GLO2 and use thereof in promoting or inhibiting inflammation or immune responses

Abstract

Disclosed are GLO2 and use thereof in promoting or inhibiting inflammation or immune responses. The present invention relates to use of a nucleic acid encoding GLO2, a GLO2 protein, or a promoter of GLO2 in the preparation of a drug for promoting inflammation or immune responses, use of an inhibitor of GLO2 in the preparation of a drug for inhibiting inflammation or immune responses, and use of a detection agent of GLO2 in the preparation of a kit for screening for a drug and / or a therapy for inflammation or immune responses.

Description

GLO2 and its application in promoting or inhibiting inflammatory or immune responses Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to GLO2 and its application in immune regulation and the diagnosis and treatment of immune-related diseases. Specifically, this invention relates to a novel immunomodulatory protein, GLO2, its specific sequence and related products, and its uses and methods of application in regulating inflammatory or immune responses (e.g., regulating antiviral innate immune responses and regulating autoimmune diseases). Background Technology

[0002] The inflammatory response triggered by pathogen infection is a core part of the body's defense mechanisms. Upon recognizing pathogens, the immune system initiates a series of defensive responses, including the release of inflammatory mediators such as cytokines and chemokines, promoting the aggregation of immune cells and the clearance of pathogens. These responses help to locally resist infection and eliminate invading pathogens. However, while the inflammatory response is crucial for clearing pathogens, excessive or prolonged inflammation can lead to tissue damage and even induce more serious diseases.

[0003] The relationship between pathogen infection and inflammation is bidirectional. Pathogens trigger inflammation by activating the immune response, but some pathogens can also cause excessive inflammatory responses by secreting toxins or interacting with host cells. For example, endotoxin reactions caused by bacteria and cytokine storms caused by viral infections can exacerbate systemic inflammation, leading to serious consequences such as sepsis or acute respiratory distress syndrome. Excessive inflammation not only fails to effectively eliminate pathogens but may also cause long-term damage to the body. In addition, some pathogens have the characteristics of latent or chronic infection, allowing them to persist in the host and cause chronic low-grade inflammation. This chronic inflammation is often associated with a variety of chronic diseases, such as gastric cancer, cirrhosis, and certain types of colon cancer. Chronic inflammation not only interferes with the normal function of the immune system but may also promote cell mutation, tissue damage, and cancer development. At the same time, some pathogens evade host immune surveillance, suppressing the immune response and causing dysregulation of the inflammatory response, thereby contributing to the progression of chronic diseases.

[0004] From a clinical perspective, the inflammatory response triggered by pathogen infection is crucial for diagnosis and treatment. Effective anti-infective therapy can reduce the pathogen burden, thereby alleviating the damage caused by inflammation. Simultaneously, treating excessive inflammation is also very important, particularly in the management of infection-induced systemic inflammatory responses and chronic inflammatory diseases. Understanding the interaction between pathogen infection and inflammation will provide more precise guidance for developing new treatment strategies and improving patient outcomes.

[0005] Despite the crucial role of inflammation in the pathogenesis of various diseases, effective treatment of chronic inflammation remains challenging in clinical practice. Firstly, the mechanisms of inflammatory responses are highly complex, involving the cross-regulation of multiple cellular and molecular signaling pathways and immune responses. Overactivation, prolonged duration, or misaligned regulation of inflammation are often key factors leading to disease development. Furthermore, in tumor immunotherapy, the use of some cell therapies or immune checkpoint inhibitors (such as PD-1 inhibitors) can cause severe inflammation-related adverse reactions such as cytokine storms, but accurately assessing and monitoring such inflammatory responses in clinical practice remains a significant challenge. Secondly, traditional anti-inflammatory treatments, such as nonsteroidal anti-inflammatory drugs (NSAIDs) or glucocorticoids, while effective in the short term, often result in long-term side effects such as gastrointestinal bleeding and immunosuppression, and these drugs may not precisely target the underlying mechanisms of chronic inflammation. Therefore, identifying new anti-inflammatory therapeutic targets and biomarkers for predicting inflammatory risk, and developing novel treatments for chronic inflammation, particularly those that specifically regulate immune cells, cytokines, and their signaling pathways, have become critical issues urgently needing to be addressed in clinical practice.

[0006] Glyoxalase II (GLO2) is an important intracellular enzyme primarily involved in the glycemic pathway. Working in conjunction with glyoxalase I (GLO1), it helps mitigate oxidative stress and glycation damage by removing toxic reactive aldehydes (such as methylglyoxal, MGO). The main function of GLO2 is to convert SD-lactylglutathione (SLG), catalyzed by GLO1, into harmless D-lactate and glutathione, thereby preventing the accumulation of harmful advanced glycation end products (AGEs). This process is crucial for maintaining cellular homeostasis, regulating glucose metabolism, and antioxidative responses.

[0007] Recent studies have shown that GLO2 plays an important role in various diseases, particularly diabetes and its complications. The accumulation of methylglyoxal (MGO) is considered a significant factor in complications such as diabetic nephropathy and diabetic retinopathy, while GLO2 reduces the toxicity of MGO and alleviates these pathological changes. Furthermore, GLO2 also exhibits potential protective effects in neurodegenerative diseases such as Alzheimer's disease, possibly by slowing nerve damage through reducing AGEs (advanced aging processes).

[0008] Furthermore, research on GLO2 has revealed its potential impact on cancer development. High expression of GLO2 in certain cancer cells helps them adapt to oxidative stress, influencing their proliferation, migration, and apoptosis. GLO2 may affect tumor cell growth and metastasis by regulating key steps in glucuronidation metabolism, thus being considered a potential target for cancer therapy. Therefore, GLO2 not only plays an important role in glucose metabolism, anti-oxidative stress, and cellular homeostasis maintenance, but also exerts a complex and far-reaching influence on the occurrence and development of various diseases, including diabetes, neurodegenerative diseases, and cancer. However, its classic function beyond detoxifying macroglobulins, particularly its role in immune and inflammatory regulation, was previously unclear. Summary of the Invention

[0009] This invention is the first to discover that changes in GLO2 expression levels and enzyme activity have immunomodulatory functions and constitute a naturally occurring negative feedback regulatory mechanism in the human body. Mechanistically, the inventors found that activation of innate immune cells in vivo leads to downregulation of GLO2 at both RNA and protein levels via the NF-κB pathway. This downregulation results in decreased intracellular GLO2 enzyme activity and the accumulation of its substrate SLG. SLG accumulation can mediate D-lactation of proteins in the cytoplasm via non-enzymatic catalysis. These lactation modifications occur on key immune function proteins such as p65, RIG-I, MAPK1, and ISG15, thereby inhibiting the positive immunomodulatory functions of these molecules. The inventors further demonstrated that GLO2 knockout can inhibit the activation and expression levels of inflammation and interferon signaling pathways, and alleviate inflammation-induced damage in vivo. Conversely, GLO2 overexpression can promote the activation and expression levels of inflammation and interferon signaling pathways, and exacerbate inflammation-induced damage in vivo, indicating that GLO2, as a novel target, can be applied to immunomodulation and the diagnosis and prediction of related diseases.

[0010] This invention provides information on GLO2 and its applications in immune regulation and the diagnosis and treatment of immune-related diseases. Specifically, it proposes the application of the GLO2 gene or its expression products (mRNA and protein) or its promoters in the preparation of products that promote inflammation and immune responses in subjects, the application of its inhibitors in the preparation of products that inhibit inflammation and immune responses in subjects, and the corresponding products. It also provides the application of detecting GLO2 or its expression products in drug screening and / or treatment regimen selection. The GLO2 of this invention can be effectively used for the selection of treatment regimens and clinical prognostic assessment for inflammatory and autoimmune diseases caused by clinical pathogen infections, as well as for personalized and targeted therapy, showing promising clinical application prospects.

[0011] In order to address the aforementioned critical issues that urgently need to be resolved, in a first aspect, this application provides the use of GLO2-encoding nucleic acids, GLO2 proteins, or GLO2 promoters in the preparation of medicaments for promoting inflammatory or immune responses.

[0012] In a second aspect, this application provides the use of GLO2 inhibitors in the preparation of medicaments for suppressing inflammatory or immune responses.

[0013] In a third aspect, this application provides the use of GLO2 detection reagents in the preparation of kits for screening drugs and / or therapies against inflammatory or immune responses. Attached Figure Description

[0014] The present application will now be described in more detail with reference to the accompanying drawings, in which:

[0015] Figure 1 shows the results of pathogen stimulation leading to downregulation of GLO2 expression level: qPCR was used to detect the GLO2 expression level of mouse macrophages after stimulation by vesicular stomatitis virus (VSV) or lipopolysaccharide (LPS) to confirm that pathogen stimulation led to downregulation of GLO2 expression level.

[0016] Figure 2 shows the upregulation of the GLO2 substrate SLG level after pathogen stimulation: LC-MS was used to detect the intracellular concentration of SLG after pathogen stimulation as shown in the figure to demonstrate that the GLO2 substrate SLG level was upregulated after pathogen stimulation.

[0017] Figure 3 shows the results of Western blot analysis of protein D-lactation levels after stimulation by different pathogens: Western blot analysis of changes in protein D-lactation levels in mouse macrophages and dendritic cells after stimulation by different pathogens. The results confirm that pathogen stimulation leads to upregulation of D-lactation modification levels in immune cells.

[0018] Figure 4 shows the results of antibody-enriched LC-MS / MS detection of protein lactation levels after VSV virus stimulation. Antibody-enriched LC-MS / MS identified changes in intracellular lactation modification sites before and after VSV stimulation, and GO and KEGG enrichment analyses were used to analyze the signaling pathways and biological pathways involved in the enrichment of these lactation modification sites. The results confirmed that cellular lactation levels were upregulated after pathogen stimulation, and that the modification sites were enriched in immune response and inflammation-related signaling pathways.

[0019] Figure 5 shows the results of transcriptomic enrichment analysis of wild-type and GLO2 knockout macrophages after VSV stimulation: Bone marrow-derived macrophages (BMDM) from GLO2 knockout (GLO2 KO) mice were isolated, with wild-type C57 mouse BMDM serving as a control. After VSV treatment, wild-type and GLO2 KO cells were collected for transcriptomic sequencing and GSEA enrichment analysis. The results confirmed that the enrichment levels of some signaling pathways related to immune responses or inflammation were downregulated in GLO2 knockout cells, indicating that GLO2 levels are correlated with the level of inflammatory immune responses.

[0020] Figure 6 shows the results of detecting inflammatory cytokine levels in wild-type and GLO2 knockout macrophages after pathogen stimulation: BMDM cells from GLO2 knockout (GLO2 KO) (Figure A) and GLO2 overexpression (GLO2 OE) (Figure B) mice were isolated, with wild-type C57 mouse BMDM cells used as a control. After treatment with VSV or LPS, cell supernatants were collected for ELISA detection of IFNb, IL6, and TNF. The results confirmed that GLO2 knockout led to a downregulation of inflammatory cytokine levels, while GLO2 overexpression led to an upregulation of inflammatory cytokine levels.

[0021] Figure 7 shows the ELISA results and survival analysis of the anti-inflammatory therapeutic effects in wild-type and GLO2 knockout mice in an acute inflammation model: Whole blood was collected from the orbital venous plexus 12 hours after mice received intraperitoneal injections of VSV or LPS. Serum was collected after centrifugation for ELISA detection of IFNb, IL6, and TNF. For the survival experiment, mice were maintained for 72 hours after the challenge and then euthanized. The results confirmed that GLO2 knockout led to a downregulation of in vivo inflammatory cytokine levels in the mouse acute infection model and enhanced survival in mice under lethal acute cytokine storm.

[0022] Figure 8 shows the ELISA results and survival analysis of the anti-inflammatory therapeutic effects in wild-type and GLO2-overexpressing mice in an acute inflammation model. Whole blood was collected from the orbital venous plexus 12 hours after mice received intraperitoneal injections of VSV or LPS. Serum was collected after centrifugation for ELISA detection of IFNb, IL6, and TNF. For the survival experiment, mice were maintained for 72 hours after the challenge and then euthanized. The results confirmed that GLO2 overexpression led to upregulation of in vivo inflammatory cytokine levels in the mouse acute infection model and attenuated survival in mice under lethal acute cytokine storm. Detailed Implementation

[0023] This application relates to the use of GLO2-encoding nucleic acid, GLO2 protein, or GLO2 promoters in the preparation of medicaments for promoting inflammatory or immune responses. In one embodiment, the GLO2 promoter may include an agent that causes GLO2 overexpression in a subject. This application may also relate to the use of an agent that causes GLO2 overexpression in the preparation of medicaments for promoting inflammatory or immune responses. This application may also relate to a method for promoting an inflammatory or immune response. In one embodiment, the method includes administering to a subject GLO2-encoding nucleic acid, GLO2 protein, or a GLO2 promoter. In one embodiment, the GLO2-encoding nucleic acid comprises DNA and / or RNA. In one embodiment, the GLO2-encoding nucleic acid comprises mRNA encoding GLO2. In one embodiment, the GLO2-encoding nucleic acid comprises cDNA. In one embodiment, the GLO2-encoding nucleic acid comprises the sequence shown in SEQ ID NO: 1, its homologous sequence, a sequence having at least 90% sequence identity with it, or a complementary sequence thereof. In one embodiment, the GLO2-encoding nucleic acid comprises a sequence having at least 95% sequence identity with the sequence shown in SEQ ID NO: 1. In one embodiment, the coding nucleic acid of GLO2 comprises a sequence having at least 96% sequence identity with the sequence shown in SEQ ID NO: 1. In one embodiment, the coding nucleic acid of GLO2 comprises a sequence having at least 97% sequence identity with the sequence shown in SEQ ID NO: 1. In one embodiment, the coding nucleic acid of GLO2 comprises a sequence having at least 98% sequence identity with the sequence shown in SEQ ID NO: 1. In one embodiment, the coding nucleic acid of GLO2 comprises a sequence having at least 99% sequence identity with the sequence shown in SEQ ID NO: 1. In one embodiment, the coding nucleic acid of GLO2 consists of the sequence shown in SEQ ID NO: 1. In one embodiment, the homologous sequence of the sequence shown in SEQ ID NO: 1 comprises a homologous sequence in non-human vertebrates. In one embodiment, the complementary sequence comprises a sequence that is at least partially complementary to the sequence shown in SEQ ID NO: 1, its homologous sequence, or a sequence having at least 90% sequence identity with it. In one embodiment, the complementary sequence comprises a sequence that hybridizes to the sequence shown in SEQ ID NO: 1, its homologous sequence, or a sequence having at least 90% sequence identity with it. In one embodiment, the GLO2 protein includes the sequence shown in SEQ ID NO: 2, its homologous sequences, or a sequence having at least 90% sequence identity with it. In another embodiment, the GLO2 protein includes a sequence having at least 95% sequence identity with the sequence shown in SEQ ID NO: 2.In one embodiment, the GLO2 protein comprises a sequence having at least 96% sequence identity with the sequence shown in SEQ ID NO: 2. In one embodiment, the GLO2 protein comprises a sequence having at least 97% sequence identity with the sequence shown in SEQ ID NO: 2. In one embodiment, the GLO2 protein comprises a sequence having at least 98% sequence identity with the sequence shown in SEQ ID NO: 2. In one embodiment, the GLO2 protein comprises a sequence having at least 99% sequence identity with the sequence shown in SEQ ID NO: 2. In one embodiment, the GLO2 protein consists of the sequence shown in SEQ ID NO: 2. In one embodiment, the homologous sequence of the sequence shown in SEQ ID NO: 2 includes homologous sequences in non-human vertebrates. In one embodiment, the GLO2 promoter may include any agent that increases GLO2 expression. In one embodiment, inflammation includes infectious diseases and / or their symptoms. In one embodiment, inflammation includes bacterial infections, viral infections, fungal infections, parasitic infections, infections caused by chemical toxins, infections caused by physical factors, and various inflammations and complications arising therefrom, as well as chronic inflammatory diseases caused by infection. In one embodiment, inflammation includes infection caused by vesicular stomatitis virus (VSV) and the various symptoms and complications thereof. In one embodiment, inflammation includes infection caused by bacterial lipopolysaccharide (LPS) and the various symptoms and complications thereof. In one embodiment, inflammation includes infection caused by herpes simplex virus (HSV) and the various symptoms and complications thereof. In one embodiment, inflammation includes infection caused by Listeria monocytogenes (LM) and the various symptoms and complications thereof. In one embodiment, inflammation includes infection caused by Sendai virus (SeV) and the various symptoms and complications thereof. In one embodiment, the immune response includes the level or activity of inflammatory cytokines and interferons. In one embodiment, the immune response includes the level or activity of signaling or effector genes or proteins in the NF-κB or interferon signaling pathway. In a preferred embodiment, the inflammatory cytokines include IL-6 and TNF-α. In a preferred embodiment, the interferons include type I interferons, such as IFN-α and IFN-β. In a preferred embodiment, the immune response includes the level or activity of IFNβ, IL-6, and / or TNF-α. In a preferred embodiment, the signal transduction or effector gene or protein includes RelA, p38, TBK1, and IRF3. In one embodiment, the drug is used on a subject suffering from inflammation or requiring modulation of an immune response. In one embodiment, the subject includes mammals. In one embodiment, the subject includes humans, non-human primates, rats, mice, cats, dogs, pigs, horses, cattle, or sheep.Medicinal agents used to promote inflammatory or immune responses may include a pharmaceutical composition comprising a GLO2 promoter and a pharmaceutically acceptable carrier. This application may also relate to a method for increasing or overexpressing GLO2. In one embodiment, the method includes knocking in an exogenous GLO2 gene into the genome of a subject.

[0024] This application also relates to the use of GLO2 inhibitors in the preparation of medicaments for suppressing inflammatory or immune responses. This application may also relate to a method for suppressing inflammatory or immune responses. In one embodiment, the method includes administering a GLO2 inhibitor to a subject. In one embodiment, the GLO2 inhibitor may include any agent that reduces GLO2 expression. In one embodiment, the GLO2 inhibitor may include an agent that reduces, knocks down, or eliminates GLO2 expression in a subject. This application may also relate to the use of agents that reduce, knock down, or eliminate GLO2 expression in the preparation of medicaments for suppressing inflammatory or immune responses. This application may also relate to a method for reducing, knocking down, or eliminating GLO2 expression. In one embodiment, the method includes knocking down or eliminating an endogenous GLO2 gene from the genome of a subject. In one embodiment, the GLO2 inhibitor includes antibodies, siRNA, miRNA, antisense oligonucleotides, antagonists, and / or blockers. In one embodiment, the GLO2 inhibitor includes N,S-Bis-Fmoc-glutathione (DiFMOC-G) or a pharmaceutically acceptable derivative, salt, or ester thereof. In one embodiment, the salt of DiFMOC-G comprises an alkali metal salt. In one embodiment, the salt of DiFMOC-G comprises sodium and potassium salts. In a preferred embodiment, the salt of DiFMOC-G comprises a sodium salt. In one embodiment, the ester of DiFMOC-G comprises C 1-6 Alkyl esters. In one embodiment, the esters of DiFMOC-G include C... 1-6Alkyl diester. In a preferred embodiment, the ester of DiFMOC-G comprises diethyl ester. In this application, the inflammatory or immune response may include the inflammatory or immune response described in any of the above embodiments. In one embodiment, suppressing the inflammatory or immune response includes treating diseases and / or symptoms associated with immune overreaction or excessive type I interferon. In one embodiment, these diseases and / or symptoms include acute inflammation caused by pathogen infection, autoimmune diseases, inhibitor rejection, allergic diseases, and cytokine storm symptoms caused by the above diseases or drug treatments. In one embodiment, autoimmune diseases include systemic lupus erythematosus, rheumatoid arthritis, systemic vasculitis, multiple sclerosis, ulcerative colitis, and / or inflammatory bowel disease. In one embodiment, cytokine storm symptoms include cytokine storm caused by clinical use of PD-1 inhibitors. In one embodiment, suppressing the inflammatory or immune response includes suppressing a lethal acute cytokine storm. In one embodiment, the medicament is used for subjects suffering from inflammation or requiring modulation of the immune response. In this application, the subject may include the subject described in any of the above embodiments. A medicament for suppressing an inflammatory or immune response may include a pharmaceutical composition comprising an inhibitor of GLO2 and a pharmaceutically acceptable carrier.

[0025] This application also relates to the use of GLO2 detection reagents in the preparation of kits for screening drugs and / or therapies against inflammatory or immune responses. This application may also relate to a method for screening drugs and / or therapies against inflammatory or immune responses. In one embodiment, the method includes using a GLO2 detection reagent. In one embodiment, the GLO2 detection reagent includes a substance for detecting a nucleic acid encoding GLO2 or a GLO2 protein. In this application, the nucleic acid encoding GLO2 or the GLO2 protein may include the nucleic acid encoding GLO2 or the GLO2 protein as described in any of the above embodiments. In this application, the inflammatory or immune response may include the inflammatory or immune response or disease and / or its symptoms as described in any of the above embodiments. Kits for screening drugs and / or therapies against inflammatory or immune responses may contain a GLO2 detection reagent and apparatus or devices for detection, such as one or more substances selected from the group consisting of: containers, instructions for use, positive controls, negative controls, buffers, auxiliaries, or solvents.

[0026] All numerical ranges provided herein are intended to clearly include all values ​​falling between the endpoints of the range and the range of values ​​between them. Features mentioned in the invention or embodiments may be combined. All features disclosed in this specification may be used in any combination form, and each feature disclosed in the specification may be replaced by any alternative feature that provides the same, equivalent, or similar purpose. Therefore, unless otherwise specified, the disclosed features are merely general examples of equivalent or similar features.

[0027] As used in this article, “containing,” “having,” or “including” includes “containing,” “mainly composed of,” “substantially composed of,” and “composed of”; “mainly composed of,” “substantially composed of,” and “composed of” are subordinate concepts of “containing,” “having,” or “including.”

[0028] The numerical ranges in this article include their endpoints as well as the specific numerical points and subranges within that range. For example, 1 to 3 includes endpoints 1 and 3, the specific integer numerical points 2 and non-integer numerical points (e.g., but not limited to: 1.2, 1.5, 1.8, 2.1, 2.3, 2.4, 2.8, etc.), and their subranges (e.g., but not limited to: 1 to 2, 2 to 3, 1 to 1.2, 1.5 to 1.8, etc.).

[0029] Example

[0030] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make appropriate modifications and variations to the present invention, and such modifications and variations are all within the scope of the present invention.

[0031] Experimental methods not specifically described in the following examples can be performed using conventional methods in the art, such as referring to *Molecular Cloning: A Laboratory Manual* (3rd edition, New York: Cold Spring Harbor Laboratory Press, 1989) or following the conditions recommended by the supplier. RNA sequencing methods are conventional in the art and can also be provided by commercial companies.

[0032] Unless otherwise stated, percentages and parts are by weight. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar or equivalent to those described herein may be applied to the methods of this invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0033] Example 1: qRT-PCR detection of the GLO2 gene

[0034] Mouse peritoneal macrophages were obtained from the peritoneal cavity of wild-type C57BL6J mice (Shanghai Bikai Laboratory Animal Co., Ltd.), counted, and prepared into 6×10⁶ cells. 5Cells were cultured in 24-well plates with a cell suspension of 100 cells / ml. 500 μl of suspension (RPMI-1640 medium) was added to each well. After overnight culture, cells were stimulated with VSV virus (laboratory-supplied) (multiple of infection = 1) or LPS (400 ng / mL) for 0, 12, and 24 hours, respectively. Cells were then lysed with 1 ml of Trizol reagent (ambion, 15596026) per well, and RNA was extracted following the Trizol reagent procedure.

[0035] After extracting total RNA from cells, 1 μg of RNA was used for reverse transcription using a high-performance reverse transcription kit (Fastagen, 220011). The reverse transcription system is as follows:

[0036] The reverse transcription program was: 37℃ for 15 min, 95℃ for 5 min, and 4℃ ± ∞.

[0037] Using the reverse transcription product as a template, real-time quantitative PCR was performed using the following primers:

[0038] The HPRT gene was used as an internal reference and was relatively quantified using the 2^-ddCT method.

[0039] The qPCR system was prepared according to the Takara TB Green Real-Time Fluorescence Kit (RR820), and quantitative PCR amplification was performed using an ABI Q6 PCR instrument. The system is as follows:

[0040] The qPCR amplification procedure is as follows:

[0041] The experimental results are shown in Figure 1. The results show that GLO2 expression decreased significantly after viral infection or LPS stimulation, with a decrease of nearly 80% at 12 h.

[0042] The above results indicate that pathogen stimulation can lead to a significant downregulation of GLO2 levels, suggesting that GLO2 may play a regulatory role in the activation of immune cells.

[0043] Example 2: Mass spectrometry detection of GLO2 substrate SLG levels after pathogen stimulation

[0044] Mouse macrophages, either unstimulated (controls) or stimulated with VSV, HSV-1 (provided by our laboratory), LPS (Sigma, L3755), or LM (provided by our laboratory) (as described in Example 1), were lysed in lysis buffer (0.5% NP 40, 50 mM Tris-HCl pH 7.4, 150 mM NaCl). Samples were then centrifuged at 13,000 rpm for 10 min at 4 °C. Proteins were precipitated by adding 20% ​​(w / v) 5-sulfosalicylic acid (final concentration 2%) and removed by centrifugation at 10,000 rcf for 5 min at room temperature. Next, 100 μl of methanol was added to 100 μl of clear supernatant, and the sample was sonicated at 4 °C for 30 min and allowed to stand for 30 min. The sample was then centrifuged at 12,000 rpm for 15 min at 4 °C, ready for liquid chromatography / mass spectrometry (LC / MS) detection. In liquid chromatography (LC), 6 μl of the clarified supernatant was separated using a Waters Acquity UPLC apparatus (equipped with an Acquity UPLC HSS Amide column (1.7 μm, 2.1 mm × 100 mm)) at a flow rate of 0.30 mL / min and a temperature of 40 °C. Solvent A was 10 mM ammonia, and solvent B was acetonitrile. Mass spectrometry (MS) analysis was performed using an AB SCIEX 5500 Qtrap-MS under the following conditions: ion source: ESI; window gas: 35 arb; collision gas: 9 arb; electrospray voltage: 4500 V; temperature: 400 °C; ion source gas 1: 45 arb; ion source gas 2: 45 arb. For SLG quantification, integration was performed using MultiQuant software, and the SLG standard curve was calculated.

[0045] The experimental results are shown in Figure 2. The results show that the intracellular SLG level was significantly upregulated after stimulation by different pathogens, with an upregulation factor of approximately 10-fold.

[0046] The above results indicate that pathogen-induced downregulation of GLO2 levels leads to the accumulation of SLG.

[0047] Example 3: Western blot and mass spectrometry detection of upregulation of lysine lactation after pathogen stimulation

[0048] For Western blotting to detect the D-lactation level of cellular proteins after pathogen stimulation, mouse peritoneal macrophages were first stimulated with different pathogens (VSV, HSV-1, LM.) as described in Example 1. Cells were then collected, and total protein was extracted using lysis buffer. Protein concentration was determined using the BCA or Bradford method. Next, an SDS-PAGE gel was prepared. The extracted protein sample was mixed with loading buffer and electrophoresed on a polyacrylamide gel. After electrophoresis, the separated proteins were transferred to a nitrocellulose membrane using wet or semi-dry transfer. After transfer, the membrane was blocked with 5% skim milk powder or BSA (bovine serum albumin) in TBS-T buffer for 1 hour to prevent non-specific binding. The membrane was then incubated overnight at 4°C with a diluted D-lactation antibody (PTM-1429, PTMbio) to ensure sufficient antibody binding to the target protein on the membrane. After incubation, wash the membrane three times with TBS-T buffer for 5 minutes each time to remove unbound antibodies. Then, incubate the membrane with HRP-labeled secondary antibody (CST) at room temperature for 1 hour. After washing, wash the membrane again with TBS-T buffer to remove unbound secondary antibody. Finally, develop the membrane using a chemiluminescence (ECL) kit or other colorimetric system, observe the bands on the membrane, and record the location and expression of the target protein.

[0049] The experimental results are shown in Figure 3. The results show that the level of protein D-lactation in cells was significantly upregulated after stimulation by different pathogens.

[0050] For mass spectrometry detection of lactation modification levels and sites, mouse macrophages (1×10⁻⁶) 7 VSV (V) or the culture medium control (C) were treated separately, and then collected and sonicated three times on ice using a high-intensity sonic processor (Scientz) in lysis buffer (8M urea, 0.5% protease inhibitor, 3μM TSA, and 50mM NAM). The lysates were then centrifuged at 13,000 rpm for 15 minutes at 4°C, and the supernatant was collected. The protein concentration of the extract was determined by the diazolic acid method (BCA method). Then, trypsin digestion, enrichment of lactylated peptides, and mass spectrometry detection were performed according to PTMbio's standard protocols.

[0051] In brief, during trypsin digestion, 2% trypsin was added and digestion was performed overnight. The peptides were reduced with 5 mM dithiothreitol at 56°C for 30 min and alkylated with 11 mM iodoacetamide at room temperature in the dark for 15 min. To enrich the lactylated peptides, the trypsin-digested peptides were dissolved in NETN buffer (100 mM NaCl, 1 mM EDTA, 50 mM Tris-HCl, 0.5% NP-40, pH 8.0) and incubated overnight at 4°C with agarose beads (PTM-1404, PTMbio) conjugated with an anti-lactyl lysine antibody. After elution, the peptides were desalted using C18 ZipTips (Millipore) according to the manufacturer's instructions. For mass spectrometry detection, the peptides were separated on a nanoElute UHPLC system (Bruker Daltonics) and subsequently analyzed using timsTOF Pro (Bruker Daltonics). Precursor ions and fragment ions were analyzed on a TOF detector in an MS / MS scan range of 100 to 1700 m / z. Precursor ions with charged states of 0 to 5 were selected for fragmentation.

[0052] To identify and relatively quantify lactylation sites, MS / MS spectral data were analyzed using MaxQuant software (v.1.6.15.0). Tandem mass spectrometry data were aligned to the mouse SwissProt database, with trypsin / P designated as the lysin, allowing a maximum of two cleavage deletions. The mass tolerance for precursor ions was set to 20 ppm for the initial search and 5 ppm for the master search, with a fragment ion mass tolerance of 0.02 Da. Amino methylation on Cys was designated as a fixed modification, while acetylation of the protein's amino terminus, Met oxidation, and lysine lactylation were designated as variable modifications. The false discovery rate (FDR) was adjusted to <1%. The tag-free quantification mode was set to LFQ, with the minimum LFQ ratio count set to 2, the minimum LFQ neighbor count set to 3, and the maximum LFQ neighbor count set to 6. The tag minimum ratio count was set to 2. The peptide used for quantification was set to Unique+Razor. Acetylation of the protein's amino terminus and Met oxidation were designated as modifications used in quantification; unmodified counterparts were discarded for protein quantification.

[0053] The experimental results are shown in Figure 4. Mass spectrometry confirmed that the number of lactation modification sites was significantly upregulated and that the modification sites were largely enriched in immune-related signaling pathways.

[0054] Example 4: Effects of GLO2 protein expression levels on inflammatory immune responses detected by transcriptomics sequencing and ELISA

[0055] The inventors first constructed C57BL6J mice with GLO2 knockout (GLO2 KO) (Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd., through CRISPR / Cas9 system to knock out the endogenous GLO2 gene from the mouse genome) and GLO2 overexpression (GLO2 OE) (Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd., through CRISPR / Cas9 system to insert the exogenous GLO2 gene fragment into the mouse genome at a specific site), and isolated their bone marrow macrophages (BMDM), using wild-type C57 mouse BMDM as a control. After treatment with VSV, wild-type and GLO2 KO cells were collected for transcriptomic analysis.

[0056] RNA-seq experiments were performed by TIANGEN Co., Ltd. according to their standard protocols. Briefly, after total RNA extraction and DNase I treatment, eukaryotic total RNA with a Poly-A structure was enriched using the TIANSeq mRNA capture kit (TIANGEN Biotechnology). Next, libraries were constructed using the TIANSeq rapid RNA library construction kit (Illumina platform) (TIANGEN Biotechnology). After RNA lysis, cDNA synthesis, end repair, addition of mononucleotide A (adenine), adapter ligation, and PCR library enrichment, the transcriptome sequencing library was constructed. During quality control, the sample libraries were quantified and their quality assessed using an Agilent 2100 bioanalyzer and an ABI StepOnePlus real-time PCR system. Finally, the libraries were sequenced using an Illumina HiSeq2000 or Illumina NovaSeq 6000, yielding approximately 150 bp paired-end sequencing reads. Differential expression analysis for each group was performed using FPKM values. Subsequent FPKM results were enriched using GSEA, and data visualization was performed using R-studio, GSEA, and GraphPad Prism 10.0.

[0057] The experimental results are shown in Figure 5. Transcriptome GSEA enrichment analysis showed that in GLO2 knockout cells, the enrichment levels of some signaling pathways related to immune responses or inflammatory responses were downregulated, indicating that the level of GLO2 is correlated with the level of inflammatory immune responses.

[0058] After treatment with VSV or LPS, cell supernatants were collected for ELISA detection. The concentrations of mouse IFNβ, IL6, or TNFα in the cell culture supernatant were determined using the IFNβ cytokine-specific VeriKine ELISA kit (PBL Interferon Source), the IL6 Quantikine ELISA kit (R&D Systems), or the TNFα Quantikine ELISA kit (R&D Systems), respectively, following the manufacturer's instructions.

[0059] The experimental results are shown in Figure 6. ELISA results confirmed that GLO2 knockout led to a downregulation of inflammatory cytokine levels, while GLO2 overexpression led to an upregulation of inflammatory cytokine levels. This further demonstrates that GLO2 levels are correlated with the level of inflammatory immune response.

[0060] Example 5: ELISA detection and survival analysis of the anti-inflammatory therapeutic effect of GL02 in a mouse acute inflammation model.

[0061] C57BL6J mice (as described in Example 4) with GLO2 knockout (GLO2 KO) and GLO2 overexpression (GLO2 OE) stimulated with LPS or VSV were used as controls. Whole blood was collected from the orbital venous plexus 12 hours after intraperitoneal injection of VSV (1×10^7 pfu / g) or LPS (80 μg / g). Serum was collected after centrifugation for ELISA detection. Serum concentrations of IFN β, IL6, or TNFα were determined using the IFN β cytokine-specific VeriKine ELISA kit (PBL Interferon Source), the IL6 Quantikine ELISA kit (R&D Systems), or the TNFα Quantikine ELISA kit (R&D Systems), respectively, following the manufacturer's instructions. For survival experiments, mice were maintained for 72 hours after the challenge and then euthanized. Survival analysis was performed using the Log-rank (Mantel-Cox) test.

[0062] As shown in Figures 7 and 8, the ELISA results confirmed that GLO2 knockout led to a downregulation of inflammatory cytokine levels in a mouse acute infection model, while GLO2 overexpression led to an increase in inflammatory cytokine levels in the same model. Survival analysis showed that GLO2 knockout enhanced survival in mice under lethal acute cytokine storm, while GLO2 overexpression reduced survival.

[0063] The GLO2 of this invention is downregulated by the NF-κB signaling pathway after activation of pattern recognition receptors on immune cells or stimulation by cytokines. The inventors further demonstrated that GLO2 knockout can inhibit the activation and expression levels of inflammation and interferon signaling pathways, and alleviate inflammation-induced damage in vivo. Conversely, GLO2 overexpression can promote the activation and expression levels of inflammation and interferon signaling pathways, and exacerbate inflammation-induced damage in vivo. Furthermore, GLO2 levels are also correlated with the severity of sepsis caused by COVID-19 infection, systemic lupus erythematosus, and pulmonary hypertension. The GLO2 of this invention can be effectively used for the selection of treatment regimens and clinical prognostic assessment for acute and chronic inflammatory diseases and autoimmune diseases, as well as for personalized and targeted therapy, showing promising clinical application prospects. This invention has also demonstrated that the small molecule drug DiFMOC-G, as an enzyme activity inhibitor of GLO2, exhibits good preventive and therapeutic effects in various inflammatory disease models (data not shown), proving that GLO2 inhibitors have good efficacy in treating inflammation and modulating immune responses.

[0064] Specifically, the inventors analyzed the expression changes of key metabolic enzymes in cells under different pathogen stimuli using transcriptomics and single-cell sequencing, and confirmed that GLO2 is the most significantly downregulated metabolic enzyme under RNA virus and LPS stimulation, and its downregulation depends on the activation of the NF-κB signaling pathway. The inventors further confirmed through experiments that its downregulation leads to the downregulation of intracellular GLO2 enzyme activity and the accumulation of its substrate SLG. The accumulation of SLG can mediate D-lactation modification of proteins in the cytoplasm in a non-enzymatic manner. These lactation modifications occur on some key immune function proteins, such as p65, RIG-I, MAPK1, ISG15, etc., thereby inhibiting the positive immune regulatory function of these molecules, avoiding excessive activation of the immune system, and thus limiting the level of inflammation. On the other hand, the inventors also explored the correlation between GLO2 expression levels and sepsis caused by SARS-CoV-2 infection, as well as autoimmune diseases such as systemic lupus erythematosus and pulmonary hypertension. They found a positive correlation between GLO2 expression levels and the severity of sepsis, and that GLO2 expression levels in samples from patients with systemic lupus erythematosus and pulmonary hypertension were significantly higher than in healthy individuals. This indicates a positive correlation between GLO2 expression levels and inflammation levels, suggesting that GLO2 can be used as an indicator for the diagnosis and prognostic analysis of inflammatory diseases. Furthermore, GLO2 can also serve as a target for the treatment of inflammation-related diseases and autoimmune diseases, achieving regulation of the immune system and thus therapeutic goals.

[0065] This invention provides novel applications for GLO2 in regulating inflammation-related diseases and autoimmune diseases. The detection of GLO2 expression levels can be used to detect inflammation-related diseases, providing valuable information for prognostic diagnosis and offering reference information for clinical treatment strategies. Targeted regulation of GLO2 expression levels or activity, including biomedical measures such as small molecule inhibitors, overexpression, and silencing, can be applied to the clinical treatment of inflammation and autoimmune diseases, achieving the goal of regulating the inflammatory immune response and ultimately treating the disease.

Claims

1. The use of GLO2-encoding nucleic acids, GLO2 proteins, or GLO2 promoters in the preparation of drugs for promoting inflammatory or immune responses.

2. The application as described in claim 1, wherein the nucleic acid encoding GLO2 comprises mRNA encoding GLO2.

3. The application of GLO2 inhibitors in the preparation of drugs for suppressing inflammation or immune responses.

4. The application as described in claim 3, wherein the GLO2 inhibitor comprises an antibody, siRNA, miRNA, antisense oligonucleotide, antagonist, and / or blocker.

5. Use of GLO2 detection reagents in the preparation of kits for screening drugs and / or therapies against inflammatory or immune responses.

6. The application of claim 5, wherein the GLO2 detection reagent comprises a substance encoding a nucleic acid or a GLO2 protein for detecting GLO2.

7. The application of any one of claims 1, 2 and 6, wherein the coding nucleic acid of said GLO2 comprises the sequence shown in SEQ ID NO: 1, its homologous sequence, a sequence having at least 90% sequence identity with it, or the aforementioned complementary sequence.

8. The application as described in claim 1 or 6, wherein the GLO2 protein comprises the sequence shown in SEQ ID NO: 2, its homologous sequence, or a sequence having at least 90% sequence identity with it.

9. The application according to any one of claims 1-6, wherein the inflammation includes infectious diseases and / or their symptoms, such as bacterial infections, viral infections, fungal infections, parasitic infections, infections caused by chemical toxins, infections caused by physical factors and their various symptoms and complications, as well as chronic inflammatory diseases caused by infection.

10. The application according to any one of claims 1-6, wherein the immune response includes the level or activity of inflammatory cytokines and interferons, and / or the level or activity of signal transduction or effector genes or proteins in the NF-κB or interferon signaling pathway, preferably, the inflammatory cytokines include IL-6 and TNF-α; the interferons include type I interferons, such as IFN-α and IFN-β; and / or the signal transduction or effector genes or proteins include RelA, p38, TBK1, and IRF3.

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