Epigenetic regulation of chemokine (CC‐motif) ligand 2 in inflammatory diseases

Abstract Appropriate responses to inflammation are conducive to pathogen elimination and tissue repair, while uncontrolled inflammatory reactions are likely to result in the damage of tissues. Chemokine (CC‐motif) Ligand 2 (CCL2) is the main chemokine and activator of monocytes, macrophages, and neutrophils. CCL2 played a key role in amplifying and accelerating the inflammatory cascade and is closely related to chronic non‐controllable inflammation (cirrhosis, neuropathic pain, insulin resistance, atherosclerosis, deforming arthritis, ischemic injury, cancer, etc.). The crucial regulatory roles of CCL2 may provide potential targets for the treatment of inflammatory diseases. Therefore, we presented a review of the regulatory mechanisms of CCL2. Gene expression is largely affected by the state of chromatin. Different epigenetic modifications, including DNA methylation, post‐translational modification of histones, histone variants, ATP‐dependent chromatin remodelling, and non‐coding RNA, could affect the ‘open’ or ‘closed’ state of DNA, and then significantly affect the expression of target genes. Since most epigenetic modifications are proven to be reversible, targeting the epigenetic mechanisms of CCL2 is expected to be a promising therapeutic strategy for inflammatory diseases. This review focuses on the epigenetic regulation of CCL2 in inflammatory diseases.

amplifying and accelerating the inflammatory cascade and is closely related to chronic non-controllable inflammation (cirrhosis, neuropathic pain, insulin resistance, atherosclerosis, deforming arthritis, ischemic injury, cancer, etc.). The crucial regulatory roles of CCL2 may provide potential targets for the treatment of inflammatory diseases. Therefore, we presented a review of the regulatory mechanisms of CCL2. Gene expression is largely affected by the state of chromatin. Different epigenetic modifications, including DNA methylation, post-translational modification of histones, histone variants, ATP-dependent chromatin remodelling, and non-coding RNA, could affect the 'open' or 'closed' state of DNA, and then significantly affect the expression of target genes. Since most epigenetic modifications are proven to be reversible, targeting the epigenetic mechanisms of CCL2 is expected to be a promising therapeutic strategy for inflammatory diseases. This review focuses on the epigenetic regulation of CCL2 in inflammatory diseases.

| BACKGROUND
When tissues are suffered from infection or injury, chemokines play a crucial role in the progress of recruiting immune cells into the inflammation area to eliminate pathogens and repair tissues. 1 Chemokines are signalling proteins whose molecular weights were small (8)(9)(10)(11)(12)(13)(14)). 2 Chemokines have been divided into the following four subfamilies according to the sequence characteristics of cysteine residues: the CC, CXC, CX3C and XC. [3][4][5] In inflammatory conditions, the imbalance of chemokines was proven to be involved in the pathogenesis of a variety of inflammation diseases, such as cardiovascular diseases, arthritis, neuropathic pain, cancer, and so on. 2,6,7 This disorder Yingyi Chen and Siyan Liu contributed equally to this work. of chemokines could lead to abnormalities in the process of tissue repair, such as delayed wound healing or excessive fibrosis, which was related to various diseases, including diabetic foot, kidney fibrosis, cirrhosis, and so on. [8][9][10] These uncontrolled inflammatory states caused by the disorder of chemokines are still a difficult problem in clinical treatment.
CC-chemokine group is the largest subfamily of chemokines and is characterized by two adjacent cysteines at the amino terminus of the protein. 11,12 Among them, CC-chemokine ligand 2 (CCL2, also known as monocyte chemokine protein-1, MCP-1) is the first chemokine to be purified and extensively studied. It is the main chemokine and activator of monocytes, macrophages, and neutrophils. 13 Under normal circumstances, appropriate responses of inflammation are conducive to pathogen elimination and tissue repair, while excessive inflammation responses are likely to result in the damage of tissues. 14 After that, cells in the injury site would express CCL2 to recruit and activate immune cells in the inflammatory area, while the infiltrated inflammatory cells could further secrete CCL2 to amplify and accelerate the inflammatory cascade. 15,16 In recent years, studies have reported that CCL2 is closely related to chronic non-controllable inflammation, such as cirrhosis, neuropathic pain, insulin resistance, atherosclerosis, deforming arthritis, ischemic injury, cancer, and so on. [17][18][19][20][21][22][23][24][25] Given the important regulatory role of chemokines in inflammation, an in-depth understanding of the regulatory mechanism of CCL2 is conducive to providing new therapeutic strategies for inflammatory diseases.
In recent years, many studies have explored the regulatory mechanism of CCL2 expression. Specifically, oxidative stress injury and virus infection could up-regulate the expression of CCL2 via NF-ĸB signalling pathways and lead to excessive fibrosis or inflammatory cascade. 26,27 In the tumour immune microenvironment, proteasome activation factor PA28γ could activate the NF-ĸB signalling pathway of oral squamous carcinoma (OSCC) cells and enhance the expression of CCL2, which promote tumour angiogenesis and reduce the survival rate. 28 In addition, HMGA2, a tumour derivative, could up-regulate the gene transcription of CCL2 in tumour-associated macrophages (TAMs) by regulating the STAT3 signalling pathway. In the state of CCL2 disorder, macrophage recruitment into the microenvironment of tumour and result in distant metastasis and reduced survival rate. 29 Moreover, in desmoplasia-associated cancers, fibroblast activation protein (FAP) could up-regulate the expression of CCL2 through STAT3 signalling pathway, which enhances the recruitment of myeloid-derived suppressor cells and promotes proliferation of tumour matrix and leads to poor prognosis. 30 From these evidences, activation of multiple signalling pathways in inflammatory diseases, such as oxidative stress, infection, or tumour, ultimately leads to transcription factors transporting into the nucleus and enhancing the gene transcription of CCL2. However, chromatin is made up of DNA, RNA, and histones in a tightly coiled structure. This creates a physical barrier to gene transcription. 31,32 Therefore, it is indispensable to investigate the accessibility of chromatin while studying the regulatory mechanism of CCL2.
In a recent couple of years, the regulatory roles of epigenetics on a variety of inflammatory diseases such as pro-neoplastic inflammation, neuropathic pain, pulpitis, and periodontitis have gradually attracted attention. [33][34][35] Epigenetics is defined as the heritable changes in gene functions that ultimately lead to phenotypic changes without changes in the DNA sequence. 36  (H3K27me3). 38 In inflammatory diseases, the activating histone modifications of CCL2 include H3K27Ac, H3K4me2/3, H3Ser10 Phosphorylation; inhibitory histone modifications include H3K27me3, H3K9me3, and so on ( Figure 1). 41,[52][53][54] Methyltransferase METTL3 in non-alcoholic steatohepatitis (NASH) could induce the deacetylation of H3K27 and H3K9 by recruiting HDAC1/2 on the CCL2 promoter, thus inhibiting the expression of CCL2 and inhibiting the progression of NASH. 55 Consistently, in the case of obesity, CCL2 expression is up-regulated when H3K27Ac enrichment is occupied on the CCL2 promoter of macrophages. In addition, Kiguchi et al. 56  Importantly, Aoi et al. 78 found that under hypoxia conditions, the levels of DNA methylation and histone H3K9 dimethylation in CCL2 enhancer and promoter region were significantly increased and jointly inhibited the expression of IL-1β-induced MCP-1 under hypoxia conditions. This discovery revealed the epigenetic mechanism of the inhibition regulation of CCL2 under hypoxia, as well as the functional correlation between DNA methylation and histone methylation in gene silencing. The close relationship between them has also been confirmed by many studies. 79,80 Some researchers believed that there might be a mutually reinforcing and self-circulating epigenetic network between DNA methylation, deacetylation and methylation of H3K9, ultimately leading to long-term inhibition of gene expression. 81 In addition, it is important to note that although DNA methylation is usually associated with gene suppression, it may also play an activation role of gene transcription. Petrus et al. 61

| METABOLIC-EPIGENOMICS REGULATION OF CCL2
In the process of inflammation, immune cells would undergo metabolize reprogramming under the challenge of pathogenic microorganisms and inflammation. 96 Cell metabolism has a significant impact on the functions of immune cells. For example, in the process of macrophage polarization or T cell activation, to deal with the biosynthesis needs of macromolecular, glucose metabolism would change from oxidative phosphonic acidification (efficient but relatively slow) to glycolysis (rapid energy supply). 25,97 In the process of metabolic reprogramming, cells metabolite, such as acetyl CoA, ATP, lactic acid, serine, SAM, and succinic acid, were reported to be closely related to a variety of inflammatory pathways. 92,97-100 These metabolites could act as substrates for the related enzymes of epigenetic modification, such as histone acetylation, histone methylation, histone lactate, DNA methylation, and ATP-mediated chromatin remodelling. 101,102 This crosstalk between metabolic reprogramming and epigenetic modification could affect the 'open' state of chromatin, to allow immune cells to accurately respond to the specific immune environment and make corresponding changes in function. In recent years, researchers have suggested that the integrated analysis of metabolism and epigenetics could be considered a new field: metabolo-epigenomics. 96 Interestingly, crosstalk between metabolism and epigenetics also plays an important role in CCL2 regulation in inflammatory environments. In the inflammatory state, immune cells undergo metabolic reprogramming, and multiple metabolic pathways, such as glycolysis and 'de novo ATP generation', significantly increase the generation of intracellular ATP. 98,99 Increased ATP will promote site-specific 'ATPdependent chromatin remodeling' which consumes the energy of ATP hydrolysis to loosen the histone contact with DNA and thus promote transcription factor binding. In acute kidney injury, Brahma-related gene 1 can continuously increase the binding of RNA polymerase II to the CCL2 gene by mediating chromatin remodelling, thus promoting hypoxic-stress-induced kidney proximal tubule CCL2 expression. 103 Besides, in recent years, it has been found that LPS-induced SAM production in the one-carbon metabolism of macrophages significantly increased and then altered the methylation status of DNA and histone of target genes. 75

| CONCLUSION
Inflammation is a defensive response toward tissue injury, which plays an important role in maintaining tissue homeostasis. 105 However, under the regulation of chemokines such as CCL2, a large number of immune cells gather at the inflammatory site, which leads to the activation of the chemoattractant-cytokine network, resulting in the amplification and persistence of the inflammatory response. 14 Due to its important role in the inflammatory cascade, CCL2 has become a therapeutic target for various inflammatory diseases such as insulin F I G U R E 4 Metabolic-epigenomics regulation of CCL2. Created with BioRender.com. resistance, atherosclerosis, ischemic injury, and cancer. In recent years, more and more studies have reported the regulation of CCL2 mediated by epigenetic mechanisms, such as histone posttranscriptional modifications (e.g., H3K27ac, H3K27me3), DNA methylation and ncRNA, and the interaction between epigenetic regulation and metabolic state of cells has been widely discussed.
Therefore, reversible epigenetic changes may become a new idea for the treatment of inflammatory diseases. However, there are still many problems that need to be solved in epigenetic regulation. First, the cell type specificity of epigenetic modification, that is, the same epigenetic change acting on different cells may cause different effects on the target genes. Secondly, there may be a mutually reinforcing and self-circulating complex epigenetic regulatory network between different epigenetic mechanisms. However, the role of this complex epigenetic network in inflammatory diseases remains to be further explored. A deeper exploration of epigenetic regulation is conducive to a more thorough understanding of inflammatory diseases, so as to provide a theoretical basis for the development of new targeted therapeutic drugs.