The Role of Interferon Regulatory Factors in Liver Diseases

The interferon regulatory factors (IRFs) family comprises 11 members that are involved in various biological processes such as antiviral defense, cell proliferation regulation, differentiation, and apoptosis. Recent studies have highlighted the roles of IRF1-9 in a range of liver diseases, including hepatic ischemia–reperfusion injury (IRI), alcohol-induced liver injury, Con A-induced liver injury, nonalcoholic fatty liver disease (NAFLD), cirrhosis, and hepatocellular carcinoma (HCC). IRF1 is involved in the progression of hepatic IRI through signaling pathways such as PIAS1/NFATc1/HDAC1/IRF1/p38 MAPK and IRF1/JNK. The regulation of downstream IL-12, IL-15, p21, p38, HMGB1, JNK, Beclin1, β-catenin, caspase 3, caspase 8, IFN-γ, IFN-β and other genes are involved in the progression of hepatic IRI, and in the development of HCC through the regulation of PD-L1, IL-6, IL-8, CXCL1, CXCL10, and CXCR3. In addition, IRF3-PPP2R1B and IRF4-FSTL1-DIP2A/CD14 pathways are involved in the development of NAFLD. Other members of the IRF family also play moderately important functions in different liver diseases. Therefore, given the significance of IRFs in liver diseases and the lack of a comprehensive compilation of their molecular mechanisms in different liver diseases, this review is dedicated to exploring the molecular mechanisms of IRFs in various liver diseases.


Introduction
Interferon regulatory factors (IRFs) are a family of transcription factors that play crucial roles in various aspects of the immune response, including the development and differentiation of immune cells, as well as the regulation of responses to pathogens [1].IRF1, the first identified IRF that induced type I interferons, exhibited strong antiviral activity against a broad spectrum of viral infections, both in an interferon-dependent and interferonindependent manner [2].IRF2 was linked to the development of various cancers like colorectal, pancreatic, and hepatocellular carcinoma (HCC), by controlling the transcription of genes such as TP53, CXCL3, Bcl-2, and Bax [3][4][5].IRF3, IRF5, and IRF7 were essential for the production of type I interferons (IFNs) upon pathogen recognition by receptors, impacting viral and bacterial infections, inflammatory responses and autoimmune diseases, cancer growth, and metastasis, and changes in the tumor microenvironment [6][7][8][9].IRF4 and IRF8 contributed to tumor immunity by modulating the functions of T cells, B cells, and NK cells [10,11].IRF6, with its unique helix-turn-helix DNA-binding motif, primarily governs processes in limb and stomatofacial development, with limited documentation on its role in regulating IFNs in higher vertebrates [12].IRF9 regulated the expression of interferon-stimulated genes (ISGs), which served as markers for type I and III interferon activation [13,14].IRF10 was only observed in fish and birds, while IRF11 seemed to be a fish-specific member of the IRF family and will not be discussed in this review [15].
Recent research has shown that IRFs play a crucial role in the advancement and exacerbation of various liver diseases, such as hepatic ischemia-reperfusion injury (IRI), alcohol-induced liver injury, Con A-induced liver injury, nonalcoholic fatty liver disease (NALFD), cirrhosis, and HCC.IRF1, a nuclear transcription factor, was involved in various liver diseases, particularly hepatic IRI and HCC.It played a crucial role in regulating gene expression during inflammation and was up-regulated in both cold and warm liver IRI.IRF1 exerted deleterious effects by inducing the expression of inflammatory mediators, but liver grafts deficient in IRF1 showed significantly reduced liver injury [16,17].Specifically, in the early post-transplantation period, IRF1 overexpression in donor livers led to elevated caspase 3 expression, whereas knockdown of IRF1 reduced caspase 8 activity, resulting in a significant reduction in hepatocyte apoptosis and liver injury.In addition, IRF1 was involved in the hepatic IRI process through the regulation of various immune cell activities (NK and T cells), cytokines (IL-6, IL-12, IL-15, IL-23), and death ligands, as well as oxidative stress [18][19][20][21].IRF1 also served as a key transcription factor that controlled gene expression in both innate and adaptive immunity, responding to type I (IFN-α/β) and type II (IFN-γ) interferons [22,23].It was involved in IFN-γ-induced apoptosis of tumor cells by triggering downstream signals like inducible nitric oxide synthase (iNOS), p21, p53, p53-upregulated modulator of apoptosis (PUMA), and Fas-related death domain [24][25][26][27][28]. Yan, Y. et al. showed that IRF1 promotes the migration of CD8 + T cells and NK and NKT cells and stimulates IFN-γ secretion in NK and NKT cells, ultimately leading to tumor cell apoptosis via the CXCL10/CXCR3 pathway [29].However, Wang, R. et al. found that gamma interferons can upregulate IRF1 expression in HCC cells, which in turn activates human endogenous retrovirus-H long terminal repeat-associating 2 (HHLA2) expression and promotes M2 polarization and macrophage chemotaxis, potentially facilitating immune evasion and HCC progression [30].IRF1 may have a dual role in HCC, which requires further experimental verification.In addition to IRF1, other members of the IRF family have also been discovered to play significant roles in various liver diseases, demonstrating diverse functions in either promoting or inhibiting these diseases.Consequently, a comprehensive understanding of the role of IRFs in liver disease can offer valuable insights into disease mechanisms and potential therapeutic targets.

Materials and Methods
A systematic search was conducted in the PubMed electronic database to gather relevant literature.Additionally, the reference lists of the primary studies included were reviewed to identify potentially eligible articles.Only articles published in English were considered, with no restrictions on publication year.The index terms encompassed various topics such as 'interferon regulatory factor', 'IRF', 'ischemia reperfusion injury', 'hepatic ischemia reperfusion injury', 'liver injury', 'hepatic injury', 'NAFLD', 'nonalcoholic fatty liver disease', 'cirrhosis', 'liver fibrosis', 'HCC', and 'hepatocellular carcinoma'.A critical evaluation was performed on all studies included in this paper.

The Structure and Function of IRFs
The eleven members of the IRF family, IRF1 to IRF11, have been identified, with their primary roles centered around transcriptional regulation in the immune system and cell growth [31][32][33].All IRFs share common domains, including an N-terminal helical DNA-binding domain (DBD) with five conserved tryptophan repeats, and a C-terminal IRF-associated domain (IAD) that facilitates protein interactions and modulates various transcriptional activities [31,34,35].Each member of the IRF family plays a distinct role in various biological processes, including pathogen response, cytokine signaling, cell growth regulation, hematopoietic development, immune cell development, differentiation, and apoptosis [36].In-depth study of the functions of IRFs is crucial for comprehending the mechanisms underlying disease occurrence and progression.
IRF1, a pleiotropic transcription factor, exhibited high responsiveness to various stimuli such as IFN, NF-κB, TBK-1, and IKK-ε, undergoing rapid and dynamic regulation upon infection [37][38][39][40].Despite the short life cycle of IRF1 mRNA and protein, it consistently expressed a variety of host defense genes and effectively drove the expression of genes related to innate immunity [31,[41][42][43].Type I/II IFN stimulated hepatocytes to release IL-7, which in turn limited IRF1, enhanced the secretion of pro-inflammatory cytokines induced by lipopolysaccharide (LPS), and reduced macrophage tolerance to LPS [44].Similar to IRF1, IRF2 competed for the same cis-elements and inhibited the function of IRF1, thereby reducing the production of IRF1-dependent pro-inflammatory genes such as IL-12, IFNβ, and iNOS, which ultimately helped in reducing hepatic IRI [45].IRF3, a crucial component of the innate immune response, was responsible for detecting and reacting to foreign antigens, thereby helping to prevent viral infections [46].Besides its role in gene transcription, IRF3 also initiated non-transcriptional and pro-apoptotic signaling pathways.Although the pro-apoptotic activity of IRF3 was beneficial in fighting viral infections, it could also lead to liver cell death and exacerbate detrimental immune responses in liver disease [47].IRF4 was an oncogenic master transcription factor that induced cancer transcriptional programs by forming unique regulatory circuits that interacted with upstream pathways and binding partners [10].The expression of IRF4 and PD-1 was positively correlated, and the overexpression of IRF4 was found to impede the proliferation and migration of HCC by suppressing the JAK2/STAT3 signaling pathway and epithelial-mesenchymal transition [48].Furthermore, IRF4 served as a crucial transcriptional regulator of lipid processing in adipocytes and acted as a suppressor of inflammation in diet-induced obesity.Reduced IRF4 expression was associated with the exacerbation of NAFLD, leading to heightened levels of inflammation and insulin resistance [49].IRF5 played a central role in inflammation and was recruited to promote inflammatory genes with the assistance of the NF-kB p65 sub-unit RelA, inducing pro-inflammatory cytokines such as IL-6, IL-12, IL-23, and TNF-a [50][51][52].Highly expressed in monocytes, macrophages, B-cells, and dendritic cells, IRF5 was implicated in various inflammatory and autoimmune diseases [51,[53][54][55].Alzaid, F. et al. showed a significant increase in IRF5 expression in hepatic macrophages of individuals with non-alcoholic fatty liver disease or hepatitis C virus infection, contributing to liver fibrosis.Interestingly, mice lacking IRF5 in myeloid cells were found to be protected from stress-induced hepatic fibrosis, suggesting the crucial role of IRF5 in hepatocellular cell death and liver fibrosis in both mice and humans [56].The tumor suppressor IRF6 was not implicated in IFN gene regulation and was notably decreased during epithelialmesenchymal transition (EMT) in gastric and pancreatic ductal adenocarcinomas.This downregulation was controlled by the transcription factor ZEB1 [57,58].In hepatocytes, IRF6 directly interacted with the promoter of the peroxisome proliferator-activated receptor γ (PPARγ) gene, leading to the inhibition of PPARγ transcription and its associated target genes involved in lipogenesis and fatty acid uptake [59].Moreover, lower IRF6 expression was observed in colorectal cancer tissues and liver metastases compared to normal tissues.Mechanistically, IRF6 may enhance cisplatin-mediated cell proliferation, migration, invasion, and apoptosis sensitivity to chemotherapy by regulating E-calmodulin and Ki67 [60].IRF7, initially recognized as a key transcription factor in the production of type I IFNs and regulation of the innate immune response, was situated on chromosome 11p15.5 in humans and chromosome 7 F5 in mice [7,61].With a diverse array of functions, IRF7 was a pivotal component in the type I/III IFNs induced signaling pathway, contributing significantly to viral infections, autoimmune diseases, and the maintenance of homeostasis [7].IRF8 played a crucial role in regulating immune cell differentiation and inducing innate immunity.It collaborated with IRF1, IRF2, IRF4, and other transcriptional regulators like TEL, PU.1, MIZ1, and E47 to modulate gene transcription by influencing the formation of DNAbinding compounds and controlling the expression of downstream target genes [62][63][64].IRF8 knockout mice showed a reduction in inflammatory cell infiltration and cytokine release, leading to improved outcomes after liver IRI.Conversely, overexpression of IRF8 was linked to worsened liver damage and functional abnormalities [65].Interestingly, in HCC cells, IRF8 overexpression demonstrated enhanced antitumor effects by potentially regulating tumor-associated macrophages and T cell levels in the tumor microenvironment [66].IRF9 interacted with peroxisome proliferator-activated receptor α (PPARα) and activated PPARα target genes to attenuate inflammation, hepatic steatosis, and insulin resistance [67].However, IRF9 also suppressed SIRT1 expression, resulting in increased p53 protein acetylation, ultimately exacerbating hepatic IRI [68].These findings suggest that the role of IRFs may vary depending on the specific liver disease being studied.

IRFs and Liver Diseases
IRFs are a group of multifunctional transcription factors that target IFN promoters and interferon-stimulated response elements (ISREs) in ISGs, activating the expression of target genes.This indicates the essential role of IRFs in a range of biological processes including antiviral defense, innate immunity, adaptive immunomodulation, cell growth, apoptosis, and homeostasis maintenance [69].Despite their known functions, the involvement of IRFs in the regulation of liver diseases has yet to be explored (Table 1).

IRFs and Liver Ischemia-Reperfusion Injury
IRI was a common complication of liver transplantation, partial hepatectomy, and hypovolemic shock.This phenomenon resulted in complex hepatocellular damage, early graft dysfunction, and an increased risk of acute and chronic rejection, ultimately resulting in poor prognosis and low patient survival rates [114,115].Treatment for IRI was primarily supportive, as there were no specific drugs or methods available.Various strategies have been explored to address liver IRI, such as ischemic preconditioning, pharmacological and surgical interventions, and gene therapy [116].Despite advancements in graft management targeting autophagy, oxidative stress, sterile inflammation, and apoptosis, the intricate pathological mechanisms of IRI remained inadequately understood.

Autophagy and Oxidative Stress
IRF1 and IRF5 expression were found to be significantly increased in the liver following exposure to IRI [75,81].IRF1 had been shown to have a detrimental impact on hepatic IRI by regulating the expression of various inflammatory mediators.Overexpression of IRF1 in donor livers led to elevated expression of caspase 3 during the early post-transplantation period.Conversely, knockdown of IRF1 resulted in reduced mRNA levels of death ligands and receptors in hepatocytes, as well as decreased caspase 8 activity, leading to a notable decrease in hepatocyte apoptosis and liver injury [16,17,20].Yu, Y. et al. demonstrated that IRF1 played a critical role in promoting P62 expression by activating autophagy through P38 phosphorylation, leading to hepatocyte death [77].Additionally, Yan, B. et al. showed that IRF1 overexpression induces autophagy, suppresses β-catenin expression, and worsens hepatic IRI [73].Furthermore, IRF1 exacerbated hepatic IRI via JNK-mediated autophagy, resulting in increased Beclin1 levels.Conversely, silencing IRF1 reduces high mobility group box 1 (HMGB1) expression and release in the liver, decreases LC3II and Beclin1 levels, and mitigates hepatic IRI [72,75].Reactive oxygen species (ROS) also played a crucial role in IRI of the liver, primarily originating from Kupfer cells and mitochondria during IRI [117].Elevated hepatic ROS levels at the onset of IRI triggered the expression of IRF1, promoting the transcription of HMGB1.Acetylated HMGB1 then activated the downstream TLR-4/NF-κB signaling pathway, resulting in the release of inflammatory mediators like TNF-α and IL-1β, worsening hepatic IRI damage [70].The inhibition of IRF1-mediated HMGB1 release and subsequent TLR activation or p38 MAPK signaling pathway inactivation was shown to prevent hepatic IRI [71,79].In addition, IRI upregulated iNOS and promoted the transcriptional activity of IRF1 through HDAC2-mediated histone H3 acetylation, which led to cell death and tissue injury [74].Moreover, IRF1 regulated the transcription of Rab27a (a guanosine triphosphatase) and extracellular vesicle secretion, leading to oxidized phospholipids activation in neutrophils and subsequent hepatic IRI [76].Thus, autophagy and oxidative stress play an important role in hepatic IRI, but it has not been clearly elucidated, and more studies are still needed.

Inflammatory Cytokines
IRI first triggers pro-inflammatory signaling cascades like TNF-α, IL-6, IFN, and NF-kB.As liver cells perish, cytokines and chemokines are produced, leading to a chemical storm that attracts neutrophils and other immune cells to the liver.This recruitment further stimulates the release of CXCL1, CXCL2, and complement, exacerbating the IRI [118].Moreover, IRI activates the innate immune system, which drives the overall development of inflammatory hepatocellular injury.Further exploration of the roles played by immune cells and inflammatory cytokines in IRI is crucial for us to design safe and effective therapeutic strategies to ameliorate IRI in patients [119].In recent years, it has been found that multiple IRF family members can promote the progression of IRI by regulating interleukin production and immune cell infiltration.Yokota, S. et al. discovered that knocking down IRF1 led to a decrease in the population of NK, NKT, and CD8 + T cells in the liver.Additionally, they observed that IRF1 increases cytotoxic effects and systemic inflammatory responses, worsening hepatic injury by controlling the expression of IL-15 and IL-15Rα mRNA in mouse hepatocytes and hepatic dendritic cells [78].Another study by Nakano, R. et al. demonstrated that overexpression of IRF1 in ApoE -/-mice exacerbated hepatic IRI injury by activating hepatic NK and T cells through IL-15 production [18].Additionally, early induction of IL-23 in hepatic IRI activated the IFN-γ/IRF1 pathway, leading to increased apoptosis and necrosis [21].Type I IFNs were found to up-regulate hepatic IRF1 expression, which in turn regulated apoptosis and induced hepatic injury after IRI.Deprivation of plasmacytoid dendritic cells (pDC) in mice after IRI resulted in milder hepatic injury, reduced levels of hepatic IL-6, TNF-α, and apoptosis, and impaired expression of IRF1 and pro-apoptotic molecules such as Fas ligand, Fas, and death receptor 5 [19].Therefore, IRF1 played a crucial role in hepatic IRI by regulating immune cells and their cytokines, making it a potential target for mitigating IRI.In addition, endogenous IRF2 was typically expressed in the liver and can be slightly upregulated by liver IRI.Overexpression of IRF2 was shown to protect against hepatic IRI and also restricted the production of IRF1-dependent proinflammatory factors such as IL-12, IFNβ [45].Loi, P. et al. found that IRF3-deficient mice exhibited heightened hepatic necrosis and increased neutrophil infiltration due to elevated expression of IL-12/IL-23p40, IL-23p19, and IL-17A mRNA, along with reduced expression of IL-27p28 mRNA [80].Wang, P.-X.et al. also found that IRF9 decreased the expression of SIRT1 and increased the level of acetyl p53, leading to a significant increase in immune cell infiltration, inflammatory cytokine levels and hepatocyte apoptosis [68].Therefore, an in-depth study of the role of IRFs in IRI can help us better understand the mechanisms of disease development and develop feasible therapeutic measures.

IRFs and Alcohol-Induced Liver Injury/Alcoholic Liver Disease
Alcoholic-induced liver injury (ALI) is typically identified by disrupted liver function, inflammatory cell accumulation, and oxidative stress.Clinically, alcoholic liver disease (ALD) is the predominant form of ALI.The abuse of alcohol leads to liver injury through various mechanisms, such as oxidative and non-oxidative ethanol metabolism, the production of oxidative stress, damage and dysfunction of mitochondria and lysosomes, endoplasmic reticulum stress, inflammation, cytokine release, and the triggering of cell death [120].Approximately 90% of individuals with alcoholism develop hepatic steatosis, with 35% of those individuals progressing to alcoholic hepatitis.Unfortunately, 40% of patients diagnosed with severe alcoholic hepatitis do not survive beyond 6 months despite receiving treatment [121].Regrettably, corticosteroids continue to be the conventional treatment for severe alcoholic hepatitis, showing no advancements in the past forty years [121,122].It is imperative to enhance our comprehension of the pathogenic mechanisms underlying ALD, particularly the liver injury it induces, to formulate more efficient treatment approaches.Luther, J. et al. discovered that mice fed alcohol showed heightened hepatic expression of the cGAS-IRF3 pathway.And they observed that the downregulation of connexin 32 (the predominant hepatic gap junction) led to a decrease in IRF3 expression, ultimately leading to a decrease in liver injury as evidenced by a notable reduction in ALT/AST levels [89].Furthermore, IRF1-mediated caspase 1 inflammatory vesicles and NOX2-dependent ROS pathways were found to exacerbate ALI and steatosis [87].In addition, excessive alcohol consumption led to dysbiosis of the gut microbiota, compromising the integrity of the intestinal epithelium and facilitating the transport of gut microbial products (e.g., LPS) to the liver, which was recognized by Toll-like receptor-4 (TLR4), ultimately leading to liver injury and subsequent ALD [88,123].Liang, S. et al. demonstrated that chronic ethanol intake and LPS injection resulted in elevated serum ALT and IL-1 levels, along with enhanced hepatic CCL5 and CXCL10 expression.In normal conditions, macrophages could facilitate IRF1 degradation through autophagy, eliminate damaged mitochondria, and restrict macrophage activation and inflammation.However, following p62 silencing or myeloid cell-specific autophagy-related 7 knockout, IRF1 accumulation occurred in autophagy-deficient macrophages and translocated into the nucleus, leading to increased expression of CCL5 and CXCL10 [88].Petrasek, J. et al. showed that systemic IRF3 knockout mice were protective against ALI, steatosis, and inflammation, but knockout of IRF3 only in parenchymal cells exacerbated ALI.Hepatic parenchymal cells were further found to be a major source of type I IFNs, whose action was dependent on TLR4/IRF3.Meanwhile, type I IFNs potentiated LPS-induced IL-10 and inhibited inflammatory cytokine production in mouse macrophages and human leukocytes.Thus, IRF3 activation in liver parenchymal cells and the resulting type I IFNs are protective against ALD by modulating the inflammatory function of macrophages [124].However, Sanz-Garcia, C. et al. found that Gao-binge ethanol exposure activated IRF3 signaling and led to liver injury.IRF3 was further found to have a non-transcriptional function and could be induced to bind to Bax in mitochondria and activate caspases 3 and 9, which in turn activated the apoptotic pathway and limited NF-κB activity [125].Therefore, IRF3 could regulate the innate immune environment of the liver and alleviate ALI/ALD by increasing apoptosis of immune cells.

IRFs and Con A-Induced Liver Injury
Conjugin A (Con A), a plant lectin isolated from the miller bean (Canavalia ensiformis), was recognized for its role as a T-lymphocyte activator in the immune response to allograft rejection, viral infections or autoimmune disorders in mammals [126].Con A stimulated the release of a variety of cytokines from immune cells (such as TNF-α, IFN-γ, GM-CSF, IL-2, IL-1β), induced oxidative stress, activated multiple signaling pathways (NF-κB, MAPK, PI3K/PDK1/mTOR, STAT3, and STAT5), and altered the Bax/Bcl-2 ratio, ultimately resulting in severe liver inflammation and hepatocyte apoptosis/necrosis [83,[127][128][129][130].However, the precise cellular mechanisms underlying liver dysfunction induced by Con A activation remained incompletely understood.Recent studies have revealed the involvement of IRFs in Con A-induced liver injury.Binding of Con A to the mannose 6-phosphate receptor of HSCs induced JAK2/STAT-1 phosphorylation and promoted IRF1 transcription, which in turn inhibited superoxide dismutase (SOD) expression and promoted JNK and caspase 3 activation, leading to oxidative stress and apoptosis in hepatocytes [82].In a study involving mice injected with Con A, elevated levels of IL-28A were observed.Deficiency in IL-28A was found to limit M1 macrophage polarization by modulating a signaling pathway that inhibits IRF5, consequently reducing the release of pro-inflammatory cytokines such as TNF-α, IL-12, IL-6, and IL-1β from M1 macrophages [83].Corilagin was shown to effectively inhibit the release of pro-inflammatory cytokines in M1 macrophages by restraining the activation of the IRF5 signaling pathway, providing protection against Con A-induced immune-mediated liver injury.This treatment also resulted in reduced expression of M1 macrophage-associated pro-inflammatory cytokines and genes, including IL-6, IL-12, and iNOS [84].Additionally, in Con A-induced hepatitis, the absence of liver X receptor α (LXRα) resulted in an increase in MDSCs in the liver, which in turn attenuated liver injury.Mechanistically, MDSCs from LXRα -/-mice exhibited significantly lower expression of IRF8, facilitating the expansion of MDSCs and effectively reducing immune injury in the liver [85].

IRFs and Post-Transplantation/Other Modes of Liver Injury
Studies have shown that increased IRF1 and IRF4 expression in patients undergoing acute rejection post-liver transplantation contributes to liver inflammation and injury [91,131,132].Moreover, treatment with tacrolimus (TCA) suppresses IRF4 expression, thereby alleviating acute rejection after liver transplantation.This effect may be mediated through the TAC-NFAT-IRF4 and BATF/JUN/IRF4 complex-IL-21 axes, with the latter inhibiting IL-12-producing Tfh cells and consequently reducing liver injury [91,92].Zhao, W. et al. also demonstrated that silencing IRF4 resulted in decreased levels of inflammatory factors such as TNF-α, IL-6, and IFN-γ and induced anti-inflammatory IL-10 levels, thereby attenuating acute liver transplant rejection in mice [133].In adult patients with acute hepatitis A caused by HAV infection, severe liver injury was observed with elevated levels of chemokines such as CXCL10, CCL4, and CCL5.However, inhibiting IRF3 expression was found to decrease CXCL10 production, thus alleviating liver injury [90].Similarly, in patients diagnosed with primary sclerosing cholangitis and primary biliary cirrhosis, bile acids promoted IRF3 phosphorylation, resulting in elevated expression of the target gene ZBP1.This ultimately exacerbated hepatic injury and fibrosis through interaction with RIP1, RIP3, and NLRP3 [86].Additionally, in an LPS-induced liver injury model in mice, IRF3 expression was increased, a transcription factor was linked to systemic inflammation, while B-HA was shown to attenuate LPS-stimulated inflammatory responses by inhibiting the activation of the TLR4 signaling pathway through the phosphorylation of IRF3 [134,135].In summary, IRFs play an important role in liver injury caused by a variety of etiological factors, and in-depth study of the molecular mechanisms of IRFs can help to understand the occurrence of liver injury and develop appropriate therapeutic strategies.

IRFs and Nonalcoholic Fatty Liver Disease
Inflammation has significant implications for metabolism, particularly in the context of obesity and NAFLD.Studies on NAFLD mice induced with a high-fat, high-fructose diet revealed the progression of simple steatosis, steatohepatitis, and hepatic fibrosis over 4, 8, and 16 weeks, respectively.The expression of IRF3 and IRF7 showed an increase at week 4, peaked at week 8, and returned to basal levels by week 16 [136].However, research on liver tissues from 11 patients with NAFLD and 11 controls did not show a significant difference in IRF3 expression [137].In mice on an HFD diet, systemic knockdown of IRF3 prevented steatosis and glycemic abnormalities, while hepatocyte-specific knockdown of IRF3 only impacted glycemic abnormalities.Mechanistically, IRF3-mediated Ppp2r1b induced an increase in PP2A activity, leading to AMPKα and AKT dephosphorylation [93].Knockdown of IRF3 in mouse livers with HFD and FFA-induced L-O2 cells resulted in reduced hepatic inflammation and apoptosis, potentially through the regulation of the NF-κB signaling pathway, inflammatory cytokines, and apoptotic signaling [94].Moreover, obese NAFLD patients show heightened hepatic IRF3 activation, which can be reversed with bariatric surgical treatment [93].In a NASH mouse model, skeletal muscle-specific IRF4 knockout mice displayed improvements in hepatic steatosis, inflammation, and fibrosis without affecting body weight.IRF4 plays a role in transcriptionally regulating FSTL1, establishing a connection between muscle and liver [93].In the HFD-induced NAFLD model, hepatic IRF6 was inhibited by promoter hypermethylation, and hepatocyte-specific transgenic mice overexpressing IRF6 exhibited attenuated steatosis and metabolic disease.Mechanistically, hepatocyte IRF6 bound directly to the promoter of the PPARγ gene and subsequently stopped transcription of PPARγ and its target genes (regulating adipogenesis and fatty acid uptake), resulting in amelioration of NAFLD progression [59].

IRFs and Liver Fibrosis
Liver fibrosis is a precursor to cirrhosis and results from extracellular mesenchymal protein deposition, activated hepatic stellate cells (HSC) are essential for the development of liver fibrosis, and liver inflammation triggered by activation of liver resident macrophages and massive leukocyte aggregation are also associated with liver fibrosisrelated acute and chronic liver injury.A study revealed a lower frequency of the AG genotype of the IRF3(-925A/G) gene in cirrhotic patients, suggesting a potential protective effect against HCV infection [138].Yu, J. et al. discovered that Gαs-coupled GPCR signaling increased IRF3 phosphorylation through cAMP-mediated PKA activation, resulting in elevated IL-33 expression, ultimately promoting HSC activation, and subsequently contributing to hepatic fibrosis progression [99].Iracheta-Vellve, A. et al. demonstrated that in CCL4-treated hepatocytes, endoplasmic reticulum (ER) stress via STING triggered TBK1 phosphorylation, followed by IRF3 phosphorylation, which then interacted with BAX in mitochondria through its BH3-only structural domain, leading to pro-apoptotic caspase 3 activation and hepatocyte apoptosis, ultimately contributing to liver fibrosis [98].However, Wu, Q. et al. found that STING increased IRF3 phosphorylation via TBK1, subsequently inhibiting CDK4/6-mediated RB hyperphosphorylation and inactivating E2F transcription factors, thereby inducing senescence in HSC cells.Interestingly, total knockdown or conditional deletion of IRF3 in HSC exacerbated liver fibrosis, indicating a dual role for IRF3 in this process that required further investigation [100].Moreover, INF-γ was found to significantly upregulate indoleamine 2,3-dioxygenase (IDO) expression through STAT1 activation, leading to tryptophan depletion and subsequent G1 cell cycle arrest.Upon release from IFN-γ-induced G1 cell cycle arrest by 1-MT treatment, HSC apoptosis was significantly increased due to enhanced IRF1 expression [97].Similarly, GRh2 attenuates hepatic inflammation and fibrosis by up-regulating IRF1 expression, which inhibits SLC7A11 and promotes HSC iron death and inactivation [96].In human hepatocyte macrophages with liver fibrosis from NAFLD or HCV infection, elevated IRF5 expression triggered the release of inflammatory cytokines and death effectors, leading to hepatocyte caspase-dependent apoptosis.Conversely, IRF5-deficient macrophages under hepatocyte stress exhibited immunosuppressive polarization, secreting IL-10 and TGFβ to support BCL2 family member-mediated anti-apoptotic signaling in hepatocytes during metabolic or toxic stress-induced liver fibrosis [56].

IRFs and Hepatocellular Carcinoma
HCC posed a significant global health challenge due to its high fatality rate [139,140].Despite undergoing combination therapies such as radiochemotherapy and immunotherapy, the 5-year survival rate for HCC remained notably low [141].IRF1 and IRF2 were essential in regulating interferon activity, where the absence of IRF1 and the increase in IRF2 expression had been associated with aggressive traits in different types of cancer.IRF1, acting as a tumor suppressor gene, enhanced the migration of CD8 + T-cells, NK cells, and NKT cells, while also stimulating IFN-γ secretion in NK and NKT cells.This activation led to apoptosis in tumor cells through the CXCL10/CXCR3 paracrine axis [29].Conversely, the downregulation of IRF2 significantly reduced invasive capacity, correlating with the decreased expression of STAT3, p-STAT3 and MMP9 [142].However, increased IRF1 mRNA expression was observed in patients with highly differentiated or early HCC.In vitro studies demonstrated that IFN-γ induced PD-L1 mRNA and protein expression by enhancing IRF1 levels in both mouse and human HCC cells, whereas IRF2 overexpression down-regulated IFN-γ-induced PD-L1 promoter activity and protein abundance.Additionally, IRF1 was found to antagonize IRF2 binding to the IRE promoter element in PD-L1, leading to the upregulation of PD-L1 in the tumor microenvironment [143].Furthermore, IRF1 was found to post-transcriptionally suppress and induce apoptosis in HCC cells by facilitating the interaction between miR-195 and the 3 ′ UTR of checkpoint kinase 1 (CHK1).However, IRF1 also enhanced PD-L1 expression by promoting STAT3 phosphorylation [106].Therefore, elevated IRF-1 expression not only triggered apoptosis in HCC cells but also boosted PD-L1 levels, shedding light on the regulation of the PD-L1/PD-1 pathway in HCC therapy.In both human and mouse HCC cells, IL-33 overexpression was shown to inhibit proliferation and decrease PD-L1 levels at the transcriptional level by enhancing the ubiquitin-dependent degradation of IRF1.This process was disrupted by E3 ligase RanBP2-mediated SUMOylation of IL-33 at Lys54 [102].Additionally, treatment with cisplatin and CHK1 inhibitors led to the upregulation of major histocompatibility complex (MHC) class I associated chains A (MICA) expression through IRF1-mediated transcriptional effects, resulting in increased infiltration of NK cells and CD8 + T cells in HCC tissues [104].FOXO1 functioned as a tumor suppressor by promoting macrophage infiltration and antitumor polarization via positive regulation of the IRF1/NO pathway.Polarized macrophages further inhibited HCC proliferation and migration by suppressing IL-6/STAT3 activation [105].NR4A1 was markedly upregulated in tumor-infiltrating NK cells, which reduced the efficacy of anti-PD-1 therapy through modulation of the IFN-γ/p-STAT1/IRF1 signaling pathway, leading to dysfunctional tumor-infiltrating NK cells [103].Moreover, high expression levels of miR-23a, miR-31, and miR-301a in HCC enhanced its progression by inhibiting IRF1 expression [107][108][109].Conversely, miR-345 showed low expression in HCC, counteracting the inhibitory impact of IRF1 through reversible trans-overexpression, thereby influencing the epithelial-mesenchymal transition process and tumor metastasis [101].Phosphatase and tensin homolog (PTEN) in HBV-associated HCC regulated the nuclear localization of IRF3 by dephosphorylating IRF3 at ser97, leading to the inhibition of the PI3K/AKT pathway and the reduction of oncogenic effects [110].IRF4 overexpression suppressed the proliferation and migration capabilities of HCC cells by inhibiting the JAK2/STAT3 signaling pathway and epithelial-mesenchymal transition [48].IRF5, identified as a tumor suppressor, exhibits down-regulated mRNA and protein expression levels in HCV-infected human hepatocytes and cells with autonomous replication of HCV RNA.Conversely, restoration of IRF5 expression hampered HCV protein translation and RNA replication [111].However, Fang, Y. et al. observed an upregulation of IRF5 expression in HCC, facilitating an oncogenic effect by enhancing glycolysis through upregulation of lactate dehydrogenase A (LDHA) expression [112].In addition, miR-424-3p reduced the interferon pathway by attenuating the transcriptional activation of STAT1/2 and IRF9 genes by SRF, which in turn enhanced matrix metalloproteinases (MMPs)-mediated ECM remodeling [113].Therefore, IRF1, IRF3, IRF5, and IRF9 are all involved in the development of HCC, and further research into the molecular mechanisms involving IRFs in carcinogenesis could facilitate the discovery of new targeted therapies.

Conclusions and Future Perspectives
In summary, IRF1, IRF2, IRF3, IRF5, IRF8, and IRF9 are involved in hepatic IRI; IRF1, IRF5, and IRF8 are involved in Con A-induced liver injury; IRF3 is involved in cholestasis-induced liver injury and HAV-induced liver injury; IRF4 is involved in liver transplantation-induced liver injury; IRF1 and IRF3 are involved in alcohol-induced liver injury; IRF3, IRF4, and IRF6 are involved in the development of NAFLD; IRF1, IRF3, and IRF5 are involved in the development of liver fibrosis; and IRF1, IRF3, IRF4, IRF5, IRF8, and IRF9 are involved in the development of HCC.Almost all members of the IRF family play a role in different liver diseases, especially IRF1, which has been the most studied.IRF7 is less well-studied in liver diseases, and its expression is only increased in certain disease processes and usually accompanied by IRF3.Although the family of IRFs plays a very important role in liver diseases, there are no targeted drugs against IRFs to treat the disease process.In addition, the same IRFs may play different functions in different liver diseases, promoting or inhibiting the disease.Therefore, further clarification of the molecular mechanisms of IRFs in liver diseases is needed in the future to guide clinical practices and the development of corresponding targeted drugs.

Table 1 .
The function and mechanism of IRFs in liver diseases.
(LTx) I/R injury via hepatocyte IL-15/IL-15Rα production which suggests that targeting IRF-1 and IL-15/IL-15Rα may be effective in reducing I/R injury associated with LTx.