New Posttranslational Modification Lactylation Brings New Inspiration for the Treatment of Rheumatoid Arthritis.

LA mainly exists in the body as two enantiomers, namely L-LA and D-LA. Although L-LA is a frequently occurring byproduct of the body’s metabolic processes, D-LA is only created in small amounts by some microbes. 39 In humans, L-LA primarily arises from converting glucose and alanine into pyruvate. 40 Glucose undergoes glycolysis and the pentose phosphate pathway to produce pyruvate. Lactate dehydrogenase (LDH) catalyses the reduction of pyruvate to L-LA. 41 LA serves as both a fuel for cells and perhaps contributes to the development of certain diseases, as well as the regulation of cell activity. 42 , 43 The potential of LA as a signalling molecule was largely overlooked until 2008, when a groundbreaking report emerged to enhance our understanding of LA-mediated signal transduction. Ge et al were the pioneers in discovering GPR81, a G protein-coupled receptor (GPCR) that interacts explicitly with LA and transmits signals into cells. 44 When GPR81 is combined with LA, α-subunits with Guanosine triphosphate-Guanosine diphosphate (GTP-GDP) immediately switch energy structure transformation occurs, which will affect the downstream signalling molecules, such as cyclic adenosine monophosphate (cAMP) and Ca 2+ . 45 Hypoxia is a fundamental metabolic change in many inflammatory diseases, and the anaerobic glycolysis pathway must provide an adenosine triphosphate (ATP) supply to sustain the energy needed for rapid cell growth. This statement aligns with previous research investigations on RA that have reported increased amounts of LA and reduced glucose in the synovial fluid. 46–48 According to reports, heightened glycolytic activity and elevated levels of LA and pyruvate contribute to the development of angiogenesis and pannus production. 49 These processes ultimately lead to the destruction of joint tissue structure in RA. LA also enhances activation and movement while promoting the growth of proinflammatory cytokines like TNF and IL, which are released by immune cells. TNF and IL-1 are the main inflammatory factors involved in RA. LA enhances the process of activating the transcription of TNF and IL-1 by increasing the transcriptional activity of NF-κB. Furthermore, LA plays a crucial function in relation to myeloid differentiation factor-2 (MD-2), the stimulation of the Toll-like receptor 4 (TLR4) community, and the activation of inflammatory genes in human U937 tissue cells. 50 Moreover, LA can be especially involved in the onset and evolution of RA by controlling the differentiation of T cells, macrophages, B cells, mesonuclear cells, and other cells and the secretion of inflammatory factors. 51 At present, many monocarboxylate transporters (MCT) inhibitors, including bindarit, 52 phloretin, 53 and other drugs regulating LDH activity such as FX11, 54 GSK2837808A 55 have been developed to treat tumour and arthritis by regulating LA. Research has demonstrated that these medications play a beneficial function in the management and enhancement of RA by regulating the transit and buildup of LA.

PTM refers to adding biochemical components to most proteins during or after protein translation. This process is crucial for regulating protein structure, localisation, and function and is characterised by its ability to change and be reversed. PTMs add a unique level of complexity to the proteome that is not dependent on DNA. They act as continuously fine-tuned regulators in various cellular processes. 17 PTMs such as citrullination, carbamylation, acetylation, ubiquitination, and methylation have been associated with RA development. 18 As is well-known, citrullinated proteins are critical in the pathogenesis of RA. Studies have found that citrulline-specific helper T cell 1 (TH1) and TH17 cells are increased in patients with RA. 19 , 20 The synovial biopsy samples from people with RA have shown a considerably more significant amount of citrullinated protein compared to the synovium of healthy individuals. 21 Anti-carbamylated proteins (anti-CarP) are specific indicators for the diagnosis of RA, and their production is related to the carbamylation of Homocitrulline. 22 , 23 Autophagy was also found to correlate with carbamylation levels in mononuclear cells from treatment-naïve patients with RA. 24 In addition, it has been found that the dysregulation of acetylation can hinder the progression of RA by decreasing the expression of forkhead box protein P3 (FOXP3), 25 and histone acetylation shows cross-reaction with ACPA in seronegative patients with RA. 26 In addition, ubiquitination can also affect the signalling of signalling factors such as tumour necrosis factor (TNF) receptor-associated factor-2 (TRAF2), TRAF6, which are directly or indirectly involved in pro-inflammatory activity and inflammation, thus aggravating the symptoms of RA. 27 Methylation modifications, including DNA and RNA methylation, are also associated with RA development. Global DNA hypomethylation has been observed in peripheral blood mononuclear cells (PBMCs) and FLS of individuals with RA. 28 The hypomethylated sites are detected in crucial genes associated with RA, such as signal transducer and activator of transcription 3 (STAT3). 29 RNA methylation has been implicated in the pathogenesis of RA in macrophages and FLS. It was found that activation of lipopolysaccharide (LPS) can increase the expression of methyltransferase-like 3 (METTL3) in macrophages and increase the total amount of N6-methyladenosine (m6A). 30 METTL3 can enhance the inflammatory response of RA FLS by stimulating the nuclear factor-κB (NF-κB) signaling pathway. 31 Drugs for PTMs, such as proteasomal inhibitors, are also being developed and tested in clinical trials. The proteasomal inhibitor has the ability to exert its effects at several levels, suppressing transforming growth factor-β (TGF-β)-activated kinase 1 (TAK1) and TRAF6 signalling pathways, possibly rendering cancerous cells prone to being affected by chemotherapy and programmed cell death. They are also thought to be anti-inflammatory agents, having, under normal conditions, been shown to block IL, cell-sticky molecules, and enzymes. 32–34 Bortezomib is a proteasomal inhibitor extensively studied in pre-clinical trials. It effectively inhibits the 26S proteasome, resulting in reduced cell proliferation and the induction of chemotherapy. Bortezomib is commonly used in the treatment of malignancies and arthritis. It is authorised by the Food and Drug Administration (FDA), effectively hinders IĸB kinase and aids in preventing the breakdown of NF-κB. This action is crucial in managing inflammatory illnesses. 35–37 Moreover, Bortezomib has been reported that it can improve joint performance for RA. 38 Evidently, many types of PTM have been found to have specific functions in the onset and progression of RA, so does the newly emerged PTM lactylation mediated by the metabolite LA also play a particular role in RA? Continuing to explore this will help to clarify the aetiology of RA and the investigation of novel pharmaceuticals, and it is believed that the exploration of the related mechanism in the lactylation will also bring new enlightenment for RA.

Rheumatoid arthritis (RA) is a persistent and inflammatory joint disease caused by the immune system attacking immunoglobulin G (IgG) and citrullinated proteins. These attacks are identified by the presence of autoantibodies known as rheumatoid factor (RF) and anti-citrullinated protein antibodies (ACPAs). The development of RA is influenced by genetic, epigenetic, and environmental factors. 1 The majority of epidemiological investigations on RA have been conducted in Western countries. These studies have revealed a prevalence of RA ranging from 0.5% to 1.0% among individuals of white ethnicity. 2 , 3 If not addressed, these conditions can manifest as small areas of tissue death, the sticking together of granulation tissue, and the formation of fibrous tissue on the joint’s surface. This can result in the gradual stiffening of the joint, its deterioration, deformities, and functional impairment. 4 Furthermore, at the advanced stage of RA, there is a heightened risk of developing respiratory, circulatory, and tumour-related illnesses, which significantly increases the likelihood of mortality. RA’s prevalence and significant ramifications have garnered considerable interest, leading to a growing study on the subject. 5 Currently, the precise cause of RA is unknown. Although numerous drugs, such as nonsteroidal anti-inflammatory drugs (NSAIDs), corticosteroids, and disease-modifying antirheumatic drugs (DMARDs), have been used, it remains undeniable that a significant number of patients may encounter adverse reactions and treatment failure, including nonresponse and limited effectiveness. 6 , 7 Therefore, it is necessary to continue exploring RA’s critical pathogenesis. Figure 1 The process of lactylation. As a substrate for lactylation, the metabolite LA can enter the nucleus to bind to histone lysine residues or bind to non-histone lysine residues in vivo to form protein modifications. In turn, it regulates gene expression and affects cellular activity. Figure 2 Network of relationships among PTMs, lactylation, LA, and RA. A variety of PTMs are related to the occurrence and development of RA. As a metabolite of glycolysis, LA exists in the synovium of the hypoxic microenvironment, which affects the progression of RA in many ways. LA accumulation was found to cause protein lactylation modification, a type of PTM, and may affect RA through critical cells and essential mechanisms of RA. Figure 3 Lactylation and critical cells and essential mechanisms of RA. (1) Lactylation modification can promote the process of converting pro-inflammatory M1 macrophages into anti-inflammatory M2 macrophages and promote the expression of M2-like genes Arg1 and Klf4. Moreover, it can inhibit the pyroptosis and antigen presentation of macrophages. (2) On the T cell side, lactylation reprograms proinflammatory Th17 cells into regulatory T cells and may additionally attenuate CD8+ T cell effector function. (3) Lactylation can promote osteogenic differentiation and enhance the expression of osteogenic genes. (4) Lactylation can enhance the expression of pro-angiogenic factors such as FGF2 and promote angiogenesis in retina and prostate cancer. (5) Lactylation can promote the proliferation, invasion and migration of human and rat FLS, endometrial stromal cells, human renal cell carcinoma cell lines, and human non-small cell lung cancer cells. (6) Lactylation can also regulate fibrosis genes such as PDGFA, affecting pulmonary, liver, placental, and heart fibrosis. The process of lactylation. As a substrate for lactylation, the metabolite LA can enter the nucleus to bind to histone lysine residues or bind to non-histone lysine residues in vivo to form protein modifications. In turn, it regulates gene expression and affects cellular activity. Network of relationships among PTMs, lactylation, LA, and RA. A variety of PTMs are related to the occurrence and development of RA. As a metabolite of glycolysis, LA exists in the synovium of the hypoxic microenvironment, which affects the progression of RA in many ways. LA accumulation was found to cause protein lactylation modification, a type of PTM, and may affect RA through critical cells and essential mechanisms of RA. Lactylation and critical cells and essential mechanisms of RA. (1) Lactylation modification can promote the process of converting pro-inflammatory M1 macrophages into anti-inflammatory M2 macrophages and promote the expression of M2-like genes Arg1 and Klf4. Moreover, it can inhibit the pyroptosis and antigen presentation of macrophages. (2) On the T cell side, lactylation reprograms proinflammatory Th17 cells into regulatory T cells and may additionally attenuate CD8+ T cell effector function. (3) Lactylation can promote osteogenic differentiation and enhance the expression of osteogenic genes. (4) Lactylation can enhance the expression of pro-angiogenic factors such as FGF2 and promote angiogenesis in retina and prostate cancer. (5) Lactylation can promote the proliferation, invasion and migration of human and rat FLS, endometrial stromal cells, human renal cell carcinoma cell lines, and human non-small cell lung cancer cells. (6) Lactylation can also regulate fibrosis genes such as PDGFA, affecting pulmonary, liver, placental, and heart fibrosis. Recent studies have demonstrated that epigenetic regulators, including histone modifications, methylation of DNA and RNA, and non-coding RNAs, are crucial in the development of RA. 8 Moreover, current research has discovered that certain metabolites within the body serve as signalling molecules in the regulation of genes. One method by which these metabolites communicate is by the chemical alteration of proteins, such as histones. It is classified as a posttranslational modification (PTM). Zhang et al have identified a new histone modification called lactylation, produced from the biological metabolite lactic acid (LA). 9 , 10 Moreover, in the synovial fluid of patients with RA, high levels of LA accumulate due to the high metabolic demand of synovial cells, making it a reliable indicator for differentiating inflammatory arthritis. 11 , 12 In turn, the LA accumulated in RA synovium acts on fibroblast-like synoviocytes (FLSs), macrophages, T cells, and other cells, becoming an amplifier of inflammation. 13 Studies also showed that LA can activate hypoxia-inducible factor-1α (HIF-1α) in bovine FLSs through the PI3K/Akt/ NF-κB signaling pathway to enhance the gene expression of pro-inflammatory factor interleukin-6 (IL-6). 11 On the other hand, With the discovery of lactylation, more and more studies also have found that lactylation may play a key role in cancer, autoimmune diseases, and other diseases by regulating gene expression in various cells, such as macrophages and T cells. 14 First discovered in macrophages, lactylation may induce macrophage M2 signature by initiating the expression of homeostasis genes. 10 The latest single-cell sequencing study found that RA lactylation score was significantly increased in patients with RA, which was positively correlated with immune cell infiltration and immune checkpoint molecular expression. The study also confirmed that core lactylation-promoting genes NDUFB3, NGLY1 and SLC25A4 were highly expressed in RA. It can be used as a potential biomarker for RA. 15 It has also been found that artemisinin can enhance the lactylation modification of pyruvate kinase M2 (PKM2) in FLSs. Moreover, this effect further promotes the binding of modifying enzyme of lactylation p300, induces cell cycle arrest in the S phase, regulates the cell cycle, and ultimately inhibits RA-mediated synovial hyperplasia and inflammation. 16 In view of the critical mechanism of LA and lactylation, in this paper, we review whether lactylation can be a potential therapeutic target for RA through the research on lactylation in RA-related cells and mechanisms and provide novel perspectives on RA development, identification, and management.

Lactylation not only has a profound influence on the pathogenesis associated with RA but also perhaps plays a key role in RA complications. Up to 6.8% of deaths in women and 9.8% of deaths in men with RA are caused by interstitial lung disease (ILD), one of the most prevalent and potentially severe extra-articular features of RA. ILD typically presents as a radiographic and pathological pattern of generalised interstitial pneumonitis, which is similar to IPF in terms of its pathogenesis and disease behaviour. At the same time, pulmonary fibrosis is a typical pathological feature of a large group of ILD that can lead to the destruction of normal lung structure, lung scarring, and eventual organ failure. 109 Lung macrophages and lung fibroblasts are important in the local inflammatory response to pulmonary fibrosis. In the fibrotic microenvironment of lung macrophages, TGF-β-induced lung fibroblasts and bronchoalveolar lavage fluid (BALF) from TGF-β- or bleomycin-induced pulmonary fibrosis mice showed a significant increase in LA content and promoted the lung fibrosis genes PDGFA, thrombospondin 1 (THBS1) and vascular endothelial growth factor A (VEGFA) by upregulating p300-mediated macrophage histone lactylation modification. 110 Arsenic exposure induced elevated levels of LA in the microenvironment of lung fibrosis tissues, and alveolar epithelial cells further promoted elevated levels of histone H3K18 lactylation through LA uptake, which activated the neuronal regeneration related protein (NREP)/TGF-β1 signalling pathway, stimulated further activation of lung fibroblasts differentiation, and promoted the disease progression of arsenic-induced pulmonary fibrosis. 111 Li et al found that PM2.5 exposure significantly increased LDH levels in murine LDH activity and LA content in lung macrophages and increased the expression of pro-fibrotic mediators by triggering glycolysis in macrophages to induce histone lactylation at the macrophage gene promoter as well as the secretion of pro-fibrotic cytokines. 112 These findings further illuminate the pathophysiology of lactylation’s crucial role in the aetiology of pulmonary fibrosis. Meanwhile, histone lactylation is vital in promoting hepatic, placental, and cardiac fibrosis. The induction of liver fibrosis is believed to be largely dependent on the activation and phenotypic modification of quiescent hepatic stellate cells (HSC). It was shown that hexokinase 2 expression boosts glycolytic activity and produces a significant amount of LA, a substrate for histone lactylation, which mediates the activation of H3K18 lactylation to change the active phenotype of HSC and encourages the onset of hepatic fibrosis. 113 Zhou et al discovered that to increase HSC activation and liver fibrosis, the m6A reader IGF2BP2 modulates histone lactylation and controls glycolytic metabolism. 114 In preeclamptic placentas, hypoxia increases the expression of pro-placental fibrosis factors FN 1 and SERPINE 1 through LA-induced histone lactylation. 115 Another study found that after myocardial infarction, LA was able to induce lactylation and nuclear translocation of Snail 1, which promoted endothelial-to-mesenchymal transition, increasing cardiac fibrosis through activation of the TGF-β/Smad 2 signalling pathway. 116 A national study conducted in Denmark noted that 14% of RA-ILD cases were diagnosed with lung disease 1–5 years before RA diagnosis and elucidated that lung inflammation plays a central role in RA disease pathogenesis. 117 Biomarkers for screening RA-ILD are not currently available in clinical practice, and their treatment criteria have not been standardised. The treatment of RA is a double-edged sword. On the one hand, it can alleviate the progression of RA-ILD. Still, at the same time, it also has the drawbacks of inducing pulmonary toxicity and increasing susceptibility to infection. Investigating the possible function of lactylation in various physiological and pathological processes may offer a novel approach for the diagnosis and treatment of RA-ILD, considering the importance of lactylation in fibrosis of the lung and other diseases’ fibrotic pathology (lactylation and critical cells and essential mechanisms of RA are summarized in Figure 3 ).

Although we have yielded new findings about lactylation, we must acknowledge that there is still much to learn about this subject and that much research on lactylation has not been fully explained. According to Zhang and associates, histone lactylation is a response to levels of LA and is brought on by the accumulation of LA. 10 However, it is unknown if histone lactylation results from LA analogues or is an inevitable consequence of LA buildup. Whether LA concentrations in the nucleus cause histone lactylation is also uncertain. Moreover, palmitoylation takes place at threonine residues and succinylation at cysteine residues. 118 Whether lactylation happens at residues of amino acids other than lysine is also unknown. At the same time, the lactylation detection of the modification and the low abundance of lactylated peptides are likely to be overshadowed by the undecorated peptide mass in mass spectrometry. However, it is believed that future use of diagnostic cycIm ions in MS/MS spectroscopy, as well as the use of more publicly accessible datasets, will help to identify relevant lactylation events. 119 In terms of epigenetic tools, enzymes that read, write, and erase lactylation are not fully revealed. More evidence has confirmed that p300 is a potential writer of lactylation. In p300-knockdown macrophages, both histone lactylation and profibrotic gene expression were reduced. 110 In HEK293T cells, a modest increase in lactylation was observed after p300 overexpression. 10 In addition to p300, other proteins have been found to be involved in writing. Through the silent p300/CBP, HMGB1 lactylation was weakened. 60 In erasing lactylation modification, Zu et al reported sirtuin 2 (SIRT2) as a potent histone lactylation eraser in vitro. 120 In addition, histone deacetylase 1–3 (HDAC1-3) and SIRT1-3 were identified as powerful erasers for lactylation in vitro. 121 Through overexpression and knockdown analysis, HDAC1 and HDAC3 were proved to have distinct functions in histone lactylation. Subsequently, HDAC2, HDAC3, HDAC8, SIRT2, and SIRT3 were also regarded as potential lactylation erasers. 122 At present, no study has revealed a reading of lactylation. Proteomic analysis of H3K18 lactylation immunoprecipitation assay by Hu et al revealed specific brahma-related gene 1 (Brg1) recruitment during reprogramming, with both H3K18 lactylation and Brg1 enriched at the promoter of genes involved in pluripotency and epithelial junction. The binding of Brg1 to H3K18 lactylation was revealed for the first time, suggesting that it may be a reader of histone lactylation. 123 Epigenetic tool enzymes are crucial in the process of lactylation. Given that the research on critical enzymes has made much progress in the acetylation-targeted therapy of tumours, 124 identifying the critical enzymes of lactylation may also provide new ideas for RA-targeted therapy. Lactylation modification acts on the upstream and downstream of the mechanism and interacts with other PTMs. Gu et al found that histone lactylation enhances alpha-ketoglutarate-dependent dioxygenase homolog 3 (ALKBH3) expression and simultaneously attenuates the formation of tumour-suppressive promyelocytic leukaemia protein (PML) condensates by removing the m1A methylation of SP100A, promoting the malignant transformation of cancers. 125 Moreover, scientists have discovered that increased doses of LA can induce the acetylation of additional lysine sites. Through the suppression of SIRT1, a known “eraser” accountable for acetylation, this process is promoted. 126 Later studies on murine BMDMs revealed that crucial phases in the LA-driven process include the phosphorylation and subsequent degradation of YAP and LATS1, upstream molecules of SIRT1. 60 It can be seen that lactylation modification is complex in the process of protein modification, and the research on it is more hopeful of bringing more updates and breakthroughs in epigenetic crosstalk. Through existing studies, it can be found that a variety of cells, including macrophages and a variety of disease-related pathological mechanisms, can be widely affected by lactylation, and these cells and mechanisms are also closely related to the occurrence and development of RA. At the same time, the mediator of lactylation, LA, also plays a promoting role in RA, revealing the possibility of lactylation in RA. It provides new ideas for researchers. Numerous medications that target histone PTMs have been used in clinical settings to treat a range of illnesses and have shown promising results. For example, medicines that increase histone crotonylation, decrease histone deacetylation by inhibiting HDACs and decrease histone methylation by inhibiting histone methyltransferases are useful for renal injury. 127 Therefore, studying the specific process of lactylation to find accurate targets is expected to become a promising treatment for RA. Shortly, more research will eventually solve the mysteries of lactylation, usher in updates in the field of RA and even more other diseases, and then be applied to clinical, bringing new hope to more patients with diseases such as RA.

One of the several pathways contributing to the pathophysiology of RA is pathological angiogenesis. Synovial tissues of patients with RA stimulated by chronic inflammation release various pro-angiogenic factors, which stimulate vascular endothelial cells to proliferate, differentiate, migrate, and promote neoangiogenesis. Neovascularisation promotes leukocyte recruitment and synovial tissue proliferation by increasing vascular permeability and more oxygen and nutrient supply. Subsequently, abnormally proliferating synoviocytes, inflammatory cells, and neovascularisation constitute an aggressive vascular opacification, which adheres to the surface of the articular cartilage, causing irreversible damage and ultimately leading to joint destruction and dysfunction. 94 In the early stage of RA, energy metabolism is characterised by aerobic glycolysis, 95 and several studies have shown that LA, as the end product of aerobic glycolysis, is closely related to neovascularisation. Tumour-derived LA promotes endothelial cell activation and angiogenesis. 96 In the retina, LA upregulates vascular endothelial growth factor expression in macrophages and promotes choroidal neovascularisation. 97 Bao et al found that both endogenous and exogenous nitric oxide (NO) induced by immune activation can induce joint angiogenesis in adjuvant-induced arthritis (AIA) rats by driving synovial hypoxia, in which the levels of NO and LA in the serum of arthritis model mice fluctuated proportionally. 98 LA produced by the endogenous glycolytic pathway is a crucial regulator of histone lactylation modification, and when LA produced by glycolysis increases, lysine lactylation also increases. It has been shown that HIF-1α lactylation mediated by MCT1 under normoxic conditions promotes the transcriptional activity of KIAA 1199, which enhances angiogenesis in prostate cancer. 99 Wuchererine impairs HIF-1α histone lactylation to inhibit Semaphorin 3A-mediated angiogenesis by inducing iron apoptosis in prostate cancer. 100 And in a study of the mechanism of retinal vascular neogenesis, the up-regulation of p300 expression led to elevated microglia YY1 lactylation modification, which in turn enhanced the transcription and expression of fibroblast growth factor 2 (FGF2), thereby promoting angiogenesis. 101 In another study, LA was found to exacerbate microvascular abnormalities in diabetic retinopathy by promoting lactylation of the histone H3K18 locus to drive the expression of the obesity-associated protein FTO. 102 Therefore, it is possible that lactylation is an important mechanism involved in angiogenesis in RA. Inhibition of neovascularisation by hindering lactylation may be effective in curbing the development of synovitis and ameliorating the arthritic symptoms of RA.