Biomarkers of Innate Immunity and Immunological Susceptibility to Viral Infection in Patients with Alcoholic Cirrhosis

Background: The harmful effect of alcohol on the immune system may be due to both a direct action of the alcohol or its metabolites on immune cells as an indirect action modifying the different mechanisms of intercellular interaction. The interplay between stimulatory (aKIR) and inhibitory (iKIR) natural killer (NK) cell receptors and their corresponding human leukocyte antigen (HLA) ligands influences the outcome of virus infection. The aim was to analyze the influence of the KIR/HLA pair genetic profile in male alcoholic cirrhosis (AC) patients with and without viral infections to find susceptibility biomarkers that can help establish the risks and prevent viral infections. Methods: A total of 281 male AC patients were analyzed. The sociodemographic characteristics, viral hepatitis C (HCV), hepatitis B (HBV), and cytomegalovirus (CMV) infections were analyzed. Genomic DNA was extracted, and genetic the KIR/HLA profiles were investigated. A total of 6 KIR genes and their corresponding ligands (HLA-C) were analyzed. Patients were grouped into two groups: with and without associated viral infection. Results: A statistically significant increase in the combination of KIR2DL2+/C1C1 was observed in male AC patients with viral infection compared to those without viral infection (45.9% vs. 24.5%, p = 0.021). The analysis of KIR2DL3+/C1+ showed a high frequency comparing healthy controls and male AC patients without virus infection (85% vs. 76.4%; p = 0.026). The analysis of KIR2DL3+/C2C2 frequency showed a statistically significant increase comparing male AC patients without viral infection and healthy controls (23.6% vs. 15%; p = 0.026). Conclusions: The genetic KIR2DL2+/C2C2 profiles may play a significant role in determining the vulnerability of male AC patients to viral infections, providing valuable insights for future research and potential therapeutic interventions.


Introduction
The harmful effect of alcohol on the immune system may be due to both a direct action of the alcohol or its metabolites on immune cells as an indirect action modifying the different mechanisms of intercellular interaction [1][2][3].Neither mechanism excludes the other, and possibly both coexist in the immunological injury associated with alcohol consumption [4,5].The optimal functioning of the immune system requires a high degree of interaction, modulation, and regulation between the different cell types that it comprises [6].According to research conducted in vitro, acute ethanol consumption in humans has been shown to either increase or reduce the activity of natural killer (NK) cells [7], depending on when the measurement is taken [8][9][10].On the other hand, the activity of NK cells obtained from chronic alcoholics can be normal, although there is associated liver disease [7][8][9].Without chronic ethanolic liver disease, NK cell activity tends to be decreased or even suppressed [10].
In patients with HCV infection, alcohol intake hastens liver damage and reduces interferon alfa's (IFN-α) anti-hepatitis C virus (HCV) activity [11].Alcohol's potential to accelerate HCV disease progression and improve HCV replication is uncertain [12][13][14].The capacity to study the relationship between alcohol and HCV replicon expression has been made possible by the availability of hepatic cells that contain the HCV replicon [11].
IFN-based regimens, a poorly tolerated medication, were formerly used to treat chronic HCV infection [15,16].Patients who actively used alcohol in the previous year were more likely to discontinue IFN-based HCV treatment during the IFN period; therefore, many medical professionals were reluctant to treat them [17].Classically, the existence of pre-transplant viral infection has been investigated in HBV, HCV, and CMV infections in relative studies in liver and bone marrow transplantation and also in liver pathology as previously published [18] where the influence of these factors in rejection and outcome is established.
On the other hand, NK cells are part of the innate immune system and play a significant role in defense against viral infections and tumor cells [19][20][21].NK cells express surfacebinding proteins (killer cell immunoglobulin-like receptors or KIRs) that bind ligands on most cells' surfaces.The interplay between stimulatory and inhibitory NK cell receptors and their corresponding human leukocyte antigen (HLA) ligands influences the outcome of acute HCV infection and other infections [22,23].Previous studies demonstrated that KIR receptor genes and HLA class I and II loci helped determine the clinical outcome of HCV infection [22][23][24][25].Infections in alcoholic cirrhosis (AC) patients significantly cause morbidity and mortality [11].
The aim was to analyze the influence of the KIR/HLA pair genetic profile in male AC patients with and without viral infections to find susceptibility biomarkers that can help establish risks and prevent viral infections.

Patient Enrollment
A retrospective analysis was done on the medical records of 281 male AC patients with and without virus infection at the University Clinic Hospital Virgen de la Arrixaca in Spain (Figure 1).The exclusion criteria were the following: children's or women's AC patients were removed due to the scarce sample size.Age-related sociodemographic information, HCV, hepatitis B (HBV), cytomegalovirus (CMV) infections, and the genetic KIR/HLA profile were investigated.
The Model for End-Stage Liver Disease score in male AC patients was 14.23 ± 0.37 (mean ± SEM).In this study, the mean age was comparable across all patients studied.Men who had a history of drinking alcohol, and had cirrhosis were required to meet the inclusion criteria.In addition, 319 age-matched male individuals were used as a healthy control cohort compared to the overall male AC population.In each case, the median scores for the two populations were similar.Before participating in the trial, each patient provided informed consent for inclusion.The HUVA Ethics Committee (PI19/01194) approved the study, which was carried out per the Declaration of Helsinki.

Alcohol Cirrhosis Diagnostic Criteria
Clinical, radiographic, and biochemical indicators were used to diagnose AC [26].In cases where the self-report of alcohol intake was negative, the relatives opinions were considered.Cirrhosis was discovered through a routine scan, ultrasound, or clinical examination, because the disease usually has no symptoms in its early stages.Other times, the illness went undiagnosed until the second stage of decompensated cirrhosis, at which point problems such as ascites, upper gastrointestinal hemorrhage, and encephalopathy manifested.As previously reported [27], cases of suspected cirrhosis were confirmed using analysis and imaging methods.

Viral Infection Diagnosis
Viral infections such as HCV, HBV, and CMV were analyzed.A qualitative immunoassay (AxSYM HCV v 3.0, Abbott, Wiesbaden-Delkenheim, Germany) was used to identify anti-HCV antibodies and ascertain the presence of HCV.If the test was positive for anti-HCV antibodies, an RNA test for HCV was performed to confirm chronic infection The manufacturer s instructions verified the results using immunoblotting technology (RIBA), real-time (RT)-PCR (REAL, C.E. Durviz, Valencia, Spain) or Roche COBAS ® Ampliprep TNAI/TaqMan ® 48 RUO Assay, Thermofisher Scientific, Hempstead, UK.
HBV infection was detected using a radioimmunological approach to measure the HBV surface antigen (HBsAg), and CMV infection was detected using an immunoassay (Liason ® CMV-IgG, DiaSorin, Saluggia, Italy) to test for anti-CMV IgG antibodies.The first diagnosis of the CMV infection was made using IgG antibodies at a concentration of 0.6 IU/mL.An RT-PCR was utilized to confirm the results of these positive samples

Alcohol Cirrhosis Diagnostic Criteria
Clinical, radiographic, and biochemical indicators were used to diagnose AC [26].In cases where the self-report of alcohol intake was negative, the relatives' opinions were considered.Cirrhosis was discovered through a routine scan, ultrasound, or clinical examination, because the disease usually has no symptoms in its early stages.Other times, the illness went undiagnosed until the second stage of decompensated cirrhosis, at which point problems such as ascites, upper gastrointestinal hemorrhage, and encephalopathy manifested.As previously reported [27], cases of suspected cirrhosis were confirmed using analysis and imaging methods.

Viral Infection Diagnosis
Viral infections such as HCV, HBV, and CMV were analyzed.A qualitative immunoassay (AxSYM HCV v 3.0, Abbott, Wiesbaden-Delkenheim, Germany) was used to identify anti-HCV antibodies and ascertain the presence of HCV.If the test was positive for anti-HCV antibodies, an RNA test for HCV was performed to confirm chronic infection The manufacturer's instructions verified the results using immunoblotting technology (RIBA), real-time (RT)-PCR (REAL, C.E. Durviz, Valencia, Spain) or Roche COBAS ® Ampliprep TNAI/TaqMan ® 48 RUO Assay, Thermofisher Scientific, Hempstead, UK.
HBV infection was detected using a radioimmunological approach to measure the HBV surface antigen (HBsAg), and CMV infection was detected using an immunoassay (Liason ® CMV-IgG, DiaSorin, Saluggia, Italy) to test for anti-CMV IgG antibodies.The first diagnosis of the CMV infection was made using IgG antibodies at a concentration of 0.6 IU/mL.An RT-PCR was utilized to confirm the results of these positive samples (LightCycler ® CMV-Quant-kit, Roche, Indianapolis, IN, USA).Although it could be questionable to use CMV IgG as the screening criteria for CMV diagnosis due to CMV IgG would need 15 days to turn positive after exposure, we reviewed all cases in our study and no missed cases were found.

KIR and HLA Typing
The QIAamp DNA Blood Mini Kit (QIAGEN, Hilden, Germany) was used to extract genomic DNA from peripheral blood, as advised by the manufacturer and previously reported [28], and quantified by NanoDrop (Thermo Scientific™, Boston, MA, USA).
KIR genotyping was conducted in patients and healthy individuals utilizing Luminex ® technology and sequence-specific oligonucleotides (PCR-SSO) (Tepnel Lifecodes, Stanford, CT, USA).This technique reveals inhibitory KIR2DL1-3 and KIR3DL1-3 genes and activates KIR2DS1-S5, KIR3DS1, and KIR2DL4.This approach failed to separate the genes KIR2DL5A and KIR2DL5B; however, it did identify KIR2DP1 and KIR3DP1 pseudogenes.Both genes were investigated, since KIR2DL2 is frequently related to KIR2DS2 and their individual effects could not be differentiated in our population.Patients were divided into two groups for this study according to the presence of inhibitory KIR genes (iKIRs) and activating KIR genes (aKIRs).Constitutive KIR genes and pseudogenes were excluded in this study.
The Dynal RELI TM SSO HLA-C Kit (Dynal Biotech ASA, Oslo, Norway) was used for genotyping HLA-I at a degree of resolution required to identify the dimorphism at position 80 on the 1-helix.The two main HLA-C KIR ligand allele groups, HLA-C1 and HLA-C2, as well as the three separate HLA-C genotypes, C1C1, C1C2, and C2C2, were determined based on this dimorphism [29][30][31].

Statistical Analysis
Demographic and outcome data were entered into a database (Microsoft Access 2.0; Microsoft Corporation, Seattle, WA, USA) and analyzed with SPSS v27.0 (SPSS software Inc., Chicago, IL, USA).All data were expressed as the mean, standard deviation, or percentage.Pearson's chi-square and two-tailed Fisher's exact tests were employed to compare classified variables between groups, while a two-sided Student t-test and the nonparametric Mann-Whitney test were used to compare mean values.The statistical significance level was set at p = 0.05.The odds ratio (OR) and 95% confidence interval (CI) were determined to evaluate relative risk.

Clinical and Sociodemographic Characteristics
The sociodemographic and main clinical characteristics of the total cohort (n = 281) of male AC patients are shown in Table 1.This study excluded pediatric and adult patients who could not have specified testing performed.The mean age was similar in all the analyzed male AC patients with and without the associated virus.There were no statistically significant differences between them.In our cohort of male AC patients, 68 patients (24.2%) with associated viral infections were observed.Table 1 shows the frequency of each virus type in male AC patients analyzed in this study.HCV infection was the most representative (61.8%), which added to the percentage of coinfection with HBV (5.9%) and CMV (2.9%), representing 70.6% of patients infected by HCV.HBV infection was represented in 20.6%.In addition, HBV occurs in a tiny percentage associated with CMV (2.9%, n = 2).CMV infection represented 5.9% of AC patients.

Analysis of KIR Genes and Their Corresponding HLA-C Ligands in AC Patients with and without Virus Infections
The frequency of each KIR gene (iKIRs and aKIRs) with their corresponding HLA-I ligands (C1, C2) in male AC patients with and without virus infection was analyzed (Table 2).The analysis of KIR2DL1+/S1− with and without its epitope (C2+/C2−) showed a similar frequency distribution in the healthy population compared with total AC patients (p = 0.817).By contrast, the analysis of KIR2DL3+ with C1+ showed a higher frequency in the control group versus total AC patients (85% vs. 75.9%,respectively); this decrease in AC patients was statistically significant.(OR = 1.801; 95% CI: 1.155-2.809,p = 0.010).Similar results were also obtained when comparing healthy controls and male AC patients without virus infection (85% vs. 76.4%;OR = 1.758; 95% IC: 1.092-2.831,p = 0.026).
Furthermore, the combination of KIR2DS5 with the C1+ ligand has also been less frequent in the total group of male AC patients compared to the control group (84.3% vs. 72.9%),although in this case without reaching statistical significance (p = 0.072).On the contrary, the combination of the C2+ ligand with KIR2DS5 appeared to increase in the group of total male AC patients compared to the control group (74% vs. 67.5%),but the difference observed was not statistically significant (p = 0.410).

Analysis of KIR Genes and Their Genotypes HLA-C in Male AC Patients with and without Virus Infections
The combination of KIR genes with HLA-C genotypes was also analyzed in male AC patients with and without viral infections and healthy controls (Table 3).The analysis showed how the KIR2DL1+/S1− with C2C2 genotype (14.9%) was diminished in healthy controls versus total AC patients (21.2%).However, the observed differences did not reach statistical significance (p = 0.159).On the other hand, no significant changes were observed for these combinations when AC patients with and without viral infection were compared (17.8% vs. 22.5%; p = 0.666).
Then, the combination of KIR2DL2 with the different HLA-C genotypes was also analyzed, and a statistically significant increase in the combination of KIR2DL2+/C1C1 was observed in AC patients with viral infection (Figure 2) compared with these patients without viral infection (45.9% vs. 24.5%,OR = 0.382; 95% CI: 0.175-0.837,p = 0.021).The analysis of KIR2DL3+ and HLA-C genotypes showed an increase in the frequency of the C2C2 genotype in total AC patients (24.1%) compared to the control population (15%), this difference being statistically significant (OR = 0.555; 95% CI: 0.356-0.866,p = 0.010).Similar results were observed when comparing AC patients without viral infection and healthy controls (OR = 0.569; 95% CI: 0.353-0.916,p = 0.026).

Discussion
This study aimed to investigate the genetic profile of the KIR/HLA pair in male AC patients with and without viral infections to uncover susceptibility biomarkers that can assist in establishing risks and preventing viral infections in AC patients as a new approach aimed at detecting the profile which reflects greater vulnerability to viral infection and worsening liver functions.
Our results showed that the most common viral infection in AC patients was HCV, followed by HBV and CMV.The relationship between alcohol and HCV is well-established, with evidence suggesting that alcohol consumption can worsen the progression and severity of HCV infection [32,33].The combined effects of alcohol and HCV on the liver can lead to increased liver damage, fibrosis, and even cirrhosis [31].Moreover, alcohol use has been shown to decrease the effectiveness of HCV treatment and increase the risk of developing hepatocellular carcinoma (HCC) in HCV-infected individuals [34,35].Therefore, healthcare professionals and individuals diagnosed with HCV must acknowledge the detrimental role of alcohol in exacerbating the disease.By abstaining from alcohol or practicing moderation, individuals can significantly improve their chances of healthier outcomes and ultimately reduce the burden of HCV [36].
On the other hand, alcohol consumption has a detrimental effect on HCV infection and the functioning of NK cells, leading to an increased risk of liver damage and disease progression [31,37,38].Evidence consistently demonstrates that alcohol induces oxidative stress, impairs immune response, and promotes HCV replication within the liver.Furthermore, alcohol interferes with the role of NK cells in recognizing and eliminating HCV-infected cells, diminishing their antiviral function [39][40][41].Evidence suggests that NK cells, through their membrane receptors, play dual roles in developing and progressing liver fibrosis, performing both profibrotic and antifibrotic actions [42].On the other hand, the role of NK cells in controlling viral infections is also known [43,44].
KIR receptors on the NK cell and their specific ligands, HLA class I (MHC-1) molecules, also play an essential role in the antifibrotic effect [45].Inhibitory KIR receptors (iKIR) present on the NK cell bind to their ligands, HLA class I (MHC-1) molecules present on the hepatic stellate cell (HSC), inhibiting its destruction.When liver damage occurs, the expression of inhibitory KIR receptors decreases; therefore, the inhibitory effect on the destruction of HSCs decreases [46].The binding of iKIR with MHC-1 sets in motion a series of mechanisms that will favor apoptosis of stellate cells [47].Our results showed a statistically significant increase in viral infection in male AC patients with the KIR2DL2+ with HLA-C1/C1 combination (Figure 2).This molecular interaction produces an inhibition of the cytotoxic function of NK cells that prevents the innate immune system from eliminating virus-infected cells.Similar results were obtained in a study with chronically infected patients with HIV-1, which identified two KIR/HLA combinations, KIR2DL2/HLA-C*12:02 and KIR2DL2/HLA-C*14:03, that impact suppression of HIV-1 replication [48].
The interaction of stimulatory and inhibitory KIRs with their corresponding HLA ligands is believed to influence the outcome of acute Hepatitis C viral (HCV) infection, leading to either chronic viremia (CV) or spontaneous viral clearance (SC).The ability of alcohol to increase HCV disease progression and improve HCV replication is unknown [12][13][14].The availability of hepatic cells containing the HCV replicon has enabled researchers to investigate the link between alcohol and HCV replicon expression [11].
Interferon-based regimens, which are poorly tolerated, were formerly used to treat chronic hepatitis C (HCV) infection [15,16].Patients who had used alcohol the previous year were more likely to abandon interferon-based HCV treatment during the interferon period; therefore, many doctors were hesitant to treat them [17].
Other studies [49,50] observed an association between inhibitory KIR genes and their specific ligands (KIR 2DL3/3DL3 with HLA-C1/C1) in patients infected with HCV but in this case related to CS.On the other hand, Azocar et al. [51] described the association of DQB1*0501 with CV.Also, new genetic interactions have been described between the KIR gene 2DL3 and HLA-DRB*1201 in SC of HCV [27].

Conclusions
In conclusion, this study allows us to expand the knowledge between innate immunity and alcoholic cirrhosis, allowing (1) to delve into the molecular mechanisms carried out by innate immunity that can trigger a greater susceptibility to viral infections in the AC patient, and (2) to search for therapeutic strategies that can prevent and mitigate the effect of the inhibition of NK cytotoxicity in the patient, since it can also make him susceptible to the development of hepatocellular carcinoma.The screening of KIR genes and HLA-C genotypes identified that KIR2DL2/C2C2 genetic profiles may play an important role in determining the vulnerability of male AC patients to viral infections, providing valuable information for future research to delve into the possibility of enhancing the activation of the cytotoxic activity of the NK cells of these patients.Funding: Our work was possible thanks to support from Instituto de Salud Carlos III (ISCIII), Spanish Ministry of Economy and Competitiveness (grant number P19/01194), and co-funding from the European Union through the European Fund of Regional Development (FEDER) with the principle of "A manner to build Europe" and project of Fundacion Mutua Madrileña Grant Number AP183152023.
Informed Consent Statement: Written informed consent has been obtained from the patient(s) to publish this paper.

Figure 1 .
Figure 1.Different types of patients studied in this study are shown in the graphic.N, total number of individuals; n is the number of individuals in each subgroup; AC, alcoholic cirrhosis.CMV: Cytomegalovirus.HBV: Hepatitis B virus.HCV: Hepatitis C virus; Other viral combinations* included HCV + HBV, HCV + CMV, HBV + CMV.

Figure 1 .
Figure 1.Different types of patients studied in this study are shown in the graphic.N, total number of individuals; n is the number of individuals in each subgroup; AC, alcoholic cirrhosis.CMV: Cytomegalovirus.HBV: Hepatitis B virus.HCV: Hepatitis C virus; Other viral combinations* included HCV + HBV, HCV + CMV, HBV + CMV.

Biomedicines 2024 , 11 Figure 2 .
Figure 2. Hypothetical scheme of the inhibition of cytotoxicity mediated by NK cells in AC patients with the genetic profile (KIR2DL2/C2C2) and high susceptibility to associated viral infection.It is shown that the presence of KIR2DL2/C2C2 in the patient produces cellular inhibition of the NK cell, which is incapable of carrying out its cytotoxic activity to eliminate viral infections.

Figure 2 .
Figure 2. Hypothetical scheme of the inhibition of cytotoxicity mediated by NK cells in AC patients with the genetic profile (KIR2DL2/C2C2) and high susceptibility to associated viral infection.It is shown that the presence of KIR2DL2/C2C2 in the patient produces cellular inhibition of the NK cell, which is incapable of carrying out its cytotoxic activity to eliminate viral infections.

Table 1 .
Characterization of the male AC study population.
n, is the number of individuals in each subgroup; AC, alcoholic cirrhosis.;CMV: Cytomegalovirus.HBV: Hepatitis B virus.HCV: Hepatitis C virus; SEM: Standard error of the mean.

Table 2 .
Analysis of KIR genes and their epitopes HLA-I ligands in male AC patients with and without virus compared with healthy controls.
N is the total number of individuals; n is the number of individuals with the presence or absence of ligand for a KIR gene.Two-tailed Fisher's exact test made comparisons.* The viruses analyzed were CMV, VHB, and VHC.P1, p value obtained by comparing total AC patients with healthy controls; P2 and P3, p value obtained by comparing AC patients with and without viral infection with healthy controls, respectively; P4, p value obtained by comparing AC patients without viral infection with AC with viral infection.a , OR = 1.

Table 3 .
Frequency of KIR genes and HLA-C genotypes in male AC patients with and without associated viruses compared with healthy controls.is the total number of individuals; n is the number of individuals with the presence or absence of the KIR gene.Two-tailed Fisher's exact test made comparisons.Each p value was obtained by comparing each ligand with the other ligands for that specific gene.P1, p value obtained by comparing total male AC patients with N

Table 3 .
Frequency of KIR genes and HLA-C genotypes in male AC patients with and without associated viruses compared with healthy controls.