A Study Of Toxoplasma Gondii Infection And Macrophage Polarization

Objective To investigate the polarization direction and cell morphological changes of rat alveolar macrophages after Toxoplasma gondii infection,as well as the changes of cellular morphology and structure.And further to study the effect of alveolar macrophage polarization on the proliferation of Toxoplasma gondii.Methods 1.Using normal NR8383 cells as a control group,macrophages were co-stimulated with LPS(100 ng/mL)or LPS(100 ng/m L)and IFN-γ(20 ng/mL).Using real-time fluorescent quantitative PCR to detect macrophage polarization factors.Western blot was used to detect the changes of the iNOS protein of M1 macrophage and determine the best inducer and induction time.The induced cells were observed and identified by scanning electron microscope and transmission electron microscope.2.Toxoplasma gondii infection-induced M1 macrophages were harvested for 24 h and48h respectively.Diff staining was used to observe the infection of Toxoplasma gondii in M1 macrophages.Scanning microscopy was used to observe M1 macrophage infection with Toxoplasma gondii.Transmission electron microscopy was used to observe the changes in the internal structure and organelles of M1 macrophages infected with Toxoplasma gondii.3.Toxoplasma gondii infected normal NR8383 cells and the cells and cell culture supernatants were collected at 0h,6h,12 h,24h,36 h and 48 h after infection.Real-time fluorescence quantification and ELISA were used to detect the changes of the polarization-related factors.Western blot was used to detect M1 and M2 types.Expression changes of macrophage marker proteins iNOS and ARG-1;Diff staining was used to observethe proliferation of Toxoplasma gondii in normal macrophages;scanning electron microscopy was used to observe the morphology changes of normal NR8383 macrophages after infection with Toxoplasma gondii;transmission electrons Microscopy was used to observe the changes in the internal structure and organelles of normal NR8383 macrophages infected with Toxoplasma gondii.Results 1.Real-time fluorescence quantitative PCR results showed that after LPS,LPS + IFN-γ stimulation of macrophages,TNF-a,INOS,CXCL10 inflammatory factor was significantly increased,reached the highest value at 6h.And further comparing the 6h inflammatory factors,the expression of LPS+IFN-γ was significantly higher than LPS alone(P<0.05).Western blot results showed that iNOS was not expressed in normal group,and the expression of iNOS in LPS+IFN-γ was higher than that in LPS alone.Under the scanning electron microscope,the burr-like synapses on the cell surface were significantly increased,and the cell volume became larger.Under the transmission electron microscope,the nucleus was pyknosis,a large number of lysosomes appeared in the cytoplasm,and the number of mitochondria increased.2.Diff staining showed that normal NR8383 macrophages showed clear karyoplasm and T.gondii could hardly detected the presence after infecting with M1 macrophages.Under the scanning electron microscope,a large number of round protrusions were observed on the surface of cells at 24 h.At 48 h after infection,the cells were disrupted.Under transmission electron microscopy observation,the cells were infected with a very small amount of Toxoplasma gondii,and the surface was raised like a rod.After 48 h infection,almost no T.gondii was present under the microscope,and there was a large amount of lysosomes and mitochondria in the cytoplasm.The nucleus was pyknosis and the cells were suspected of necrosis.3.Toxoplasma gondii infection of normal NR8383 cells,Diff staining results showed that Toxoplasma gondii can proliferate in the cell,and over time,the number also increasedsignificantly.Scanning electron microscopy showed that the surface of the infected cells had protrusions.Under the transmission electron microscope,the surface of the cell was raised like a rod,a small amount of lysosomes and a large amount of mitochondria appeared in the cytoplasm,and the cells were suspected to have apoptosis.RT-qPCR results showed that after T.gondii infection,the relative expression levels of M2-related genes TGF-β1 and ARG-1 in NR8383 cells increased from 6h after infection,reached the highest value at 24 h,and then the expression level began to decline,but remained high.In normal group,the relative expression level of CCL17 increased from 24 h,reached the highest at 36 h,but decreased at48 h but still higher than that of normal group;the relative expression of CCL18 increased from 12 h,increased at 36 h,decreased at later time but still high In the normal group(P<0.05).M1-type related factors increased at individual time points,while other time points did not change significantly compared with the normal group.Western blot results showed no expression of iNOS,ARG-1 began to increase after infection for 12 hours(P<0.01),and remained unchanged until 48 hours(P<0.01).Conclusion 1.Successfully constructed macrophages polarization model in vitro.2.M1 type macrophages can significantly inhibit Toxoplasma gondii proliferation,cells necrosis occurred.3.Toxoplasma gondii infection induced macrophage polarization into M2 type,cell apoptosis.