CN111494610B

CN111494610B - New use of IFN-lambda in Toxoplasma gondii infection

Info

Publication number: CN111494610B
Application number: CN202010453637.1A
Authority: CN
Inventors: 陈金铃; 刘逸凡; 申潭
Original assignee: Nantong University
Current assignee: Nantong University
Priority date: 2020-05-26
Filing date: 2020-05-26
Publication date: 2023-02-17
Anticipated expiration: 2040-05-26
Also published as: CN111494610A

Abstract

The invention discloses an application of IFN-lambda in preparing a medicament for treating or preventing Toxoplasma gondii infection. The invention also discloses a composition for preventing or treating Toxoplasma gondii infection, which is characterized in that the active ingredient of the composition is IFN-lambda. The invention further discloses the application of IFN-lambda in inhibiting Toxoplasma gondii infection, which comprises the following aspects: (1) preparing pregnant mice; (2) preparing different groups of model groups; (3) Monitoring the weight change of the pregnant mice, and observing the abortion rate of the pregnant mice, the weight of fetal mice and the size of fetal mice; (4) structural changes of placentas of different pregnant mouse groups; (5) detecting the expression condition of CK functional molecules in the placenta; (6) After the Toxoplasma gondii and JEG-3 cells are co-cultured, the expression of the Toxoplasma gondii SAG1 is detected by PCR. The invention provides a new way and a new method for treating Toxoplasma gondii infection, and solves the problem that bad pregnancy caused by Toxoplasma gondii infection cannot be treated in the prior art.

Description

New use of IFN-lambda in Toxoplasma gondii infection

Technical Field

The invention belongs to the technical field of biological medicine, relates to a new application of IFN-lambda in Toxoplasma gondii infection, and particularly relates to an application of IFN-lambda in preparing a medicine for treating or preventing Toxoplasma gondii infection, a composition for preventing or treating Toxoplasma gondii infection and an application of IFN-lambda in inhibiting Toxoplasma gondii infection.

Background

Toxoplasma gondii (Toxoplasma agndii) is an important opportunistic pathogenic parasite. Individuals with normal immunity are mostly in a recessive infection state after being infected with toxoplasma gondii. Toxoplasma infections are a significant cause of death in patients with impaired or defective immune function, such as AIDS, organ transplantation and malignancies. Infection in pregnant women can affect the development of the fetus, resulting in congenital toxoplasmosis such as abortion, teratocarcinosis, dead fetus, premature birth, birth defects, etc. In recent years, the number of pets (particularly cats) kept in urban areas has been increasing rapidly, and as a result of such reasons as preference for poor eating habits such as wild animals, more and more people are exposed to the risk of toxoplasma infection. Statistically, about 10 million people worldwide are infected with toxoplasma, with pregnant women having an infection rate of 10% to 27.5%, and about 9 million newborns are threatened by toxoplasma infection each year. As a large population, china has a great challenge on how to effectively prevent and treat congenital toxoplasmosis.

Interferons (IFNs) are largely classified into type 3: type I interferons (IFN-alpha and IFN-beta), type II interferons (IFN-gamma) and type III interferons (IFN-lambda). Type I IFNs (IFN-alpha and IFN-beta) are mainly antiviral, and induce the expression of various interferon activated genes (ISGs) through related signal paths, thereby playing the role of antivirus. Type II interferons (IFN-. Gamma.) are important cytokines that inhibit the proliferation of Toxoplasma gondii. After the toxoplasma tachyzoite invades the body, macrophages are stimulated to generate IL-12, and NK cells and T cells are further activated to secrete IFN-gamma. IFN-gamma then induces expression of IFN-gamma-industrigenes, which in turn directly kills tachyzoites that are parasitic in the host cell. However, during pregnancy, IFN- γ production and excessive secretion are major factors in the development of poor pregnancy. IFN-gamma recruitment of CD49b ⁺ NK simultaneously regulates the expression of Ly-49 receptor of NK cells, resulting in the occurrence of abortion. IFN-gamma up-regulates turnnecrosisfactor-alpha (TNF-alpha) and down-regulates matrixmetalloprotenases-2 and-9 (MMP-2 and MMP-9) expression in the rat abortion model, further aggravating abortion. In human pregnancy, the effect of IFN- γ on causing poor pregnancy has also been confirmed, and T cells of fetal origin promote uterine contraction by secreting IFN- γ and TNF- α, thereby causing the occurrence of premature labor. Thus, IFN- γ does not protect the mother during pregnancy against toxoplasma infection, maintaining normal pregnancy.

IFN- λ (type III interferon) has a relatively independent and specific ability to fight pathogen infection. The IFN-lambda receptor is composed of Interleukin 28receptor alpha (Interleukin-28receptor alpha, IL-28R alpha) and Interleukin 10receptor beta (Interleukin-10 receptor beta, IL-10R beta), wherein the IL-10R beta is widely present in cells and tissues, and the IL-28R alpha is mainly expressed on the surfaces of epithelial cells, neutrophils and hepatocytes. Receptor surfaceThe limitations mean that IFN- λ exerts a relatively independent and specific antipathogenic effect in certain tissues. The human IFN- λ family consists of IFN- λ 1, IFN- λ 2, IFN- λ 3 and IFN- λ 4, while the mouse has only two functional IFN- λ, IFN- λ 2and IFN- λ 3. In the research of rotavirus, researchers find that IFN-lambda 2 can obviously inhibit the replication of rotavirus in intestinal tracts of young mice, and the knockout of IFN-lambda receptors can promote the proliferation of the rotavirus in mice. Muir et al first suggested that IFN-. Lambda.1 a could inhibit the proliferation of Hepatitis C Virus (HCV). IFN-. Lambda.1a inhibits HCV replication rapidly (within 12 hours) and achieves a similar effect to IFN-. Alpha.within 24 hours. However, the rate of the patients with the complications such as thrombocytopenia and neutropenia is obviously reduced. Therefore, IFN-. Lambda.1a is expected to be clinically useful as a substitute for IFN-. Alpha.for HCV therapy. Recaccal, et al, found CCR2 by studying antifungal immune response using Aspergillus fumigatus (Af) as a model ⁺ Depletion of monocytes reduces the ability of neutrophils to inhibit the growth of invasive fungi. IFN-. Lamda.2/3 acts directly on neutrophils to activate their antifungal response, whereas neutrophils specifically deficient in IFN-. Lamda.receptors die of invasive aspergillosis. Transfer of CCR2 by adoptive ⁺ Monocytes or IFN-. Lambda.2/3 were effective in treating mice depleted of neutrophil CCR 2. Therefore, IFN-. Lambda.2/3 is a key regulator of neutrophils and exerts antifungal effects. Cryptosporidium parvum (c. Parvum) is an important opportunistic pathogenic protozoa causing clinical manifestations mainly of diarrhea. Exogenous IFN- λ 3 reduces insect load in Intestinal Epithelial Cells (IECs), restores transmembrane resistance (TEER), and resists c. Although a large number of experimental data support the ability of IFN- λ to inhibit the proliferation of viruses, fungi and Cryptosporidium parvum, whether IFN- λ inhibits the proliferation of Toxoplasma gondii is under further investigation.

Disclosure of Invention

The purpose of the invention is as follows: the invention provides the use of IFN-lambda in the manufacture of a medicament for the treatment or prevention of Toxoplasma gondii infection, a composition for the prevention or treatment of Toxoplasma gondii infection, the use of IFN-lambda in the inhibition of Toxoplasma gondii infection; to solve the problems in the prior art.

In view of the above objects, the present invention provides the use of IFN- λ for the manufacture of a medicament for the treatment or prevention of Toxoplasma gondii infection.

Further, the IFN- λ intervention inhibits the proliferation of toxoplasma gondii.

The embodiment of the invention also provides a composition for preventing or treating Toxoplasma gondii infection, which is characterized in that the active ingredient of the composition is IFN-lambda.

Further, the composition comprises IFN- λ and one or more pharmaceutically acceptable carriers.

Wherein the carrier comprises pharmaceutically acceptable diluents, excipients, fillers, binders, absorption promoting agents, surfactants and synergists.

The invention further provides the use of IFN- λ for inhibiting toxoplasma gondii infection, which comprises:

(1) Preparing a pregnant mouse: closing the female mouse and the male mouse at 5 pm in the first night, and detecting a white vaginal suppository at 7 pm the next day; if the vaginal suppository is detected, determining the pregnancy period of the mouse to be E0.5;

(2) Grouping the pregnant mice in the step (1) and preparing models of different pregnant mice groups, wherein the specific grouping is as follows: 1. pregnant mice were not infected with toxoplasma gondii group; 2. pregnant mice infected with toxoplasma gondii group; 3. infecting toxoplasma gondii group after pre-treating IFN-lambda 24 h;

(3) Monitoring the weight change of the pregnant mice, and observing the abortion rate of the pregnant mice, the weight of the fetal mice and the size of the fetal mice;

(4) HE staining to observe structural changes of the placenta of different pregnant mouse groups;

(5) Performing immunofluorescence detection on the expression condition of CK functional molecules in the placentas of different pregnant mice;

(6) After the Toxoplasma gondii and JEG-3 cells are co-cultured, PCR is carried out to detect the expression of the Toxoplasma gondii SAG1 in each cell model group. The cell model groups are specifically grouped as follows: 1. cells were not infected with toxoplasma gondii group; 2. infection of cells with toxoplasma gondii group; 3. the cells are stimulated by Toxoplasma gondii and IFN-lambda together; 4. infecting toxoplasma gondii group after IFN-lambda is pretreated for 24 h; 5. cells with Toxoplasma gondii and IFN-gamma co-stimulation group; 6. cells were infected with Toxoplasma gondii 24h after pretreatment with IFN-. Gamma..

Further, in the step (2), the IFN-lambda pretreatment process for Toxoplasma gondii infected pregnant mice: pregnant mice were injected with IFN- λ at a concentration of 2 μ g/ml on day 9.5 of gestation, and infected with Toxoplasma gondii on day 10.5; then injecting IFN-lambda into pregnant mice every day until 18.5 days of gestation; on day 18.5, fetal mouse size was measured by hip length x frontal diameter of occipital, while observing miscarriage rates; collecting each tissue sample in the mother mouse and the fetal mouse, and carrying out subsequent experiments.

Further, in the step (4), the HE staining process: washing the slide with tap water for 3min; washing with distilled water for 1min; placing the slide in a solution of 3% TritonX-100 (precooled at 4 deg.C) and allowing to permeate for 5min; staining with hematoxylin for 10min; washing for 30s by tap water; 2% hydrochloric acid ethanol differentiation for 20s; washing with tap water for bluing for 5-10min; eosin staining for 30s-2min; gradient dehydrating 50%, 70%, 80%, 95% and anhydrous alcohol for 1-3min; the xylene I and the xylene II are transparent for 1-3min; sealing the neutral resin sheet, and shooting an image under a microscope;

further, the cellular immunofluorescence detection process in the step (5): collecting mouse placenta tissue, and making into 6-8 micrometer slices; when the slices are dyed, the slices are dried for 15 minutes at room temperature; then soaking the sample in PBS for 10 minutes to remove OCT;0.5 percent TritonX-100 percent for 20min at room temperature; blocking the slices for 1h at room temperature by using PBS containing 10% goat serum; dropping the diluted primary antibody on a glass slide, and placing the glass slide in a refrigerator at 4 ℃ for incubation overnight; after washing with PBS for 3 times, dripping the diluted fluorescent anti-di-antibody on a slide, and incubating for 90min at room temperature in a dark place; after washing with PBS for 3 times, dripping Hoechst dye on a slide, and incubating for 15min at room temperature in a dark place; after 3 times of PBS washing, 50% glycerol mounting; and (3) shooting images by using laser confocal technology.

Further, in the step (6), the process of detecting the number of Toxoplasma gondii: collecting tissue or cell, adding Trizol to the tissue or cell of proper weight for lysis, extracting RNA, and reverse transcribing with Oligo (dT) 18 to obtain cDNA with reverse transcription kit; freezing and storing at minus 80 ℃; after 10 times diluted toxoplasma gondii infects JEG3 cells, the extracted RNA is reversely transcribed into cDNA, and the cDNA is used as a template to be amplified on a fluorescence quantitative PCR instrument to establish a standard curve; reaction conditions are as follows: pre-denaturation at 95 ℃ for 3min; 40 cycles: 95 ℃ for 15s;60 ℃ for 1min; the calculated relative amount of toxoplasma gondii SAG1 corresponds to the standard curve.

The technical scheme of the invention has the following beneficial effects:

(1) According to the invention, multiple groups of pregnant mouse models are prepared, and multiple groups of pregnant mouse model experiments prove that IFN-lambda intervention can obviously reduce the abortion rate caused by toxoplasma infection, reduce the damage of mouse placenta caused by toxoplasma infection, and up-regulate the expression of functional molecules such as CK in the placenta; meanwhile, after the toxoplasma gondii and the JEG-3 cells are co-cultured, the expression of the toxoplasma gondii SAG1 in each cell culture model group is detected by PCR, and the inhibition of the IFN-lambda intervention on the proliferation of Toxoplasma gondii is verified, so that the invention verifies that the IFN-lambda intervention can treat the bad pregnancy caused by Toxoplasma gondii and inhibit the proliferation of Toxoplasma gondii, and provides a theoretical basis for treating Toxoplasma gondii infection.

(2) The invention provides application of IFN-lambda in inhibiting Toxoplasma gondii infection, a medicament for preventing or treating Toxoplasma gondii infection, provides a new way and method for treating Toxoplasma gondii infection, and solves the problem that bad pregnancy caused by Toxoplasma gondii infection cannot be treated in the prior art.

Drawings

FIG. 1 is a graph showing the abortion of a group of pregnant mice not infected with Toxoplasma gondii, a group of pregnant mice infected with Toxoplasma gondii, and a group of pregnant mice infected with Toxoplasma gondii after IFN-. Lambda.pretreatment for 24h in example 1 of the present invention;

FIG. 2 is a graph showing the damage of the placenta of mice infected with Toxoplasma gondii after the pregnancies are not infected with Toxoplasma gondii, infected with Toxoplasma gondii and pretreated with IFN-. Lambda.24 h in example 2 of the present invention;

FIG. 3 is a graph showing the expression of placental CK in a group of Toxoplasma gondii infected with pregnant mice, and a group of Toxoplasma gondii infected with pregnant mice pretreated with IFN-. Lambda.24 h in example 3 of the present invention;

FIG. 4 is a graph showing the expression of Toxoplasma SAG1 in example 4 of the present invention, after co-culture of Toxoplasma gondii and JEG-3 cells, the cells were not infected with Toxoplasma gondii group, the cells were co-stimulated with Toxoplasma gondii and IFN-. Gamma., the cells were infected with Toxoplasma gondii group after 24h of pretreatment of IFN-. Lambda., the cells were infected with Toxoplasma gondii group, the cells were co-treated with Toxoplasma gondii and IFN-. Gamma., and the cells were infected with Toxoplasma gondii group after 24h of pretreatment of IFN-. Gamma..

Detailed Description

In order to make the technical problems, technical solutions and advantages to be solved by the present invention clearer, the following detailed description is given with reference to specific embodiments.

Experimental Material

1.1, JEG-3 cell line was purchased from ThermoFisher scientific.

1.2 Trizol reagent was purchased from Invitrogen, USA.

1.3, reverse transcription reagents were purchased from Bio-Rad, USA.

1.4 SYBRGreenMasterMix, taqMan Universal MasterMix, available from ThermoFisher scientific, USA.

1.5 primers were synthesized by Sigma, USA.

1.6 fetal bovine serum was purchased from Hyclone.

1.7 pancreatin and MEM were purchased from ThermoFisher scientific, USA.

Preparation of the experiment

At least 24 male mice and 48 female mice were divided into 24 groups, each group including 2 female mice and one male mouse, 2 female mice and 1 male mouse were housed at 5 pm in the evening before, and white vaginal plugs were detected 7 pm the day after. If vaginal emboli are detected, the pregnancy time of the mouse is determined to be E0.5 (embryonicary 0.5).

The 24 groups of pregnant mice were grouped and prepared into different groups of model groups, which were specifically grouped as follows: 1. pregnant mice were not infected with toxoplasma gondii group (normal pregnant group); 2. pregnant mice infected the toxoplasma gondii group; 3. infecting toxoplasma gondii group (toxoplasma gondii and IFN-lambda co-treatment group) after pre-treating IFN-lambda for 24 h; at least 4 mice were pregnant in each group and the experiments in examples 1-3 below were performed.

Example 1 IFN-Lambda intervention significantly reduced the efflux rates caused by Toxoplasma gondii infection

In E10.5, the pregnant mouse is injected with 1X 10 abdominal cavity ⁴ Toxoplasma tachyzoite was killed on E18.5 days. The change of the weight of the pregnant mice is monitored, and the abortion rate of the pregnant mice, the weight of the fetal mice and the size of the fetal mice are observed. The toxoplasma gondii is infected in the middle pregnancy period, and the weight of the pregnant mice is in a descending trend compared with the weight of the pregnant mice. Killing on day E18.5, as shown in fig. 1, when pregnant mice were infected with toxoplasma gondii in the toxoplasma gondii group, the toxoplasma gondii infection in the toxoplasma gondii group resulted in an abortion rate of about 58.9% compared to that of pregnant mice that were not infected with toxoplasma gondii group, and after pregnant mice were pretreated with IFN- λ for 24h, the abortion rate of pregnant mice infected with toxoplasma gondii group was down-regulated, thus indicating that the abortion rate of pregnant mice was significantly down-regulated after IFN- λ intervention. These results suggest that: IFN- λ improves poor pregnancy outcome resulting from Toxoplasma gondii infection.

Example 2 IFN-Lambda reduction in Toxoplasma damage caused by Toxoplasma infection

After the pregnant mice were infected with toxoplasma at day E10.5, mouse placentas were collected at day E18.5 and formalin-fixed for HE staining to observe structural changes of the placentas. The normal structure of mouse placenta is divided into a periostracum layer (Decidua, DE), a Junction Zone (JZ) and a maze zone (LabyrinthZone, LZ). Toxoplasma gondii infection in the pregnant mouse toxoplasma gondii-infected group destroyed the normal structure of placenta, as shown by thinning of the periostracum layer and connecting region, accompanied by a significant decrease in the number of cells in the labyrinthine region of the placenta, as shown in FIG. 2, suggesting decreased supply of nutrients to the placenta and gas exchange failure. And the pregnant mouse is infected with toxoplasma gondii group after IFN-lambda is pretreated for 24h, the thicknesses of the decidua layer and the connecting area are close to the normal mouse placenta after the intervention of IFN-lambda, and simultaneously, the cell number in the labyrinth area is obviously increased. These results indicate that toxoplasma infection can disrupt the normal architecture of the mouse placenta, while IFN- λ can significantly reduce placental damage.

Example 3 IFN-. Lambda.intervention upregulates the expression of functional molecules like CK in the placenta

Cytokeratin (CK) is a marker protein for mouse placental trophoblast cells. In the pregnant mouse toxoplasma infected group, the number of CK in the mouse placenta was significantly decreased compared to the expression of a large amount of CK observed in the mouse placenta in the normal pregnant group (uninfected toxoplasma group), indicating that toxoplasma can decrease the number of mouse placental trophoblast cells. As shown in figure 3, after IFN-lambda is pretreated for 24h, the pregnant mice are infected with Toxoplasma gondii group, and after IFN-lambda dry prognosis, CK expression is obviously increased, which indicates that IFN-lambda can reduce placenta damage and improve placenta function.

Example 4 IFN-Lambda intervention inhibits Toxoplasma gondii proliferation

In this example, the inhibition of the proliferation of Toxoplasma gondii by IFN- λ intervention was verified using an in vitro experiment in cell culture, wherein the cell model groups in the in vitro experiment of this example are specifically grouped as follows: 1. cells were not infected with toxoplasma gondii group; 2. infection of cells with toxoplasma gondii group; 3. the cells are stimulated by Toxoplasma gondii and IFN-lambda together; 4. infecting toxoplasma gondii group after IFN-lambda is pretreated for 24 h; 5. cells with Toxoplasma gondii and IFN-gamma co-stimulation group; 6. cells were infected with toxoplasma gondii 24h after pretreatment with IFN-. Gamma..

The process of infecting the JEG-3 cell line with Toxoplasma gondii is as follows: culturing JEG-3 cell line in cell culture flask, and culturing in MEM complete medium containing 10% fetal calf serum at 37 deg.C for 5% CO ₂ Culturing in a cell culture box; replacing culture solution every other day for the cells, and carrying out cell passage or carrying out subsequent experiments when the cells reach 80% of fusion degree; 2 x 10 ⁵ The JEG-3 cells/well were plated on a 6-well plate, and after 24 hours of culture in a cell incubator, toxoplasma gondii infected the JEG-3 cell line at a multiplicity of infection of 2.

In this example, JEG-3 cells were co-cultured with Toxoplasma gondii, and real-timePCR detected SAG1 expression in each cell model group. IFN-gamma and Toxoplasma gondii act on JEG-3 cells together in vitro, after co-culture for 48h, cell culture supernatant is collected, and the expression of the Toxoplasma gondii SAG1 is detected by using the Real-timePCR method. As a result, the cells, the toxoplasma gondii and IFN-gamma co-stimulation group and the cells are infected by the toxoplasma gondii group as two positive control groups after 24 hours of pretreatment of IFN-gamma, wherein the IFN-gamma has the capacity of inhibiting the proliferation of the toxoplasma gondii. Similarly, the cells significantly reduced SAG1 expression by IFN- λ in the Toxoplasma gondii and IFN- λ co-stimulated group, as shown in FIG. 4, while the expression of SAG1 in Toxoplasma gondii infected with Toxoplasma gondii was further reduced 24h after the cells were pretreated with IFN- λ.

These results indicate that IFN- λ inhibits Toxoplasma gondii proliferation in vitro.

While the foregoing is directed to the preferred embodiment of the present invention, it will be appreciated by those skilled in the art that various changes and modifications may be made therein without departing from the principles of the invention as set forth in the appended claims.

Claims

Use of IFN- λ for the manufacture of a medicament for the treatment or prevention of toxoplasma gondii infection, wherein said IFN- λ intervenes in the inhibition of the proliferation of toxoplasma gondii and in the treatment of poor pregnancy caused by toxoplasma gondii.