other glial cells, neurons and even someperipheral immune cells that permeate blood-brain barrier also participate ininflammatory reaction in CNS. Therefore, further research need to be carried out toexplain the function played by microglia in viral encephalitis caused by PHEV.