Method for regulating directional differentiation of helper T cell 17 and application thereof

Abstract

The invention belongs to the field of immunology, and particularly relates to a method for regulating directional differentiation of helper T cell 17 (Th17) and application of the method. The method for regulating and controlling the directional differentiation of the Th17 cells into the Th1 cells is provided firstly, specifically, the activity of a nucleolus composite pore associated protein 4 gene is inactivated or reduced, so that the directional differentiation of the Th17 cells into the Th1 cells is influenced. The invention also provides a pharmaceutical composition, which is a protein and / or a compound and / or RNA for inhibiting the gene function of the gene for directionally differentiating the helper T cell 17 into the Th1 cell. According to the application, the differentiation trend of Th17 to Th1 cells is regulated and controlled on the translation level by knocking out the Noc4L gene by utilizing the regulation and control function of the nucleolus composite pore associated protein 4 in the Th17 cells. The application can provide a new way and means for developing novel nucleic acid drugs to treat autoimmune diseases caused by inflammatory cytokines.

Description

Technical Field

[0001] This application belongs to the field of immunology, and in particular relates to a method for regulating the directed differentiation of helper T cells 17 and its application. Background Technology

[0002] Naive CD4 + Upon antigen stimulation, the T cell antigen receptor (TCR) on the surface of T cells differentiates into different helper T cells under the induction of corresponding cytokines. These induced helper T cells can also adjust their function and phenotype according to the constantly changing cytokine environment, thus exhibiting functional plasticity. Among them, CD4 + T cells differentiate into helper T cells 17 (Th17) under the co-induction of transforming growth factor-β (TGF-β) and interleukin-6 (IL-6). Th17 cells are one of the newly discovered helper T cell subsets in recent years, exhibiting a dual function. On the one hand, they help the host resist pathogenic bacterial and fungal infections, playing a crucial role in mucosal immunity; on the other hand, they are closely related to the occurrence and progression of autoimmune diseases. Furthermore, Th17 cells are highly unstable and plastic, capable of altering their intralineage phenotype while also acquiring characteristics of Th1, Th2, T follicular helper T cells (Tfh), or regulatory T cells. Therefore, Th17 cells can differentiate into other helper T cell types under the in vivo inflammatory environment or in vitro induction by corresponding cytokines. The most typical example is in the experimental autoimmune encephalomyelitis (EAE) model, where Th17 cells differentiated in vivo transform into Th1 cells and secrete large amounts of inflammatory cytokines such as interferon-γ (IFN-γ) and granulocyte-macrophage colony-stimulating factor (GM-CSF), which may trigger an inflammatory response in the central nervous system.

[0003] The plasticity of Th17 cells is determined by a series of intracellular and extracellular signals, including lineage-specific transcription factors, chromatin remodeling, key cytokines, environmental factors, and metabolic levels. Therefore, regulating Th17 cell differentiation can modulate the secretion of inflammatory cytokines. Although research on the regulation of Th17 cell plasticity is extensive, it primarily focuses on the transcriptional level, with very little research at the post-transcriptional level. Summary of the Invention

[0004] In view of this, this application aims to explore the specific regulation of Th17 cell plasticity changes at the posttranscriptional level.

[0005] The primary objective of this application is to provide a method for regulating the directed differentiation of Th17 cells into Th1 cells, the method comprising inactivating or reducing the expression activity of genes on the genome that affect the directed differentiation of Th17 cells into Th1 cells.

[0006] Furthermore, this gene is the nucleolar complex pore-associated protein 4 gene.

[0007] Furthermore, specific methods for inactivating or reducing the function of nucleolar complex pore-associated protein 4 include, but are not limited to, knocking out or silencing the nucleolar complex pore-associated protein 4 gene on the genome.

[0008] A second objective of this application is to provide a pharmaceutical composition comprising a protein and / or compound and / or RNA that inhibits the gene function of a gene that directs helper T cells 17 to differentiate into Th1 cells.

[0009] Furthermore, the gene that influences the directed differentiation of Th17 cells into Th1 cells is the nucleolar complex pore-associated protein 4 gene.

[0010] Specifically, the pharmaceutical composition is used to treat or prevent autoimmune diseases mediated by inflammatory cytokines secreted by Th17 cells, including but not limited to multiple sclerosis.

[0011] Specifically, inflammatory cytokines include interleukin-17, interferon-γ, granulocyte-macrophage colony-stimulating factor, and tumor necrosis factor.

[0012] The delivery methods of the pharmaceutical composition include, but are not limited to, oral or injectable administration and targeting mammals.

[0013] Beneficial effects of the invention

[0014] In recent years, the role of Th17 cells in autoimmune diseases has received increasing attention, and controlling the transition between anti-inflammatory and pro-inflammatory Th17 cells is a key aspect of future treatments for these diseases. This application utilizes the function of nucleolar complex pore-associated protein 4 (Noc4L gene), a ribosomal biogenetic factor, in Th17 cells. By inactivating the Noc4L gene, the differentiation of Th17 cells into Th1 cells can be regulated at the post-transcriptional translational level. This application can provide new pathways and methods for developing novel nucleic acid-based drugs to treat autoimmune diseases caused by inflammatory cytokines. Attached Figure Description

[0015] The above features and advantages of this application will become more apparent from the following detailed description taken in conjunction with the accompanying drawings, wherein:

[0016] Figure 1 The following is shown in Il17a according to this application.Cre A schematic diagram illustrating the principle that IL-17A-expressing cells can be permanently labeled in R26YFP mice using the enhanced yellow fluorescent protein gene.

[0017] Figure 2 This invention illustrates the detection of CD4 in mice by flow cytometry according to the present application. + T cells are divided into: IL-17A cells that do not express IL-17A. – YFP – T cells expressing IL-17A that has not yet been labeled by YFP. + YFP – T cells, IL-17A expressing IL-17A and labeled with YFP. + YFP + T cells and IL-17A cells that expressed IL-17A and were labeled by YFP but subsequently lost IL-17A expression. – YFP + Results of T cell activity.

[0018] Figure 3 The Noc4l according to this application is shown fl / fl A schematic diagram of the mouse construction strategy.

[0019] Figure 4 This demonstrates the PCR amplification of CD4 according to this application. + YFP – T cells and CD4 + YFP + Agarose gel electrophoresis image of wild-type (WT) and loxp strands in T cells.

[0020] Figure 5 This application demonstrates the flow cytometry detection of Il17a. Cre .R26YFP.Noc4l fl / fl Mice and their littermates Il17a Cre Naive CD4 in the spleen and peripheral lymph nodes of .R26YFP control mice + Results of T cell activity.

[0021] Figure 6 The YFP levels of Noc4L knockout mice were shown between 48 and 120 hours after the Noc4L gene was specifically knocked out according to this application. + Cell proliferation and YFP in control mice + Comparison results of cell proliferation.

[0022] Figure 7The results of flow cytometry analysis show that, according to this application, the specific knockout of the Noc4L gene in Th17 cells resulted in a decrease in the number of Th17 cells and a slowdown in early proliferation between 48 and 120 hours.

[0023] Figure 8 shows the flow cytometry analysis of 6-8 week old Il17a cells according to this application. Cre .R26YFP.Noc4l fl / fl Mice and their littermates Il17a Cre IL-17A in the spleen, peripheral lymph nodes, and colon of R26YFP control mice + YFP + Th17 cells, IL-17A – YFP + Th17 and IL-17A + YFP – Changes in the proportion of T17 cells; among which ( Figure 8A CD4+ is found in the spleen, peripheral lymph nodes, and colon of mice. + The expression of IL-17A and YFP in T cells, Figure 8B () represents the statistical proportion of IL-17A+YFP– cells in the spleen, peripheral lymph nodes, and colon of mice.

[0024] Figure 9 shows the flow cytometry detection of Il17a according to this application. Cre .R26YFP.Noc4l fl / fl Mice and Il17a Cre YFP in the spleen and lymph nodes of R26YFP control group mice + Expression results of cell chemokine receptor CCR6 and proliferation-related molecule Ki-67; among which ( Figure 9A The expression of CCR6 and Ki-67 in the spleen and lymph nodes of mice is shown in the figure. Figure 9B () represents the statistical proportions of CCR6 and Ki-67 in the spleen and lymph nodes of mice, respectively.

[0025] Figure 10 A schematic diagram of the process for constructing a mouse model of autoimmune encephalomyelitis according to this application is shown.

[0026] Figure 11 This application demonstrates the monitoring of Il17a. Cre .R26YFP.Noc4l fl / fl Mice and their littermates Il17a Cre Clinical scoring results obtained from the activity status of .R26YFP.

[0027] Figure 12 shows the flow cytometry detection of Il17a according to this application. Cre.R26YFP.Noc4l fl / fl Mice and their littermates Il17a Cre CD4 in the spleen and central nervous system (CNS) of R26YFP control mice + Changes in the number of T cells Figure 12A ) and proportion statistics ( Figure 12B ).

[0028] Figure 13 shows the flow cytometry detection of Il17a according to this application. Cre .R26YFP.Noc4l fl / fl Mice and their littermates Il17a Cre YFP in the spleen and central nervous system of R26YFP control mice + Changes in the number of Th17 cells ( Figure 13A ) and proportion statistics ( Figure 13B ).

[0029] Figure 14 shows the peak of experimental autoimmune encephalomyelitis according to this application, Il17a Cre .R26YFP.Noc4l fl / fl Mice and their littermates Il17a Cre YFP in the spleen and central nervous system of R26YFP control mice + The results of Th17 cells transforming into Th1 cells. Figure 14(A-1) shows the expression changes of IL-17A and IFN-γ, and Figure 14(A-2) shows the statistical proportions of IL-17A and IFN-γ; Figure 14(B-1) shows the expression changes of GM-CSF in the early stage of the disease, and Figure 14(B-2) shows the statistical proportions of GM-CSF; Figure 14(C-1) shows the expression changes of TNF-α in the early stage of the disease, and Figure 14(C-2) shows the statistical proportions of TNF-α.

[0030] Figure 15 shows the flow cytometry detection of Il17a according to this application. Cre .R26YFP.Noc4l fl / fl Mice and their littermates Il17a Cre .R26YFP control mice YFP + The expression results of T-bet and TCF-1 in cells; among which ( Figure 15A The expression of T-bet and TCF-1 in the spleen and central nervous system of mice is shown in the figure. Figure 15B () represents the statistical proportions of T-bet and TCF-1 in the spleen and central nervous system of mice, respectively. Detailed Implementation

[0031] Terminology Definition

[0032] Unless otherwise defined, all terms used herein have the same meaning as understood by one of ordinary skill in the art. For definitions and terminology in this field, those skilled in the art may refer to Current Protocols in Molecular Biology (Ausubel). The abbreviations for amino acid residues are the standard 3-letter and / or 1-letter codes used in this field to refer to one of the 20 commonly used L-amino acids.

[0033] The term "naive T cells" refers to T cells that have matured and left the thymus but have not yet encountered antigens. Naive T cells need to travel from the blood to secondary lymphoid tissue to find their antigens.

[0034] The term "loxp" refers to a 34bp sequence, consisting of an approximately 13bp inverted repeat sequence and an 8bp spacer sequence in the middle. The approximately 13bp spacer is the recognition site of Cre recombinase, which recognizes and processes this sequence, while the middle 8bp spacer determines the directionality of the loxp.

[0035] The term "Cre recombinase" refers to a type I topoisomerase of bacterial phage P1, which is also a tyrosine recombinase. It recognizes the 34 bp loxP site and catalyzes site-specific recombination between loxP sites. This enzyme does not require energy cofactors, and Cre-mediated recombination quickly reaches equilibrium between the substrate and the reaction product.

[0036] The term "STOP" sequence can prevent downstream genes from being transcribed, similar to a transcription stop site.

[0037] The term "l17aCre" mice refers to mice that express a one-codon modified Cre recombinase on the mouse endogenous Il17 gene. This targeted mutation eliminates the expression of Il17a, thereby facilitating fate tracking of IL-17A secretion during helper T cell activation.

[0038] The term "Gt(ROSA)26Sor" refers to a non-coding gene consisting of three exons located on mouse chromosome 6. Because this region is highly susceptible to gene insertion, and because no functionally expressed protein has been found there, coupled with the ease of homologous recombination, proteins inserted into this region can be well expressed without affecting the expression and function of other endogenous genes. It is widely expressed in all cell types and developmental stages, so the mouse Rosa26 locus is often used as a safe site for gene targeting.

[0039] The term "CCR6 chemokine receptor" refers to a G protein-coupled chemokine receptor that binds tightly to CCL20 to form a pair of chemokine ligands with high affinity and "specificity." Numerous studies have shown that CCR6 is associated with a variety of inflammatory diseases and plays an important role in the occurrence and development of various digestive system tumors.

[0040] The term "Ki-67" refers to a nuclear antigen that appears in the G1, S, G2, and M phases of cells. Due to its short half-life, it can accurately reflect the proliferative activity of cells.

[0041] To enable those skilled in the art to more clearly understand the technical solutions of this application, the following embodiments and test examples will be used to further describe this application in detail. These embodiments and test examples are for illustrative purposes only and are not intended to limit the scope of this application. The test materials used in the embodiments of this application are all conventional test materials in the art, which can be purchased through commercial channels or prepared by existing technical methods.

[0042] Example 1: Obtaining mice with Noc4L gene specifically knocked out in Th17 cells and their phenotypic analysis (construction of Il17a) Cre .R26YFP.Noc4l fl / fl Mouse model)

[0043] In order to conduct research in Th17 cells, Il17a, purchased from Jackson Lab, will be used. Cre Mice (Il17acre mice are numbered 016879 on The Jackson Laboratory website) were mated with Rosa26-stop-eYFP mice to obtain Il17a. Cre Rosa26-stop-eYFP mice possess a stop sequence flanked by a loxp (locus of X(cross)-over in P1) followed by an enhanced yellow fluorescent protein gene (eYFP) inserted at the Gt(ROSA)26Sor site. When hybridized with mice expressing Cre recombinase, the stop sequence is deleted upon recognition by Cre recombinase, and the two loxps break and reassemble into a new loxp fragment, thus allowing the expression of eYFP to be observed. Therefore, in Il17a Cre In R26YFP mice, YFP can permanently label cells expressing IL-17A. Figure 1 ) IL-17A and YFP can reduce CD4+ in mice. + T cells are divided into: IL-17A cells that do not express IL-17A. – YFP –T cells expressing IL-17A that has not yet been labeled by YFP. + YFP – T cells, IL-17A expressing IL-17A and labeled with YFP. + YFP + T cells and IL-17A cells that expressed IL-17A and were labeled by YFP but subsequently lost IL-17A expression. – YFP + T cells ( Figure 2 ), including IL-17A – YFP + T cells are considered to be highly plastic Th17 cells, which are prone to transforming into other helper T cells.

[0044] Then, Il17a Cre R26YFP mice and Noc4lfloxed mice (nucleolar complex pore-associated protein 4 knockout mouse model, abbreviated as No4l) fl / fl Mating yields Il17a Cre .R26YFP.Noc4l fl / fl Mice, including Noc4l fl / fl Mouse construction strategy ( Figure 3 This involves designing two Loxp sites, one on each side of the intron at the third exon of the Noc4L gene. The Cre recombinase specifically recognizes these Loxp sites, thereby specifically knocking out the third exon of the Noc4L gene. This allows for the specific knockout of the Noc4L gene in Th17 cells. Since this application primarily targets YFP... + The study used Th17 cells to verify YFP. + The knockout efficiency of the Noc4L gene in Th17 cells was determined using a flow cytometer (manufacturer: BD, model: FACS ARIA III) from Th17a cells. Cre .R26YFP.Noc4l fl / + YFP was isolated from mice + and YFP – CD4 + T cells were analyzed using PCR with primers for Noc4l floxed mouse genotyping to detect the expression of DNA loxp in the genomes of both cell populations. The primers and PCR amplification conditions used are as follows:

[0045] PCR reaction system

[0046]

[0047] PCR reaction procedure

[0048]

[0049] Agarose gel electrophoresis images show that on CD4 + YFP – Both WT (wild-type) and loxp chains can be detected in T cells, while in CD4... + YFP + Only one strand of the WT can be detected in T cells. Figure 4 ), proving in YFP + The Noc4L gene can be successfully knocked out in Th17 cells.

[0050] Example 2: Study on YFP in mice caused by Noc4L gene deletion + Changes in the number of Th17 cells

[0051] For 6-8 week old Il17a Cre .R26YFP.Noc4l fl / fl Mice and their littermates Il17a Cre Phenotypic analysis was performed on R26YFP control mice. First, we compared the appearance of the two groups of mice and found that, compared with the control group, the Noc4L gene-specific knockout mice did not show significant changes in body weight, individual development, or activity level. Next, we obtained the spleen and peripheral lymph nodes from both groups of mice and sorted out Naive CD4 cells. + T cells ( Figure 5 After counting, the spleens and peripheral lymph nodes of mice in the control and experimental groups were then analyzed. CD4+ T cells were mixed at a 1:1 ratio and labeled with Cell Trace (CellTrace Violet Cell Proliferation Kit, Invitrogen). The mixed cells (2 x 10 cells per well) were then... 6 (Number of lymphocytes) were seeded into 24-well plates coated one day in advance. Under Th17 cell polarization conditions (coating conditions for lymphocyte in vitro culture: anti-CD3 (clone: ​​2C11) diluted to 2 μg / mL with 1×PBS, anti-CD28 (clone: ​​PV1) to 1 μg / mL, 500 μL / well; Th17 cell polarization conditions: 1640 complete medium + 20 ng / mL IL-6 + 0.5 ng / mL TGF-β + 10 μg / mL anti-IFN-γ), cells were cultured for 48 h, 72 h, 96 h, and 120 h, respectively. The expression of CD45.1 / CD45.2 was used to distinguish between the two cell types using flow cytometry (model: LSR Tortessa, manufacturer: BD). YFP was detected starting the next day. +The proportion of cells was measured every other day until day five. It was found that, under in vitro co-culture conditions, compared to the control mice, the Noc4L gene-specific knockout mice had significantly higher YFP levels. + The proportion of cells decreased slightly ( Figure 6 YFP levels were detected in mice in the experimental and control groups under co-culture conditions. + Cell proliferation over time was studied, and it was found that YFP cells lacking the Noc4L gene proliferated more rapidly than the control group. + Th17 cell proliferation was slightly slowed, with the difference mainly observed in the early stages of proliferation. These results indicate that specific knockout of the Noc4L gene still has an effect on activated cells; in Th17 cells, specific knockout of the Noc4L gene leads to a decrease in the number of Th17 cells and a slowdown in early proliferation. Figure 7 ).

[0052] Example 3. Study on the effects of Noc4L gene deletion on the generation, migration, and proliferation of Th17 cells in mice under homeostatic conditions.

[0053] 1.Il17a Cre .R26YFP.Noc4l fl / fl The production of IL-17A in mice was not affected.

[0054] Due to YFP + Th17 cell production is mediated by IL-17A expression; therefore, it is necessary to investigate whether the deletion of the Noc4L gene affects Th17 cell production in mice. Flow cytometry was used to detect IL-17A expression in 6-8 week old mice. Cre .R26YFP.Noc4l fl / fl Mice and their littermates Il17a Cre CD4+ in the spleen, peripheral lymph nodes, and colon of .R26YFP control mice + The expression of IL-17A and YFP in T cells, and the effect of IL-17A + YFP – The proportion of cells was statistically analyzed. The results showed that, compared to the control group mice, Il17a... Cre .R26YFP.Noc4l fl / fl Mouse IL-17A + YFP + Th17 cells and IL-17A – YFP + The proportion of Th17 cells decreased, but IL-17A... + YFP – The proportion of T cells did not change significantly. Figure 8A (and B) indicates that the deletion of the Noc4L gene does not affect the production of Th17 cells under homeostatic conditions.

[0055] 2. Noc4L gene deletion does not affect the migration and proliferation of Th17 cells under homeostatic conditions.

[0056] Further investigation is needed to determine whether knocking out the Noc4L gene will affect YFP under steady-state conditions. + Migration and proliferation of Th17 cells. Flow cytometry detection of Th17a. Cre .R26YFP.Noc4l fl / fl YFP in the spleen and lymph nodes of mice and control mice + The expression of cell chemokine receptor CCR6 and proliferation-related molecule Ki-67 was investigated. The results showed that, compared with control mice, Noc4L gene-specific knockout mice had significantly higher levels of YFP in the spleen and lymph nodes. + There was no significant difference in the expression of CCR6 and Ki-67 in Th17 cells. Figure 9A (and B). These results indicate that the deletion of the Noc4L gene does not affect the migration and proliferation of Th17 cells under steady-state conditions.

[0057] Example 4. Study of Il17a Cre .R26YFP.Noc4l fl / fl Changes in the severity of autoimmune encephalomyelitis in mice

[0058] 1. Establishment of an autoimmune encephalomyelitis (EAE) mouse model

[0059] Th17 cells play a crucial role in the pathogenesis of autoimmune diseases, a prime example being an inducible multiple sclerosis mouse model of experimental autoimmune encephalomyelitis (EAE). Therefore, this study utilizes an EAE inflammatory mouse model to further investigate the effects of specifically knocking out the Noc4L gene on Th17 cells in an inflammatory environment. Figure 10 ).

[0060] Il17a was given on day 0. Cre .R26YFP.Noc4l fl / flMice and their littermate control mice were immunized with myelin oligodendrocyte glycoprotein (MOG35-55 peptide) plus complete Freund's adjuvant (CFA). Mice were then injected with pertussis toxin (PTX) on days 0 and 2, respectively. From day 8 onwards, the condition of both groups of mice was monitored, and their neurological function was pathologically scored, generally into 5 grades: Grade 1: Tail weakness; Grade 2: Tail weakness and hind limb weakness; Grade 3: Complete hind limb paralysis and forelimb weakness; Grade 4: Severe paralysis of all four limbs, unable to recover after passive rolling; Grade 5: Mice in a near-death state. Subsequently, during the peak of EAE (early acute exacerbation) at days 16-20, mice were dissected, and lymphocytes from the spleen and central nervous system of both experimental and control groups were obtained. Phenotypic analysis of the mice was performed using flow cytometry. Figure 11 By comparing the disease progression during EAE induction in two groups of mice, it was found that littermate control mice developed severe central nervous system disorders under EAE-induced experimental autoimmune encephalomyelitis, while Noc4L gene-specific knockout mice showed almost no disease. This indicates that knocking out the Noc4L gene in Th17 cells can, to some extent, prevent the formation of autoimmune diseases in mice under inflammatory conditions.

[0061] 2.Il17a Cre .R26YFP.Noc4l fl / fl Reduced inflammatory cell infiltration in the central nervous system of mice

[0062] In normal mice, the blood-brain barrier typically results in a low number of lymphocytes in the central nervous system. However, during the pathogenesis of acute exacerbation of erythema eelata (EAE), the blood-brain barrier is disrupted, allowing a large number of lymphocytes to enter the central nervous system and trigger inflammation. Therefore, we aim to investigate the effect of Noc4L gene deletion on lymphocytes in the central nervous system. During the peak of EAE disease, we examined the effects of Il17a... Cre .R26YFP.Noc4l fl / fl and its nestmate Il17a Cre R26YFP control mice were dissected, and CD4 counts in the spleen and central nervous system of both groups of mice were detected by flow cytometry. + T cells and YFP + The proportion and number of Th17 cells were measured. Results showed that, compared to control mice, Noc4L gene-specific knockout mice had significantly higher levels of CD4+ cells in their spleens. + The number and proportion of T cells did not change significantly, but CD4+ cells in the central nervous system... + The number and proportion of T cells were significantly reduced. Figure 12A And B). Further analysis of YFP +Changes in Th17 cells revealed that, compared to control mice, Noc4L gene-specific knockout mice (YFP) showed increased activity in both the spleen and central nervous system. + The number and proportion of Th17 cells were significantly reduced. Figure 13A And B). Among them, Figure 13B The statistical chart also included trends in cell changes in peripheral lymph nodes, and the results were consistent with those in the spleen. Furthermore, compared to steady-state conditions, YFP... + The reduction in Th17 cells was even more pronounced. These results demonstrate that, in the EAE model, Noc4L gene deletion can reduce inflammatory cell infiltration in the central nervous system.

[0063] 3. Detection of the effect of Noc4L gene deletion on Th17 cell function

[0064] Because a large amount of experimental data has shown that Th17 cells exhibit plasticity in their effector program during the development of autoimmune diseases—that is, Th17 cells can transform into Th1 cells, accompanied by a decrease in the secretion of the cytokine IL-17A and an increase in the production of pro-inflammatory cytokines such as IFN-γ and GM-CSF—we used flow cytometry to detect IL-17A secretion during the peak incidence of experimental autoimmune encephalomyelitis (EAE). Cre .R26YFP.Noc4l fl / fl and nest Il17a Cre .R26YFP control group mice YFP + The expression of Th17 cell-related cytokines interleukin IL-17A and interferon IFN-γ was investigated and the results were statistically analyzed. The results showed that during the peak of EAE, a higher proportion of YFP was observed in the spleen and central nervous system of control mice. + Th17 cells are transforming into Th1 cells, accompanied by a significant increase in the expression of the cytokine IFN-γ. Compared to control mice, Th17a... Cre .R26YFP.Noc4l fl / fl YFP in mouse spleen and central nervous system + Th17 cells did not show a significant transformation towards Th1 cells, IL-17A – IFN-γ + The proportion of T cells was significantly reduced, and IL-17A... + IFN-γ + The proportion of T cells was slightly reduced, and IL-17A... + IFN-γ – The proportion of T cells increased ( Figure 14A-1 And 14A-2). Furthermore, in the early stages of EAE, Il17a was detected by flow cytometry. Cre .R26YFP.Noc4lfl / fl and nest Il17a Cre YFP in the spleen and peripheral lymph nodes of R26YFP control mice + The expression of inflammatory cytokines, granulocyte-macrophage colony-stimulating factor GM-CSF, and tumor necrosis factor-α in Th17 cells was found to be significantly higher than that in control mice. The study also revealed that Th17 cells lacking the Noc4L gene showed significantly higher GM-CSF levels. Figure 14B-1 The expression of 14B-2 and TNF-α was also reduced. Figure 14C-1 (and 14C-2). These results demonstrate that the deletion of the Noc4L gene in Th17 cells can, to some extent, prevent the expression of inflammatory cytokines in Th17 cells, thereby protecting mice and reducing the risk of developing severe autoimmune diseases.

[0065] Example 5. Study on the stability of Th17 cells in Noc4L gene-deleted mice

[0066] Previous studies have shown that in mouse models of autoimmune diseases, Th17 cells can be divided into two subsets based on metabolic and functional heterogeneity: pathogenic Th17 cells and non-pathogenic Th17 cells. Non-pathogenic Th17 cells are relatively stable, exhibiting stem cell-like characteristics, high expression of the transcription factor TCF-1, and low levels of anabolism. Pathogenic Th17 cells, on the other hand, are prone to transforming into Th1 cells, highly expressing the transcription factor T-bet, and exhibiting higher metabolic activity. Therefore, this study investigates whether specific knockout of the Noc4L gene in Th17 cells affects Th17 cell lineage development. Flow cytometry was used to detect the peak incidence of EAE (autoimmune acute leukemia) in the 1117a subpopulation. Cre .R26YFP.Noc4l fl / fl YFP in the spleen and central nervous system of mice from the same littermate control group + Expression of TCF-1 and T-bet in Th17 cells. Results showed that YFP was found in the spleen and central nervous system of control mice. + Th17 cells highly express T-bet and have low TCF-1 expression levels. Compared with control mice, Noc4L gene-specific knockout cells showed increased YFP expression in the spleen and central nervous system. + Cellular T-bet expression was significantly reduced, while TCF-1 expression was significantly increased. Figure 15A (and B). This indicates that the deletion of the Noc4L gene can, to some extent, prevent Th17 cells from differentiating into pathogenic directions, causing them to remain in a stem cell-like state with low metabolic activity.

[0067] In summary, the role of Th17 cells in autoimmune diseases has received increasing attention, and understanding the mechanisms of the transition between anti-inflammatory and pro-inflammatory Th17 cells is a future direction for the treatment of autoimmune diseases. Specific ribosomes and post-transcriptional translational regulation provide a new perspective on the plasticity changes of Th17 cells. By monitoring the dynamic changes of ribosomes and the interactions between proteins or between proteins and RNA, the mechanisms of autoimmune diseases caused by pathogenic differentiation of Th17 cells can be explored at the translational level.

[0068] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A method of modulating the directed differentiation of T helper 17 cells into Thl cells, characterized in that, inactivating or reducing the expression activity of a gene on the genome that affects the directional differentiation of helper T cells 17 into Th1 cells; preferably, the gene is the nucleolar pore complex associated protein 4 gene.

2. The method of claim 1, wherein, Methods for inactivating or reducing the expression activity of the nucleolar pore complex associated protein 4 include, but are not limited to, knocking out or silencing the nucleolar pore complex associated protein 4 gene on the genome.

3. A pharmaceutical composition comprising a protein and / or a compound and / or an RNA that inhibits the gene function of a gene that affects the directional differentiation of helper T cells 17 into Th1 cells.

4. The pharmaceutical composition of claim 3, wherein, The gene that affects the directional differentiation of helper T cells 17 into Th1 cells is the nucleolar pore complex associated protein 4 gene.

5. The pharmaceutical composition of claim 3, wherein, The delivery mode of the pharmaceutical composition includes, but is not limited to, oral or injection.

6. The pharmaceutical composition according to any one of claims 3-5, characterized in that, Any pharmaceutical adjuvant that can be used in the pharmaceutical composition is also included.

7. Use of the pharmaceutical composition according to any one of claims 3-5 in the treatment or prevention of autoimmune diseases mediated by inflammatory cytokines secreted by helper T cells 17.

8. The pharmaceutical composition of claim 7, wherein, The inflammatory cytokines include interleukin-17, interferon-γ, granulocyte-macrophage colony-stimulating factor, and tumor necrosis factor.

9. The subject of the pharmaceutical composition according to claim 7 is a mammal.

10. Use according to claim 7, characterized in that, The autoimmune diseases include, but are not limited to, multiple sclerosis.

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