Influence of polymorphisms in TNF-α and IL1β on susceptibility to alcohol induced liver diseases and therapeutic potential of miR-124-3p impeding TNF-α/IL1β mediated multi-cellular signaling in liver microenvironment

Background and aims Alcoholic liver disease (ALD) is the leading cause of the liver cirrhosis related death worldwide. Excessive alcohol consumption resulting enhanced gut permeability which trigger sensitization of inflammatory cells to bacterial endotoxins and induces secretion of cytokines, chemokines leading to activation of stellate cells, neutrophil infiltration and hepatocyte injury followed by steatohepatitis, fibrosis and cirrhosis. But all chronic alcoholics are not susceptible to ALD. This study investigated the causes of differential immune responses among ALD patients and alcoholic controls (ALC) to identify genetic risk factors and assessed the therapeutic potential of a microRNA, miR-124-3p. Materials and methods Bio-Plex Pro™ Human Chemokine analysis/qRT-PCR array was used for identification of deregulated immune genes. Sequencing/luciferase assay/ELISA detected and confirmed the polymorphisms. THP1 co-cultured with HepG2/LX2/HUVEC and apoptosis assay/qRT-PCR/neutrophil migration assay were employed as required. Results The combined data analysis of the GSE143318/Bio-Plex Pro™ Human Chemokine array and qRT-PCR array revealed that six genes (TNFα/IL1β/IL8/MCP1/IL6/TGFβ) were commonly overexpressed in both serum/liver tissue of ALD-patients compared to ALC. The promoter sequence analysis of these 6 genes among ALD (n=322)/ALC (n=168) samples revealed that only two SNPs, rs361525(G/A) at -238 in TNF-α/rs1143627(C/T) at -31 in IL1β were independently associated with ALD respectively. To evaluate the functional implication of these SNPs on ALD development, the serum level of TNF-α/IL1β was verified and observed significantly higher in ALD patients with risk genotypes TNF-α-238GA/IL1β-31CT+TT than TNF-α-238GG/IL1β-31CC. The TNF-α/IL1β promoter Luciferase-reporter assays showed significantly elevated level of luciferase activities with risk genotypes -238AA/-31TT than -238GG/-31CC respectively. Furthermore, treatment of conditioned medium of TNF-α/IL1β over-expressed THP1 cells to HepG2/LX2/HUVEC cells independently showed enhanced level of ER stress and apoptosis in HepG2/increased TGFβ and collagen-I production by LX2/huge neutrophil infiltration through endothelial layer. However, restoration of miR-124-3p in THP1 attenuated such inter-cellular communications and hepatocyte damage/collagen production/neutrophil infiltration were prohibited. Target analysis/luciferase-reporter assays revealed that both TNF-α/IL1β were inhibited by miR-124-3p along with multiple genes from TLR4 signaling/apoptosis/fibrogenesis pathways including MYD88, TRAF3/TRADD, Caspase8/PDGFRA, TGFβR2/MCP1, and ICAM1 respectively. Conclusion Thus, rs361525(G/A) in TNF-α and rs1143627(C/T) in IL1β gene may be used as early predictors of ALD susceptibility among East Indian population. Impeding overexpressed TNF-α/IL1β and various genes from associated immune response pathways, miR-124-3p exhibits robust therapeutic potential for ALD patients.


Introduction
Alcohol is one of the oldest environmental insults that causes chronic liver diseases with a wide spectrum of manifestations including steatosis or fatty liver to steatohepatitis, fibrosis and cirrhosis (1,2).Although the early stages of ALD are reversible with cessation of alcohol, cirrhosis or severe alcoholic hepatitis often lead to death (3).Despite significant advancement in understanding the disease biology, lack of any early predictor for high-risk individuals and unavailability of FDA approved therapy for end-stage liver diseases, ALD is still a major risk of liver-related mortality worldwide.
ALD is associated with chronic inflammatory responses due to changes in intestinal microbiome composition and infiltration of pathogen associated molecular patterns (PAMPS) such as bacterial lipo-polysaccharides (LPS), bacterial DNA into the portal circulation.These activate Kupffer cells (KC) through pattern recognition receptor (PRRs), mostly TLR4 and TLR9 and produce pro-inflammatory cytokines such as tumour necrosis factor TNF-a, IL-1b, IL-6, GM-CSF and chemokines such as MCP-1, MIP-1a, etc. through NF-kb (4,5).Subsequently, intracellular signaling begins through both MyD88 dependent IRAK4/IRAK1-TRAF6-MAPK-NF-kb-TNFa axis and MyD88 independent TRAM-TRIF-TRAF3/ TRAF6-IRF3/NF-kb pathways (6).This TNF-a signaling through TLR4 axis in alcoholic hepatitis has been markedly appreciated but anti-TNF-a therapy often associated with hepatotoxicity (7), and bacterial/fungal infection (8).Diverse attempts have been executed to develop a potential molecule inhibiting inflammatory responses to alcohol using mice models.Seminal studies with knock out mice of MCP1/IRF3/TLR4/caspase-I have revealed that these mice are protected from various stages of ALD including steatosis, steatohepatitis, and fibrosis as inflammasome dependent signaling in KCs is inhibited (9)(10)(11).Albeit, the IL1 receptor antagonist showed promising results (12,13), further studies are required prior to its therapeutic application.
In recent decades, the contribution of microRNAs (miRNAs) in physiological and pathological liver function; its potential as diagnostic marker and therapeutic target for liver disease management have been established (27).The role of deregulated miRNAs in hepatocytes and KCs in rodents fed with alcohol depicted MiR-125b, let7 and miR-21 restrict functions of TNF-a, IL6, and TLR4 pathways by blocking NF-kb mediated IL10 production (28-30).Chunyan Ma et al. have reported that miR-124 attenuates immune signaling in alveolar epithelial cells by repressing multiple proteins in TLR6 signaling pathways including TLR6/MyD88/TRAF6 (31).In light of these findings, using clinical specimens, and in vitro experiments, this study was intended to assess the genetic makeup of deregulated immune genes considering alcoholic individuals with single ethnic group from India and its impact on the expression of genes to gain an insight into the pathogenesis of the disease.Subsequently, therapeutic potential of miR-124-3p was evaluated considering its binding sites on the 3'UTR of TNF-a/IL-1b/IL8/MCP1 along with the genes from TLR4 pathways MYD88/TRAF3/TRADD/TRAF6, fibrotic growth factor receptors TGFBR2/PDGFRA and leucocyte adhesion protein ICAM1.

Inclusion of samples
A total of 490 consecutive subjects belonging to a single ethnic group (Bengali) with history of significant ethanol consumption (more than 80 g/day for more than 10 years) (16, 19) was enrolled for the study from the indoor and outdoor of Department of Hepatology at IPGME&R, Kolkata, East India and Department of Medicine at AGMC, Tripura, North-East India.Diagnosis of ALC (n=168) was based on ultra-sonographic evidence of normal liver with normal level of ALT/AST, while cirrhotic ALD patients (n=322) were diagnosed with portal hypertension, esophageal/ gastric varices with/without ascites and steatohepatitis patients with fatty liver, elevated level of ALT but no histological sign of cirrhosis.Following the same criteria, 100 ALD patients were included in the validation cohort.Healthy individuals (n=10) having normal ALT/AST were also included.
Although samples were included from two different regions of India having similar ethnicity, both the groups exhibited same pattern of allelic distribution, hence samples were clubbed.

Exclusion of samples
Subjects from different ethnic background, positive for anti-HBsAg, Anti-HCV, or HIV seropositive, having co-morbidities like diabetes mellitus, and unwilling to comply with the study protocol were excluded.

Collection of blood
Blood (5ml) was collected from every participant in EDTA-vial and used for isolation of chromosomal DNA (32), PBMC and neutrophil using histopaque 1077 (Sigma).Demographic, clinical and biochemical data of non-alcoholic normal, ALC and ALD patients are presented in Tables 1A and 1B.

Non-alcoholic steatohepatitis
Non-alcoholic steatohepatitis (NASH) patients (n=50) with comparable age, sex and having no other liver diseases as verified histologically were included from the Department of Hepatology, IPGME&R, Kolkata, India (Supplementary Table 1).

Collection of liver tissues
Explanted liver tissues were collected from patients undergone orthotopic liver transplantation due to alcoholic cirrhosis (n=4) from Centre for Liver and Biliary Sciences, Indraprastha Apollo Hospital, New Delhi, India and confirmed with histological, and radiological evidences.
Normal liver tissues (n=10) were collected from gastrointestinal surgery clinic of IPGME&R, Kolkata, during cholecystectomy of gall bladder carcinoma (GBC) patients as routine analysis to test liver metastasis.Liver tissues with normal architecture (n=4) were included in the study after histological verification.Tissues were collected in RNA later (ThermoFisher, Cat.#AM7020) and preserved at -80 0 C for future use.

Transcriptome data analysis
RNAseq data of alcoholic hepatitis (n=5) vs. normal liver tissue (n=5) (GSE143318) was retrieved from https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE143318 and the data was analyzed using DEseq2 package to obtain differential gene expression (DEG).DEG with log 2 fold change>2 and P adjusted value <0.05 were considered for further pathway analysis using Kyoto Encyclopedia of Genes and Genomes (KEGG).

Bio-Plex Pro ™ Human chemokine analysis
A BioPlex ProHuman chemokine Screening Panel, 48 plex (Bio-Rad, # 120072831) was used to detect the levels of forty serum chemokines following the manufacturer's protocol at National Institute of Biomedical Genomics, Kalyani, India.The data were analyzed using BioPlex® 200 Multiplexing Platform.DGE were performed as mentioned earlier and subjected to KEGG pathway analysis.

Identification of transcription factors binding to the promoter
The total promoter sequence of TNF-a and IL1ß were obtained from the University of California Santa Cruz (UCSC) Genome Browser, selecting the Human GRCh37/hg19 genome assembly.The entire promoter sequence was scanned using TFBIND tool for the transcription factor binding site.ChIP Seq (https://genome.ucsc.edu/)data was used for validation.

RNA isolation, reverse transcription and quantitative real-time PCR
Liver tissue (1mg) was homogenized and RNA was isolated using Trizol (ThermoFisher, # 15596026).About 2.5mg/500 ng of total RNA was used to synthesize cDNA for mRNA/miRNA using Revertaid Reverse Transcriptase (ThermoFisher, #MAN0012885)/ miSCRIPT PCR starter kit (Qiagen, #218193) respectively.Expression of immune related genes was assessed in ABI QuantStudio 7 real-time PCR machine using SYBR green (Roche).Only upregulated genes in each sample were further considered for genotyping.Primers used for quantification of mRNAs and miRNAs are presented in Supplementary Table 2A.

Chromosomal DNA preparation and method of genotyping
Chromosomal DNA was isolated using salting out method from 3ml of EDTA-blood (14, 32).Variations in the promoter reǵion was determined by sequencing with Big-Dye Terminator v3.1 (ThermoFisher, #4337455) on ABI Prism 3100 Genetic Analyzer followed by multi-sequence alignment using Sequencher software.
Lipofectamine 2000 (ThermoFisher, #11668019) and x-tremeGENE HP transfection reagents (Sigma, #6366236001) were used for transfection in different cell lines as required.THP1 cells were treated with LPS (100ng/ml) for 12 hours after transfection.HepG2/THP1/LX2/HUVEC were either directly transfected with required plasmids or incubated with conditioned media (CM) of transfected THP1 cells (1:1 ratio) for 48 hrs.Each experiment was performed in duplicate and repeated three times.

ELISA of secreted cytokines
Secreted levels of TNF-a/IL1b in the serum of ALD/ALC individuals, and in cell culture supernatant were determined using ELISA following the manufacturer's protocol (Ray BioTech, #P01375 and Abclonal, #RK00001).

LPS treatment of PBMC
About 2x10 6 PBMCs were seeded on 6 well plates.After 24 hours, cells were either stimulated with LPS (100ng/ml) for 6 hours or left untreated and subsequently total RNA was isolated.

Apoptosis assay
Apoptotic cells were quantified in BD-FACS VERSE using Annexin V and PI staining kit (Invitrogen, #556547) after 48 hours of transfection following manufacturers' protocol.Early (Annexin v-FITC + ) and late apoptotic cells (Annexin V-FITC + PI + ) were considered for quantification of total apoptotic cells.

Trans-endothelial migration assay
HUVEC cells (5x10 4 cells/well) were seeded on the collagen precoated upper well of the Boyden Chamber (Cell BioLabs, 3mm pore size, #CBA-103) and either directly transfected or conditioned media (CM) of pTNF-a/Anti-TNF-a anti-miR-124-3p oligo transfected in THP1 cells (1:1) were added.The lower chamber was filled with 30% FBS containing media and freshly isolated neutrophil (10 6 cells) was added on the upper chamber and number of cells migrated to the lower chamber was counted at different time points (2-24 hrs) using hemocytometer.

Statistical analysis
All the statistical tests were performed using SPSS version 10.0 and Graphpad prism.Hardy-Weinberg Equilibrium test was done for each SNP to determine whether the SNP present in this population is in equilibrium or influenced by any evolutionary forces.The association of genotype and allele frequencies were calculated using Pearson's chisquare test (14, 19).Unpaired two tailed Student's t-test was performed for comparison of expression data.T-test and Mann Whitney U test was employed for biochemical parameters as required.The parameters were presented either as mean ± SD (for normally distributed parameters) or as median and minimum to maximum distribution with range of the datasets (for skewed distributed parameters).Individual predictors were determined using multiple logistic regression analysis with significantly associated SNPs in Univariate analysis.P-values were corrected using Benjamini−Hochberg multiple testing correction.P adjusted <0.05 was considered as significant.

Demographic, clinical and biochemical characteristics of the study subjects
This study has included 490 individuals in discovery cohort (ALD=322 and ALC=168) and 150 individual in validation cohort (ALD=100 and NASH=50).In addition, 10 healthy individuals were incorporated.Explanted Liver tissues were also collected from four ALD patients.Liver biopsies from gall bladder carcinoma patient (n=10) with normal histology were considered as normal (n=4).

Selection of overexpressed ALD susceptible immune genes
Despite having similar amount of alcohol consumption, all alcoholics are not susceptible to ALD suggesting genetic variations play crucial role here.To identify the genes responsible for this differential behavior, overexpressed genes in ALD were identified after analysis of the transcriptome data, GSE 143318 (Alcoholic hepatitis vs. Control).It was followed by KEGG pathway analysis and revealed that the chemokine signaling pathways were the most upregulated pathways in ALD (Figure 1A).Furthermore, our Bio-Plex Pro ™ Human Chemokine Panel, 40-Plex assay with serum of ALD (n=28) and ALC (n=20) individuals also disclosed cytokine-cytokine receptor signaling and chemokine signaling pathways were the highest upregulated pathways in ALD (Figure 1B).Thus, we focused on upregulated immune genes only and genes were selected after validation in the liver tissue of ALD (n=4) and ALC (n=4) using customized qRT-PCR expression array which included immune genes associated to alcohol induced steatohepatitis and cirrhosis such as TLR4, IFN-a, CD14, MCP1, IL8, TGF-b, TNF-a, IL6, IL18, IFN-g, IL22, IL17, IL10, CCR2 and complements C5 and C3 as found in the published literatures (19,34,35) and in the above two analyses.After comparative analysis of the above two arrays, six genes TNF-a, IL6, MCP1, IL1b (P value<0.05) and TGF-b, IL8 (border line significant in liver tissue) were found up-regulated in both the serum and liver tissue samples of all the ALD patients tested compared to ALC (Figures 1C, D), which suggested their significant contribution in the ALD development.

Association of SNPs in the immune genes with ALD
Now, to find the reason of ALD development in only 10-30% of chronic alcoholics, the serum cytokine data of all the six genes in ALC group was carefully investigated (Figure 1C) and observed that

Impact of SNPs on the expression of TNF-a and IL1b genes in ALD Patients
Now, to determine the impact of two SNPs on the expression of TNF-a and IL1b, normal individuals carrying the above SNPs were tested by qRT-PCR and ELISA.To our surprise, the expression of TNF-a and IL1b were noted significantly higher in the liver tissue of the normal individuals carrying rs361525GA at -238 (n=6) than GG (n=9) and rs1143627CT at -31(n=9) than CC (n=6) genotype respectively (Figures 2A, B).ELISA data of normal individuals with risk allele rs361525GA and rs1143627CT also showed higher level of TNF-a and IL1b than GG and CC allele respectively (Figures 2C,  D).These data suggest that aforementioned SNPs in the promoter of TNF-a and IL1b might be contributed to the disease pathogenesis in susceptible individuals.
Next, to understand the mechanism of overexpression of TNFa and IL1b in susceptible individual, UCSC-ChIP-seq data was analyzed and observed a NFkb binding site at -239 position in the TNF-a promoter.Thus, Luciferase reporter assay with the TNF-a promoter constructs carrying either -238GG or -238AA genotype  was performed in presence or absence of LPS and p65-NFkb subunit.It was observed that genotype "AA" showed higher luciferase activity than GG (Figures 2E, F) indicating that the presence of "A" at -238 loci might enhance the binding affinity of NFkb to that site.Similarly, PBMC isolated from normal individual having either -31C/C or -31C/T allele in IL1b and subsequent qRT-PCR data depicted that the individual with -31C/T allele had significantly higher expression of IL1b than -31C/C at IL1b loci (T/T allele was absent in this population), in both presence or absence of LPS (Figure 2G).Next, when HepG2 cells were transfected with either pIL1b-31TT or pIL1b-31CC plasmids, the expression of IL1b was noted higher in presence pIL1b-31TT than pIL1b-31CC by qRTPCR and ELISA as well (Figure 2H).It is worthy to be noted that a new TATA box appears when the C allele replaces with T allele at -31 loci of IL1b promoter.So, binding of more TATA box binding protein plausibly induced the expression of IL1b in CT genotype carrying individuals.
In 2000, Hill et al, have reported that alcohol/LPS stimulates the expression of the nuclear factor NFkb, which induces synthesis of TNF-a (36) and TNF-a and IL1b trigger a wide range of intracellular signaling pathways, which require IL6, MCP1, ICAM1 and TGF-b.To our surprise, multiple NFkb binding sites were noted in the promoter regions of all these six genes which were significantly altered in our cohort (Supplementary Figure 1A).The data was validated in THP1 cells treated with LPS and observed overexpression of TNF-a, IL1b, MCP1, IL8 and NFkb while the expression of each gene was also synergistically increased upon overexpression of TNF-a in HepG2 cells treated with LPS (Supplementary Figures 1B, C).
Thus, our data suggest that LPS induces expression of TNF-a and/or IL1b and individual carrying either rs361525G/A in TNF-a or rs1143627C/T in IL1b produces excessive amount of TNF-a and IL1b in the liver milieu.Now, TNF-a signaling through TLR4 pathway activates multiple intracellular as well as intercellular signaling cascades that lead to progression of liver diseases from steatosis to steatohepatitis, fibrosis and cirrhosis (13).

Selection of miR-124-3p that targets both TNF-a and IL1b
In purpose to abolish the effect of increased level of TNF-a and IL1b upon alcohol exposure, extensive bioinformatics analysis was performed using Targetscan, miRDB and miRwalk to select a common miRNA targeting both the genes.Here, a few more selection criteria were employed such as the selected miRNA was not abundant in the liver, and it was unaltered upon alcohol exposure.Following above criteria, miR-124-3p was selected which was an anti-inflammatory miRNA.The read count of miR-124-3p was evaluated from next generation sequencing data of ALD vs. normal liver tissue and it was quite low (Read Count<5, data not shown).
Next, the data obtained in bioinformatics analysis was validated by 3'-UTR-luciferase reporter assays using both wildtype (WT) and miR binding site mutant (MT) 3'UTR-Luciferase constructs.Thus, HepG2 cells were co-transfected with premiR-124 and WT/MT 3'UTR-luciferase constructs of TNF-a and IL1b.After 48h of transfection, a significant reduction in the luciferase activity was observed with WT construct and with increasing doses of miR-124-3p while the MT-luciferase constructs showed no response to miR-124-3p.Furthermore, ELISA data with the conditioned media (CM) of miR-124-3p overexpressed HepG2 cells depicted that the restoration of miR-124-3p in HepG2 suppressed the level of TNF- Frontiers in Immunology frontiersin.orga and IL1b by 2.5 folds and 4 folds respectively (Figures 3A-F).So, these data clearly suggested that the binding of miR-124-3p the 3'UTR of TNF-a and IL1b.

Effect of miR-124-3p on TNF-a and IL1b induced ER stress and apoptotic cell death
Upon alcohol consumption, the gut derived bacterial endotoxin LPS mediates inflammation through CD14 and TLR4 to initiate MyD88 dependent and TRIF signaling cascade to activate NF-kB which regulates TNF-a and IL1b secretion from activated Kupffer Cells (KCs) (11).TNF-a binds to the TNF-a receptor (TNFR1) on the hepatocytes and activates TNFa receptor-associated death domain (TRADD) mediated extrinsic apoptosis pathway (37).Here, HepG2 cells were incubated with the CM of the LPS treated THP1 cells transfected with pTNF-a/Anti-TNF-a oligo/pre-miR-124/anti-miR-124-3p and apoptotic death of HepG2 cells was quantified in FACS acquiring AnnexinV-FITC + /AnnexinV-FITC + PI + cells.Apoptotic cell death was noted higher in presence of TNF-a compared to the mock while it was reduced upon treatment with anti-TNF-a/miR-124 and anti-miR-124-3p treatment showed opposite data (Figure 4A).This experiment was also repeated in HepG2 cells directly transfected with the cDNA of TNF-a/pIL1b-CC(low IL1b)/pIL1b-TT(high IL1b)/pre-miR-124 independently in presence of LPS and observed higher apoptosis level upon overexpression of both TNFa, and pIL1b-TT than mock/pIL1b-CC and restoration of miR-124 in the milieu also suppressed the apoptotic death (Figure 4B).
Apart from TNFR1 mediated cell death, Alcohol-induced endoplasmic reticulum (ER) stress is now established as an important contributor to apoptotic cell death in ALD (38).Thus, expression of major ER chaperon protein GRP78, two transmembrane signaling molecules ATF6 and IRE1 were quantified in HepG2 cells treated with the CM of TNF-a cDNA, Anti-TNF-a, pre-miR-124, anti-miR-124-3p transfected THP1 cells or HepG2 cells were directly transfected with cDNA of TNFa, pIL1b-CC(low IL1b)/pIL1b-TT(high IL1b) and pre-miR-124.All the ER stress markers were significantly overexpressed in the presence of TNF-a and pIL1b-TT compared to the vector and pIL1b-CC which were dropped upon treatment with either anti-TNF-a or miR124 and again re-established upon anti-miR-124-3p treatment (Figures 4C, D).

Effect of miR-124-3p on TNF-a and IL1b induced neutrophil infiltration through endothelial barrier
Neutrophil infiltration in the liver is a hallmark of alcoholic hepatitis.The chemokines/cytokines such as IL8, MCP1, MIP2 etc. released by the LPS exposed KCs behave as chemotactic molecule for neutrophils to migrate to the site of inflammation (40).Thus, to mimic the milieu, HUVEC cells seeded on the collagen-coated upper well of Boyden chamber was treated with the CM of TNF-a cDNA/Anti-TNF-a/pre-miR-124/Anti-miR-124-3p transfected THP1 cells or directly transfected with cDNA of TNF-a/pIL1b-CC (low IL1b)/pIL1b-TT (high IL1b)/pre-miR-124 and freshly isolated neutrophils were added on top of it.Infiltrated neutrophils were counted in the lower chamber and found higher in TNF-a/IL1b overexpressed condition than the controls which was decreased upon treatment with anti TNF-a/miR-124 and reversed when treated with Anti-miR-124-3p (Figure 5C).Similar data was obtained upon direct transfection of plasmids in HUVEC cells (Figure 5D).

Mechanism of function of miRNA-124-3p
Next to understand the mechanism of function of miR-124-3p, the other target genes from TLR4 signaling axes were retrieved using Bioinformatics analysis.It is noteworthy to be mentioned that miR-124-3p was found to target various genes involved in LPS mediated inflammatory signaling pathways such as MyD88, TRAF6 and TRAF3; TRADD, Caspase8; IL8, MCP1, ICAM1 and TGFBR2 and PDGFRA (Supplementary Table 3).The data was validated by 3'-UTR luciferase assays with WT/MT luciferase constructs of each target gene and the data has confirmed the binding of miR-124-3p to TRADD, Caspase8, PDGFRA, TGFbR2, IL8, ICAM1, and MCP1 (Figure 6).Binding of miR-124-3p to the 3'UTR of other genes has been reported (31).
Cumulatively, these data suggest that alcohol sensitizes liver macrophages to secrete TNF-a and IL1b in liver microenvironment.Failure to control such situation triggers vicious activation of downstream cytokine cascade through MyD88 dependent and independent pathways leading to hepatocyte death, stellate cell activation and neutrophil infiltration.This causes progression of fibrosis and cirrhosis.Restoration of miR-124-3p in this milieu may resist the intercellular communications and withheld multiple downstream activation pathways suggesting its robust therapeutic potential for inflammatory diseases including ALD (Figure 7).

Discussion
study has presented several important findings.Firstly, a genetic study was conducted to determine the factors responsible for the susceptibility of only 10-30% of chronic alcoholics to the development of ALD and a novel approach was employed in the search of the risk factors for ALD.Unlike other association study, GSE143318 dataset, plasma cytokine profiling and a tissue qRT-PCR array of immune genes were used and only differentially expressed cytokines with high fold alteration both in the liver tissues and serum of the ALD patients were considered for this study.Among the six overexpressed cytokines, only two sequence variations were noted in the promoter region of TNF-a (rs361525G/A) and IL1b (rs1143627C/ T) genes with higher frequency among ALD patients than ALC.Enrichment of TNF-a and IL1b in the liver microenvironment is associated with these variations independently.However, several in vitro assays were performed to comprehend the mechanism of development of ALD upon overproduction of TNF-a and IL1b.Lastly, a novel bioinformatics approach was employed to identify a common miRNA, which not only targeted TNF-a and IL1b genes but also several other cytokines, receptors and cargo molecules involved in LPS mediated TLR4 inflammatory signaling pathways.Binding of the miRNA to each of the target was verified by series of in vitro assays.
Unlike other complex genetic diseases, chronic ethanol drinkers are not always susceptible to develop ALD, although steatosis or fatty liver is commonly observed.Only one-third of the chronic drinkers develop steatohepatitis.Hence, the complex interplay between environment and host genetic makeup is the likelihood of ethanol dependence and also disease development.In this regard, numerous case-control and genome-wide association studies (GWAS) have identified and independently confirmed association of polymorphism(s) in myriad of genes including ethanol metabolism (ADH, ALDH, CYP2E1) (14, 15), oxidative stress response (GSTT1, GSTM1, MnSOD) (18), immune response (TNF-a, IL1b, IL10, CD14, TLR4, CTLA4) (19, 20), and fibrosis associated factors (TGFb, MMP3) (19).Several issues such as low sample size, single centered study, inappropriate characterization of samples, no replication in an independent cohort and most importantly lack of functional characterization in disease developmental pathways have limited the authentication of the identified SNPs.To overcome these limitations, the preliminary risk factor association study was designed with six differentially expressed immune genes, which were overexpressed in liver tissues and also higher level was noted in the serum of the ALD patients suggesting alteration of these genes could be the predictor of the disease.Subsequently case-control association study revealed rs361525 (G/A) at -238 of TNF-a and rs1143627(C/T) at -31 in IL1b were significantly associated with ALD as observed in discovery cohort of 322 patients, validation cohort of 100 patients and 50 patients in a non-linked NASH group.Hence, these three tiers of verification enhance the degree of positive correlation of those two SNPs with the disease.Furthermore, comprehensive functional validation of each SNP in the disease progression pathways has strengthened our study.
Here, we have found that a high level of TNF-a and IL1b in the serum of chronic alcoholic with either -238GA or -31CT genotype correlated well with their liver disease status.Interestingly, our data suggest healthy individuals with those two variations were having more TNF-a and IL1b in the serum indicating their susceptibility towards inflammatory diseases including ALD.NF-kb is the major transcription factor (TF) that triggers inflammatory response upon alcohol exposure.Excess binding of this TF to the -238A allele in the TNF-a promoter triggered expression of this cytokine in ALD patients which led to unrestricted activation of downstream IL6, IL8, MCP1, ICAM1 etc.As a result of it, peripheral neutrophils The graphical model shows the impact of increased level of TNF-a and IL1b on various cells present in the liver milieu and the therapeutic potential of miR-124-3p.The figure was generated using BioRender.com.infiltrated to the damage site through endothelial layers (40).Binding of TNF-a to TRADD also induced extrinsic apoptosis pathways and ROS mediated death liver cells, which in turn stimulated production of TGFb and PDGF that signals to stellate cells to secrete fibrogenic collagens and other extracellular matrix proteins (ECM).
Given that, miRNAs are very well studied as a critical player in immune suppression, several seminal studies have shown it as a novel anti-inflammatory therapeutic agent.For example, miR-146 targets key adaptor molecules TRAF6 and IRAK1 in TLR/NFkb mediated immune response pathways and high expression of this miRNA has been reported in Psoriasis causing inflammatory disorder (41).Whereas miR-124 promotes microglial immune suppression by targeting TRAF6 and p65 subunit of NFkb (42).It also reduces BCG induced immune responses by reducing the production of proinflammatory molecules NF-kb/IL-6/TNF-a/IL-1a/IL-8/IL-12a/IFN-b as it attenuates TLR6/MyD88/TRAF6 in alveolar epithelial cells (31).Corticosteroid and TNF-a inhibitors are tested for the treatment of alcoholic hepatitis but the results are controversial (43,44) as both the molecules inhibit liver regeneration (45) and enhance the rate of bacterial infection.
Interestingly, this study suggests that miR-124 could diminish various inflammatory responses that are characteristics of several inflammatory diseases including ALD by abolishing both MyD88 dependent and independent inflammatory pathways.It has myriads of targets in different cells present in liver microenvironment such as MYD88/TRAF6/TRAF3/TNF-a/IL1b/MCP1/IL8 mostly in Kupffer cells; TRADD/Caspase8 in hepatocytes; PDGFRA/ TGFBR2 on Stellate cells and ICAM1 on endothelial cells.In vitro co-culture assays had confirmed the robust potential of miR-124 in controlling the intercellular crosstalk executed during initiation and progression of ALD.The CM of THP1 with restored miR-124 expression and with low TNF-a and IL1b showed reduced level of apoptosis and ER stress in HepG2 cells, inhibited neutrophil infiltration through endothelial layers and reduced activation of Stellate cells followed by restricted fibrosis.Thus, by maintaining intracellular protein homeostasis, miRNA targeting multiple signaling proteins shared in diverse cellular pathways and serves as an efficient means to control robust signal activation or over activation of pro-inflammatory response.A recent study showed that depletion of neutrophil specific miR-223 protects mice from ALD (46) as it attenuates several macrophages associated with chemokines and cytokines such as TNF-a/MCP-1/MIP-1b/MIP-2 by downregulating p47 phox .
Cumulatively, our clinical data and comprehensive functional analysis revealed that SNPs, rs361525 (G/A) at -238 in TNF-a and rs1143627(C/T) at -31 in IL1b are significantly associated with ALD.Upon exposure to alcohol, risk allele carrying individuals are having activation of LPS/CD14 mediated TLR4 signaling pathways which lead to overproduction of proinflammatory cytokines.These are the key players in the pathogenesis of ALD.Hence, these SNPs are two independent predictors for ALD among East Indian population and could be used in clinical practice to identify the susceptible individual who may develop ALD in future.Furthermore, in vitro assays and clinical data suggest that miR-124 is a potent immune modulator which remains unaltered between ALD and ALC individuals.But, exogenous overexpression of this miRNA could restrict multiple inflammatory cascades in ALD simultaneously and thus exhibit protective role in the disease management.Indeed liver-specific delivery of miRNA in human may be a challenge in order to generate better therapeutics for ALD but it is now an emerging field with a lot of hopes.Therapy with miRNA can achieve earlier unreachable medical benefits compared to conventional mono targeted therapeutics.

1 (
FIGURE 1(A) GSE 143318 data was analyzed using DEseq2 packages to identify differentially expressed genes in ALD (n=5) vs.Control (n=5) liver tissue samples and KEGG pathway analysis was performed with significantly upregulated genes.(B) KEGG pathway analysis with differential gene expression data obtained from BioPlex ProHuman chemokine Panel assay using serum sample of Alcoholic control (ALC, n=20) and Alcoholic liver disease (ALD, n=28) patients, (C) Presentation of commonly altered genes in serum and liver tissue (D) Unsupervised hierarchical heatmap with qRT-PCR array data performed with liver tissue samples from ALC and ALD patients.p<0.05 was considered significant and *** means p<0.001.

TABLE 1A Demographic
, biochemical and clinical features of discovery cohort consist of ALC and ALD patients.

TABLE 1B Demographic
, biochemical and clinical features of normal and ALD patient whose liver tissues were collected.
this group had significantly lower level of serum cytokines albeit they were exposed to same volume of alcohol compared to the ALD patients.A group of ALC individuals were noted with cytokines level below the detection limit.To understand this disparity in expression, the entire promoter regions of all the above six genes were sequenced in 322 ALD and 168 ALC TABLE 2A Allele frequency of rs361525 in TNF-a and rs1143627, rs16944 in IL1b among the study population.
TABLE 2B Univariate and Multiple logistic regression analysis to determine the independent risk factors for development of Alcoholic liver diseases.