MicroRNA-122 protects against interferon-α-induced hepatic inflammatory response via the Janus kinase–signal transducer and activator of transcription pathway

Abstract

Significant overlap in the epidemiology and coinfection of chronic hepatitis B virus (HBV) and hepatitis C virus (HCV) has been identified, which accelerates the development of severe liver cirrhosis and hepatocellular carcinoma worldwide. Interferon-α (IFN-α), a cytokine with antiviral properties, exerts profound physiological effects on innate immunity by regulating interferon-stimulated genes (ISGs) within cells. However, the underlying mechanism of IFN-α in hepatic inflammation remains to be fully elucidated. Here, we utilized LO2 cells treated with the recombinant IFN-α protein and conducted microRNA (miR) sequencing. MiR-122-3p and miR-122-5p_R+1 were the most enriched miRNAs involved in the pathogenesis of IFN-α-induced inflammatory responses and were significantly downregulated by IFN-α treatment. Furthermore, we identified interferon induced protein with tetratricopeptide repeats 1 (IFIT1) as a potential target gene of miR-122. IFN-α markedly increased the expression of proinflammatory cytokines and fibrogenic genes but decreased the mRNA expression of ISGs. Additionally, IFN-α significantly activated the NF-κB p-p65, MAPK p-p38, and Jak/STAT pathways to trigger inflammation. Importantly, supplementation with a miR-122 mimic significantly alleviated IFN-α-induced inflammation and induced IFIT1 expression in LO2 cells. Conversely, the suppression of miR-122 markedly exacerbated the inflammatory response triggered by IFN-α. Furthermore, silencing IFIT1 via an siRNA elicited an inflammatory response, whereas IFIT1 overexpression ameliorated hepatic inflammation and fibrosis in a manner comparable to that induced by IFN-α treatment. Taken together, our findings suggest that miR-122 and its target, IFIT1, reciprocally regulate the inflammatory response associated with IFN through the Jak/STAT pathway.

Introduction

Hepatitis refers to liver inflammation caused by various infectious viruses and noninfectious agents [1]. Chronic hepatitis B virus (HBV) and hepatitis C virus (HCV) infections have become major public health challenges of this decade because of their significant risks of causing cirrhosis and hepatocellular carcinoma [2]. Notably, HBV and HCV coinfection is associated with a more severe clinical course than infection with either virus alone because of their similar modes of transmission [3]. HCV replication typically predominates in the context of HBV/HCV coinfection [4], which chronically activates the inflammatory response and subsequent liver fibrosis [5]. Interferons (IFNs) are first-generation antiviral medications used for treating HCV [6]. However, the antiviral efficacy of IFNs is limited by the viral load and the presence of different virus genotypes, which also cause severe side effects during clinical use [7]. Therefore, identifying the underlying mechanisms of IFNs in liver inflammation is essential.

Interferons (IFNs) are a class of cytokines used in the clinical treatment of viral infections and immune diseases because of their antiviral and antiproliferative effects [8]. Compelling evidence indicates that IFN-α regulates inflammatory cascade reactions and immune activation by mediating the Janus kinase (Jak)–signal transducer and activator of transcription (STAT) pathway, the nuclear factor kappa B (NF-κB) pathway, and the mitogen-activated protein kinase (MAPK) pathway [9]. Plasmacytoid dendritic cells are the primary sources of IFNα, which is located on chromosome 9 and signals through a shared type I IFN (IFN-I) heterodimeric receptor complex composed of the IFN-α receptor (IFNAR) 1 and 2 subunits [10]. Upon the binding of IFN-α to IFNAR1/2, a cascade of signaling events is initiated through the JAK/STAT pathway, which contributes to the activation of IFN-stimulated genes (ISGs) to modulate immune responses [11]. Pioneering evidence has demonstrated that IFN-α effectively inhibits HBV replication by accelerating the decay of HBV replication-competent nucleocapsids [12]. Previous research has indicated that exogenously expressed ISG56, also known as IFN-induced protein with tetratricopeptide repeats 1 (IFIT1), suppresses HCV replication in hepatoma cells, whereas IFIT1 knockdown exacerbates HCV replication [13]. Furthermore, the knockdown of IFIT1 promotes intracellular HBV replication in IFN-α-treated hepatoma cells by increasing the activity of the HBV S gene promoter [14]. Notably, IFIT1 has been reported to negatively regulate the inflammatory signaling pathway in lipopolysaccharide (LPS)-activated macrophages [15], suggesting a potential role for IFIT1 in the innate immune response.

In recent decades, mammalian microRNAs (miRNAs) have been reported to be involved in various biological processes and the innate immune system [16]. Clinical evidence has revealed a significant reduction in miR-122 expression in NASH patients, and silencing miR-122 markedly elevates cholesterol biosynthesis-associated and lipogenic gene expression in cultured hepatoma cells [17]. Clinical evidence has revealed a significant reduction in miR-122 expression in NASH patients, and silencing miR-122 markedly elevates cholesterol biosynthesis-associated and lipogenic gene expression in cultured hepatoma cells [18]. Notably, miR-122 has been reported to directly target the HCV genomic RNA and promote HCV replication, while the silencing of miR-122 in liver cells results in a significant loss of autonomously replicating HCV RNA [19]. The insufficiency of miR-122 is associated with liver disorders, as the inhibition of miR-122 significantly suppresses hepatic inflammation in vivo and in vitro [20]. However, the regulatory mechanisms of miR-122 in interferon-associated inflammatory immune responses have not yet been studied.

Considering the potential roles of miR-122 and IFN-induced ISGs in the hepatic immune response, we aimed to investigate the regulatory mechanisms of miR-122 and IFIT1 in IFN-α-induced hepatic inflammation. In the present study, we treated LO2 cells with recombinant IFN-α protein and performed miRNA sequencing. MiR-122-3p and miR-122-5p_R+1 were the most enriched miRNAs involved in the pathogenesis of the IFN-α-induced inflammatory response. Furthermore, we identified IFIT1 as a potential target gene of miR-122 and showed that miR-122 and IFIT1 reciprocally regulate the IFN-associated inflammatory response.

Materials and methods

Cell culture and cell viability measurement

The human fetal hepatocyte line LO2 was cultured in Dulbecco’s modified Eagle’s medium (DMEM)-high glucose with 10% (vol/vol) fetal bovine serum (SN202104, Hakata, Shanghai, China) and a 1% penicillin–streptomycin solution (15140-122, Gibco, USA). The LO2 cells were maintained in a humidified atmosphere (5% CO2 and 95% air) at 37°C. For IFN-α treatment, LO2 cells were treated with 1, 5, 10, 20, 40, 50, 100 or 200 ng/mL recombinant IFNα protein (Sigma, USA) for 24 h. The cytotoxicity of IFN-α to LO2 cells was measured with a methylthiazoltetrazolium (MTT) assay, as previously described [21].

MicroRNA sequencing

LO2 cells were treated with 10 ng/mL recombinant IFN-α protein for 24 h, and total RNA was collected and sequenced via Illumina sequencing technology (LC-Bio Technology Co., Ltd., Hangzhou, China). Briefly, microRNA libraries were constructed and sequenced using the Illumina TruSeq Small RNA Preparation Kit, with cluster generation on Illumina’s Cluster Station and sequencing on an Illumina GAIIx, according to the vendor’s instructions [22]. ACGT101-miR (LC Sciences, USA) was utilized for the subsequent analysis of raw data, including the removal of adapter dimers, junk, low complexity, common RNA families (rRNA, tRNA, snRNA, and snoRNA), and repeats. Unique sequences were mapped to specific species precursors in miRBase 22.0 using BLAST (Basic Local Alignment Search Tool) to identify known miRNAs and novel 3p- and 5p-derived miRNA candidates. The miRNA–target gene binding sites were predicted with TargetScan (v6.0) and miRanda (v3.3a). The differentially expressed miRNAs, identified based on normalized deep-sequencing counts, were analyzed using the R package limma. Student’s t test was used to compare differences between two groups, which were analyzed using the R package limma. The Benjamini–Hochberg method was employed for p value correction, which was applied through the topTable function in limma. The indicated miRNA target genes were screened using adjusted p values, which were applied to control the false discovery rate (FDR). A threshold of adjusted p < 0.05 was set to identify the target genes for the IFN-α and control groups (Table 1). The visualization of differentially expressed miRNAs was performed with R software (version 4.4.0).

Table 1 miRNA-target genes list

miRNA	KEGG pathway	Target gene	adjusted p value
hsa-miR-122-5p_R+1	Hepatitis C	Ifit1	2.37E-06
hsa-miR-122-5p_R+1	Hepatitis C	Irf7	4.48E-03
hsa-miR-122-5p_R+1	Hepatitis C	Jak1	4.48E-03
hsa-miR-122-3p	Hepatitis B	Il6	5.68E-03
hsa-miR-122-3p	Hepatitis B	Irf7	5.68E-03
hsa-miR-122-5p_R+1	Hepatitis C	Irf7	5.68E-03
has-miR-122-3p	Hepatitis B	Ifnar1	5.68E-03
hsa-miR-122-3p	JAK-STAT signaling pathway	Ifnar1	1.39E-02
hsa-miR-122-3p	PI3K-Akt signaling pathway	Ifnar1	1.65E-02
hsa-miR-122-3p	Hepatitis C	Ifnar1	1.65E-02
hsa-miR-122-3p	Influenza A	Ifnar1	1.65E-02

MiR-122 mimic, miR-122 inhibitor and siRNA transfection

LO2 cells were cultured in 12-well plates for 24 hours until they reached approximately 70% confluence. For the miR-122 mimic treatment, the cells were transfected with 50 nM commercial miR-122 mimic for 24 h and then treated with 10 ng/mL recombinant IFN-α protein for 24 h. The nontargeted sequence of the miR-122 mimic was 5'-UUCUCCGAACGUGUCACGUTT-3', while the target sequence of the miR-122 mimic was 5'-UGGAGUGUGACAAUGGUGUUUG-3'. For miR-122-targeted inhibitor transfection, 100 nM miR-122-targeted inhibitor with the following sequence was pretransfected for 24 h: 5'-CAAACACCAUUGUCACACU-3'. Then, cells were cotreated with 10 ng/mL recombinant IFN-α protein for 24 h. For small interfering RNA (siRNA)-mediated gene silencing, LO2 cells were transfected with 50 nM nontarget siRNA or Ifit1-target siRNA for 24 h and then incubated with 10 ng/mL recombinant IFN-α protein for 24 h. Nontarget siRNA (si-Ctrl) and Ifit1 siRNA-targeted (si-Ifit1) sequences were prepared separately as follows: 5'-UUCUCCGAACGUGUCACGU-3' (si-Ctrl), 5'-CCUUGGAAUACUACACUCA-3' (si-Ifit1). The specific miR-122-NC, miR-122-mimic, miR-122-targeted inhibitor, and si-ctrl or si-Ifit1 were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China) and transfected with the riboFECT^TM CP Transfection Kit (RiboBio, Guangzhou, China), as previously described [23].

Plasmid transfection

The pLV3-CMV-IFIT1(human)-3xFLAG-Puro plasmid was purchased from MIAOLING BIOLOGY (Wuhan, China). LO2 cells were cultured in 12-well plates for 24 hours until they reached approximately 70% confluence. LO2 cells were transfected with IFIT1 plasmids using the RiboFECT^TM CP Transfection Kit (RiboBio, Guangzhou, China) to overexpress IFIT1, according to the manufacturer’s protocol. Twenty-four hours after transfection, the cells were treated with or without 10 ng/mL recombinant IFN-α protein for 24 h.

Quantitative real-time PCR

Total RNA was extracted from LO2 cells using TRIzol reagent (Vazyme, Nanjing, China) according to the manufacturer’s instructions. cDNA was synthesized using a reverse transcriptase kit (Tsingke Co., Ltd., Beijing, China). Quantitative real-time PCR (qPCR) was performed on a CFX Connect Optics Module (Bio-Rad, Hercules, CA, USA) using Fast qPCR Mix (SYBR Green, Tsingke Co., Ltd., Beijing, China) [24]. The comparative threshold cycle (2^–ΔΔCt) method was used to calculate the relative gene expression, as previously described [25]. The values were normalized to those of GAPDH. The primer sequences are described in Table 2.

Table 2 Primers sequences

Name	Forward Primer	Reverse Primer
h. IL-6	5'-GGAAATCGTGGAAATGAG-3'	5'-GCTTAGGCATAACGCACT-3'
h. IL-1β	5'-AGGCTCCGATGAACAA-3'	5'-AAGGCATTAGAAACAGTCC-3'
h. TNF-α	5'-GTGGAACTGGCAGAAGAGGCA-3'	5'-AGAGGGAGGCCATTTGGGAAC-3'
h. MCP1	5'-CCCCAGTCACCTGCTGTTAT-3'	5'-TGGAATCCTGAACCCACTTC-3'
h. CCL5	5'-TGTTTGTCACTCGAAGGAACCG-3'	5'-TGGGGGTCAGAATCAAGAAACCC-3'
h. CXCL10	5'-ATGACGGGCCAGTGAGAATGAGG-3'	5'-GCACTGCACAAGAAGATGCG-3'
h. IFIT1	5'-GCGCTGGGTATGCGATCTC-3'	5'-CAGCCTGCCTTAGGGGAAG-3'
h. IFNAR1	5'-ATTTACACCATTTCGCAAAGCTC-3'	5'-TCCAAAGCCCACATAACACTATC-3'
h. IFNAR2	5'-TCATGGTGTATATCAGCCTCGT-3'	5'-AGTTGGTACAATGGAGTGGTTTT-3'
h. IRF3	5'-CCTTCTTTCATTCCCTCTGTGAC-3'	5'-CCCAACCTTTTGACCCTTTTTAT-3'
h. IRF7	5'-CCCAGCAGGTAGCATTCCC-3'	5'-CCCAGCAGGTAGCATTCCC-3'
h. MiR-122	5'-TTGAATTCTAACACCTTCGTGGCTACAGAG-3'	5'-TTAGATCTCATTTATCGAGGGAAGGATTG-3'
h. TGF-β1	5'-GGCCAGATCCTGTCCAAGC-3'	5'-GTGGGTTTCCACCATTAGCAC-3'
h. α-SMA	5'-AAAAGACAGCTACGTGGGTGA-3'	5'-GCCATGTTCTATCGGGTACTTC-3'
h. COL1A1	5'-GAGGGCCAAGACGAAGACATC-3'	5'-CAGATCACGTCATCGCACAAC-3'
h. PAI-1	5'-TGATGGCTCAGACCAACAAG-3'	5'-CAGCAATGAACATGCTGAGG-3'
h. GAPDH	5'-ACATGGCCTCCAAGGAGTAAGAA-3'	5'-GGGATAGGGCCTCTCTTGCT-3'

Western blot

Proteins from LO2 cells were collected and homogenized in radioimmunoprecipitation (RIPA) lysis buffer containing protease and phosphatase inhibitors (Roche Diagnostics, USA), as described previously [26]. The primary antibodies used in the present study were anti-IFIT1 (abs149003) (Absin, China), anti-phospho-nuclear factor kappa light chain-enhancer of activated B cells (NF-κB) p65 (Ser276) (ab106129) (Abcam, USA), anti-NF-κB (#3034), anti-GAPDH (#2118) (Cell Signaling Technology, USA), anti-phospho-p38 mitogen-activated protein kinase (p38 MAPK) (Thr180/Tyr182) (ER2001-52), anti-p38 MAPK (ET1702-65), anti-phospho-signal transducer and activator of transcription 1 (STAT1) (Ser727) (ET1611-20), anti-STAT1 (ET1606-39) (Huabio Co., Ltd., Hangzhou, China), anti-phospho-Janus kinase 1 (JAK1) (Tyr1022) (310040), anti-JAK1 (310108) (Zenbio Co., Ltd., Chengdu, China) and β-Actin (GB12001) (Servicebio Co., Ltd., Wuhan, China). The procedures used for western blotting were described previously [27]. The intensity of the bands in the autoradiograms was measured using ImageJ software.

Statistical analysis

All the data are presented as the means ± SEMs (means ± standard errors of the means). Differences between the mean values were assessed using one-way analysis of variance (ANOVA) followed by the Student–Newman–Keuls test. p values < 0.05 or 0.01 were considered statistically significant.

Results

Differential expression of miRNAs in IFN-α-treated LO2 cells

An MTT assay was performed to determine the toxicity of the recombinant IFN-α protein in LO2 cells (Fig. 1A). Compared with the blank treatment, the IFN-α treatment significantly inhibited the proliferation of LO2 cells at concentrations ranging from 40 ng/mL to 200 ng/mL at 24 h (Fig. 1A). The changes in miRNAs secreted by LO2 cells treated with IFN-α treatment were explored by conducting miRNA sequencing on LO2 cells treated with 10 ng/mL recombinant IFN-α protein for 24 h (Figs. 1, 2). The Pearson correlation coefficient between the ctrl and IFN-α-treated groups showed a significant difference (Fig. 1B). Principal coordinate analysis (PCoA) clearly differentiated the ctrl- and IFN-α-treated groups, with the IFN-α-treated group exhibiting a significant deviation from the control group (Fig. 1C). Notably, 14 human-annotated miRNAs, such as miR-122-3p, miR-122-5p_R+1, miR-6728-p5_1ss18CA, miR-6728-p3_1ss18CA, miR-1934-p3_1ss9TA, miR-4508_L-1R+2, and miR-1291, were significantly decreased, whereas 13 human-annotated miRNAs, such as miR-299b-3p_R+4 and miR-101b-p5, were markedly increased by IFN-α treatment (Fig. 1D). Importantly, Gene Ontology (GO) analysis revealed that IFN-α treatment significantly altered several biological processes (Supplementary Fig. 1A). The most enriched GO functions included signal transduction, transcriptional regulation, and G protein-coupled receptor signaling pathways (Supplementary Fig. 1A). Additionally, the most enriched pathways were related to protein phosphorylation, protein ubiquitination, transmembrane transport, and the Wnt signaling pathway (Fig. 1E). Furthermore, the functions related to cellular components such as the membrane, cytoplasm, and nucleus were enriched in the IFN-α-treated group (Supplementary Fig. 1A). Additionally, molecular functions, including protein binding, metal ion binding, and transcriptional regulation, were significantly affected (Supplementary Fig. 1A).

Fig. 1  Identification of differentially expressed miRNAs in LO2 cells.

(A) The cell viability of LO2 cells treated with different concentrations of IFN-α for 24 h by MTT assay. (B) The Pearson correlation. (C) Principal component analysis (PCA) analysis. (D) Volcano plot analysis of miRNA sequencing between Control and IFN-α groups. (E) The top 20 enriched biological process Go functions. Data are expressed as means ± SEM, n = 5, **p < 0.01 vs. blank control group.

Fig. 2  Identification of differentially expressed miRNAs and its-targeted genes in LO2 cells.

(A) Heatmap of relative miRNA expression based on miRNA sequence (p < 0.05 or log2 fold change >1 or <–1). (B) KEGG analysis of differentially expressed significant miRNAs in response to IFN-α treatment. The top 20 of the most enriched pathways were shown. (C) mRNA expression of miR-122, TNF-α, IL-1β, IL-6, MCP1, CCL5, CXCL10, IFIT1, IFNAR1, IFNAR2, IRF3, IRF7, TGF-β1, α-SMA, COL1A1 and PAI-1 in the LO2 cells treated by 10 ng/mL IFN-α for 24 h, n = 3. (D, E) Western blotting analysis (D) and quantification data (E) of IFIT1, p-Jak1, Jak1, p-p38, p38, p-p65, p65, p-STAT1, STAT1, and β-actin protein in the LO2 cells treated by 10 ng/mL IFN-α for 24 h, n = 3. Data are expressed as means ± SEM, *p < 0.05, **p < 0.01 vs. blank control group.

IFN-α downregulates the expression of miR-122 and ISGs

Among the significantly changed miRNAs, human miR-122-3p, miR-122-5p_R+1, miR-6728-p3_1ss18CA, miR-6728-p5_1ss18CA, miR-640-p5_1ss16CG, miR-22-3p, miR-22-3p, miR-9259-p3_1ss5TA, miR-1934-p3_1ss9TA, miR-345-5p_L-1R-1, miR-1291, miR-4508_L-1R+2, and miR-221-3p were significantly downregulated by the recombinant IFN-α protein treatment in LO2 cells (Fig. 2A). Conversely, IFN-α significantly upregulated the transcript levels of human miR-221-3p, miR-3969-p5_1ss16TG, miR-299b-3p_R+4, miR-3058-5p_R-2, and miR-6978-3p (Fig. 2A). We subsequently performed Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis to identify the cellular pathways affected by these altered miRNAs (Supplementary Fig. 1B and Fig. 2B). The results revealed that the most significantly altered miRNAs were enriched in pathways related to cellular processes, environmental information processing, genetic information processing, human diseases, metabolism, and organismal systems (Supplementary Fig. 1B). Notably, the top 20 enriched pathways implicated in the effects of IFN-α on liver disorders included the JAK-STAT signaling pathway, hepatitis B, hepatitis C, the TGF-β signaling pathway, and the mTOR signaling pathway (Fig. 2B).

Given the significant enrichment of human miR-122-3p and miR-122-5p_R+1 following IFN-α treatment, we performed a miRNA target prediction analysis (Table 1). The results indicated that several interferon-stimulated genes (ISGs) and cytokines, including Ifit1, Irf7, Jak1, Il6, and Ifnar1, were predicted target genes of miR-122-3p and miR-122-5p_R+1 (Table 1). These targets are enriched in several KEGG pathways, including hepatitis B, hepatitis C, the JAK-STAT signaling pathway, the phosphatidyl-inositol 3-kinase/serine-threonine kinase (PI3K/AKT) signaling pathway, and influenza A (Table 1). We further verified the miRNA sequencing data by conducting a qPCR analysis to identify the potential effects of IFN-α on the immune inflammatory response and hepatic fibrosis (Fig. 2C). IFN-α administration substantially elevated the mRNA expression of pro-inflammatory cytokines, including tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β), IL-6, and monocyte chemoattractant protein-1 (MCP1), and chemokines such as C-C motif chemokine ligand 5 (CCL5) and C-X-C motif chemokine ligand 10 (CXCL10) (Fig. 2C). Also, several fibrogenic genes such as TGF-β1, alpha-smooth muscle actin (α-SMA), collagen type I alpha 1 chain (COL1A1) and plasminogen activator inhibitor 1 (PAI-1) were dramatically increased by IFN-α in LO2 cells (Fig. 2C). Consistent with sequencing data, IFN-α treatment significantly decreased the mRNA level of miR-122, and its target ISG genes, including IFIT1, IFNAR1, IFNAR2, IRF3 and IRF7 (Fig. 2C). In addition, these results were consistent with the decreased phosphorylation levels of nuclear factor kappa B (NF-κB) p65 and mitogen-activated protein kinase (MAPK) p38 in LO2 cells treated with IFN-α (Fig. 2D, E). IFN-α treatment also significantly reduced the IFIT1 protein levels and markedly increased the levels of p-Jak1 and p-STAT1 in LO2 cells (Fig. 2D, E).

An miR-122 mimic alleviates the IFN-α-induced inflammatory response

LO2 cells were transfected with a miR-122 mimic for 24 h, followed by treatment with 10 ng/mL recombinant IFN-α protein for an additional 24 h to assess the potential function of miR-122 in the IFN-α-induced inflammatory response (Fig. 3). The results revealed that treatment with the miR-122 mimic significantly increased the level of miR-122. It effectively attenuated the inflammatory response induced by IFN-α, which was evidenced by a notable reduction in the mRNA expression levels of TNF-α, IL-1β, IL-6, MCP1, CCL5, and CXCL10 compared to cells treated solely with IFN-α (Fig. 3A). The mRNA levels of fibrogenic genes were significantly decreased by the miR-122 mimic treatment (Fig. 3B). Notably, miR-122 mimic also significantly increased the mRNA expressions of ISGs, such as IFIT1, IFNAR1, IFNAR2, IRF3 and IRF7 (Fig. 3C). Compared with the group treated with IFN-α alone, the miR-122 mimic group also presented significantly increased levels of IFIT1 and proinflammatory-related proteins (Fig. 3C–E).

Fig. 3  Effects of miR-122 mimic on inflammatory response induced by IFN-α in LO2 cells.

LO2 cells transfected with or without a miR-122 mimic for 24 h followed by treatment with 10 ng/mL of IFN-α for an additional 24 h. RNA and proteins were collected to perform qPCR and western blot analysis. (A) mRNA expression of miR-122, TNF-α, IL-1β, IL-6, MCP1, CCL5 and CXCL10, n = 3. (B) mRNA expression of TGF-β1, α-SMA, COL1A1 and PAI-1, n = 3. (C) mRNA expression of IFIT1, IFNAR1, IFNAR2, IRF3 and IRF7, n = 3. (D, E) Western blotting analysis (D) and quantification data (E) of IFIT1, p-Jak1, Jak1, p-p38, p38, p-p65, p65, p-STAT1, STAT1, and β-actin protein, n = 3. Data are expressed as means ± SEM, *p < 0.05, **p < 0.01 vs. blank control group.

Inhibition of miR-122 promotes inflammation and fibrosis through Jak/STAT pathway

LO2 cells were transfected with the appropriate miR-122 inhibitor to further investigate the role of miR-122 in the development of inflammation induced by IFN-α, particularly its mechanism regulating IFN-α-associated ISGs (Fig. 4). Initially, the expression of miR-122 was significantly suppressed by the miR-122 inhibitor. Its expression was further reduced following the administration of IFN-α (Fig. 4A). In contrast to the findings observed in the miR-122 mimic-treated group, a notable increase in the mRNA and protein expression of proinflammatory cytokines, chemokines, and fibrogenic genes was observed upon treatment with the miR-122 inhibitor (Fig. 4A–C). Compared with the normal control group, treatment with IFN-α alone significantly suppressed the mRNA expression of ISGs-related genes. This suppression was further enhanced by supplementation with the miR-122 inhibitor, affecting genes such as IFIT1, IFNAR2, and IRF7 (Fig. 4D). Consistently, IFN-α treatment alone significantly increased the levels of p-Jak1 and p-STAT1 in LO2 cells, which was further aggravated by miR-122 inhibitor treatment (Fig. 4E, F). Additionally, the IFIT1 protein level increased under these conditions (Fig. 4E, F), suggesting the crucial role of IFIT1 in alleviating the effects of miR-122 on IFN-α-induced inflammation by regulating the Jak-STAT pathway.

Fig. 4  Effects of miR-122 inhibitor on inflammatory response induced by IFN-α in LO2 cells.

LO2 cells transfected with or without a miR-122 inhibitor for 24 h followed by treatment with 10 ng/mL of IFN-α for an additional 24 h. RNA and proteins were collected to perform qPCR and western blot analysis. (A–D) mRNA expression of miR-122, TNF-α, IL-1β and IL-6 (A), TGF-β1, α-SMA, COL1A1 and PAI-I (B), MCP1, CCL5, CXCL10 and IFIT1 (C), IFNAR1, IFNAR2, IRF3 and IRF7 (D), n = 3. (E, F) Western blotting analysis (E) and quantification data (F) of IFIT1, p-Jak1, Jak1, p-p38, p38, p-p65, p65, p-STAT1, STAT1, and β-actin protein, n = 3. Data are expressed as means ± SEM, *p < 0.05, **p < 0.01 vs. NC-control group; ^#p < 0.05, ^##p < 0.01 vs. miR-122 inhibitor-treated group.

MiR-122 and its target Ifit1 reciprocally regulated inflammatory response

We transfected LO2 cells with an siRNA targeting IFIT1 for knockdown to further confirm the role of IFIT1 in regulating the miR-122-induced amelioration of the inflammatory response (Fig. 5). As shown in Fig. 5, siRNA-mediated knockdown of IFIT1 markedly reduced the mRNA levels of IFIT1 and miR-122, while significantly increasing the mRNA and protein expression of pro-inflammatory and fibrogenic indicators (Fig. 5A–C). However, knocking down IFIT1 failed to affect the IFN-α-activated inflammatory response and hepatic fibrosis (Fig. 5A–C). Additionally, knockdown of IFIT1 effectively decreased the mRNA levels of ISGs, similar to the effects induced by IFN-α on LO2 cells (Fig. 5D). Consistently, Ifit1 deficiency also increased the phosphorylation levels of components of the Jak/STAT pathway (Fig. 5E, F), demonstrating the potential effects of miR-122 and its target IFIT1 on ameliorating the IFN-α-induced inflammatory response.

Fig. 5  Effects of IFIT1 knockdown on inflammatory response induced by IFN-α in LO2 cells.

LO2 cells transfected with or without a siRNA for IFIT1 for 24 h followed by treatment with 10 ng/mL of IFN-α for an additional 24 h. RNA and proteins were collected to perform qPCR and western blot analysis. (A–D) mRNA expression of IFIT1, miR-122, TNF-α and IL-1β (A), TGF-β1, α-SMA, COL1A1 and PAI-I (B), IL-6, MCP1, CCL5 and CXCL10 (C), IFNAR1, IFNAR2, IRF3 and IRF7 (D), n = 3. (E, F) Western blotting analysis (E) and quantification data (F) of IFIT1, p-Jak1, Jak1, p-p38, p38, p-p65, p65, p-STAT1, STAT1, and β-actin protein, n = 3. Data are expressed as means ± SEM, *p < 0.05, **p < 0.01 vs. si-Ctrl group.

IFIT1 overexpression counteracted IFN-α-induced inflammation and hepatic fibrosis

Given the potential protective effects of IFIT1 on the IFN-α-mediated inflammatory response and hepatic fibrosis, we further overexpressed IFIT1 in LO2 cells (Fig. 6). The overexpression efficiency of IFIT1 was confirmed by a significant increase in both the IFIT1 mRNA and protein levels (Fig. 6A, E). Notably, compared with IFN-α treatment alone, IFIT1 overexpression significantly increased the level of miR-122 and counteracted IFN-α-induced increase in the expression of proinflammatory and fibrogenic markers (Fig. 6A–C). In addition, the mRNA levels of ISGs-related genes were also significantly increased by IFIT1 overexpression, which counteracted the increase in the mRNA level of IRF3 following IFN-α treatment (Fig. 6D). In contrast to the results of IFIT1 knockdown, IFIT1 overexpression significantly suppressed the IFN-α-induced activation of the NF-κB p65, MAPK p38, and Jak/STAT signaling pathways (Fig. 6E, F), further illustrating the protective effects of IFIT1 on IFN-α-induced inflammation.

Fig. 6  Effects of IFIT1 overexpression (OE) on inflammatory response induced by IFN-α in LO2 cells.

LO2 cells transfected with or without a IFIT1 plasmid for 24 h followed by treatment with 10 ng/mL of IFN-α for an additional 24 h. RNA and proteins were collected to perform qPCR and western blot analysis. (A–D) mRNA expression of IFIT1, miR-122, TNF-α and IL-1β (A), TGF-β1, α-SMA, COL1A1 and PAI-I (B), IL-6, MCP1, CCL5 and CXCL10 (C), IFNAR1, IFNAR2, IRF3 and IRF7 (D), n = 3. (E, F) Western blotting analysis (E) and quantification data (F) of IFIT1, p-Jak1, Jak1, p-p38, p38, p-p65, p65, p-STAT1, STAT1, and β-actin protein, n = 3. Data are expressed as means ± SEM, *p < 0.05, **p < 0.01 vs. mock-Ctrl group. # means p < 0.05 vs. OE-IFIT1-Ctrl group; ## means p < 0.01 vs. OE-IFIT1-Ctrl group.

Discussion

Patients with coinfections typically experience a faster rate of fibrosis progression to cirrhosis, as well as a greater risk of developing hepatic decompensation and hepatocellular carcinoma, than do those with chronic HBV or HCV infection alone [28, 29]. The advent of DAAs has supplanted the traditional use of IFN-α, revolutionizing the treatment of HCV worldwide [30]. However, concerns regarding HBVr and related immune responses have emerged with the clinical use of DAAs to treat chronic HCV patients [30, 31]. IFN-α, initially identified as an antiviral factor that disrupts viral replication in mammalian cells, is secreted by infected cells to activate the innate immune response and increase cytokine production [32]. Originally identified for its antiviral properties, IFN-α also acts as a multifunctional immunomodulatory cytokine, significantly influencing the cytokine cascade and displaying both pro- and anti-inflammatory characteristics [33]. However, the mechanism of action of IFN-α in hepatic inflammation remains to be elucidated. Pegylated interferon-α (PEG-IFN-α), in which IFN-α is attached to polyethylene glycol (PEG), is an approved therapy for chronic HBV infection [34]. A comparative analysis of 50 HCV-positive subjects revealed a significant increase in transforming growth factor beta (TGF-β) expression after receiving the PEG-IFN-α intervention, suggesting a risk of developing fibrosis after receiving interferon therapy [35]. Elevated expression levels of the proinflammatory cytokine TNF-α and suppressor of cytokine signaling 3 (SOCS-3) were observed in individuals who did not respond to DAAs, suggesting a potential link between the inflammatory response and insulin resistance following anti-HCV therapy [35]. Thus, understanding the potential mechanisms by which IFN-α regulates immune and inflammatory responses is essential for providing theoretical support for future combination therapy with interferons and DAAs in patients with HCV. Moreover, IFN-α administration markedly induces the innate immune response by increasing the phosphorylation levels of STAT1/STAT2 in peripheral blood mononuclear cells (PBMCs) collected from HBV-infected patients [36]. IFN-α exerts antiviral effects by inducing the transcription of ISGs through the activation of the JAK-STAT signaling pathway [11]. In human hepatoma cells, IFN-α promotes the expression and phosphorylation of STAT1 and the expression of ISGs, including Isg15, IFIT1, and MX dynamin-like GTPase 1 (Mx1), in HG23 cells [37]. In the present study, we employed the recombinant IFN-α protein to treat LO2 cells. We found that IFN-α significantly increased the levels of proinflammatory cytokines and chemokines and activated proinflammatory signaling pathways, such as the NF-κB p65, p38 MAPK, and Jak/STAT signaling pathways. Notably, the IFN-α-induced chemokine CXCL10 has been reported to be correlated with the degree of organ damage [38]. Current advances indicate that proinflammatory cytokines can activate hepatic stellate cells (HSCs), the main effector cells involved in hepatic fibrogenesis, thereby perpetuating hepatic inflammation and fibrosis [39]. Our miRNA sequencing results showed that the TGF-β signaling pathway was significantly enriched in the IFN-α-treated group and that the expression of key fibrogenic genes was markedly elevated by IFN-α treatment in LO2 cells. These findings suggest that the IFN-α-induced cytokine storm further triggers hepatic fibrosis. Overall, IFN-α activates inflammatory pathways by inducing the expression of proinflammatory and chemotactic proteins, triggering inflammatory immune responses and subsequent hepatic fibrosis. However, the specific regulatory mechanisms involved remain to be elucidated.

In recent decades, intrahepatic miRNAs have emerged as crucial players associated with a variety of liver disorders, including hepatic inflammation, steatosis, cirrhosis, and liver cancer [16, 40]. Among the most abundant miRNAs, miR-122 is a highly conserved liver-enriched miRNA, representing 70% and 52% of the total hepatic miRNAs in adult mice [41] and humans [42], respectively. Emerging evidence has revealed the noncanonical role of miR-122 in the pathogenesis of HCV infection through its interaction with two seed sequence binding sites located at the 5'-end of the HCV genomic RNA [19]. A preclinical study reported that a locked nucleic acid (LNA)-modified antisense miR-122 oligonucleotide achieved chronic and stable suppression of HCV viremia in chimpanzees [43]. The suppression of target mRNAs containing miR-122 seed sites inhibited interferon-regulated genes and provoked an HCV-induced liver inflammatory response [43]. We conducted miRNA sequencing to elucidate the role of miRNAs in the IFN-α-associated innate immune response and identified miR-122 as the most significantly enriched miRNA in LO2 cells. Notably, its expression was markedly suppressed following IFN-α administration. Several significantly altered ISGs were predicted as potential target genes of miR-122, and the expression of both miRNAs and their target genes was notably enriched in pathways associated with HBV, HCV, and proinflammatory signaling. A previous study demonstrated that the deletion of miR-122 led to hepatosteatosis, hepatic inflammation, and the development of tumors resembling hepatocellular carcinoma (HCC) in mice [18]. MiR-122 knockout mice exhibit hyperactivation of oncogenic pathways and hepatic infiltration of inflammatory cells, which produce protumorigenic cytokines, including IL-6 and TNF [18]. In this study, treatment with the miR-122 mimic notably reduced the expression of proinflammatory cytokines and chemokines induced by IFN-α and significantly suppressed the activation of proinflammatory signaling pathways. Conversely, inhibition of miR-122 effectively activated inflammation and exacerbated the IFN-α-associated inflammatory response. In addition, the upregulation of chemokines caused by miR-122 deficiency led to the recruitment of inflammatory cells within the liver [18]. We observed that elevated levels of chemokines, such as CCL5 and CXCL10, could be directly induced by either miR-122 loss or IFN-α-related inflammatory responses. These findings underscore the regulatory role of chemokines in the effects of miR-122-associated hepatic inflammation.

Our miRNA sequencing screen aimed at identifying target genes that regulate IFN-α-induced inflammation in LO2 cells revealed a negative regulatory role for IFIT1. IFIT1 mediates the negative feedback regulation of virus-triggered induction of type I IFNs via the Jak/STAT signaling pathway [44]. In a previous study, overexpression of IFIT1 significantly inhibited virus-triggered activation of IRF3, NF-κB, and the IFN-β promoter, whereas silencing of IFIT1 abolished these effects [45]. IFIT1 has been traditionally studied as an antiviral factor. However, the depletion of IFIT1 was reported to cause a dramatically increased viral load and subsequent ISG response [45], potentially triggering an inflammatory response. We identified IFIT1 as one of the potential target genes of miR-122, and the miR-122 mimic significantly induced the mRNA and protein expression of IFIT1 in IFN-α-treated LO2 cells. Previously, IFIT1 was shown to downregulate TNF-α expression in LPS-treated macrophages while upregulating IFN-β1 expression, acting downstream of IRF3 activation [15]. Our results consistently showed that IFN-α inhibited the mRNA expression of IFIT1 and downstream IRF3, whereas the miR-122 mimic significantly promoted IFIT1 expression in LO2 cells. The downregulation of the inflammatory response in IFIT1-depleted cells may be attributed to several mechanisms, predominantly involving Myd88-dependent activation of the NF-κB and MAPK pathways in macrophages [46]. In IFIT1-silenced LO2 cells, significant activation of proinflammatory signaling pathways, including the NF-κB p65, MAPK p38, and Jak/STAT pathways, was observed, suggesting the potential anti-inflammatory role of IFIT1 in IFN-α-induced inflammation. Notably, miR-122 deficiency inhibited IFIT1 and triggered inflammation, suggesting a reciprocal regulatory relationship between miR-122 and IFIT1 in mediating interferon-associated inflammation. Conversely, IFIT1 overexpression has been previously reported to suppress LPS-induced inflammation by decreasing the levels of proinflammatory cytokines and inhibiting the NF-κB p65 signaling pathway [47]. Consistent with these findings, we found that IFIT1 overexpression counteracted IFN-α-induced inflammation and hepatic fibrosis. However, further research is needed to elucidate the interaction between miR-122 and IFIT1.

In conclusion, this study elucidated how miR-122 and its target IFIT1 regulate the IFN-α-related inflammatory response (Graphical Abstract). First, miR-122-3p and miR-122-5p_R+1 were downregulated in LO2 cells treated with the recombinant IFN-α protein, based on miRNA sequencing data. The recombinant IFN-α protein markedly induced an inflammatory response in LO2 cells, accompanied by decreased levels of miR-122 and IFIT1. MiR-122 mimic supplementation exacerbated IFN-α-triggered inflammation. Notably, several ISGs, including IFIT1, IRF7, and IFNAR1, which are predominantly enriched in HBV, HCV, and proinflammatory signaling pathways, were identified as target genes of miR-122. Notably, the silencing of either miR-122 or IFIT1 activated inflammatory signaling pathways in a manner comparable to that of IFN-α treatment alone, suggesting the reciprocal regulatory roles of miR-122 and IFIT1 in the interferon-activated inflammatory response. These findings underscore the crucial roles of miR-122 and IFIT1 in maintaining liver homeostasis during interferon intervention, with significant therapeutic implications, including the potential use of miR-122-IFIT1 delivery for HCV-infected patients, and provides potential targets for combination therapy with interferons and DAAs for treating HCV.

Graphical Abstract  This study demonstrates the mechanism underlying the miR-122 and its target, IFIT1, reciprocally regulate the inflammatory response associated with IFN through the Jak/STAT pathway. Recombinant IFN-α protein markedly induced inflammatory response and hepatic fibrosis in LO2 cells through activating NF-κB p65, p38 MAPK, and Jak/STAT signaling pathways. IFN-α administration substantially elevated the mRNA expression of pro-inflammatory cytokines, including TNF-α, IL-1β, IL-6, and MCP1, and chemokines such as CCL5 and CXCL10. The inflammatory response activates the transcriptional level of fibrogenic genes such as TGF-β1, α-SMA, COL1A1 and PAI-1 in LO2 cells. MicroRNA-122 (miR-122) emerged as the most enriched miRNA involved in the pathogenesis of IFN-α-induced inflammatory responses, which were significantly downregulated by IFN-α treatment. IFN-induced proteins with tetratricopeptide repeat 1 (IFIT1) as a potential target gene of miR-122. MiR-122 and its target, IFIT1, reciprocally regulate the inflammatory response.

A major limitation of the current study is the use of an immortalized cell line, which cannot fully capture the IFN-α-associated immune activation and hepatic inflammation observed in humans or in experimental animals. Future investigations are needed to validate the current findings in mouse models and human-derived hepatocytes. In this study, we found that IFN-α promotes hepatic inflammation in LO2 cells, with miR-122 and IFIT1 jointly regulating IFN-α-induced inflammation and hepatic fibrosis. However, future research should focus on the roles and underlying mechanisms of miR-122 and IFIT1 in viral activation responses, providing new theoretical insights into the clinical antiviral effects of IFN-α.

Author Contributions

F.L., B.L., S.X., collected samples, acquired, and analyzed data. F.L., and X.L., wrote the manuscript. Y.N. and X.L., contributed to the study concept, design and revised the manuscript.

Funding

This study was supported by the Zhejiang Provincial Natural Science Foundation (Q21H190009), National Key R&D Program of China (2020YFE0204300).

Disclosure

The authors declare that there are no conflicts of interest.

References

• 1  The Lancet Infectious Diseases (2022) Explaining the unexplained hepatitis in children. Lancet Infect Dis 22: 743.
• 2   Alberts  CJ,  Clifford  GM,  Georges  D,  Negro  F,  Lesi  OA, et al. (2022) Worldwide prevalence of hepatitis B virus and hepatitis C virus among patients with cirrhosis at country, region, and global levels: a systematic review. Lancet Gastroenterol Hepatol 7: 724–735.
• 3   Airewele  NE,  Shiffman  ML (2021) Chronic hepatitis B virus in patients with chronic hepatitis C virus. Clin Liver Dis 25: 817–829.
• 4   Raimondo  G,  Brunetto  MR,  Pontisso  P,  Smedile  A,  Maina  AM, et al. (2006) Longitudinal evaluation reveals a complex spectrum of virological profiles in hepatitis B virus/hepatitis C virus-coinfected patients. Hepatology 43: 100–107.
• 5   Matsuzaki  K,  Murata  M,  Yoshida  K,  Sekimoto  G,  Uemura  Y, et al. (2007) Chronic inflammation associated with hepatitis C virus infection perturbs hepatic transforming growth factor beta signaling, promoting cirrhosis and hepatocellular carcinoma. Hepatology 46: 48–57.
• 6   Fried  MW,  Shiffman  ML,  Reddy  KR,  Smith  C,  Marinos  G, et al. (2002) Peginterferon alfa-2a plus ribavirin for chronic hepatitis C virus infection. N Engl J Med 347: 975–982.
• 7   Ye  J,  Chen  J (2021) Interferon and hepatitis B: current and future perspectives. Front Immunol 12: 733364.
• 8   Pestka  S,  Langer  JA,  Zoon  KC,  Samuel  CE (1987) Interferons and their actions. Annu Rev Biochem 56: 727–777.
• 9   Ji  L,  Li  T,  Chen  H,  Yang  Y,  Lu  E, et al. (2023) The crucial regulatory role of type I interferon in inflammatory diseases. Cell Biosci 13: 230.
• 10   Hoffmann  HH,  Schneider  WM,  Rice  CM (2015) Interferons and viruses: an evolutionary arms race of molecular interactions. Trends Immunol 36: 124–138.
• 11   Levy  DE,  Darnell  JE Jr (2002) Stats: transcriptional control and biological impact. Nat Rev Mol Cell Biol 3: 651–662.
• 12   Xu  C,  Guo  H,  Pan  XB,  Mao  R,  Yu  W, et al. (2010) Interferons accelerate decay of replication-competent nucleocapsids of hepatitis B virus. J Virol 84: 9332–9340.
• 13   Raychoudhuri  A,  Shrivastava  S,  Steele  R,  Kim  H,  Ray  R, et al. (2011) ISG56 and IFITM1 proteins inhibit hepatitis C virus replication. J Virol 85: 12881–12889.
• 14   Pei  R,  Qin  B,  Zhang  X,  Zhu  W,  Kemper  T, et al. (2014) Interferon-induced proteins with tetratricopeptide repeats 1 and 2 are cellular factors that limit hepatitis B virus replication. J Innate Immun 6: 182–191.
• 15   John  SP,  Sun  J,  Carlson  RJ,  Cao  B,  Bradfield  CJ, et al. (2018) IFIT1 Exerts Opposing Regulatory Effects on the Inflammatory and Interferon Gene Programs in LPS-Activated Human Macrophages. Cell Rep 25: 95–106.E6.
• 16   Lodish  HF,  Zhou  B,  Liu  G,  Chen  CZ (2008) Micromanagement of the immune system by microRNAs. Nat Rev Immunol 8: 120–130.
• 17   Cheung  O,  Puri  P,  Eicken  C,  Contos  MJ,  Mirshahi  F, et al. (2008) Nonalcoholic steatohepatitis is associated with altered hepatic MicroRNA expression. Hepatology 48: 1810–1820.
• 18   Hsu  SH,  Wang  B,  Kota  J,  Yu  J,  Costinean  S, et al. (2012) Essential metabolic, anti-inflammatory, and anti-tumorigenic functions of miR-122 in liver. J Clin Invest 122: 2871–2883.
• 19   Jopling  CL,  Yi  M,  Lancaster  AM,  Lemon  SM,  Sarnow  P (2005) Modulation of hepatitis C virus RNA abundance by a liver-specific MicroRNA. Science 309: 1577–1581.
• 20   Hu  Y,  Peng  X,  Du  G,  Zhang  Z,  Zhai  Y, et al. (2022) MicroRNA-122-5p inhibition improves inflammation and oxidative stress damage in dietary-induced non-alcoholic fatty liver disease through targeting FOXO3. Front Physiol 13: 803445.
• 21   Lu  X,  Bao  X,  Huang  Y,  Qu  Y,  Lu  H, et al. (2009) Mechanisms of cytotoxicity of nickel ions based on gene expression profiles. Biomaterials 30: 141–148.
• 22   Ma  L,  Lyu  W,  Song  Y,  Chen  K,  Lv  L, et al. (2023) Anti-inflammatory effect of clostridium butyricum-derived extracellular vesicles in ulcerative colitis: impact on host microRNAs expressions and gut microbiome profiles. Mol Nutr Food Res 67: e2200884.
• 23   Wang  Y,  Chang  L,  Zhai  J,  Wu  Q,  Wang  D, et al. (2017) Generation of carbamoyl phosphate synthetase 1 reporter cell lines for the assessment of ammonia metabolism. J Cell Mol Med 21: 3214–3223.
• 24   Ni  Y,  Zheng  L,  Zhang  L,  Li  J,  Pan  Y, et al. (2024) Spermidine activates adipose tissue thermogenesis through autophagy and fibroblast growth factor 21. J Nutr Biochem 125: 109569.
• 25   Ma  L,  Shen  Q,  Lyu  W,  Lv  L,  Wang  W, et al. (2022) Clostridium butyricum and its derived extracellular vesicles modulate gut homeostasis and ameliorate acute experimental colitis. Microbiol Spectr 10: e0136822.
• 26   Ni  L,  Zhuge  F,  Yang  S,  Ma  L,  Zheng  A, et al. (2021) Hydrolyzed chicken meat extract attenuates neuroinflammation and cognitive impairment in middle-aged mouse by regulating M1/M2 microglial polarization. J Agric Food Chem 69: 9800–9812.
• 27   Ni  Y,  Ni  L,  Ma  L,  Wang  Z,  Zhao  Y, et al. (2021) Neuroprotective effects of ProBeptigen/CMI-168 on aging-induced cognitive decline and neuroinflammation in mice: a comparison with essence of chicken. Acta Biochim Biophys Sin (Shanghai) 53: 419–429.
• 28   Crockett  SD,  Keeffe  EB (2005) Natural history and treatment of hepatitis B virus and hepatitis C virus coinfection. Ann Clin Microbiol Antimicrob 4: 13.
• 29   Konstantinou  D,  Deutsch  M (2015) The spectrum of HBV/HCV coinfection: epidemiology, clinical characteristics, viralinteractions and management. Ann Gastroenterol 28: 221–228.
• 30   Bhattacharya  D,  Aronsohn  A,  Price  J,  Lo Re  V, AASLD-IDSA HCV Guidance Panel (2023) Hepatitis C guidance 2023 update: AASLD-IDSA recommendations for testing, managing, and treating hepatitis C virus infection. Clin Infect Dis 2023: ciad319.
• 31   Ghany  MG,  Morgan  TR, AASLD-IDSA Hepatitis C Guidance Panel (2020) Hepatitis C guidance 2019 update: American association for the study of liver diseases-infectious diseases society of America recommendations for testing, managing, and treating hepatitis C virus infection. Hepatology 71: 686–721.
• 32   Ivashkiv  LB,  Donlin  LT (2014) Regulation of type I interferon responses. Nat Rev Immunol 14: 36–49.
• 33   Tilg  H (1997) New insights into the mechanisms of interferon alfa: an immunoregulatory and anti-inflammatory cytokine. Gastroenterology 112: 1017–1021.
• 34   Lucifora  J,  Xia  Y,  Reisinger  F,  Zhang  K,  Stadler  D, et al. (2014) Specific and nonhepatotoxic degradation of nuclear hepatitis B virus cccDNA. Science 343: 1221–1228.
• 35   Farooq  M,  Rauf  M,  Tahir  F,  Manzoor  S (2021) A comparative analysis of interferons and direct-acting antivirals on the expression of genes involved in hepatitis C pathogenesis. J Med Virol 93: 6241–6246.
• 36   Nishio  A,  Bolte  FJ,  Takeda  K,  Park  N,  Yu  ZX, et al. (2021) Clearance of pegylated interferon by Kupffer cells limits NK cell activation and therapy response of patients with HBV infection. Sci Transl Med 13: eaba6322.
• 37   Yu  P,  Li  Y,  Wang  Y,  Peppelenbosch  MP,  Pan  Q (2020) Lipopolysaccharide restricts murine norovirus infection in macrophages mainly through NF-kB and JAK-STAT signaling pathway. Virology 546: 109–121.
• 38   Geneva-Popova  MG,  Popova-Belova  SD,  Gardzheva  PN,  Kraev  KI (2022) A study of IFN-alpha-induced chemokines CCL2, CXCL10 and CCL19 in patients with systemic lupus erythematosu. Life (Basel) 12: 251.
• 39   Hammerich  L,  Tacke  F (2023) Hepatic inflammatory responses in liver fibrosis. Nat Rev Gastroenterol Hepatol 20: 633–646.
• 40   Bandiera  S,  Pfeffer  S,  Baumert  TF,  Zeisel  MB (2015) miR-122—a key factor and therapeutic target in liver disease. J Hepatol 62: 448–457.
• 41   Lagos-Quintana  M,  Rauhut  R,  Yalcin  A,  Meyer  J,  Lendeckel  W, et al. (2002) Identification of tissue-specific microRNAs from mouse. Curr Biol 12: 735–739.
• 42   Girard  M,  Jacquemin  E,  Munnich  A,  Lyonnet  S,  Henrion-Caude  A (2008) miR-122, a paradigm for the role of microRNAs in the liver. J Hepatol 48: 648–656.
• 43   Lanford  RE,  Hildebrandt-Eriksen  ES,  Petri  A,  Persson  R,  Lindow  M, et al. (2010) Therapeutic silencing of microRNA-122 in primates with chronic hepatitis C virus infection. Science 327: 198–201.
• 44   Terenzi  F,  Hui  DJ,  Merrick  WC,  Sen  GC (2006) Distinct induction patterns and functions of two closely related interferon-inducible human genes, ISG54 and ISG56. J Biol Chem 281: 34064–34071.
• 45   Li  Y,  Li  C,  Xue  P,  Zhong  B,  Mao  AP, et al. (2009) ISG56 is a negative-feedback regulator of virus-triggered signaling and cellular antiviral response. Proc Natl Acad Sci U S A 106: 7945–7950.
• 46   Suslov  A,  Boldanova  T,  Wang  X,  Wieland  S,  Heim  MH (2018) Hepatitis B virus does not interfere with innate immune responses in the human liver. Gastroenterology 154: 1778–1790.
• 47   Jiang  W,  Zhang  Y,  Sheng  Y,  Liu  M,  Du  C, et al. (2023) Overexpression of IFIT1 protects against LPS-induced acute lung injury via regulating CCL5-p65NF-kappaB signaling. Int Immunopharmacol 114: 109485.