The complement system, an essential component of innate immune response, consists of approximately 60 proteins that work in concert to eliminate pathogens and regulate immune responses. Complement activation occurs through three principal pathways: classical, lectin, and alternative, all converging to form the membrane attack complex, which induces cell lysis. Beyond its conventional role in pathogen defense, the complement system is integral to inflammation, immune complex clearance, and the modulation of adaptive immunity. In liver diseases, its influence extends further, with growing evidence suggesting that complement components function as immunometabolic factors that fuel MASLD progression. Population studies reveal that circulating complement components rise in tandem with MASLD prevalence and severity, with overactivation implicated in hepatic steatosis, inflammation, fibrosis, and cirrhosis [8]. Given that the liver is the primary site for the synthesis of most complement proteins [9], hepatic dysfunc-tion profoundly affects complement activity. As evidence supporting the complement system’s pivotal role in MASLD development and progression grows, targeted studies are imperative to unravel the ways that complement pathway activation and immune cell interactions drive MASLD pathophysiology.

Another burgeoning area of MASLD research is in epigenetics, a fundamental aspect of biological regulation, enabling organisms to modulate gene expression and adapt to environmental stimuli without altering the primary DNA sequence. Central to this regulation is DNA methylation, which fine-tunes gene expression, facilitating organismal adaptation, establishment of cellular identity, and proper development. Methylation, particularly within CpG islands, can obstruct transcription factor binding, recruit repressive methyl-binding proteins, and effectively silence gene expression [10]. This modification serves as a critical link between cellular processes and the genetic code, driving long-term changes in cellular function and metabolic pathways. DNA methylation is also a powerful predictor, providing accurate estimates of biological age and life expectancy across various cells, including blood and organs [11]. Notably, obesity accelerates epigenetic aging in the liver [12], indicating that DNA methylation mediates some of the complex interactions between cardiometabolic factors and hepatic dysfunction.

The exploration of DNA methylation as a driving force in MASLD development and progression, as well as a disease biomarker, has a long history [13]. Our research [14] and studies by others [15] have identified altered hepatic DNA methylation patterns in individuals with MASLD. Similar alterations have been observed in blood methylation profiles [13]. These profiles offer valuable mechanistic insights. For example, reductions in epithelial cell estimates, alongside elevated immune cell estimates, correlate with advancing fibrosis in individuals with MASLD, suggesting that these cellular changes may partly explain the variations in DNA methylation observed across different stages of fibrosis [14]. The role of the methylome in mediating hepatocellular injury and progressive pathology in MASLD remains an exciting research area. Despite its potential, targeted investigations into the intersections of DNA methylation, hepatic dysfunction, and specific biological systems—such as the complement system—remain limited.

In this issue, Magdy et al. [16] report the results of an extensive investigation of the DNA methylome of complement system genes in relation to MASLD, leveraging diverse data sources. The authors utilized DNA methylation profiles from liver biopsy specimens from 106 individuals with accompanying clinical and histological data, RNA sequencing (RNA-seq) data from a separate cohort of 92 individuals, single nucleus RNA-seq (snRNA-seq) data from the Gene Expression Omnibus archive involving 47 individuals at various stages of MASLD progression, and gene expression and methylation data from a mouse model of MASH. The initial methylation analysis revealed 277,484 differentially methylated positions (DMPs), with 106,116 hypermethylated and 171,368 hypomethylated CpGs in MASLD patients compared to controls. Focusing on 277 DMPs within 61 selected complement genes, they discovered 14 genes had no DMPs, 16 were exclusively hypermethylated, seven were only hypomethylated, and 24 exhibited both types of methylation. Notably, fibrosis severity was associated with increasing hyper- or hypomethylation across these complement genes.

RNA-seq analysis showed that 143 of the 277 DMPs inversely correlated with complement gene expression, while 35 showed a positive correlation. The latter were linked to heterochromatin regions and lower transcriptional activity, whereas inversely correlated DMPs were prominent in MASH samples and localized to active promoter regions and strong enhancers in hepatocytes. The most significant DMPs included six hypermethylated genes (C1R, C1S, C3, C6, C4BPA, SERPING1) and three hypomethylated genes (C5AR1, C7, CD59). These methylation patterns were strongly associated with MASLD histological severity and were validated in an independent cohort of predominantly European ancestry.

Further examination of gene expression in MASLD revealed that C1R, C1S, C3, C4BPA, and SERPING1 were significantly downregulated in MASH, with C6 showing decreased expression in MASL, with an even greater reduc-tion in MASH. In contrast, C5AR1, C7, and CD59 were upregulated in MASH. These expression trends correlated with liver steatosis, lobular inflammation, and ballooning, suggesting that methylation of complement genes is primarily influenced by MASH status, while gene expression is largely affected by methylation status.

SnRNA-seq analysis highlighted the highest expression of complement genes in hepatocytes, prompting a focused differential expression analysis in these cells. Across all disease stages, the six complement genes showed differential expression, with significant downregulation in endstage disease, correlating with disease progression. Specifically, C1R, C3, C1S, SERPING1, and C4BPA were markedly downregulated in end-stage hepatocytes, consistent with patterns seen in lobular inflammation, ballooning, and fibrosis.

The study also explored zonation patterns of complement gene expression in hepatocytes, utilizing zonal subpopulations defined by the differential regulation of certain genes. In healthy liver tissue, complement genes showed similar expression levels in periportal and pericentral populations. However, in MASLD, these patterns differed significantly, with substantial downregulation of the six complement genes in central-zone hepatocytes, especially in end-stage disease.

In a complementary mouse model study, C57Bl6 mice fed a MASH diet supplemented with sucrose exhibited significant hypermethylation and downregulation of C1ra, C3, C4bpa, and Serping1, alongside hypomethylation and upregulation of C5ar1 in liver, mirroring the findings in human liver samples.

Collectively, these results provide compelling evidence of epigenetic alterations in complement genes linked to MASLD progression. Integrative analysis of DNA methylome and transcriptome data from Korean patients with MASLD revealed consistent patterns of hypermethylation and downregulation of C1R, C1S, C3, C6, C4BPA, and SERPING1, as well as hypomethylation and upregulation C5AR1, C7, and CD59, observed in individuals of European ancestry and a mouse MASH model. The study also established a correlation between complement gene expression and liver zonation, underscoring the complex interplay between complement regulation and hepatic pathophysiology.

These findings provide significant insights into the epigenetic modifications of complement genes in MASLD, but they also prompt questions that warrant further exploration. While the data suggest a correlation between altered methylation of complement genes and disease severity, causation remains unclear. It is yet to be determined whether changes in DNA methylation of complement genes directly contribute to MASLD pathogenesis—impacting inflammation, immune response, and fibrosis—or if they are a consequence of progressive liver damage and metabolic disturbances characteristic of the disease. This observation aligns with the authors’ finding that methylation was influenced by MASH status, while gene expression was affected by methylation status. Longitudinal studies and functional experiments are essential to clarify these relationships, with an emphasis on tracking methylation changes and disease progression over time to identify potential points of intervention.

Additionally, the snRNA-seq data focused on hepatocytes due to the highest expression of complement genes being observed in this cell population. This was a reasonable and strategic first step, given the hepatocytes’ dominant contribution to the bulk DNA methylation and RNAseq data. However, MASLD and MASH are characterized by significant changes in the liver’s cellular composition, especially with increasing inflammation and fibrosis, which could influence bulk tissue-derived data. As Magdy et al noted, three complement genes (C7, C5AR1, and CD59) were predominantly expressed in non-hepatocyte cells and exhibited hypomethylation and upregulation in MASLD samples. Given the liver’s diverse cellular composition, further single-cell analysis is necessary to clarify these findings.

Lastly, studies focusing on methylation and RNA expression do not account for changes in protein levels, a critical consideration for the complement system, which is activated through a proteolytic cascade leading to effector protein amplification. Understanding the relationship between methylation, mRNA expression, and protein levels of the nine complement genes in the liver, particularly in MASLD, is of significant interest. Elevated circulating levels of C3, C4, C5, complement factor B, and acylation stimulating protein (ASP) have been reported in MASLD patients compared to controls [17]. Furthermore, the levels of complement components in the blood may not originate solely from the liver. Adipose tissue C3 and its proteolytic cleavage products, C3a and ASP, have been implicated in adipose tissue inflammation and insulin resistance [18], potentially impacting MASLD development and progression. Adipose tissue also exhibits distinct epigenetic regulation of C3, with C3 methylation negatively associated with BMI and C3 mRNA linked with insulin resistance, consistent with protein data. The liver data from Magdy et al. [16] suggest complex roles and regulation of the complement system in MASLD, obesity, and metabolic dysfunction.

The study by Magdy et al. [16] underscores the role of DNA methylation in regulating complement genes and its correlation with MASLD severity. While this research significantly advances our understanding of the interplay between epigenetic regulation and immune responses in MASLD, it also emphasizes the need for further investigations to uncover the underlying mechanisms and inform potential therapeutic strategies.