Abstract
Interleukin-6 (IL-6) is a multifunctional cytokine with a wide range of biological functions, playing a key role in inflammatory response, immune regulation, metabolic regulation, and various disease processes. The IL-6 related mouse model is a core tool for studying the physiological and pathological functions of this molecule. This article systematically reviews the construction strategies and identification methods of IL-6 mouse models, covering two categories: genetic engineering models (including gene knockout, conditional overexpression, and knock in models) and inflammation induced models (such as lipopolysaccharide induced sepsis models, collagen induced arthritis models, etc.), and summarizes the identification indicators and method system of the models from multiple dimensions such as phenotype characteristics, molecular biology, biochemical immunology, and histopathology. This review aims to provide a systematic reference for researchers in related fields in the selection, construction, and identification of IL-6 mouse models.
**Keywords * *: interleukin-6; Mouse model; Gene knockout; Conditional overexpression; Model identification
1. Introduction
Interleukin-6 (IL-6) is a multifunctional cytokine produced by multiple cell types, playing a key role in immune response, inflammation regulation, hematopoietic function, metabolic balance, and neuroprotection. Studies have shown that the abnormal activation of IL-6 signaling pathway is closely related to the occurrence and development of rheumatoid arthritis, inflammatory bowel disease, atherosclerosis, diabetes retinopathy, multiple sclerosis, multiple tumors and other diseases. However, the role of IL-6 is highly complex - it may exhibit dual functions of pro-inflammatory or anti-inflammatory under different cellular sources and physiological or pathological conditions.
The mouse model is an indispensable tool for studying the biological functions of IL-6 and its role in diseases. By using genetic engineering to construct IL-6 overexpressing or knockout mice, and inducing inflammation related models with chemical or biological agents, researchers can explore the functional mechanisms of IL-6 in different disease backgrounds at the in vivo level, and evaluate the effectiveness and safety of targeted IL-6 treatment strategies. This article will systematically introduce the current research progress of IL-6 mouse models from two dimensions: model construction strategies and model identification methods, in order to provide methodological references for related research.
2、 Construction method of IL-6 mouse model
According to different research purposes, IL-6 related mouse models can be roughly divided into two categories: one is a genetic modification model constructed through genetic engineering, and the other is an inflammatory disease model induced by chemical or biological agents.
1. Genetic engineering mouse model
1.1 IL-6 gene knockout (KO) model
IL-6 gene knockout mice are the most classic tool for studying IL-6 function. The most widely used currently is the IL-6 KO mouse model constructed by targeted knockout of exons 2 to 5 of the Il6 gene (strain name C57BL/6Smoc-Il6em1Smoc). This type of mouse homozygous (Il6 ⁻/⁻) can survive and reproduce normally, but exhibits significant deficiencies in response to various viruses and inflammatory reactions to tissue damage or infection. Taking the experiment of inducing liver injury with carbon tetrachloride (CCl ₄) as an example, wild-type mice showed significant upregulation of Il6 mRNA and serum IL-6 protein levels after CCl ₄ stimulation, while IL-6 KO mice showed no detectable expression of IL-6 regardless of stimulation, confirming the effectiveness of knockout.
In addition to complete knockout, researchers have also developed conditional reversible knockout models. For example, IL6-DIO-KO mice constructed using double inverted open reading frame (DIO) technology exhibit a systemic IL-6 knockout phenotype under normal conditions, but can be reactivated for IL-6 expression through Cre recombinase. This type of model has unique advantages in studying the functional recovery effects of IL-6.
1.2 IL-6 Conditional Overexpression Model
To elucidate the specific role of IL-6 derived from specific cells in diseases, researchers constructed multiple conditional overexpression mouse models using the Cre loxP system. For example, Knopp et al. hybridized IL-6 overexpressing mice with LysM-Cre mice to obtain LysM-IL-6OE mice that specifically overexpress IL-6 in myeloid cells. The model mice aged 8 to 12 weeks can spontaneously develop inflammatory colitis, significant impairment of endothelial dependent aortic dilation function, elevated levels of aortic reactive oxygen species, and dysfunction of resistance vessels. The bone marrow transplantation experiment further confirmed that these vascular dysfunctions are driven by bone marrow-derived IL-6 and are dose-dependent.
Similarly, Chicherina et al. constructed a transgenic mouse model overexpressing human IL-6 (hIL-6) in CX3CR1 ⁺ myeloid cells induced by tamoxifen. This study confirms that in the context of systemic inflammation induced by lipopolysaccharide (LPS), the level of hIL-6 in the serum of transgenic mice is significantly elevated, and high levels of transgenic expression are detected in both the heart and lungs. This model provides a powerful tool for studying the systemic effects of IL-6 in chronic inflammation.
In addition, significant progress has been made in constructing a humanized IL-6 conditional expression model using CRISPR/Cas9 technology. Nidadavolu et al. established a TetO-hIL6 knock in mouse model using a donor vector containing a tetracycline response element (TRE) promoter, which can induce hIL-6 expression in adult mice by administering water containing doxycycline. After induction, mice showed weakened phenotypes such as decreased grip strength, weakened running ability, increased frequency of falls, and decreased basal body temperature, providing a new platform for studying the role of IL-6 in aging and related metabolic disorders.
1.3 Cell specific IL-6 receptor knockout model
The IL-6 signal works through two main modes: classical signal transduction (via membrane-bound IL-6 receptors) and trans signal transduction (via soluble IL-6 receptors). To distinguish the specific functions of these two signaling pathways, researchers constructed a cell specific Il6ra knockout model. For example, Weng et al. used Cre loxP technology to construct M ü ller glial cell specific Il6ra knockout mice to study the specific role of IL-6 trans signal transduction in diabetes retinopathy.
In a broader immunological study, Gogoleva et al. constructed mice with conditionally inactivated IL-6 in CX3CR1 ⁺ cells (including microglia) or CD11c ⁺ dendritic cells to investigate the role of IL-6 from different sources in experimental autoimmune encephalomyelitis (EAE). The results indicate that IL-6 derived from microglia has both pathogenic and protective functions in the central nervous system, while IL-6 derived from dendritic cells mainly participates in regulating the balance between regulatory T cells and Th17 cells.
2. Inflammatory induction model
In addition to genetic engineering models, inflammation models induced by chemical or biological agents are also important tools for IL-6 research. This type of model is commonly used to study the dynamic changes of IL-6 in acute or chronic inflammation and its role in specific diseases.
2.1 Lipopolysaccharide induced sepsis model
Lipopolysaccharide is the main component of the outer membrane of gram-negative bacteria and an effective stimulant that activates Toll like receptor 4 and induces strong inflammatory responses. The classic method for constructing a sepsis model is to pre treat mice with low-dose LPS (400 μ g/kg) for 8 hours, and then stimulate them with high-dose LPS (10 mg/kg) to successfully establish a sepsis model. In this model, the serum IL-6 levels in mice continued to increase over time after stimulation - from (239.7 ± 21.5) pg/mL at 0 hours to (2989.5 ± 84.7) pg/mL at 16 hours, while the control group only had (56.5 ± 1.8) pg/mL, indicating a positive correlation between IL-6 levels and disease severity.
In the context of special research, researchers also combine LPS stimulation with physical environmental factors. For example, Zhang Lijun et al. placed 8-week-old male C57BL/6 mice in a low pressure and low oxygen chamber to simulate high-altitude hypoxia conditions at an altitude of 6000 meters, and established a high-altitude hypoxia inflammatory brain injury model by intraperitoneal injection of 5 mg/kg LPS. Compared with simple high-altitude hypoxia or simple LPS treatment, the combined treatment group had the highest expression levels of IL-6, TNF - α, and IL-10, and the most severe brain tissue damage.
2.2 Collagen induced arthritis model
Collagen induced arthritis (CIA) is one of the most commonly used animal models for studying rheumatoid arthritis. The classic modeling method is to fully emulsify bovine or chicken type II collagen with complete Freund's adjuvant, inject it subcutaneously into the tail root of DBA/1J mice for initial immunization, and reinforce it again 21 days later. After modeling, the mice had red and swollen feet on about 28 days, and the swollen thickness of feet on 35 days was significantly higher than that of the control group, and the incidence rate was as high as 90% on 62 days.
In the context of C57BL/6J mice, induction can also be achieved by emulsifying a mixture of chicken type II collagen and complete Freund's adjuvant. In this model, the serum IL-6 levels of the model mice were significantly increased, and inflammatory cell infiltration, synovial proliferation, and cartilage structure damage were observed in the synovial tissue. The expression of p-STAT3 protein was significantly upregulated in the synovial tissue, indicating that the IL-6/STAT3 signaling pathway plays an important role in the pathogenesis of arthritis.
2.3 Other inflammatory induction models
LPS can also be used to induce other types of mouse inflammation models. For example, stimulation of C57BL/6 mice with dextran sulfate sodium (DSS) or LPS can successfully establish an inflammatory bowel disease (IBD) model. In asthma models, mouse asthma models can be established by sensitization and stimulation with ovalbumin (OVA), and the serum IL-6 levels and ovalbumin specific IgE levels in bronchoalveolar lavage fluid of the model animals are significantly increased.
In addition, Wang et al. established a novel mouse model of persistent inflammation, immune suppression, and catabolic syndrome (PICS): sepsis was induced using a modified cecal ligation and puncture (CLP) method, followed by daily injection of 2 mg/kg dexamethasone to maintain the inflammatory response. On the 14th day, the IL-6 level in the model group mice increased by about 3000% compared to the CLP group alone, while TNF - α and IL-1 β increased by about 400% and 300%, respectively. Body weight and muscle mass significantly decreased.
3、 Identification method of IL-6 mouse model
Model validation is a crucial step in ensuring the reliability of experiments and the reproducibility of results. The identification of IL-6 mouse models usually involves multiple dimensions such as gene level confirmation, protein expression detection, disease phenotype evaluation, and histopathological analysis.
1. Molecular biology identification
**Genotyping identification: For genetically engineered mice, the target gene modification must be confirmed through genotype identification after the model construction is completed. Common methods include PCR amplification of target gene sequence and agarose gel electrophoresis analysis to distinguish wild type, heterozygote and homozygote. For the conditional model constructed through the Cre loxP system, it is necessary to verify the expression and activity of Cre recombinase.
**Gene expression level detection: Real time fluorescence quantitative PCR (qRT PCR) is a commonly used method for detecting Il6 mRNA expression levels. Taking the IL-6 KO model as an example, researchers found through liver qPCR detection that IL-6 KO homozygous mice did not detect Il6 mRNA expression before and after CCl ₄ stimulation, while wild-type mice showed significant upregulation of Il6 mRNA after stimulation. For overexpression models, qRT PCR is required to verify the expression level of transgenic genes in the target tissue.
2. Protein level identification
**ELISA detection: Enzyme linked immunosorbent assay is the gold standard method for quantitatively detecting IL-6 protein levels in serum or tissue homogenates. In the LPS induced sepsis model, researchers found through ELISA dynamic monitoring that the serum IL-6 level in mice gradually increased with the progression of sepsis, reaching 2989.5 pg/mL at 16 hours. In the CIA model, ELISA detection showed that the levels of inflammatory factors such as IL-6, IFN - γ, MCP-1 in the serum of model mice were significantly higher than those in the control group.
**Western blot * *: For signal pathway research, Western blot can be used to detect the expression and activation status of downstream signaling molecules of IL-6. For example, in the CIA mouse model, researchers detected the expression level of p-STAT3 protein in joint synovial tissue and found that the model group was significantly higher than the normal group.
**Immunohistochemistry and immunofluorescence: These techniques can be used to detect the in situ expression and distribution of IL-6 and its receptors in specific tissues or cell types. For example, in the identification of rheumatic mouse models, quantitative analysis of CD68 positive cells can serve as an important indicator for evaluating synovitis.
3. Phenotypic feature identification
**Survival and reproductive ability: Classic IL-6 knockout homozygous mice can survive and reproduce normally without obvious underlying phenotypes. However, in certain overexpression models, high levels of IL-6 overexpression may lead to adverse consequences - for example, transgenic mice overexpressing hIL-6 in the macrophage monocyte lineage may exhibit early postpartum mortality, which researchers speculate may be related to the impact of hIL-6 on hematopoietic function.
**Physiological and behavioral indicators: In the TetO-hIL6 humanized frailty model, mice induced to express hIL-6 showed multiple behavioral and physiological changes: significant decrease in grip strength (p=0.003), decreased running ability (p=0.02), 40% increase in treadmill drop rate (p=0.001), and significant decrease in basal body temperature (p<0.001)). these="" phenotypic="" changes="" provide="" direct="" evidence="" for="" the="" pathogenic="" role="" of="" il-6="" in="" age-related="" functional="" decline.="">
**Morphological assessment: In arthritis models, commonly used morphological identification indicators include grading of joint swelling degree (0-4 level scoring system), measurement of paw volume, and arthritis index (AI) score. In the CIA model of subcutaneous injection of CII collagen, the mice in the model group showed redness and swelling of their paws 28 days after the first immunization, and the thickness of paw swelling was significantly higher than that of the control group 35 days later.
4. Histopathological identification
Histopathological analysis is an important step in verifying the phenotype of model diseases. For arthritis models, joint slices need to be stained with hematoxylin eosin (HE) to evaluate the severity of synovitis (on a 0-3 point scale) and the degree of bone erosion (on a 0-5 point scale). Typical pathological changes in CIA include narrowing of the joint cavity, synovial tissue proliferation, extensive infiltration of inflammatory cells, and varying degrees of damage to cartilage structure.
In the sepsis model, researchers need to perform HE staining pathological examination on organs such as the heart, liver, kidneys, lungs, and spleen. Research has found that in the early stages of sepsis, the heart and liver are the first organs to show damage, manifested as pathological changes such as myocardial fiber edema and hepatocyte degeneration and necrosis. In the high-altitude hypoxic inflammatory brain injury model, HE staining showed characteristic changes such as cell swelling, widened intercellular spaces, vascular proliferation, and neuronal shrinkage with nuclear consolidation and deep staining in the cortex and hippocampus.
5. Multi omics analysis
With the development of omics technology, transcriptomics and metabolomics analysis are increasingly being applied for in-depth identification of IL-6 mouse models. In the TetO-hIL6 model, researchers conducted RNAseq analysis on whole blood and found that after 6 weeks of hIL-6 induction, pro-inflammatory markers were significantly upregulated, and cell proliferation and metabolic pathways also underwent significant changes. Metabolomics analysis further showed significant changes in key metabolites such as ATP (reduced by 56%), pyruvic acid (reduced by 35%), alpha ketoglutarate (reduced by 47%), and succinic acid (increased by 306%) in the plasma of model mice. These multi omics data provide unprecedented depth and breadth in understanding the systemic effects of IL-6.
4、 Application and selection strategy of models
Different IL-6 mouse models are suitable for different types of research questions. The IL-6 systemic knockout model is suitable for studying the overall effects of IL-6 deficiency on physiological and pathological processes. However, due to the complexity of IL-6 function and its widespread expression in multiple cell types, complete knockout may lead to compensatory mechanisms, thereby affecting the interpretation of the results. The conditional overexpression or knockout model provides a higher resolution option for studying the function of IL-6 derived from specific cells.
In terms of disease mechanism research, induction models such as CIA and EAE are widely used in the study of autoimmune disease mechanisms and drug screening. The LPS induced sepsis model is suitable for studying the dynamic changes of IL-6 in acute inflammatory response. Humanized IL-6 models (such as TetO-hIL6 and NSG+hIL6 mice) have unique advantages in studying human specific IL-6 function, testing targeted human IL-6 therapeutic drugs, and constructing tumor patient derived xenograft models.
V. Conclusion
After decades of continuous development, the construction methods of IL-6 mouse models have become increasingly mature and diversified. From the initial complete gene knockout model to the current spatially and temporally specific expression regulation system, researchers have an increasingly rich research toolbox. At the same time, model identification methods have evolved from single phenotype observation to a multidimensional evaluation system that integrates molecular biology, histopathology, behavior, and multi omics analysis.
In the face of increasingly refined research demands, the future development direction of IL-6 mouse models will include: further improving the tissue-specific regulatory system to analyze the functional differences of IL-6 from different cell sources; Develop a report mouse that can simultaneously monitor IL-6 signaling activity to track its dynamic changes in vivo in real-time; And expand the application of humanized models in preclinical drug evaluation. These advances will drive the biological research of IL-6 to a deeper level, providing a solid theoretical foundation for precise treatment strategies targeting IL-6.
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