IL-10 and IL-18: Key players in liver damage associated with hepatitis A virus infection

Abstract

BACKGROUND:

Hepatitis A virus infection is a health threat with multiple transmission patterns across areas, It is evaluated using immune response markers IL-10 and IL-18, along with molecular and biochemical diagnostic methods for accurate diagnosis.

OBJECTIVE:

The association between liver damage and interleukin-10 and interleukin-18 levels in people with hepatitis A virus infection as indications of the risk of acute liver failure.

METHODS:

110 samples were collected from Iraqi individuals from both sexes and different age groups $\leqslant$ 1 to $\geqslant$ 25, including 60 patients and 50 healthy people. All samples were collected from a hospital in Diwaniyah city, and the infection was confirmed by antiHAV IgM titers and One-Step RT-PCR. ELISA was used to determine the levels of IL-10 and IL-18, while Biochemical tests measured for alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST) and total serum Bilirubin (TSB) in serum.

RESULTS:

In this study, IL-10 levels were higher in HAV patients (0.12 $\pm$ 0.06 ng/L) compared to controls (0.11 $\pm$ 0.04 ng/L), but the difference was not significant ( $p=$ 0.17). Conversely, IL-18 levels were significantly elevated in patients (1.41 $\pm$ 0.71) versus controls (0.58 $\pm$ 0.35) ( $p=$ 0.00). Biochemical tests showed significantly elevated levels in HAV patients: ALT (170.18 $\pm$ 117.67 vs. 21.25 $\pm$ 7.41), AST (183.05 $\pm$ 128.13 vs. 26.00 $\pm$ 7.69), ALP (607.68 $\pm$ 214.93 vs. 202.02 $\pm$ 121.35), and TSB (2.77 $\pm$ 2.5 vs. 0.55 $\pm$ 0.14) (all $p<$ 0.001). These findings underscore the potential of IL-10 and IL-18 as biomarkers for HAV severity and highlight their role in liver injury.

CONCLUSION:

Our study highlights the important roles of IL-10 and IL-18 in acute hepatitis A and reveals their impact on the immune response and liver damage. Elevated levels of IL-10, IL-18 and Biochemical tests are associated with disease severity, suggesting their potential as biomarkers and therapeutic targets to improve the management of HAV infection.

Keywords

HAV IL-10 IL-18 one step-PCR ALF

1. Introduction

Table 1
Distribution of patients and control groups according to age group

Age group	Patient		Control		Total		$P$ value
	No.	%	No.	%	No.	%
1–5	16	76.2%	5	23.8%	21	100.0%	0.00 NS
6–10	18	66.7%	9	33.3%	27	100.0%
11–15	13	43.3%	17	56.7%	30	100.0%
16–20	6	35.3%	11	64.7%	17	100.0%
21–25	2	28.6%	5	71.4%	7	100.0%
$>$ 25	5	62.5%	3	37.5%	8	100.0%
Total	60	54.5%	50	45.5%	110	100.0%

Figure 1.

Collection of Blood samples from patients and healthy people.

Hepatitis A virus (HAV) is a non-enveloped positive-strand RNA picornavirus that spreads by feces or intimate contact with infected individuals [1]. The average incubation time is 28 days (range 15–50 days), and persons are infectious (dropping virus in feces) from two weeks before symptoms to one week after jaundice [1]. The medical condition is characterized by jaundice, however presentation is exceedingly diverse and more severe in older people. Jaundice occurs in less than 10% of children under 5 years old and in approximately 75% of adults [1]. Hand hygiene is essential in controlling contagion [2, 3]. HAV can remain infectious on hands for more than 4 hours and is easily transmitted to food during handling [4]. Transmission within families is extremely widespread, with secondary attack rates in susceptible household contacts ranging from 12 to 34% [5]. Secondary attack rates are 3–28% in nurseries or day care centers and 3–50% in primary schools, indicating widespread transmission among young children [5], viral hepatitis A is sometimes associated with acute liver failure, ALF is a syndrome rather than a particular disease with multiple plausible causes. Viral hepatitis is a prominent cause, however the link between self-limited and ALF hepatitis A is still poorly understood [6]. Therefore, in our current study, we did not limit our investigation to liver function tests as indicators of liver damage. We also focused on the immunological indicators of the probability of infection in liver damage that may lead to ALF. We analyzed the concentrations of interleukin-10 (IL-10) and interleukin-18 (IL-18) in patients with viral hepatitis A and compared them with healthy volunteers.

Table 2

RT-PCR program to detect the Hepatitis A virus

Stage	Temp, ^∘C	Time	Fluorescence detection	Cycle repeats
Hold	50	30 min	$-$	1
Hold	95	15 min	$-$	1
Cycling	95	5 s	$-$	10
Cycling	60	20 s	$-$	10
Cycling	72	15 s	$-$	10
Cycling 2	95	5 s	$-$	40
Cycling 2	60	30 s	FAM, JOE/HEX/Cy3	40
Cycling 2	72	15 s	$-$	40

IL-10 is an anti-inflammatory cytokine that regulates the immune system by inhibiting pro-inflammatory cytokine production, suppressing T cell activation, and promoting regulatory T cell differentiation. Its role in viral infections often involves preventing excessive immune responses and tissue damage. However, specific data on IL-10’s effects during HAV infection are limited, necessitating further research to understand its influence on disease severity and progression [7].

IL-18, a pro-inflammatory cytokine, enhances the immune response by stimulating interferon-gamma (IFN- $\gamma$ ) production, activating natural killer (NK) cells, and promoting a strong cellular immune response [8]. Despite its recognized role in other viral infections, IL-18’s specific contributions to HAV infection have not been thoroughly investigated. Understanding IL-18’s role in HAV infection could provide insights into potential therapeutic strategies. To investigate the causes that lead to acute liver failure due to hepatitis A virus, this study aims to explore the roles of IL-10 and IL-18 in HAV infection and their association with liver damage that may progress to acute liver failure. By integrating cytokine marker estimation, comprehensive biochemical profiling, and Real-Time PCR for HAV detection, we seek to unravel the pathophysiological mechanisms linking HAV infection to ALF. This comprehensive research endeavor aims to enhance our understanding of disease dynamics and potentially pave the way for more effective diagnostic, prognostic, and therapeutic strategies to mitigate HAV-induced ALF [6].

2. Materials and method

A total of 110 Iraqi individuals participated in this study, with 60 being infected with the Hepatitis A virus (Patient group) and 50 serving as apparently healthy controls. The blood samples were collected from the patient’s group who were attended AL-Diwaniyah hospital in AL-Diwaniyah city. The age of patients ranged from $\leqslant$ 1 to $\geqslant$ 25 years (Table 1).

For sample collection, five milliliters of blood samples were collected from patients infected with the hepatitis A virus (HAV) using a single-use syringe as shown in (Fig. 1). The drawn blood was immediately divided into two parts, the first part (3 milliliters) was transferred into a gel activator tube and then left to stand at room temperature for 10 minutes to give the clot activator included in the tube time to respond before being centrifuged. at 3500 rpm for 10 minutes to do the biochemical and serological tests immediately, and then 120 $\mu$ L of the serum was dispensed into one separate plain tubes and storage at $-$ 20^∘C until the immunological tests were carried out, whereas the second part of the blood (2 ml) was transferred into a gel activator tube and then left to stand at room temperature for 10 minutes to give the clot activator included in the tube time to respond before being centrifuged 3500 rpm for 10 minutes. Then, the serum was distributed into one separate plain tube and extracted of RNA of the HAV and then storage at $-$ 20^∘C until the exposure of HAV by one step RT-PCR within 48 h.

2.1 Extraction of RNA

The RNA was extracted from serum samples of the hepatitis A virus patients using the QIAamp viral RNA mini kit (Qiagen/Germany) used according to manufacturer instruction and protocol. The serum samples were thawed and processed using a QIAamp Viral RNA Mini Kit. Each sample was combined with buffer AVL containing carrier RNA, followed by incubation and the addition of ethanol. The mixture was then applied to a QIAamp mini-column and subjected to a series of centrifugation steps with buffers AW1 and AW2 to wash the RNA. Finally, the viral RNA was eluted with buffer AVE and stored at $-$ 20^∘C until further use.

2.2 Detection of hepatitis a virus by one step real time – polymerase chain reaction

Table 3
The addition of reaction mix in RT-PCR

10 *(N $+$ 1) $\mu$ l	RT-PCR-mix-1-TM
5.0 *(N $+$ 1) $\mu$ l	RT-PCR-mix-2
0.5 *(N $+$ 1) $\mu$ l	Taq Polymerase
0.25 *(N $+$ 1) $\mu$ l	RT-G-mix-2
0.25 *(N $+$ 1) $\mu$ l	M-MLV Revertase

HAV Real-TM is Real-Time amplification test for the qualitative detection of Hepatitis A (HAV) RNA in clinical specimens (plasma, feces, etc.) and water. HAV Real-TM Test (Sacace, Italy). The kit HAV Real-TM allows to detect HAV in 100% of the tests with a sensitivity of not less than 500 copies/ml. It is based on three major processes: isolation of HAV RNA from specimens, one-step reverse transcription of the RNA and Real Time amplification of the cDNA following the Real Time PCR Program (Table 2) by (LineGeneK^®, China). HAV detection by the PCR is based on the amplification of pathogen genome specific region using specific primers primers are specific to the manufacturer only (and detection via fluorescent dyes. These dyes are linked with probes of oligonucleotides which bind specifically to the amplified product. The real-time PCR monitoring of fluorescence intensities allows the accumulating product detection without reopening of reaction tubes after the PCR run. The total reaction volume is 25 $\mu$ l, the volume of RNA sample is 10 $\mu$ l and the reaction mix was made by the following addition in (Table 3), and then 15 $\mu$ l of prepared reaction mix was added into each tube and 10 $\mu$ l of RNA samples that obtained at the stage of RNA isolation also added by using tips with aerosol filter and mix carefully. Two controls was prepared for each panel: 10 $\mu$ l of RNA-buffer added to the tube labeled PCR Negative Control and 10 $\mu$ l of Pos Control cDNA HAV(C+) added to the tube labeled C pos. The results analyzed were interpreted by the device software through the presence of crossing of fluorescence curve with the threshold line. cDNA of HAV is detected on the JOE(Yellow)/HEX/Cy3 channel.

2.3 HAV IgM test device by VIDASHAVM

All samples underwent HAV rapid testing for diagnosis, and IgM titers against the hepatitis A virus were measured using HAV IgM test device by VIDASHAVM (BioMerieux, France). VIDAS HAV IgM is an automated qualitative test for use on VIDAS family instruments detecting IgM directed against coronavirus A (HAV) after immunocapture, in human serum or plasma (heparin or EDTA), using ELFA (Enzyme Customized) technology, fluorescent examination). Instruments detecting IgM directed against coronavirus A (HAV) after immunocapture, in human serum or plasma (heparin or EDTA), using ELFA (Enzyme Customized) technology fluorescent examination.

2.4 Measurement levels IL-10 and IL-18 for both patients and healthy people

The serum (IL-10, IL-18) levels in the samples were measured using Sandwich-ELISA kits (Bioassay Technology Laboratory, China )by ELISA system(Asys, Irland). This kits is an Enzyme-Linked Immunosorbent Assay (ELISA). The plate has been pre-coated with a human IL antibody. IL present in the sample is added and binds to antibodies coated on the wells and then a biotinylated human IL antibody is added and binds to IL in the sample. Then Streptavidin-HRP is added and binds to the biotinylated IL antibody. After incubation, unbound streptavidin-HRP has washed away during a washing step. The substrate solution is then added and color develops in proportion to the amount of human IL. The reaction is terminated by the addition of an acidic stop solution and absorbance is measured at 450 nm.

2.5 Measurement levels biochemical tests for both patients and healthy people

All the serum samples that were collected from the HAV patients and healthy people were subjected to the biochemical test which alanine aminotransferase (ALT), aspartate aminotransferase (AST). They were tested by using kits from (Biosystem, Spain) and measured by Spectrophotometer (CE7200, England).

2.6 Statistical analysis

Data were collected and analyzed using appropriate statistical methods, Statistics package for Social Science (SPSS), version 25 for windows software was used for statistics analysis. The data are normally distributed and expressed as mean $\pm$ standard deviation (SD). The data are normally distributed and analyzed by using frequency and percentage distribution.

$\displaystyle{\%}=\text{frequency}/n*100$

Student’s $t$ -test were performed to analyze the statistical significance of difference between group (1 and 2), Significant was considered whenever the $p$ value was equal or less than (0.05).

2.7 Ethics consideration

The research received approval from the Ethics Committee of the institute of Genetic Engineering and Biotechnology for Postgraduate Studies, Baghdad university. The official permission certificate is Ref: EC/HT/2010 at 22/12/2022. Having obtained a comprehensive comprehension of the research, every participant willingly signed the informed consent form.

Table 4
Distribution of mean and Sd of the IgM titer for patient according to the gender and age group

Variable	Mean $\pm$ Sd	$P$ value
Gender
Male	1.73 $\pm$ 0.63	0.17 NS
Female	1.96 $\pm$ 0.61
Age group
1–5	1.79 $\pm$ 0.66	0.82 NS
6–10	1.85 $\pm$ 0.56
11–15	1.97 $\pm$ 0.6
16–20	1.67 $\pm$ 0.63
21–25	0.85 $\pm$ 0.24
$>$ 25	1.97 $\pm$ 0.76

3. Results

3.1 Serological methods

All the samples of hepatitis A virus patients (Patients group) and apparently healthy individuals (Control group) were subject to the HAV rapid test for diagnosis of HAV infection and by measure IgM titer against the hepatitis A virus by Minividas. The results of the present study showed that all the hepatitis A virus patients were positive for HAV rapid test and anti HAV IgM titer while all the serum samples of the apparently healthy individuals were negative for the HAV rapid test and anti HAV IgM titer. In current study results indicated that the mean and standard deviation for patients was (1.819 $\pm$ 0.625) while the normal range for Titer of IgM is (0.4–0.5), by One-Sample $T$ -test Statistics there are significant differences between patient and control the $p<$ 0.001 it was less than sig $=$ 0.05. While there are no significant differences at the level of (Age groups and Gender), $P$ values $=$ (0.17, 0.82) respectively (Table 4).

Table 5
Distribution of mean and Sd of one-step RT-PCR for patients group according to gender

Gender	Mean $\pm$ Sd
Male	25.88 $\pm$ 1.87
Female	25.61 $\pm$ 1.47
Total	25.81 $\pm$ 1.72
$P$ value	0.81 NS

3.2 Detection of hepatitis A virus by one-step RT-PCR

The results of HAV detection using one-step RT-PCR revealed that 50 of the HAV patients tested positive, with an average age of (25.88 $\pm$ 1.87) for males and (25.61 $\pm$ 1.47) for females. There was no significant difference in HAV infection rates between genders, as shown in (Table 5). This study confirms the reliability and utility of RT-PCR in diagnosing HAV infection, demonstrated by the positive detection of HAV RNA in all patient samples.

Table 6
The results of IL-10 and IL-18 of patients and control group

Variables	IL-18	IL-10
	(Mean $\pm$ Sd)	(Mean $\pm$ Sd)
Status
Patient	1.41 $\pm$ 0.71	0.12 $\pm$ 0.06
Control	0.58 $\pm$ 0.35	0.11 $\pm$ 0.04
$P$ value	0.00^*	0.17 NS
Age groups
1–5	1.13 $\pm$ 0.77	0.12 $\pm$ 0.03
6–10	1.2 $\pm$ 0.79	0.11 $\pm$ 0.03
11–15	0.96 $\pm$ 0.7	0.11 $\pm$ 0.03
16–20	0.87 $\pm$ 0.46	0.14 $\pm$ 0.1
21–25	0.6 $\pm$ 0.36	0.13 $\pm$ 0.04
$>$ 25	1.22 $\pm$ 0.87	0.13 $\pm$ 0.05
$P$ value	0.274 NS	0.161 NS
Gender
Male	1.06 $\pm$ 0.67	0.11 $\pm$ 0.03
Female	1.01 $\pm$ 0.76	0.12 $\pm$ 0.06
$P$ value	0.691 NS	0.525 NS
Total	1.03 $\pm$ 0.71	0.12 $\pm$ 0.05

3.3 Levels of IL-10 and IL-18 for both patients and healthy people

All the (60) hepatitis A virus patients (Patients group) and (50) healthy individuals (Control group) of the current study were subjected to detect the IL-10 and IL-18 levels by the enzyme-linked immune sorbent assay (ELISA) method using Human IL-10 and IL-18 Elisa kit. The results of IL-10 showed that indicated at (Patient, Control) the difference was not statistically significant $P$ value $=$ 0.17 the difference was statistically significant $=$ 0.05. Also, The results of IL-10 estimation showed that the concentrations of IL-10 in hepatitis A virus patients that range (0.12 $\pm$ 0.06 ng/L) were higher than the interleukin-10 concentrations in apparently healthy individuals that range (0.11 $\pm$ 0.04 ng/L). In addition, the results of IL-10 estimation showed that the concentrations of interleukin-10 in the females of acute hepatitis A virus patients was (0.12 $\pm$ 0.06 ng/L), while the concentration of IL-10 in males of acute hepatitis A virus patients which was (0.11 $\pm$ 0.03). The results showed that the difference was not statistically significant at the level of (Age groups and Gender), $P$ values $=$ (0.161, 0.525) respectively. The results of statically analysis of IL-10 estimation for the patients and control groups revealed that there were significant differences between the hepatitis A virus patients who have a mean value and standard deviation (0.12 $\pm$ 0.06) which was higher than the mean value and standard deviation of apparently healthy individuals which was (0.11 $\pm$ 0.04) and the $P$ value $=$ 0.17 which it was more than sig $=$ 0.05 (Table 6). While, IL-18 the results showed that indicated at (Patient, Control) it was found that the difference was statistically significant $p<$ 0.001 it was less than sig $=$ 0.05. Also, the results of IL-18 estimation showed that the concentrations of IL-18 in hepatitis A virus patients that range (1.41 $\pm$ 0.71) were higher than the IL-18 concentrations in apparently healthy individuals that range (0.58 $\pm$ 0.35). In addition, the results of IL-18 estimation showed that the concentrations of IL-18 in the females of acute hepatitis A virus patients were (1.01 $\pm$ 0.76), while the concentration of IL-18 in males of acute hepatitis A virus patients which was (1.06 $\pm$ 0.67). The results of statically analysis of IL-18 estimation for the patients and control groups revealed that their difference was statistically significant (The $P$ value $=$ 0.00 which it is less than sig $=$ 0.05). These results showed that their difference was not statistically significant at the level of (Age groups and Gender), $P$ values $=$ (0.274, 0.691) respectively (Table 6).

3.4 Levels of biochemical tests for both patients and healthy people

Table 7
Distribution of liver function tests according to status, age group and gender

Variables	ALT (GPT)	AST (GOT)	ALP	TSB
	(Mean $\pm$ Sd)	(Mean $\pm$ Sd)	(Mean $\pm$ Sd)	(Mean $\pm$ Sd)
Status
Patient	170.18 $\pm$ 117.67	183.05 $\pm$ 128.13	607.68 $\pm$ 214.93	2.77 $\pm$ 2.5
Control	21.25 $\pm$ 7.41	26 $\pm$ 7.69	202.02 $\pm$ 121.35	0.55 $\pm$ 0.14
$P$ value	0.00^*	0.00^*	0.00^*	0.00^*
Age group
1–5	105.99 $\pm$ 83.01	117.72 $\pm$ 91.64	568.57 $\pm$ 296.15	1.53 $\pm$ 1.3
6–10	141.48 $\pm$ 150.36	167.13 $\pm$ 166.06	476.83 $\pm$ 251.85	2.35 $\pm$ 2.88
11–15	83.91 $\pm$ 92.14	89.01 $\pm$ 110.36	348.81 $\pm$ 226.97	1.72 $\pm$ 2.07
16–20	85.8 $\pm$ 98.47	82.83 $\pm$ 93.72	353.94 $\pm$ 294.01	1.6 $\pm$ 1.92
21–25	32.69 $\pm$ 26.42	37.33 $\pm$ 18.35	238.79 $\pm$ 190.43	0.53 $\pm$ 0.16
$>$ 25	127.83 $\pm$ 168.09	119.82 $\pm$ 125.8	449.31 $\pm$ 246.26	1.94 $\pm$ 2.66
$P$ value	0.197 NS	0.068 NS	0.013^*	0.455 NS
Gender
Male	115.52 $\pm$ 114.83	126.43 $\pm$ 125.37	455.48 $\pm$ 260.55	1.77 $\pm$ 1.94
Female	87.41 $\pm$ 112.98	94.58 $\pm$ 118.72	386.05 $\pm$ 278.05	1.75 $\pm$ 2.39
$P$ value	0.199 NS	0.175 NS	0.182 NS	0.952 NS
Total	102.48 $\pm$ 114.32	111.66 $\pm$ 122.81	423.29 $\pm$ 269.81	1.76 $\pm$ 2.15

The biochemical tests, including total serum bilirubin (TSB), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP), were conducted on 60 hepatitis A virus (HAV) patients and 50 healthy individuals. The results showed that patients infected with HAV had elevated levels of ALT, AST, ALP, and TSB compared to the control group. Statistical analysis of these results, based on sample condition (Patient, Control), is presented in (Table 7).

4. Discussion

4.1 HAV detection

The tests results of antiHAV IgM test device by VIDASHAVM were Positive, and the healthy volunteers we tested by rapid test and antiHAV IgM were Negative (Table 4). The results of the current study agree with the results of study conducted [9] showed that the HAV test device is a rapid visual immunoassay for the qualitative. While, the IgM antibodies titer that formed against the HAV presumptive detection of HAV in human serum or plasma specimens and is intended for use as an aid in the diagnosis of HAV infection [10, 11].

Figure 2.

Detection of hepatitis A virus by one step RT-PCR in patients group) one of the runs in detection of HAV).

The detection of HAV using Real Time polymerase chain reaction (RT-PCR) represents a critical component of diagnostic strategies for HAV infection. RT-PCR is widely recognized for its high sensitivity and specificity in detecting viral nucleic acids, including HAV RNA, in clinical specimens [12, 13]. The Present study confirms the reliability and utility of RT-PCR in diagnosing HAV infection, as evidenced by the positive detection of HAV RNA in all patient samples (Fig. 2). The early detection of HAV RNA by RT-PCR facilitates timely diagnosis and initiation of appropriate management strategies for patients with HAV infection [14]. The confirmation of HAV infection through RT-PCR underscores the importance of molecular methods in enhancing diagnostic accuracy and guiding clinical management decisions.

4.2 IL-10 concentration

Initially recognized as a Th2 cytokine secreted by CD4 cells, IL-10 was also known as an inhibitor of cytokine synthesis. It was proposed that IL-10 could affect the Th1 response indirectly. It is recognized that a wide variety of cells can produce IL-10, including dendritic cells, B cells, macrophages, CD4 T cells, CD8 T cells, NK cells, adaptive T cells, and regulatory T cells (15) have reported that IL-10 can be produced by a range of liver cell types, such as hepatocytes, sinusoidal endothelial cells, Kupffer cells, hepatic stellate cells, and liver-associated lymphocytes. According to [16] members of the IL-10 family are crucial for immunological tolerance and hepatic immunosuppression in liver disease.

According to these previous findings, IL-10 levels were frequently higher in hepatitis individuals than in healthy controls [17, 18] and according to [19], hepatitis C patients exhibited low levels of IL-10, indicating significant viral control and the killing of liver cells by pro-inflammatory cells because it produces the lowest quantities of IL-10.

Figure 3.

IL-10 Standard curve that measured by ELISA in HAV patients.

IL-10 is essential for controlling immunological responses and promoting the body’s removal of bacteria and viruses. Appropriate Th2 system secreted IL-10 levels are directly correlated with immune system strength. In the present investigation, we discovered that hepatitis A patients’ IL-10 levels were higher than those of healthy controls (Fig. 3) (Table 6). The increase, especially in the early stages when symptoms first appear, points to a well-regulated immune response to the virus. On the other hand, a strong Th1 response, IL-18 is necessary during acute HAV infection and can aid in viral clearance. However, persistently high IL-10 levels can lead to severe liver failure. It is important to recognize that hepatitis A triggers a dual immune response. Conversely, a Th2 response, shown by high IL-10 levels, may be the cause of acute infection that progresses to acute liver failure. According to [20], IL-10 is released to stop the inflammatory response and stop immune-mediated aggravation of liver injury. On the other hand, sustained increase of IL-10 may reduce the likelihood of severe liver failure by attenuating the Th1 response, which would impede effective viral clearance. As long as the virus is active, our results match with research by [21] that demonstrated IL-10 levels increased with an increase in viral load and peak liver damage.

In humans, polymorphisms in the IL-10 gene on chromosome 1 are connected with IL-10 production. The degree of IL-10 secretion influences the vulnerability to hepatitis caused by this highly variable gene. Immunity against viral infections is conferred by polymorphisms that support appropriate IL-10 production, whereas less IL-10 production is associated with heightened susceptibility. The patient’s genotype should be taken into account when treating hepatitis; poor IL-10 producers may benefit from greater cytokine quantities, while strong IL-10 producers may benefit from lower quantities [19].

Figure 4.

Standard curve of IL-18 that measured by ELISA in HAV patients.

4.3 IL-18 concentration

IL-18 is a pleiotropic inflammatory cytokine initially identified in the mouse liver, known to activate T and NK cells [22]. The results of our investigation on IL-18 levels showed a significant difference between the groups of patients with hepatitis A virus and the control group (Fig. 4). This is consistent with research by Kim et al. [23] that discovered those with acute hepatitis brought on by HAV infection had noticeably higher levels of IL-18. Significant IL-18 levels were found in the hepatocytes and macrophages of patients with acute hepatitis A, there was a group of our patients who were aged $>$ 25 (Table 6), The concentration of interleukin 18 was high and higher than the concentrations of the remaining patients, while interleukin 10 was also high. They had very severe symptoms. We noticed their physical weakness, the color of their skin and the whites of their eyes very yellow. Also, liver enzymes were high, and this is certainly due to damage resulting from the immune response and also due to the continued activity of the virus and this is due to what was reported in this study by Belkaya et al. [24]. The consequence is abundant and uncontrollably produced IL-18 being devoured by NK cells, which killed both HAV-infected and uninfected hepatocytes. If therapy is delayed, the liver may suffer damage or even fail. Our study also identified greater IL-18 levels in hepatitis A patients than in controls, indicating the potential toxicity of unregulated IL-18 in the liver [24].

Our findings align with the study by [25], which suggests that IL-18 and IFN-gamma are involved in the pathogenesis of acute hepatic injury in humans. Particularly, elevated serum levels of IL-10 may be predictive of improved outcomes for these patients. This indicates that monitoring IL-10 levels could serve as a valuable prognostic marker in acute hepatitis cases.

Understanding the interactions of IL-10 and IL-18 in HAV infection is critical for designing tailored therapeutics that can control the immune response, boosting viral clearance while minimizing liver damage. More research is needed to determine the precise causes and potential treatments for acute hepatitis A.

4.4 Biochemical tests levels

Numerous studies have aimed to develop simple and inexpensive biochemical markers as alternatives to liver biopsy. In HAV-positive patients, routine biochemical blood tests such as ALT, AST, ALP, TSB, and measurement of serum HAV RNA levels are typically conducted [12, 26]. Our findings revealed elevated levels of these biochemical markers in hepatitis A patients, aligning with previous research [27, 6], which demonstrated significant associations between viral hepatitis and increased levels of AST, ALT, ALP, and TSB. Additionally, there was a correlation between improved immune cell parameters and biochemical tests, reinforcing the connection between immune response and liver function in hepatitis A virus infection and our results indicate the presence of a Positive correlation between all liver function tests, the P values are equal to zero, and there are less than 0.05. When the Pearson Correlation close to 1. The indicate a perfect positive linear relationship: As one variable increases, the other variable also increases proportionally, the strongest correlation was between (ALT and AST), the Pearson Correlation $=$ 0.906, while the weakest relationship is between (TSB and ALP) which equal to 0.405 (Table 7). ALT, AST concentrations in patients serum are very sensitive indicator of hepatocellular injury [28].

All liver functions were high only for patients, There were significant differences in ALP levels among different age groups. The reported p-value is 0.013, which is less than sig $=$ 0.05, indicating statistically significant differences in ALP levels across different age categories (Table 7). ALP is generally higher in children and adolescents due to the increased osteoblastic activity associated with the bone growth that is why it is not a strong parameter to the acute liver failure [29].

5. Conclusion

In the end, our findings shed light on the immunological and biochemical changes generated by hepatitis A virus (HAV) infection. We found substantial variations in IL-10 and IL-18 levels between HAV patients and controls, which is consistent with previous research findings. In particular, the starting of elevated levels of IL-10 in patients with acute hepatitis A indicate that this molecule is necessary for activating an effective immune response. Thus, lowering IL-10 levels by different means may have a major effect on how quickly a disease progresses. Moreover, IL-10 levels show promise as a marker for HAV and assessment of therapy response. Our findings suggest a common immune response to viral infections, which is in line with findings in other kinds of hepatitis. On the other hand, IL-10 inhibition may increase IL-18’s pro-inflammatory actions, worsening liver damage in HAV patients. Likewise, the noticeably higher Levels of IL-18 highlight its part in the pathophysiology of acute hepatitis A. Notably, our data demonstrated consistent IL-10 and IL-18 responses throughout various demographic groups, underscoring the immune response’s resilience. Furthermore, the elevated liver function tests among HAV patients that have been reported are consistent with the body of research that links viral hepatitis to hepatocellular injury. These results highlight the complex relationship that exists between immune dysregulation and liver damage in HAV infection, underscoring the need for additional study to clarify underlying mechanisms and create focused treatment approaches. Our research clarifies the pro-inflammatory characteristics of interleukin-18 in acute hepatitis A virus infection, in contrast to the anti-inflammatory activity of interleukin-10. In summary, immune reactions have a significant impact in increasing the possibility of infection in acute liver failure, not only the elevation of liver enzymes. These parameters are also considered an indication for understanding the association of hepatitis with acute liver failure.

Author contributions

Conception: Zahraa A. Hussein and Saif D. Al-Ahmar.

Methodology: Zahraa A. Hussein and Saif D. Al-Ahmar.

Data collection: Zahraa A. Hussein and Saif D. Al-Ahmar.

Interpretation or analysis of data: Zahraa A. Hussein.

Preparation of the manuscript: Zahraa A. Hussein.

Revision for important intellectual content: Zahraa A. Hussein.

Supervision: Zahraa A. Hussein.

Data availability

The data substantiating the results of this study are available and can be retrieved from the authors on demand.

Ethical approval

Required approval was achieved by the institute according to the Helsinki Declaration.

Funding

There was no financial support.

Footnotes

Acknowledgments

Extended deepest gratitude to all the participants who generously contributed their time and insights to this study. Special thanks are extended to the Al-Diwanyah Teaching Hospital and all physicians for their valuable contribution and support.

Conflict of interest

No conflict of interest was declared by the authors.

References

Hawker

. Reintjes

Ekdahl

Edeghere

and Van Steenbergen

J.E.

, Communicable disease control and health protection handbook. 4 Edition, John Wiley & Sons, 2019, p. 480.

Mbithi

J.N.

Springthorpe

V.S.

and Sattar

S.A.

, Comparative in vivo efficiencies of hand-washing agents against hepatitis A virus (HM-175) and poliovirus type 1 (Sabin), Appl Environ Microbiol 59(10) (1993), 3463–9. doi: 10.1128/aem59.10.3463-3469.1993.

Bidawid

S.J.M.

and Sattar

S.A.

, Contamination of Foods by Food Handler: Experiments on Hepatitis a Virus Transfer to Food and Its Interruption, Applied and Environmental Microbiology 66(7) (2001), 2759–2763.

Mbithi

J.N.

Springthorpe

V.S.

Boulet

J.R.

and Sattar

S.A.

, Survival of hepatitis A virus on human hands and its transfer on contact with animate and inanimate surfaces, J Clin Microbiol 4 (1992), 757–63. doi: 10.1128/jcm30.4.757-763.1992.

Public Health England, Public Health Control and Management of Hepatitis A – 2017 Guidelines. Public Health England, (2017), 84.

Shammout

Alhassoun

and Rayya

, Acute Liver Failure due to Hepatitis A Virus, CaseRep. Gastroenterol 15(3) (2021), 927–932. doi: 10.1159/000514393.

Iyer

S.S.

and . , Role of interleukin 10 transcriptional regulation in inflammation and autoimmune disease, Crit Rev Immunol. 32(1) (2012), 23–63. doi: 10.1615/critrevimm.unolv32.i1.30.

Ihim

S.D.

Abubakar

Zian

Sasaki

Saffarioun

Maleknia

and Azizi

, Interleukin-18 cytokine in immunity, inflammation, and autoimmunity: Biological role in induction, regulation, and treatment. Front Immunol. 13 (2022), 919973. doi: 10.3389/fimmu2022.919973.

Utsumi

Yano

Amin

Lusida

Soetjipto

Hotta

and Hayashi

, Acute hepatitis due to hepatitis A virus subgenotype IA as an imported infectious disease from Indonesia, Kobe J. Med. Sci. 60(2) (2014), E43–47.

10.

Juniastuti

D.W.

Nihayatussa’adah

M.A.

Yamani

L.N.

Utsumi

Sustini

and Lusida

M.I.

, Analysis of genetic and serology of hepatitis A virus infection during and after outbreak in two junior high schools in Surabaya, Indonesia. J Med Virol. 91(6) (2019), 1048-1055. doi: 10.1002/jmv.25403.

11.

Gowda

Desai

Hull

Avinash

Vernekar

and Kulkarni

, A review on laboratory liver function tests, The Pan African Medical Journal 3 (2009), 112–123.

12.

Lemon

S.M.

Ott

J.J.

Van Damme

and Shouval

, Type A viral hepatitis: A summary and update on the molecular virology, epidemiology, pathogenesis and prevention. J Hepatol (2017), S0168–8278(17)32278-X. doi: 10.1016/jjhep.2017..

13.

Mustafa

Al-Samarraie

and Ahmed

, Molecular techniques of viral diagnosis, Science Archives 1(3) (2020), 89–92. doi: 10.47587/SA2020.1304.

14.

Pauly

and Lilia

, Point-of-Care testing for hepatitis viruses: A Growing need, Life 13(12) (2023), 2271. doi: 10.3390/life13122271.

15.

Pestka

Krause

C.D.

and Walter

M.R.

, Interferons, interferon-like cytokines, and their receptors, Immunol Rev 202(1) (2004), 28–32. doi: 10.1111/j0105-2896.2004.00204.x.

16.

Caparrós

and Francés

, The Interleukin-20 Cytokine Family in Liver Disease, Front. Immunol. 9 (2018), 1155. doi: 10.3389/fimmu2018.01155.

17.

Zhang

L.J.

Zheng

W.D.

Chen

Y.X.

Huang

Y.H.

Chen

Z.X.

Zhang

S.J.

Shi

M.N.

and Wang

X.Z.

, Antifibrotic effects of interleukin-10 on experimental hepatic fibrosis, Hepatogastroenterology 54(79) (2007), 2092–8.

18.

Özgüler

Akbulut

H.H.

and Akbulut

, Evaluation of Interleukin-10; Levels in Patients Diagnosed with Chronic Hepatitis. West Indian Med J 64(2) (2015), 71–5. doi: 10.7727/wimj2014.154.

19.

Afzal

Zain

Sumra

A.A.

Ali

Akhtar

Ashfaq

Parveen

Anwar

and Andleeb

, Role of Interleukin 10 in Hepatitis C Viral Clearance and Distribution of its Polymorphism in Pakistani Population, International Journal on Emerging Technologies 12(1) (2021), 217–224.

20.

Bourdi

Masubuchi

Reilly

T.P.

Amouzadeh

H.R.

Martin

J.I.

George

J.W.

and Shah

A.G.

Pohl

. Protection against acetaminophen-induced liver injury and lethality by interleukin 10: role of inducible nitric oxide synthase, Hepatology 35(2) (2002), 289–98. doi: 10.1053/jhep2002.30956.

21.

Das

Ellis

Pallant

Lopes

A.R.

Khanna

Peppa

Chen

Blair

Dusheiko

Gill

Kennedy

P.T.

Brunetto

Lampertico

Mauri

and Maini

M.K.

, IL-10-producing; regulatory B cells in the pathogenesis of chronic hepatitis B virus infection, J Immunol 189(8) (2012), 3925–35. doi: 10.4049/jimmunol1103139.

22.

Kaplanski

, Interleukin-18: Biological properties and role in disease pathogenesis. Immunol. Rev. 281(1) (2018), 138–153. doi: 10.1111/imr12616.

23.

Balin

S.J.

Pellegrini

Klechevsky

Won

S.T.

Weiss

D.I.

Choi

A.W.

Hakimian

Ochoa

M.T.

Bloom

B.R.

Lanier

L.L.

Stenger

and Modlin

R.L.

, Human antimicrobial cytotoxic T lymphocytes, defined by NK receptors and antimicrobial proteins, kill intracellular bacteria. Sci Immunol. 3(26) (2018), eaat7668. doi: 10.1126/sciimmunolaat7668.

24.

Belkaya

Michailidis

Korol

C.B.

Kabbani

Cobat

Bastard

Lee

Y.S.

Hernandez

Drutman

de Jong

Y.P.

Vivier

Bruneau

Béziat

Boisson

Lorenzo-Diaz

Boucherit

Sebagh

Jacquemin

Emile

J.F.

Abel

Rice

C.M.

Jouanguy

and Casanova

J.L.

, Inherited IL-18BP deficiency in human fulminant viral hepatitis, J Exp Med. 216(8) (2019), 1777–1790. doi: 10.1084/jem20190669.

25.

Yumoto

Higashi

Nouso

Nakatsukasa

Fujiwara

Hanafusa

Yumoto

Tanimoto

Kurimoto

Tanaka

and Tsuji

, Serum gamma-interferon-inducing factor (IL-18) and IL-10 levels in patients with acute hepatitis and fulminant hepatic failure, Journal of Gastroenterology and Hepatology 17(3) (2002), 285–294. doi: 10.1046/j.1440-1746.200202690.x.

26.

Kalas

M.A.

Chavez

Leon

Taweesedt

P.T.

and Surani

, Abnormal liver enzymes: A review for clinicians, World J Hepatol 13(11) (2021), 1688–1698. doi: 10.4254/wjh.v13i11.1688.

27.

Palewar

M.S.

Joshi

Choudhary

Das

Sadafale

and Karyakarte

, Prevalence of Hepatitis A virus (HAV) and Hepatitis E virus (HEV) in patients presenting with acute viral hepatitis: A 3-year retrospective study at a tertiary care Hospital in Western India, Journal of Family Medicine and Primary Care 11(6) (2022), 2437–2441. doi: 10.4103/jfmpcjfmpc_1746_21.

28.

Thuener

, Hepatitis A and B infections, Prim Care. 44 (2017), 621–629.

29.

Lowe

Sanvictores

and John

, Alkaline Phosphatase. In Stat Pearls. Treasure Island, Stat Pearls Publishing, 2021.

IL-10 and IL-18: Key players in liver damage associated with hepatitis A virus infection

Abstract

BACKGROUND:

OBJECTIVE:

METHODS:

RESULTS:

CONCLUSION:

Keywords

1. Introduction

Table 1 Distribution of patients and control groups according to age group

2.1 Extraction of RNA

2.2 Detection of hepatitis a virus by one step real time – polymerase chain reaction

Table 3 The addition of reaction mix in RT-PCR

2.4 Measurement levels IL-10 and IL-18 for both patients and healthy people

2.5 Measurement levels biochemical tests for both patients and healthy people

2.6 Statistical analysis

2.7 Ethics consideration

Table 4 Distribution of mean and Sd of the IgM titer for patient according to the gender and age group

3.1 Serological methods

Table 5 Distribution of mean and Sd of one-step RT-PCR for patients group according to gender

Table 6 The results of IL-10 and IL-18 of patients and control group

3.4 Levels of biochemical tests for both patients and healthy people

Table 7 Distribution of liver function tests according to status, age group and gender

4.1 HAV detection

4.4 Biochemical tests levels

5. Conclusion

Author contributions

Data availability

Ethical approval

Funding

Footnotes

Acknowledgments

Conflict of interest

References

Table 1
Distribution of patients and control groups according to age group

Table 3
The addition of reaction mix in RT-PCR

Table 4
Distribution of mean and Sd of the IgM titer for patient according to the gender and age group

Table 5
Distribution of mean and Sd of one-step RT-PCR for patients group according to gender

Table 6
The results of IL-10 and IL-18 of patients and control group

Table 7
Distribution of liver function tests according to status, age group and gender