Immune regulation metabolite screening by immune system humanized mouse intestinal flora disturbance model

Abstract

The invention relates to a method for screening and identifying metabolites and florae with a human immune regulation effect, which comprises the following steps: (1) establishing immune system humanized mice with multiple intestinal types, and analyzing the change of the florae, the metabolites and the immune system through intestinal flora sequencing, serum metabolome sequencing, flow cytometry and transcriptome sequencing; (2) determining the correlation and sequence between flora related metabolites and the change of the immune system through bioinformatics analysis; and (3) confirming the regulation and control relationship between the candidate metabolites and the immune subgroups in the step (2) through biological experiments. By means of the screening method, the chemical small molecule metabolites for regulating and controlling the human immune cells in the normal physiological state can be screened on a large scale, and the chemical small molecule metabolites can be developed into immunotherapy drugs or used as cell therapy drug culture system components. The screened propionic acid can promote cytotoxicity of T cells and CAR-T and secretion of related cytokines in the aspect of function regulation of the T cells, and the metabolite 2-hydroxybutyric acid is highly positively correlated with the Treg cells and can promote generation and functions of the Treg cells.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to screening immune regulatory metabolites using a mouse intestinal flora disturbance model with humanized immune system mice, and the application of the screened immune regulatory metabolites. Background Art

[0002] Existing technologies have found that intestinal flora disturbances are closely associated with the occurrence of various immune imbalance diseases, such as autoimmune diseases, tumors, and metabolic syndrome. For example, by exploring the intestinal flora and its metabolites, probiotics and natural products that can be used to treat Treg cell-deficient immunodeficiency diseases have been discovered. 16S rDNA sequencing technology was used to detect the intestinal flora of mice with autoimmune diseases mediated by Treg cell deficiency; metabolomics technology was used to detect changes in the metabolome profile of the intestinal flora of model mice; and molecular biology and gene knockout mice were used to study the molecular mechanisms of the effects of probiotics and metabolites. The results showed that the intestinal flora of mice with Treg cell-deficient immunodeficiency diseases were severely disturbed. Based on the changes in the intestinal flora and metabolome profile, Lactobacillus reuteri (L. reuteri) and the metabolite inosine were selected to treat Treg cell-deficient mice. L. reuteri and inosine inhibited the autoimmune diseases caused by Treg cell deficiency by activating the adenosine receptor A2AR. Conclusion The intestinal flora contains a large number of microorganisms and produces many unique metabolites, which can serve as an important source of new natural medicines for the discovery of new probiotics and active natural products for the treatment of autoimmune diseases, tumors and other diseases.

[0003] However, existing technologies lack a humanized mouse model—a model mouse with a human immune system—to study the relationship between microbiota and metabolites and immune regulation under normal physiological conditions. However, significant differences exist between the mouse and human immune systems, making it difficult to replicate normal human immune responses in mice. Consequently, studies of the relationship between metabolites and normal human immune responses are difficult to investigate.

[0004] In addition, the differences in intestinal flora imbalance status between general intestinal flora imbalance mouse models are not sufficient to provide sufficient sample abundance, which increases the difficulty of distinguishing the correlation between metabolites and immune regulatory effects under normal physiological conditions.

[0005] Therefore, there is an urgent need in the existing technology to simulate the real conditions of the human body to study the effects of metabolites on the human immune function, while improving the sample differences, so as to more accurately understand the human immune regulatory effects of metabolites. Summary of the Invention

[0006] To address the deficiencies in the prior art, the present application provides a method for screening and identifying metabolites and microbiota with immune regulatory effects, the method comprising the following steps: (1) establishing humanized immune system mice with multiple intestinal types, and analyzing changes in intestinal microbiota, metabolites, and the immune system through intestinal microbiota, serum metabolites, and immune system detection methods; (2) determining the correlation and ranking between different bacterial species, serum metabolites, and human immune subpopulations through bioinformatics analysis; and (3) confirming the regulatory relationship between the candidate metabolites and human immune subpopulations in step (2) through biological experiments.

[0007] As certain embodiments of the present application, the humanized immune system mice in step (1) include one or more of PBMC humanized mice, HSC humanized mice, and Hu-BLT model humanized mice. As certain embodiments of the present application, the humanized immune system mice in step (1) are one or more; by using multiple humanized immune system mice for screening, complementarity can be achieved, thereby increasing the probability of successfully screening metabolites with immunomodulatory effects.

[0008] As certain embodiments of the present application, the humanized immune system mouse in step (1) is an HSC humanized mouse; the HSC humanized mouse is an immunodeficient mouse transplanted with human hematopoietic stem cells and reconstructed with human immune cells.

[0009] In order to study the regulation of human immune cells by microbiota metabolites, this application constructed humanized immune system mice with different intestinal types, and studied the changes in their metabolites and immune system respectively by targeted metabolic sequencing and flow cytometry. The present invention uses a humanized immune system mouse model with human cells or tissues, which can simulate the dynamics of human immunity and become a screening metabolite with immunoregulatory effects. The humanized immune system mouse model has built a "bridge" between basic research and clinical transformation, and has been widely used in the fields of tumors, infections, etc.

[0010] As one of the main immune killer cells, the regulation of T cell function plays an important role in maintaining immune balance and tumor killing.

[0011] Common immune system humanized mouse models are based on immunodeficient mice. They are transplanted with mature human peripheral blood mononuclear cells (PBMCs), human hematopoietic stem cells (HSCs), or fetal thymus and fetal liver and inoculated with human bone marrow hematopoietic stem cells for immune reconstruction, thereby reconstructing the human immune system in immunodeficient mice.

[0012] As certain embodiments of the present application, the serum metabolome in step (1) includes a targeted metabolome or a non-targeted metabolome, and the immune system detection method includes flow cytometry, single-cell sequencing, and mass spectrometry.

[0013] As certain embodiments of the present application, the number of humanized mice with immune systems having different intestinal flora described in step (1) is one or more.

[0014] As certain embodiments of the present application, the number of humanized immune system mice with different intestinal floras in step (1) is multiple, and these humanized immune system mice with different intestinal floras have multiple intestinal types.

[0015] Enterotype, or "enteric microbial typing," is a classification based on the dominant bacterial genera of the gut microbiome combined with the functional characteristics of the entire gut microbiome. Different enterotypes have distinct microbial structures and functional genes. It can be used as an effective method to distinguish between human gut microbes.

[0016] As certain embodiments of the present application, the intestinal type includes multiple types of familiar flora, humanized flora, single bacterial colonization, sterile, dysbacterial flora, and pathological intestinal type.

[0017] As certain embodiments of the present application, the enterotype includes two, three, four, five, or six of the following: familiar flora, humanized flora, single bacterial colonization, sterile, dysbacterial, and pathological enterotypes.

[0018] As certain embodiments of the present application, there are multiple humanized immune system mice with different intestinal flora in step (1), and these humanized immune system mice with different intestinal flora have two, three, four, five, six, seven, eight, nine, ten, or more intestinal types, familiar flora, humanized flora, single bacterial colonization or sterility.

[0019] As certain embodiments of the present application, the intestinal type includes the intestinal type of HIS mice raised in a specific pathogen-free environment (SPF), the intestinal type of HIS mice treated with long-term life-cycle antibiotic combination therapy (ABA), the intestinal type of HIS mice with dysbiosis (DYS) caused by periodic antibiotic use, and the intestinal type of HIS mice transferred to a low-barrier clean-level mouse facility (CLT).

[0020] As certain embodiments of the present application, in step (1), SPF mice with humanized immune system are used as controls.

[0021] As certain embodiments of the present application, the metabolites are selected from metabolites associated with intestinal flora or metabolites of the body affected by changes in intestinal flora.

[0022] As certain embodiments of the present application, the immunodeficient mice include one or more of nude mice, NOD scid mice, RAG1KO mice, RAG2KO mice, Il2rgKO mice, and severely immunodeficient mice.

[0023] As certain embodiments of the present application, the severely immunodeficient mouse is a mouse comprising at least one of T cell deficiency, B cell deficiency, and NK cell deficiency.

[0024] As certain embodiments of the present application, the severely immunodeficient mouse is one or more of NOG mouse, NSG mouse, NCG mouse, NPG mouse, NKG mouse, BRG mouse, and their derivative strains.

[0025] As certain embodiments of the present application, the severely immunodeficient mouse is selected from NBSGW mouse, NSGW41 mouse, NOG-EXL mouse, NCG-M mouse, NCG-FLT3-KO mouse, NCG-X-TSLP mouse, NCG-hIL6 mouse, NCG-X-hIL15 mouse, NKG mouse, NKG-hIL15 mouse, NKG-hIL6 mouse, NCG-X mouse, NCG-MHC-dKO mouse, BALB / cRag2-null IL-2Rγc-null mouse, NOD.Cg-Prkdc scid Il2rg tm1Wjl / SzJ Mouse, NOD-Prkdc em26Cd52 Il2rg em26Cd22 / Nju One or more of mice, NPG mice, NPG-B2M mice, BRGSF mice, BRGS mice, BRGST mice, and derivative strains thereof.

[0026] As certain embodiments of the present application, the intestinal flora detection method includes one or more of 16s rRNA sequencing, whole genome sequencing, and metagenomic sequencing.

[0027] Metagenome sequencing studies the entire microbial community in a specific habitat using high-throughput sequencing technology. It does not require the isolation and cultivation of microorganisms, breaking away from the technical limitations of microbial isolation and cultivation in traditional research. Instead, it extracts the total DNA of environmental microorganisms for research and obtains the total genome information of environmental microorganisms to study the community structure, species classification, system evolution, gene function, and metabolic pathways of environmental microorganisms. Metagenome sequencing technology has promoted the development and utilization of microbial resources and accelerated the in-depth research of the microecological scientific research process.

[0028] As certain embodiments of the present application, the changes in the immune system in step (2) include one or more of changes in T cell function, changes in B cell function, changes in natural killer cells, changes in hematopoietic stem cells, changes in neutrophils, changes in basophils, changes in eosinophils, changes in monocytes, and changes in macrophages.

[0029] As certain embodiments of the present application, the human immune subpopulations in step (2) include CMP (common myeloid progenitor cells), GMP (granulocyte macrophage progenitor cells), MEP (megakaryocytic erythroid progenitor cells), HSC (hematopoietic stem cells), LMPP (lympho-myeloid primed progenitors), MPP (multipotent progenitors), IgA + B cells, IgG + B cells, IgM + B cells, naive B cells, plasmablasts, transitional B cells, FOXP3 + T cells, IL-2 + T cells, IL-4 + T cells, IL-13 + T cells, IL-17A + T cells, IL-21 + T cells, IL-22 + T cells, IFN-γ + T cells, TNF-α + One or more T cells.

[0030] As certain embodiments of the present application, the biological experiment in step (3) includes one or more of cell experiments, animal experiments, and clinical experiments.

[0031] As certain embodiments of the present application, the cell experiment includes using the candidate metabolites to treat immune cell subpopulations and detecting changes in the immune cell subpopulations, wherein the changes in the immune cell subpopulations include changes in the number, function, phenotype of immune cells and changes in interactions between immune cells.

[0032] As certain embodiments of the present application, the animal experiment includes using the candidate metabolites to treat humanized experimental animals and detecting changes in the immune cell subpopulations, physiology, biochemistry, and health status of the experimental animals.

[0033] As certain embodiments of the present application, the clinical trial comprises administering the candidate metabolite to a patient and detecting changes in the patient's immune cell subpopulations, physiology, biochemistry, and health status.

[0034] As certain embodiments of the present application, the cell experiment includes treating T cells or CAR-T cells with propionic acid and detecting changes in IFN-γ secretion of the T cells or CAR-T cells.

[0035] As certain embodiments of the present application, the animal experiment comprises treating humanized mice with propionic acid, and detecting the IFN-γ levels in the peripheral blood and spleen of the humanized mice.

[0036] As certain embodiments of the present application, the animal experiment includes treating tumor-bearing mice with propionic acid and CAR-T cells, and detecting the tumor volume and weight of the tumor-bearing mice.

[0037] As certain embodiments of the present application, the cell experiment comprises treating T cells with 2-hydroxybutyric acid and detecting changes in the number of Treg cells.

[0038] As certain embodiments of the present application, the cell experiment comprises treating T cells with γ-linolenic acid and detecting changes in the number of Treg cells.

[0039] As certain embodiments of the present application, the animal experiment includes treating mice treated with high-dose IL-2 with 2-hydroxybutyric acid to detect the alleviation of cytotoxicity and changes in the number of Treg cells brought about by high-dose IL-2.

[0040] As certain embodiments of the present application, the correlation analysis in step (2) includes statistical analysis or artificial intelligence algorithm analysis.

[0041] As certain embodiments of the present application, the statistical analysis includes one or more of Pearson correlation analysis, Spearman correlation analysis, Kendall Tau correlation analysis, and Point-Biserial correlation analysis.

[0042] As certain embodiments of the present application, the correlation analysis is Pearson correlation analysis and Spearman correlation analysis.

[0043] As certain embodiments of the present application, the artificial intelligence algorithm analysis includes one of linear regression, logistic regression, decision tree, naive Bayes, support vector machine (SVM), ensemble learning, K-nearest neighbor algorithm, K-means algorithm, neural network, and deep reinforcement learning (DQN).

[0044] As certain embodiments of the present application, the metabolites in step (2) are selected from suberic acid, phenylpyruvic acid, octanoic acid, caproic acid, azelaic acid, malonic acid, phenyllactic acid, D-xylose, hyodeoxycholic acid, mandelic acid, deoxycholic acid, 3-hydroxyphenylacetic acid, L-aspartic acid, D-xylulose, L-tyrosine, methylsuccinic acid, glutaric acid, D-glucose, L-α-aminobutyric acid, indoleacetic acid, acid), dimethylglycine, aminocaproic acid, tauroursodeoxycholic acid, taurocholic acid, taurochenodeoxycholic acid, propanoic acid (PA), taurohyodeoxycholic acid, L-histidine, taurodeoxycholic acid, propionylcarnitine, 3-indolepropionic acid, butyric acid, 4-hydroxyphenylpyruvic acid, sarcosine, 3-hydroxyisovaleric acid, citramalic acid, 2-hydroxybutyric acid,2-HB), Nicotinic acid, D-Fructose, Oxoglutaric acid, Beta-Hyodeoxycholic Acid, Eicosapentaenoic acid EPA, 8-11-14-Eicosatrienoic acid, Docosapentaenoic acid 22n-6, Linoleic acid, β-Alanine, 2-Hydroxy-2-methylbutyric acid, γ-Linolenic acid, Cis- and trans-Cinnamic acid, Carnitine, Glyceric acid.

[0045] As certain embodiments of the present application, the metabolites positively correlated with the proportion of T cells secreting IFN-γ in step (2) include propionic acid, taurohyodeoxycholc acid, tauroursodeoxycholic acid, taurocholic acid, taurochenodeoxycholicacid, histidine, indolepropionic acid, chenodeoxycholicacid, gluconolactone, and one or more of their derivatives.

[0046] As certain embodiments of the present application, the metabolites negatively correlated with the proportion of T cells secreting IFN-γ include one or more of myristoleic acid, methylglutaric acid, ketoglutaric acid, nicotinic acid, and derivatives thereof.

[0047] As certain embodiments of the present application, with Foxp3 + Metabolites positively correlated with changes in Treg cells include one or more of 2-hydroxybutyric acid, butyric acid, citramalic acid, β-hydroxyisovaleric acid, 4-hydroxyphenylpyruvic acid, taurochenodeoxycholic acid, and their derivatives.

[0048] As certain embodiments of the present application, with Foxp3 + Metabolites negatively correlated with changes in Treg cells include one or more of β-alanine, 2-hydroxy-2-methylbutyric acid, γ-linolenic acid, and their derivatives.

[0049] As certain embodiments of the present application, metabolites positively correlated with the proportion of T cells secreting IFN-γ include propionic acid and its derivatives. As certain embodiments of the present application, metabolites negatively correlated with the proportion of T cells secreting IFN-γ include nicotinic acid and its derivatives.

[0050] As certain embodiments of the present application, with Foxp3 + Metabolites positively correlated with changes in Treg cells include 2-hydroxybutyrate and its derivatives.

[0051] As certain embodiments of the present application, with Foxp3 + Metabolites negatively correlated with Treg cell changes include γ-linolenic acid and its derivatives.

[0052] As certain embodiments of the present application, propionic acid is positively correlated with one or more of enhancing the secretion capacity of IFN-γ and TNF-a of T cells, enhancing the secretion capacity of IFN-γ and TNF-a of CAR-T cells, and increasing the tumor-killing ability of CAR-T cells.

[0053] Spearman correlation analysis revealed a high correlation between T cells secreting IFN-γ. Further in vitro and in vivo experiments confirmed that the bacterial metabolite propionic acid can regulate T cell function by promoting T cell cytotoxicity and the secretion of related cytokines, enhancing the secretion of IFN-γ and TNF-a by T cells, and promoting the anti-tumor ability of CAR-T cells.

[0054] As certain embodiments of the present application, 2-hydroxybutyrate is positively correlated with the production and function of human Treg cells, as well as one or more of the inhibition of T cell expansion, toxic side effects, slowing of weight loss, and increased proportion of Treg cells brought about by high-dose IL2.

[0055] As certain embodiments of the present application, γ-linolenic acid is associated with the inhibition of the generation of human Treg cells.

[0056] Pearson correlation analysis revealed a high positive correlation between 2-hydroxybutyrate and Treg cells. The metabolite, 2-hydroxybutyrate, can promote the generation and function of Treg cells and inhibit T cell proliferation and toxic side effects caused by high-dose IL-2 injection in humanized mice. The functions of these small molecule metabolites on T cells have never been reported.

[0057] As certain embodiments of the present application, the immunoregulatory effect is an immunoregulatory effect under normal physiological conditions of the human body.

[0058] In this article, the immune regulation function under normal physiological conditions of the human body refers to the immune regulation function under non-autoimmune disease conditions.

[0059] The present application also provides the use of propionic acid and its derivatives in the preparation of immune-regulating products, wherein the immune regulation includes enhancing the secretion capacity of IFN-γ and TNF-a of human T cells, enhancing the secretion capacity of IFN-γ and TNF-a of human CAR-T cells, and promoting one or more of the tumor-killing ability of human CAR-T cells.

[0060] As certain embodiments of the present application, the immunomodulatory product is a drug for immunomodulation under normal physiological conditions of the human body. In this context, normal physiological conditions of the human body refer to a state of non-immune disease.

[0061] As certain embodiments of the present application, the product includes one or more of medicines, health products, and foods.

[0062] As certain embodiments of the present application, the medicine includes a biological product containing or producing propionic acid and its derivatives.

[0063] As certain embodiments of the present application, the biological product includes a culture medium for culturing cell therapy drugs.

[0064] As certain embodiments of the present application, the biological product that produces propionic acid and its derivatives includes a bacterial agent. As certain embodiments of the present application, the bacterial agent includes probiotics, engineered bacteria, oncolytic bacteria, prebiotics that promote intestinal flora to produce propionic acid and its derivatives, and symbiotics composed of probiotics and prebiotics that promote probiotics to produce propionic acid and its derivatives.

[0065] The present application also provides the use of 2-hydroxybutyric acid and its derivatives in the preparation of immune-regulating products, wherein the immune regulation includes promoting the production of Treg cells, inhibiting T cell proliferation, toxic side effects, slowing down weight loss, and increasing the proportion of Treg cells caused by high-dose IL2.

[0066] As certain embodiments of the present application, the immunomodulatory product is an immunomodulatory product under normal physiological conditions of the human body.

[0067] As certain embodiments of the present application, the product includes one or more of medicines, health products, and foods.

[0068] As certain embodiments of the present application, the medicine includes a biological product containing 2-hydroxybutyric acid and its derivatives.

[0069] As certain embodiments of the present application, the biological product includes a culture medium for culturing cell therapy drugs.

[0070] As certain embodiments of the present application, the biological product that produces 2-hydroxybutyric acid and its derivatives includes a bacterial agent. As certain embodiments of the present application, the bacterial agent includes probiotics, engineered bacteria, oncolytic bacteria, prebiotics that promote intestinal flora to produce 2-hydroxybutyric acid and its derivatives, and symbiotics composed of probiotics and prebiotics that promote probiotics to produce 2-hydroxybutyric acid and its derivatives.

[0071] As described above, the humanized immune system mouse intestinal flora disturbance model of the present invention is used to screen immune regulatory metabolites, which has the following beneficial effects:

[0072] The present invention can screen microbial metabolites on a large scale through the intestinal microbial disturbance model of humanized immune system mice, targeted metabolomics and flow cytometry;

[0073] The method of the present invention simulates the characteristics of human immune dynamics and can screen metabolites with immune regulatory effects under normal physiological conditions of the human body;

[0074] The present invention discovered for the first time that after human T cells were treated with propionic acid, the secretion of IFN-γ and TNF-a was enhanced, and that after CAR-T cells were treated with propionic acid, the secretion of IFN-γ and TNF-a was enhanced, and its tumor killing effect was enhanced. In addition, after human T cells were treated with 2-hydroxybutyric acid, the production of Treg cells increased, the related immunosuppressive function was enhanced, and the T cell expansion, toxic side effects and weight loss caused by high-dose IL2 were inhibited, expanding the role of small molecule compounds in the regulatory function of immune cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0075] Figure 1 It was shown that propionic acid treatment promoted IFN-γ secretion;

[0076] Figure 2 It shows that injection of propionic acid into humanized immune system mice promotes IFN-γ secretion by T cells;

[0077] Figure 3It was shown that propionic acid treatment of CAR-T promoted its IFN-γ secretion;

[0078] Figure 4 It shows that propionic acid treatment of CAR-T promotes its killing of tumors;

[0079] Figure 5 It shows that propionic acid-treated CAR-T inhibits tumor growth and weight;

[0080] Figure 6 It shows that propionic acid treatment of CAR-T promotes its killing of tumors and the secretion of IFN-γ and TNF-a in TILS;

[0081] Figure 7 It shows that treatment of T cells with propionic acid promotes their differentiation into Th1 cell-related pathways;

[0082] Figure 8 It was shown that 2-HB (2-hydroxybutyrate) treatment in vitro promoted the generation of Treg cells;

[0083] Figure 9 Shows that injection of 2-hydroxybutyrate into mice with humanized immune systems alleviates the cytotoxicity caused by high-dose IL-2;

[0084] Figure 10 The study showed that the injection of 2-hydroxybutyrate into mice with humanized immune systems alleviated the cytotoxicity caused by high-dose IL-2 and increased the proportion of Treg cells.

[0085] Figure 11 It shows that 2-hydroxybutyrate treatment of T cells promotes changes in their Treg cell-related transcriptional profiles;

[0086] Figure 12 A heat map showing the association between metabolites screened by the method for screening metabolites with immunomodulatory effects in Examples 1 to 2 of the present application and immune system regulation is shown;

[0087] Figure 13 A heat map showing the association between microorganisms and metabolites screened by the method for screening metabolites with immunomodulatory effects in Examples 1 and 2 of the present application is shown;

[0088] Figure 14 The design flow chart shows how to start with humanized immune system mice, then establish three humanized immune system mouse models with different microbiotas, study and confirm the connection between metabolites and the immune system, and apply the research results. DETAILED DESCRIPTION

[0089] In order to make the technical means, creative features, objectives and effects achieved by the present invention easier to understand, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0090] [Experimental Materials]

[0091]

[0092]

[0093]

[0094]

[0095] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods. Unless otherwise specified, the materials, reagents, etc. used in the following examples are all commercially available.

[0096] Example 1

[0097] Construction of three humanized mice with different immune systems based on intestinal flora

[0098] To establish a microbiota-depleted (antibiotic-induced microbiota depletion, abbreviated herein as ABX) HIS mouse model, parental NCG mice were fed an antibiotic cocktail containing 1 g / L ampicillin (A100339, Sangon Biotech), 1 g / L metronidazole (A600633, Sangon Biotech), 1 g / L neomycin (A610366, Sangon Biotech), and 0.5 g / L vancomycin (A600983, Sangon Biotech) in their drinking water, with free access to water. The offspring of these mice were used to establish HIS mice and were given antibiotics throughout their lives, with antibiotics changed twice weekly.

[0099] To simulate bacterial dysbiosis induced by brief antibiotic administration, SPF-reared, humanized mice with a humanized immune system were gavaged with an antibiotic cocktail twice daily for one week. After a two-week interval, the cocktail was administered again for one week, and then again two weeks later for the experiment. This model is referred to as the dysbiosis model. The antibiotic cocktail consisted of ampicillin (100 mg / kg), vancomycin (50 mg / kg), neomycin (100 mg / kg), and metronidazole (100 mg / kg). A control group of SPF mice received only water by gavage.

[0100] Under conventional conditions, SPF-raised humanized immune system mice were transferred to clean-level animal rooms (CL) for 4 weeks to establish a natural flora exposure model, which is referred to as the clean level transfer (CLT) model.

[0101] The three humanized immune system mouse models with different microbiota differences mentioned above each reflect different degrees of microbiota imbalance. By using multiple mouse models with different degrees of microbiota imbalance, the distribution of different bacterial species in the microbiota is changed, the abundance of microbiota and metabolites in the test samples is increased, and changes in microbiota, metabolism, and immunity are caused for correlation analysis.

[0102] The corresponding control SPF mice were all raised in SPF facilities.

[0103] Example 2:

[0104] The three types of HIS mice provided a rich sample resource for correlation analysis between gut bacteria, microbial metabolites, and immune cell types, with SPF mice used as controls. Correlation analysis between immune subsets and gut bacteria and related metabolites was the primary approach used in this study to identify human immunomodulatory bacteria and metabolites. Notably, correlations between immune subsets and metabolites were more numerous and stronger than those between immune subsets and the microbiome. This observation supports the view that microbiome regulation of the host immune system occurs primarily through metabolic effects.

[0105] Starting with humanized immune system mice, we then established three humanized immune system mouse models with different microbiotas to study and confirm the connection between metabolites and the immune system. See the design flow chart for applications based on the research results. Figure 14 .

[0106] The correlation matrix between bacteria and immune responses revealed associations between 25 bacterial genera and 21 immune phenotypes.

[0107] Applicants conducted analyses to identify correlations between potential immunomodulatory microorganisms, immune cells, and metabolites. The metabolite-immune matrix, consisting of 51 metabolites, was positively or negatively correlated with 21 different immune cell phenotypes.

[0108] Among them, the proportion of T cells secreting IFN-γ is positively correlated with multiple metabolites, including propionic acid, taurohyodeoxycholic acid, tauroursodeoxycholic acid, taurocholic acid, taurochenodeoxycholic acid, histidine, indolepropionic acid, chenodeoxycholic acid, and gluconolactone, and negatively correlated with myristoleic acid, methylglutaric acid, ketoglutaric acid, and niacin. In addition, 2-HB is closely related to the levels of Treg cells and IL-2+ T cells (see Table 2). Figure 1 A and Figure 8 A).

[0109] With Foxp3 + Metabolites associated with changes in Treg cells include 2-hydroxybutyrate, butyrate, citramalic acid, β-hydroxyisovaleric acid, 4-hydroxyphenylpyruvic acid, taurochenodeoxycholic acid, β-Alanine, 2-Hydroxy-2-methybutyric acid, and γ-linolenic acid.

[0110] The degree of dysbiosis in these three humanized immune system mice ranged from severe dysbiosis to moderate dysbiosis and finally to mild dysbiosis. The samples used for screening were complementary, which increased the abundance of metabolites detected that have a regulatory effect on immunity.

[0111] The heat map of the association between metabolites and immune system regulation screened by the above animal models and methods is shown in Figure 12 The correlation heat map of the screened microorganisms and metabolites is shown in Figure 13 .

[0112] Example 3:

[0113] The results in Example 2 were further verified by performing Spearman correlation analysis on the correlation between metabolites and T cells. Figure 1 , it can be seen that T cells secreting IFN-γ have the strongest positive correlation with PA and the strongest negative correlation with niacin.

[0114] After culturing T cells with different concentrations of metabolites for 3 days, the cell supernatants were collected and stored at -80°C. Cytokines were detected (see Figure 2 A). IFN-γ concentration was determined using human IFN-γ ELISA MAXTM (Biolegend, USA, Cat: 430104) according to the manufacturer's instructions. Flow cytometry was used to further analyze the IFN-γ levels after PA treatment, and it was found that treatment with 2 mM propionic acid promoted T cell IFN-γ secretion ( Figure 2 B).

[0115] For short-term PA treatment of SPF HIS mice, PA (60 mg / kg) was administered daily via intravenous (IV) injection for 1 week. For GF-HIS mice, PA (200 mM) was provided via drinking water for 1 month, with water changed every 3 days. IFN-γ levels were measured in peripheral blood and spleen. Figure 3 It can be seen that short-term PA treatment can promote the secretion of IFN-γ by T cells. Further PA treatment of CAR-T cells and then co-culture with corresponding HEP-12 tumor cells revealed that PA treatment can promote the secretion of IFN-γ by CAR-T cells ( Figure 4 AC).

[0116] To further verify the tumor-suppressing effect of CAR-T cells in vivo after PA treatment, the applicant first subcutaneously injected 3 million HEP-12 tumor cells into 6-8 week old NCG mice. When the tumor size reached 300 mm 3 Around 24 hours after CAR-T cell injection, mice were randomly divided into three groups. The three groups were injected intratumorally with 1 million CAR-T cells, mock CAR-T cells, and PA-treated CAR-T cells in 25 μl PBS, respectively. Tumor growth was monitored daily, and mice were sacrificed for indicator analysis 8 days after CAR-T cell injection ( Figures 5-6 ). It can be seen that the PA-treated CAR-T cells stimulated the secretion of IFN-γ by CAR-T cells ( Figure 6 ), and reduced tumor volume and weight ( Figure 5 B-C), namely, propionic acid treatment of CAR-T promotes its killing of tumors and the secretion of IFN-γ and TNF-a by CAR-T cells in TILS. Transcriptome analysis of PA-treated T cells was performed to reveal its mechanism of action. Figure 7 .

[0117] Example 4

[0118] The correlation between metabolites and Treg cells was analyzed, and the positive and negative correlations obtained by screening were verified in vitro. After culturing T cells with different concentrations of metabolites for 3 days, the proportion of Treg cells after treatment was detected by flow cytometry ( Figure 8).from Figure 8 As shown in A~B and C~D, as the concentration of 2-hydroxybutyrate increases, the number of human Treg cells increases, or 2-hydroxybutyrate promotes the production of human Treg cells. Figure 8 As shown in A to B and E to F, γ-linolenic acid treatment inhibits the generation of Treg cells.

[0119] The HDIL2 mouse model was generated using the hydrodynamic injection method outlined previously. In summary, the human IL-2 cDNA clone in the vector pckv-6-xl4 (Origene) was purified using the GoldHiEndoFree Plasmid Maxi Kit (Cat ID: CW2014M, CoWin Biosciences). Subsequently, HIS mice were weighed and injected with 50 μg of IL-2 plasmid using a 27-gauge needle. For 2-HB treatment, HIS mice were started on a daily dose of 100 mg / kg 2-HB two days prior to hydrodynamic injection (see protocol). Figure 9 A) and continued injections for a full week, while a control group received only PBS. Mouse body weight was monitored daily and flow cytometry was performed at the end of the experiment. Transcriptome sequencing was used to analyze CD4 T cells treated with 2-HB for 3 days in vitro to elucidate its mechanism of action.

[0120] 2-HB treatment results are shown in Figure 9 B, It can be seen that the injection of 2-hydroxybutyrate into humanized immune system mice alleviated the cytotoxicity caused by high doses of IL-2, and the weight loss of mice was reduced. At the same time, the injection of 2-hydroxybutyrate into humanized immune system mice alleviated the cytotoxicity caused by high doses of IL-2 and increased the proportion of Treg cells ( Figure 10 A~C). 2-Hydroxybutyrate treatment of T cells promoted changes in their Treg cell-related transcriptional profiles ( Figure 11 ).

[0121] The above description is merely a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Although the present invention has been disclosed as a preferred embodiment as above, it is not intended to limit the present invention. Any technician familiar with this profession can make slight changes or modifications to equivalent embodiments of the methods and technical contents disclosed above without departing from the scope of the technical solution of the present invention. However, any simple modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A method for screening and identifying metabolites and bacterial flora with human immune regulation effects, characterized in that: The method comprises the following steps: (1) Establish humanized mice with various intestinal types and immune systems, and analyze changes in the intestinal flora, metabolites, and immune system through intestinal flora, serum metabolites, and immune system detection methods; (2) determine the correlation and ranking between different bacterial species, serum metabolites, and human immune subsets through bioinformatics analysis; and (3) confirming the regulatory relationship between the candidate metabolites and immune cell subsets in step (2) through biological experiments.

2. The method according to claim 1, characterized in that The humanized immune system mice in step (1) include one or more of PBMC humanized mice, HSC humanized mice, and Hu-BLT model humanized mice; Preferably, the humanized immune system mouse is an HSC humanized mouse; the HSC humanized mouse is an immunodeficient mouse transplanted with human hematopoietic stem cells, and human immune cells are reconstructed; Alternatively, the serum metabolome in step (1) includes a targeted metabolome or a non-targeted metabolome, and the immune system detection method includes flow cytometry, single cell sequencing, and mass spectrometry; Alternatively, the number of humanized immune system mice with different intestinal flora described in step (1) is one or more; Alternatively, the number of humanized immune system mice with different intestinal flora in step (1) is multiple, and these humanized immune system mice with different intestinal flora have multiple intestinal types, including multiple types of familiar flora, humanized flora, single bacterial colonization, sterile, dysbacteriosis, and pathological intestinal types; Alternatively, in step (1), SPF immune system humanized mice are used as controls; Alternatively, the metabolome is selected from metabolites related to the gut microbiota or metabolites of the organism affected by changes in the gut microbiota; Preferably, the immunodeficient mice include one or more of nude mice, NOD scid mice, RAG1 KO mice, RAG2 KO mice, Il2rg KO mice, and severely immunodeficient mice; Preferably, the severely immunodeficient mouse is a mouse comprising at least one of T cell deficiency, B cell deficiency, and NK cell deficiency; Preferably, the severely immunodeficient mouse is one or more of NOG mouse, NSG mouse, NCG mouse, NPG mouse, NKG mouse, BRG mouse, and their derivative strains; More preferably, the severely immunodeficient mouse is selected from the group consisting of NBSGW mouse, NSGW41 mouse, NOG-EXL mouse, NCG-M mouse, NCG-FLT3-KO mouse, NCG-X-TSLP mouse, NCG-hIL6 mouse, NCG-X-hIL15 mouse, NKG mouse, NKG-hIL15 mouse, NKG-hIL6 mouse, NCG-X mouse, NCG-MHC-dKO mouse, BALB / c Rag2-null IL-2Rγc-null mouse, NOD.Cg-Prkdc scid Il2rg tm1Wjl / SzJ Mouse, NOD-Prkdc em26Cd52 Il2rg em26Cd22 / Nju One or more of mice, NPG mice, NPG-B2M mice, BRGSF mice, BRGS mice, BRGST mice, and their derivative strains; The intestinal type includes the intestinal type of HIS mice raised in a specific pathogen-free environment, the intestinal type of HIS mice treated with long-term combined antibiotics throughout their life cycle, the intestinal type of HIS mice with dysbacteriosis caused by periodic antibiotic use, and the intestinal type of HIS mice transferred to a low-barrier cleanliness level mouse facility; Preferably, the intestinal flora method includes one or more of 16s rRNA detection, whole genome sequencing, and metagenomic sequencing.

3. The method according to claim 1, characterized in that The changes in immune cell subpopulations in step (2) include one or more of changes in T cells, B cells, natural killer cells, hematopoietic stem cells, neutrophils, basophils, eosinophils, monocytes, and macrophages; Alternatively, the biological experiment in step (3) includes one or more of a cell experiment, an animal experiment, and a clinical experiment; Preferably, the human immune subpopulations in step (2) include CMP, GMP, MEP, HSC, LMPP, MPP, IgA + B cells, IgG + B cells, IgM + B cells, naive B cells, plasmablasts, transitional B cells, FOXP3 + T cells, IL-2 + T cells, IL-4 + T cells, IL-13 + T cells, IL-17A + T cells, IL-21 + T cells, IL-22 + T cells, IFN-γ + T cells, TNF-α + One or more of T cells; Preferably, the cell experiment comprises treating an immune cell subpopulation with the candidate metabolite and detecting changes in the immune cell subpopulation. More preferably, the changes in the immune cell subpopulation include changes in the number, function, phenotype of the immune cells and changes in the interactions between the immune cells. Preferably, the animal experiment includes treating humanized experimental animals with the candidate metabolites and detecting changes in immune cell subpopulations, physiology, biochemistry, and health status of the experimental animals; Preferably, the clinical trial comprises administering the candidate metabolite to a patient and detecting changes in the patient's immune cell subpopulations, physiology, biochemistry, and health status; More preferably, the cell experiment comprises treating T cells or CAR-T cells with propionic acid, and detecting changes in IFN-γ secretion of the T cells or CAR-T cells; More preferably, the animal experiment comprises treating humanized mice with propionic acid, and detecting the IFN-γ levels in the peripheral blood and spleen of the humanized mice; More preferably, the animal experiment includes treating tumor-bearing mice with propionic acid and CAR-T cells, and detecting the tumor volume and weight of the tumor-bearing mice; More preferably, the cell experiment comprises treating T cells with 2-hydroxybutyric acid to detect changes in the number of Treg cells; More preferably, the cell experiment comprises treating T cells with γ-linolenic acid to detect changes in the number of Treg cells; More preferably, the animal experiment comprises treating mice treated with high-dose IL-2 with 2-hydroxybutyric acid, and detecting the alleviation of cytotoxicity and changes in the number of Treg cells brought about by high-dose IL-2.

4. The method according to claim 1, wherein The correlation analysis in step (2) includes statistical analysis or artificial intelligence algorithm analysis; Preferably, the statistical analysis includes one or more of Pearson correlation analysis, Spearman correlation analysis, Kendall Tau correlation analysis, and Point-Biserial correlation analysis; Preferably, the correlation analysis is Pearson correlation analysis and Spearman correlation analysis; Preferably, the artificial intelligence algorithm analysis includes one of linear regression, logistic regression, decision tree, naive Bayes, support vector machine, ensemble learning, K-nearest neighbor algorithm, K-means algorithm, neural network, and deep reinforcement learning.

5. The method according to claim 1, wherein The metabolites in step (2) are selected from suberic acid, phenylpyruvic acid, octanoic acid, hexanoic acid, azelaic acid, malonic acid, phenyllactic acid, D-xylose, hyodeoxycholic acid, mandelic acid, deoxycholic acid, 3-hydroxyphenylacetic acid, L-aspartic acid, D-xylulose, L-tyrosine, methylsuccinic acid, glutaric acid, glucose, L-α-aminobutyric acid, indoleacetic acid, dimethylglycine, aminocaproic acid, tauroursodeoxycholic acid, taurocholic acid, taurochenodeoxycholic acid, propionic acid, taurine and deoxycholic acid. Bile acid conjugates, L-histidine, taurodeoxycholic acid, propionylcarnitine, 3-indolepropionic acid, butyric acid, 4-hydroxyphenylpyruvic acid, sarcosine, 3-hydroxyisovaleric acid, citramalic acid, 2-hydroxybutyric acid, niacin, D-fructose, ketoglutaric acid, beta-hyodeoxycholic acid, eicosapentaenoic acid, 8-11-14-eicosatrienoic acid, docosapentaenoic acid, linoleic acid, beta-alanine, 2-hydroxy-2-methylbutyric acid, gamma-linoleic acid, cis- and trans-cinnamic acids, carnitine, glyceric acid.

6. The method according to claim 1, characterized in that The metabolites positively correlated with the proportion of T cells secreting IFN-γ in step (2) include one or more of propionic acid, taurohyodeoxycholic acid, tauroursodeoxycholic acid, taurocholic acid, taurochenodeoxycholic acid, histidine, indolepropionic acid, chenodeoxycholic acid, gluconolactone, and derivatives thereof; Metabolites negatively correlated with the proportion of T cells secreting IFN-γ include one or more of myristoleic acid, methylglutaric acid, ketoglutaric acid, niacin, and derivatives thereof; With Foxp3 + Metabolites positively correlated with changes in Treg cells include one or more of 2-hydroxybutyric acid, butyric acid, citramalic acid, β-hydroxyisovaleric acid, 4-hydroxyphenylpyruvic acid, taurochenodeoxycholic acid, and their derivatives; With Foxp3 + Metabolites negatively correlated with changes in Treg cells include one or more of β-alanine, 2-hydroxy-2-methylbutyric acid, γ-linolenic acid, and their derivatives; Preferably, the metabolites positively correlated with the proportion of T cells secreting IFN-γ include propionic acid and its derivatives, and the metabolites negatively correlated with the proportion of T cells secreting IFN-γ include nicotinic acid and its derivatives; Preferably, with Foxp3 + Metabolites positively correlated with Treg cell changes include 2-hydroxybutyric acid and its derivatives; Preferably, with Foxp3 + Metabolites negatively correlated with Treg cell changes include γ-linolenic acid and its derivatives.

7. The method according to claim 6, characterized in that Propionic acid is positively correlated with one or more of enhancing the secretion capacity of IFN-γ and TNF-a of T cells, enhancing the secretion capacity of IFN-γ and TNF-a of CAR-T cells, and increasing the tumor-killing ability of CAR-T cells.

8. The method according to claim 6, characterized in that 2-Hydroxybutyrate is positively correlated with the production and function of human Treg cells, as well as inhibiting T cell expansion, toxic side effects, slowing weight loss, and increasing the proportion of Treg cells caused by high-dose IL2. Alternatively, γ-linolenic acid is associated with the suppression of the generation of human Treg cells.

9. Use of propionic acid and its derivatives in the preparation of immunomodulatory products, characterized in that: The immune regulation includes one or more of enhancing the secretion capacity of IFN-γ and TNF-a of human T cells, enhancing the secretion capacity of IFN-γ and TNF-a of human CAR-T cells, and promoting the killing ability of human CAR-T cells against tumors; Preferably, the product includes one or more of medicines, health products, and foods; Preferably, the drug comprises a biological product containing or producing propionic acid and its derivatives; More preferably, the biological product comprises a culture medium for culturing cell therapy drugs; More preferably, the biological product that produces propionic acid and its derivatives includes a bacterial agent. Further preferably, the bacterial agent includes probiotics, engineered bacteria, oncolytic bacteria, prebiotics that promote intestinal flora to produce propionic acid and its derivatives, and symbiotics composed of probiotics and prebiotics that promote probiotics to produce propionic acid and its derivatives.

10. Use of 2-hydroxybutyric acid and its derivatives in the preparation of immunomodulatory products, characterized in that: The immune regulation includes promoting the production of Treg cells, inhibiting T cell proliferation, toxic side effects, slowing down weight loss caused by high-dose IL2, and increasing the proportion of Treg cells. Preferably, the product includes one or more of medicines, health products, and foods; Preferably, the drug comprises a biological product containing 2-hydroxybutyric acid and its derivatives; More preferably, the biological product comprises a culture medium for culturing cell therapy drugs; More preferably, the biological product that produces 2-hydroxybutyric acid and its derivatives includes a bacterial agent. Further preferably, the bacterial agent includes probiotics, engineered bacteria, oncolytic bacteria, prebiotics that promote intestinal flora to produce 2-hydroxybutyric acid and its derivatives, and symbiotics composed of probiotics and prebiotics that promote probiotics to produce 2-hydroxybutyric acid and its derivatives.

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