| Background and PurposeCryptococcus neoformans (Cn), a yeast fungus with bacterial capsule, exists widely in soil and pigeon droppings and infects low immunity and immunocompromised populations mainly. Its characteristic of addicted to the central system cause central nervous system infection easily, especially among HIV correlation cryptococcal meningitis (Cm) which lead to high mortality rate. Consequently, the research on Cm, the worst fungal infection complication of AIDS, has become a hot topic in the study of HIV and fungi.Respiratory inhalation is the main pathogenic pathway of Cn infection.The pathogen pass arcoss the pulmonary alveoli-capillary by asymptomatic pulmonary infection, and arrive the blood circulation and other deep tissues, especially the central nervous system. Blood-brain barrier (BBB) is consists of close-connected brain microvasclar endothelial cells (BMEC), only through which can Cn cause meningitis. In our previous work, we found that Cn is able to induce actin cytoskeleton rearrangement in HBMEC and reallocation of CD44on membrane surface.Polysaccharide capsule is the primary virulence factor of Cn. The research of Ambrose Jong et al. indicates that hyaluronic acid synthase, which is encoded by CPS1gene, synthesizes hyaluronic acid(HA).HA is able to combine with CD44, a main adhesion molecule on the surface of HBMEC,which is close relate to Cn’s adhesion and adhesion to BMEC. HA is a cytoplasmic component of almost all mammalian cells,and also may be involved in the processes of tumor invasion and metastasis. Currently,we have utilized chromosome homologous recombination technology to establish a CPSI gene deletion strain TYCC645#32, a CPS1gene anaplerosis strain CPIP, and a no expressing HA strain TYCC645#32compared to B4500FO2. The decrease of adhesion and invasion rate in BMEC suggest that HA is the ligand of CD44, and is a critical virulence factor in the process of Cn’s adhesion and adhesion to BMEC as well. There are various kinds of receptors of HA on BMEC, such as CD44, RHAMM and Ivd4,among which CD44is the primary one.CD44is mainly distributed in the lipid rafts which flows between cell membrane and cytoplasm.This kind of flow membrane structure of lipid rafts may be the basis of its signal transduction mechanism.After infected by Cn, CD44molecule in BMEC will re-distribute to lipid rafts and reassociate from cytoplasm to cytomembrane. After a series of signal transduction, the following-up actin cytoskeleton rearrangement result in Cn’s entry to host cell.Besides, adhesion molecule CD44, a widely distributed cell surface receptor,is expressed in multiple types of cells, involved in a variety of cell’s immune process,such as adhesion between cells and substrates, cells and cells, and cell’s migration, survival, and differentiation. HA binding domain and a common across membrane structure is the conserved domain of CD44. We found that Cn CPSI wild strain stimulates a overexpression of CD44in BMEC and a large amount of CD44can be defected at the bonding point. Furthermore, the interactions between white blood cells and endothelial cells also rely on CD44/HA.Mononuclear phagocytic system is the first defensive line of immunoreaction. Mononuclear phagocyte is able to phagocytose and kill Cn, while Cn can survive in phagocytes as it has a variety of virulence factor (e.g.polysaccharide capsule).When mononuclear phagocytes function disorder shows up,the feature described above is possoible to turn to a tool assisting Cn spread to deep tissue from blood,especially in HIV infected patients.Currently, a large amount of evidences are put forward to support the existence of Trojan mechanism in blood-brain barrier model. Animal studies have shown that after injecting cryptococcus incubated together with mononuclear cells into a mouse, brain fungus load of the mice has significantly increased compared with the ones that injected cryptococcus only. HIV-1gp41-190can induce the redistribution and high expression of lipid rafts and CD44in HBMEC and further promote the adhesion and invasion of Cn to HBMEC.Chemotaxis migration experiment is a very good experimental method to study the interaction and movement between mononuclear cell and BMEC at Cn infection. The experimental apparatus is a goblet shaped membrane filter with particular pore diameter. Spread the BMEC on the membrane to imitate the blood-brain barrier. This method is widely used in various researches such as co-culture, cell chemotaxis, cell migration and invasion etc.The purpose of this research is to establish a vitro blood-brain barrier model by using human brain microvasclar endothelial cell (HBMEC), through the experiments of adhesion and chemotaxis migration to study the impact of Cn infected HBMEC on mononuclear cell adhesion and migration through blood brain barrier, and the impact of the single action of HIV-1gp41-190or the co-stimulatory with Cn to HBMEC on mononuclear cell migration through blood brain barrier; by using the vitro adhesion and chemotaxis migration experiment, compare the change of mononuclear cell migration before and after using CD44inhibitor and antibody to approve that CD44is the key molecule that affect mononuclear cell migration; by using the immunofluorescence technique, compare CD44expression on cell surface under the influence of HIV-1gp41-I90and Cn synergistic effect on HBMEC induction.Methods1. Using monocyte adhesion experiment to detect the monocyte adhesion status after HBMEC being infected by Cn, HIV-1gp41-I90:use HBMEC to pave96-hole plate106cell/hole24hours before the experiment. Dilute the strains of fungi to be used in the experiment with EM culture medium to108cfu/ml for use and stimulate single layer of HBMEC under various conditions mentioned as below:incubate for3h with different dosage of Cn, HIV-1gp41-I90; incubate for different time with the same dosage of Cn, HIV-1gp41-I90; incubate for3h with different strains of bacteria with the same amount; after the incubation of fungi or HIV-1gp41-190, incubate Bikunin or anti-CD44antibody and then incubate for1h after adding THP-1. Count the number of THP-1cells that adhere to HBMEC and calculate the adhesive rate.2. Monocyte chemotactic migration experiment to detect the mononuclear cell migration after HBMEC being infected by Cn, HIV-1gp41-I90:pave106cell to Millicell (Millicell,12μm pore diameter) to culture for3-5days. Perform the chemotaxis experiment when the TEER value is tested within200-300Ωcm2. Stimulate the HBMEC in Millicell under various conditions mentioned as below: incubate for3h with different dosage of Cn, HIV-1gp41-190; incubate for different time with the same dosage of Cn, HIV-1gp41-I90; incubate for3h with different strains of bacteria with the same amount; after the incubation of fungi or HIV-1gp41-I90, incubate Bikunin or anti-CD44antibody and then incubate for3h after adding THP-1. Count the number of cells that migrated to the bath solution and calculate the migration rate.3. Using immunofluorescence to detect the CD44expression after HBMEC being infected by Cn, HIV-1gp41-I90:culture HBMEC on a24-hole plate. Culture different bacteria quantity of wild strains B4500FO2for3h and observe the impact of different bacteria quantity infecting HBMEC; Culture the bacteria quantity of B4500FO2for30min,2h and3h respectively to observe the impact of different incubation time to CD44expression; incubate HBMEC for3h with0.5×106cfu/mL of B4500FO2, gp41-I9020μg/mL and20μg/mL of gp41together with5×106cfu/mL of Cn and observe CD44expression status by means of immunofluorescent staining.Results1. The adhesion and migration effect in THP-1cell after HBMEC being infected by Cn are related to fungal infection time and quantity of the bacteria.After HBMEC being infected by different dosage sets (106,5×106and5×107cfu/mL) of wild strains of B4500FO2, the induced THP-1cell relative adhesion rates are136±6.7%,165±6.4%and197±9.6%respectively and the migration rates are28.875±2.21%,29.625±1.15%and36.55±1.95%respectively. They show significant difference compared with the control group, P<0.05; after HBMEC being infected by different infection time sets (1h,2h, and6h) of wild strains of B4500FO2, the induced THP-1cell relative adhesion rates are165±6.4%,221±9.8%and320±14.7%; for incubation time sets (1h,2h,3h,6h,12h and24h), the corresponding migration rates are23.42±1.39%,27.925±0.34%,29.625±1.15%,39.575±3.55%,45.055±6.11%and50.77±2.83%respectively. They show difference compared with the control group, P<T0.05; and the different is obvious for6-24h sets, P<0.01.2. The adhesion and migration effect in THP-1cell after HBMEC being infected by Cn can be blocked to different level by CD44blocker, Bikunin and antibody.After applying different concentration of blocker Bikunin (0.1nM,1nM,5nM and20nM), the relative adhesion rates are82±4%,48±3.4%,37±3.4%and30±3.9%respectively and the migration rates are21.625±1.22%,19.45±2.22%,13.675±0.64%and12.725±1.61respectively; after applying different concentration of anti-CD44antibody, the relative adhesion rates are91±3.6%,77±3.3%,58±4.5%and32±3.9%and the migration rates are21.775±1.86%,21.775±1.86%,13.875±1.99%and10.075±1.15%respectively. There is obvious difference between each set and the control group, P<0.01.3. The adhesion and migration effect in THP-1cell after HUM EC being infected by Cn are related to Cn capsule HA.The induced relative adhesion rates of THP-1after infecting endothelial cells by HA wild strains, lacking strains and anaplerosis strain are165±6.3%,126±5.8%and158±6.9%respectively. There is obvious difference comparing with the wild strain, control group and lacking strain, P<0.01; the migration rates are30.575±1.99%,23.85±1.79%and26.825±1.67%. There is obvious difference comparing with the wild strain, control group, P<0.01and there is obvious difference comparing with the wild strain and HA lacking strain, P<0.05.4. The Cn quantity and infection time affect CD44expression on HBMEC cell.By adopting the fluorescently labeled antibody detection technology, we found that with the increasing of fungal concentrations and incubating time, CD44expression on cell membrane will enhance and the signals will concentrate to the cell membrane. Different incubating times also have great influence to CD44expression. The fluorescence signal will become stronger for2h and3h sets compared with the control group and concentrate to the cell membrane.5. The adhesion and migration effect in THP-1cell after HBMEC being infected by Cn are related to the dosage and fungal infection time.High concentration improves and longer incubating time of HIV-1gp41-190incubating HBMEC can promote the adhesion and migration of THP-1. The induced relative adhesion rates of THP-1under4concentration groups of HIV-1gp41-190(0.2,2,20and200μg/mL) are107±0.5%,117.5±9.5%,150±2.6%and235±15%. When the concentration reaches to2μg/mL, the relative adhesion rate show obvious difference comparing with the control group, P<0.05; When the concentration reaches to20-200μg/mL, the relative adhesion rate show obvious difference comparing with the control group, P<0.01; the migration rates are21.575±4.47%,24.483±3.16%,29.05±3.48%and30.6±3.97%respectively, there is obvious difference compared with the control group when the concentration is within20-200μg/mL, P<0.05.6. The adhesion and migration can effect in THP-1cell after HBMEC being infected by HIV-1gp41-I90enhanced Cn.The induced relative adhesion rates of THP-1being infected by4groups namely PBS, Cn, HIV-1gp41-I90and HIV-1gp41-I90+Cn are100±0%,186.5±7.47%,150.7±2.8%and228.6±29.3%respectively, there is obvious difference for each group comparing with the control group, P<0.01; the relative adhesion rate show obvious difference when compared with HIV-1gp41-I90+Cn group and single Cn group, P<0.01; the relative adhesion rate show extremely obvious difference when compare with HIV-1gp41-I90+Cn group and single HIV-1gp41-190group, P<0.001; From the migration experiment, the migration rate has been significantly improved (P<0.01) compared with control group no matter for gp41, Cn single action on HBMEC or combined action of HIV-1gp41and Cn. According to multiple comparison between groups results of repeated measurement data analysis of variance, there is obvious difference comparing gp41+Cn group and control group and single gp41group (P<0.01).7. The induced transmigration of THP-1after HBMEC being infected by HIV-1gp41-190may enhance Cn is closely related to CD44molecule.The induced migration rates of THP-1after4concentration groups of Bikunin (0.1nM,1nM,5nM and20nM) restraining HIV-1gp41-190enhanced Cn infection of endothelial cells are19.21±3.71%%,15.22±2.99%,12.75±1.9%and11.725±4.84%respectively. There is obvious difference comparing5nM and20nM concentration group with the control group, P<0.01. Besides, it has been detected through CD44Immunofluorescence that the CD44fluorescence signal expressed on endothelial cells is the strongest when at HIV-1gp41-I90+Cn group.Statistical analysisData are shown in mean±standard deviation. The statistical approach adopts one-way ANOVA of SPSS13.0statistical software and variance analysis of repeated measurement data, transformation of variables at heterogeneity of variance. The difference is statistically significant when P<0.05.Conclusion1. Cn’s in vitro infection of BMEC could induce the adhesion and migration of THP-1.2. The adhesion and transmigration of THP-1induced by Cn’s in vitro infection of BMECs may be related to CD44receptor. 3. The interaction between Gp41-190and BMECs could induce the adhesion and transmigration of THP-1.4. HIV-1gp41-I90enhanced the adhesion and transmigration of THP-1in BMECs infected with Cn.5. The chemotaxis and transmigration of THP-1in BMEC infected with Cn may be related to CD44receptor. |
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