| BackgroundToxoplasma gondii is an intracellular parasitic Protozoa that infects a wide variety of hosts. Nearly one billion people worldwide have been infected with Toxoplasma gondii at present. According to the routes of infection, there are two kinds of infection which is acquired infection and congenital infection. Acquired infection occurs due to ingesting ainimal meats with cysts of the parasite which are not cooked, raw milk, raw eggs or eating food with Toxoplasma gondii oocysts in cat feces.In recent years, researches have shown that acquired infections of Toxoplasma gondii are increasing, which can cause full pigment film inflammatory or necrosis retinal inflammatory. Heavy disorder of pigment film inflammatory can make retinal detachment, glaucoma and endophthalmitis leading to blindness. As Toxoplasma gondii infection rates constantly increase, the pathogenesis and the effective treatment of Toxoplasmosis ophthalmopathy has cause widely concerning. After infected by Toxoplasma gondii, cells of our bodies play a strong resistance of immune effects to Toxoplasma, such as T lymphocytes, natural killer cells, and macrophages. According to the different function, T cells can be divided into subgroups of CD4+T cells and CD8+T cells. Most CD4+T cells are Th cells (helper T cells). According to different cytokine production and biological function, traditional CD4+T cells are divided into subgroups of Thl and Th2type cells, they control and restraint in the immune process. Researches have shown that when acute Toxoplasma infection occurs in eyes, the concentration of y-interferon (IFN-y) and tumor necrosis factor-alpha (TNF-a) secreted by Thl cells in aqueous humor significantly increased, and the concentration of interleukin-10(IL-10) secreted by Th2cells significantly decreased. IFN-y and TNF-a plays very important roles in macrophage activation and killing parasites, which are the main active elements of the body fighting against Toxoplasma gondii. But excess IFN-y and TNF-a can cause excessive immune response and increase inflammatory response of the organization or even cause tissue necrosis. IL-10can reduce the secretion of IFN-y and TNF-a. Researches showed that IL-10played an important role in the balance of protective immunity and immune pathology. Then, early in the acute infection of Toxoplasma gondii Whether intravenous injection of IL-10can affect the concentration of IFN-y or TNF-a in peripheral blood and aqueous humor and further reduce the inflammation of the eyes or not?CD4+CD25+T cells (Treg) were discovered different from Thl and Th2cells in recent years. As a kind of regulatory T cells, they play an important role in the regulation of immune function of pathogen infecting disease by secreting IL-10and other cytokines. Thl7is another type of CD4+T cell subsets different from Thl and Th2cells, secreting IL-17, studies have shown that Th17cells play an important role in mediating inflammatory responses and pathogenesis of autoimmune disease. Under normal circumstances, the number of T cells and subsets in the surrounding tissue is relatively stable, the change of absolute number and ratio of the total number of T cells or subsets can be regarded as immune abnormalities and the occurrence and development of certain diseases. Distribution of Treg/Th17imbalance occurred in many disease processes. In acute Toxoplasma infection in a mouse model, Treg/Th17balance in the spleen is broken. Whether the Treg/Th17imbalance can further elaborate in pathogenesis of Toxoplasma gondii infection or not? There are no reports in the literature. Studies in vitro have shown that IL-10can reduce number of Th17through Treg. Then, Whether IL-10by intravenous injection affect on the splenic Treg/Th17balance or not in acquired toxoplasmosis ophthalmopathy model?ObjectiveTo understand Treg/Th17imbalance in mice spleen and effects of IL-10on murine ocular toxoplasmosis, this research established acquired toxoplasmosis ophthalmopathy C57BL/6mouse model, detected percentage of Treg and Thl7cells to total CD4+cells in mice spleen infected by Toxoplasma gondii and analysis the relationship between Treg/Th17balance and Toxoplasma gondii infection of the eye. By exogenous cytokine IL-10intervention, we discussed the effects of IL-10on Treg/Th17balance, the concentration of IFN-y and TNF-a in vivo and effects on retinal inflammation of murine ocular toxoplasmosis.MethodsOne hundred and sixty C57BL/6mice were divided into four groups:forty mice in the control group, forty mice in the infected group (experiment A),forty mice in the injecting0.01ug IL-10group (experiment B),and forty mice in the injecting0.1ug IL-10group (experiment C).102(0.2ml) tachyzoites of Toxoplasma gondii virulent strain (RH strains) were given by intraperitoneal injection of each mouse in experiment group, while each mouse in control group was injected with saline. On2d and4d after infection parasites, each mouse was given0.01μg I1-10by tail vein injection (dissolved in200ul sterile saline) in experimental B group, and each mouse was given0.1μg I1-10by tail vein injection (dissolved in200ul sterile saline) in experimental C group. After six days of infection, all mice were sacrificed and removed the eyes. Histological sections of the four groups were detected. Peripheral blood was collected. The DNA of Toxoplasma gondii in eyes and serum was detected.The mouse spleens were removed in control group and experimental groups to detect percentage of Treg and Th17cells to total CD4+cells by flow cytometry. The concentration of IFN-γ and TNF-α in eyes and serum were detected in experimental groups.Results1. PCR detection. P30gene could be detected in experiment groups on the sixth day.2. Retinal change in histological slices of differerent groups. Toxoplasma and apparent inflammation were watched in retinal slices in experiment A and B group.Toxoplasma and light inflammation were watched in retinal slices in experiment C group.3. Concentration of IFN-γ and TNF-α in aqueous humor. Compared with the concentration of IFN-γ (12.3pg/ml))and TNF-α (1165.2pg/ml)of aqueous humor in the control group, the concentration of IFN-γ (4531.4pg/ml)and TNF-α (1572.3pg/ml) in aqueous humor was higher in experiment A group; Compared with experiment A group, the concentration of IFN-γ (3838.7pg/ml) and TNF-α (1431.1pg/ml) in aqueous humor was lower in experiment B group; Compared with experiment A group, the concentration of IFN-γ (2482.7pg/ml) and TNF-α (1248.1pg/ml) in aqueous humor was lower in experiment C group.4. Concentration of IFN-y and TNF-α in serum. Compared with the concentration of IFN-γ (16.4±3.1pg/ml) and TNF-a (8.7±2.1pg/ml) in serum of the control group, the concentration of IFN-y (455.7±39pg/ml) and TNF-a (14.6±2.2pg/ml) in serum was higher in experiment A group, The difference was statistically significant (P<0.05); Compared with experiment A group, the concentration of IFN-y (389.8±42pg/ml) and TNF-a (13.1±1.7pg/ml) in serum was lower in experiment B group, The difference was statistically significant (P<0.05); Compared with experiment A group, the concentration of IFN-y (265.7±27pg/ml) and TNF-a (12.1±1.9pg/ml) in serum was lower in experiment C group. The difference was statistically significant (P＜0.05).5. The percentage of Treg cells to total CD4+cells in spleen. Compared with control group (39.16±12.53) in the spleen, the percentage of Treg cells to total CD4+cells in the experiment A group (18.41±5.32) was lower. The difference was statistically significant (P＜0.05). Compared with experiment A group, the percentage of Treg cells to total CD4+cells in the experiment C group (31.27±8.47) was higher. The difference was statistically significant (P<0.05).6. The percentage of Th17cells to total CD4+cells in spleen. Compared with control group (9.41±7.54) in the spleen, the percentage of Th17cells to total CD4+cells in the experiment A group (33.15±8.37) was higher. The difference was statistically significant (P<0.05). Compared with experiment A group, the percentage of Th17cells to total CD4+cells in the experiment C group (14.57±9.32) was lower. The difference was statistically significant (P<0.05).7. Ratio of Treg/Th17in spleen. Compared with control group (4.26±1.23) in the spleen, the ratio of Treg/Th17in experiment A group (0.54±0.17) was lower. The difference was statistically significant (P＜0.05). Compared with experiment A group, the ratio of Treg/Th17in experiment C group (2.33±1.16) was higher. The difference was statistically significant (P<0.05).ConclusionAcquired toxoplasmosis ophthalmopathy model was established by intraperitoneal injection of virulent strain of Toxoplasma gondii (RH strain) infecting C57BL6mice. Lesions were mainly concentrated in the retina. Nested-PCR test had high specificity and sensitivity. After infection with Toxoplasma gondii, the concentrations of IFN-y and TNF-a in serum and aqueous humor increased, which might be associated with inflammation of the eye infected by Toxoplasma gondii. Percentage of Treg cells to total CD4+cells decline in spleen, Percentage of Th17cells to total CD4+cells increased, the ratio of splenic Treg/Th17significantly decrease and broke the normal immune response. By tail vein injection of IL-10, The concentration of IFN-γ and TNF-a in the aqueous humor and serum were lower, the ratio of Treg/Th17became higher, and retinal inflammation decreased. Thus we speculated that by changing Treg/Th17balance and making the concentration of IFN-γ and TNF-a lower in body, exogenous IL-10could reduce inflammation of eye infected by Toxoplasma gondii in mice. |
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