Effect Of Cryptococcus Neoformans On OXPHOS And Apoptosis In Microglial Cells Of Mice

Cryptococcus neoformans (C. neoformans), which is a common clinical opportunisticpathogenic fungal pathogen with neurotropic tendency， typically affects individuals withimpaired immunity function leading to leathy Cryptococcal meningitis andmeningoencephalitis. In China mainland, it usually causes seemly immunocompententpeople infected. C. neoformans is the only encapsuled fungi and could be certified into4serotypes according to the capsular agglutination test. Out of which serotype A is the mostpopular type in China mainland.Phagocytic effector cells take a very important part in host immunity defense againstC. neoformas. As the macrophage in central nervous system (CNS), microglial cells exertprotective effect as the pathogen comes across the blood brain barrier (BBB) entering brain.It was documented that microglia could improve immunity agaist the yeast by enhancedMHC-Ⅱ expression on the cell surface in the presence of INF-γ. To the contrast,Cryptococcus neoformans could survice in microglia and envetually cause the cell lysis incase of abense of actived cytockines.Energy metabolism is an important biochemical process for the host cell survival, andplays an important role in maintening cell activities. Studies have suggested that the energymetabolism of host cells are impaired during due to pathogen infection and even lead toapoptosis or necrosis. Embedded in the lipid bilayer of the mitochondrial inner membrane,the oxidative phosphorylation (OXPHOS) system is the final biochemical pathway inenergy production of the cells. Many studies have demonstrated that pathogens can impaireenergy metabolism of host cells mainly by inhibiting oxidative phosphorylation. It was nodoubt that OXPHOS plays a vital role in the maintenance of intracellular homeostasis andcell normal activity. Due to not only providing energy for the host cell activities butalso the prodiment role of hub point in apoptosis, mitochondrial as the oragn of cellenergy metabolism is very important in the process of induction of host cell death.Research on animal Cryptococcal meningitis demonstrated that necrosis is not thecharacteristics of host cells in the C. neoformans granuloma but inflammatory cells in theinflammatory organ undergoing apoptosis. At the same time, the microglia exert muchmore obvious phagocytosis effect on the acapsular C. neoformans and the one withsmaller capsule than on the wild type, suggesting that capsule may anti phagocytosis by inducing apoptosis. Our group found that acapsular cryptococcal strain CAP64can alsoinduce apoptosis in microglia in vitro suggesting that other virulence factors of C.neoformans can also induce apoptosis in the host cells, but the specific mechanism is stillunclear.We speculate that C. neoformans could affect the activity of microglial cells byimpairing OXPHOS of the host with the result of inducing host cells apoptosis. In theexperiment, we take wild type of serotype A H99and isogenic acapsular strain CAP64asobjectives to detect the change in OXPHOS level and mechanism of apoptosis in microgliainfected with the pathogen.The whole of study consists of four parts as follow:Part I Effect of Cryptococcus neoformans capsule GXM on OXPHOS and apoptosis ofBV-2cells.GXM has been regarded as an important regulator of the host cell immunity, which isone of the most important virulence of C. neoformans. This part of the study was carriedout on the basis of obtained purified GXM aiming at observation the effect of thecompound on oxidative phosphorylation and induction of apoptosis in microglia. We takethe method of CCK-8to test the effect of GXM inhibition the vitality of microglial cell invitro. The experiment of GXM induced apoptosis in microglia was conducted withpretreatment the cells with TNF-αand IFN-γ to mimic an inflammatory environment invivo. Annexin V-fluorescein isothiocyanate/propidium iodide double label method wasused to detect the apoptosis rate of microglia in every treatment group. A commercialminim ATP content kit was taken to detect the ATP concentration of the cells aftertreatment with different factors at different time.The results in the experiment demonstrate that GXM at the dose of50ug/ml couldinhibit the vitality and the ability of ATP generation in microglia. the rate of apoptosistested by flow cytometry suggested that GXM could not enhance the ratio of apoptosis inmicroglia while the ration increased in the presence of GXM together with IFN-γ whichwas detected in the group of GXM with TNF-α. According to the result, we speculate thatIFN-γwas one of the cytokines produced after stimulation by C. neoformans, which couldnot only kill the pathogen and but also the host cells.PartⅡ Effect of Cryptococcus neoformans on the function of OXPHOS in microglia.OXPHOS is the last procedure of energy metabolism in host cells and the vital step tomaintain life activity. Many studies demonstrated that the energy metabolism was impairedduring pathogen infection. The differential expressed genes in OXPHOS pathway were detected using gene chip technique and confirmed using RT-PCR. The expression level ofgenes NDUFA4AND ATPase6V were reduced compared with the one in control group.The reduced concentration of ATP tested in follow up experiment also confirmed the effectof C. neoformans on the energy metabolism of host cells. The level of ATP concentrationin H99group and CAP64showed a significant difference of P<0.05compared with thecontrol group, which dictated that the C. neoformans capsule did not take an indispensiblerole in the effect of inhibition on energy metabolism of microglial cells.That both the wild and acapsule mutant C. neoformans strains could induce apoptosisin microglia in vitro certified the fact that other ingredients of the pathogen fungi besidesthe capsule could also induce host cells apoptosis. The effect induce apoptosis in microgliamaybe was exerted by the yeast directly or through inducing microglia produceinflammatory cytokines and then promote apoptosis in host cells. Energy metabolism wasaffected by the reduced OXPHOS effect due to the impaired electron transfer chain afterinfection with C. neoformans. The inevitable consequence of decreased energy metabolismwas the reduction in immunity activity of host cells. In a conclusion the effect of C.neoformans on host energy metabolism was a possible mechanism for the pathogen eitherto cause disease in host or avoid the immunity attack from the host cells.Part Ⅲ Study on the mechanism for Cryptococcus neoformans induce microgliaapoptosis in vitroInducing apoptosis in host cells is a very effective and important mechanism forpathogen to avoid host immunity attack and damage it. C. neoformans also could exert thiskind of effect on host cells. Taken the results obtained from the Part II into consideration,we speculate that mitochondria take a very important part in the process of C. neoformansinducing host cells apoptosis.Western-blot method was taken to detect the expression difference of the key proteinsin the mitochondrial apoptosis pathway including: Caspase3, Caspase9, Bcl-2and Bax.The release of Cyt c was also detected using Western-blot method. the results ofWestern-blot showed that the expression level of Cyt c in the mitochondria decreasedcompared with the control group while the level in cytoplasm increase indicating that theCyt c was released into cytoplasm from mitochondira. Following the differentialexpression of other proteins because of cascade effect induced by the release of Cyt c. Theapoptosis associated protein was activated and the expression level increased and theapoptosis inhibition protein was inactivated. All of the results indicated that Cryptococcus neoformans could induce microglia cell apoptosis via mitochondria pathway.Part Ⅳ Study on the mechanism of apoptosis of the inflammatory cells inCryptococcal meningitis mouse modelIntranasal inhalation through the respiratory tract to build a mouse central nervoussystem C. neoformans infection model, using pathological and infected tissue homogenatessmear test to make sure if the model is successfully built. Western-blotting was used todetect the releasing of inflammatory Cyt C of host brain tissue after C. neoformansinfection. Cyt C expression level increased in the cytoplasm of the experimental groupcompared with the control group, the mitochondrial Cyt expression level decreasedcompared with the control group; experimental group Caspase3,9and Bax expressionlevel increased than those in the control group, however, Bcl-2expression level comparedwith the control group decreased. Successfully constructed mice C. neoformans meningitisinhalation model provided the animal research model for natural infection of analog C.neoformans.Western-blotting to detect the releasing of inflammatory Cyt C of host brain tissueafter Cryptococcus neoformans infection, we found that Cyt C expression levels in thecytoplasm of the experimental group compared with the control group increased, themitochondrial Cyt expression level decreased compared with the control group;experimental group Caspase3,9and Bax expression level increased than those in thecontrol group, Bcl-2expression level compared with the control group decreased. The thirdpart of the study in vitro suggested that Cryptococcus neoformans induce host cellapoptosis through the mitochondrial pathway.