Toxoplasma Gondii Induced Microglia Activation And The Inhibitory Effect Of Minocycline

Background and Objective Toxoplasma gondii is an obligate intracellular protozoanparasite, which is capable of infecting many warm-blooded mammals including humans.Either congenital or acquired infection constitutes a serious threat to human health, suchas hydrocephalus, mental retardation, headache and epilepsy. Particularly in the patientswith immune deficiency, brain cysts rupture and bradyzoites convert to tachyzoites andget into replication. Consequently, the status of T. gondii latent infection is activated,leading to the occurrence of fatal Toxoplasma encephalitis. The recent study found thatthe different genotypes of T. gondii were related to the occurrence, development andoutcome of the disease. In North America and Europe, the major genotypes of T. gondiiare Type I, Type II and Type III. But in China, the dominant genotype is Chinese1type.Microglia is the main innate immune component in the central nervous system,including resting and activation states. It plays an important role in the pathologicaldamage of the brain. During the infection, injury and other stimuli, microglia can beactivated, followed by differentiation and proliferation, and secrete large amounts ofneurotoxic factors, which play the function of immune protection. The excessiveactivation, however, of microglia can also injure the neurons and surrounding tissues.The effect of T.gondii infection on microglia remains unclear. The present study aims toinvestigate the variation of microglia (BV-2cells) infected with T. gondii (TgCtwh6strain, Chinese1genotype) and the inhibitory effect of minocycline on BV-2cells. Theresults will provide an experimental basis for the prevention and treatment ofToxoplasma encephalopathy.Methods BV-2cells were inoculated with T. gondii in transwell followed by exposure to minocycline. The ordinary medium was used as control group, the metabolites groupwas infected with Toxoplasma TgCtwh6and the inhibitor group by adding minocycline.The morphology of BV-2cells was observed under inverted microscope. The flowcytometry was carried out to assess the condition of cell apoptosis. The production ofNO was determined with the Griess test. ELISA（enzyme-linked immuno sorbent assay）was taken to check the production of IL-1β, IL-6, and TNF-α. The transcription of IL-1β,IL-6, TNF-α and iNOS mRNA level was examined by RT-PCR. The expression ofiNOS was detected by Western blotting.Results Compared with the control, BV-2cells of the metabolites group inoculated withT. gondii were presented in round and pseudopodia were remarkably reduced, whichindicated the retarded cell movements. This change can be reduced by minocyclineadministration. The results of flow cytometry showed that BV-2cells appear to beapoptotic at the condition that the BV-2cells to parasites ratio is1:1at24h. BV-2cellsinoculated with T. gondii could induce35.72%of BV-2cells apoptosis when the BV-2cells to T. gondii ratio reaches1:5at48h. BV-2cells inoculated with T. gondii couldsecrete cytokines. The expression and transcription of IL-1β, IL-6, and TNF-α, andiNOS were all gradually enhancd in both protein level and mRNA level with prolongedincubation time; the increase of NO production was also noted. The cytokine profilesstudied were significantly attenuated after treatment of the host cells with the microgliaactivation inhibitant minocycline.Conclusion BV-2cells inoculated with T.gondii could secrete proinflammatorycytokines, such as IL-1β, IL-6and TNF-α. Minocycline inhibited the production ofthese cytokines, suggesting a therapeutic potential in help with the treatment oftoxoplasmosis.