Nonalcoholic fatty liver disease (NAFLD) has become the most common cause of chronic liver disease in the world (25%) (1, 2), however, the epidemiology and clinical characteristics of the disease in Brazil and South America are still poorly known (3). NAFLD comprises a spectrum of disease ranging from simple steatosis (NAFL) to nonalcoholic steatohepatitis (NASH), cirrhosis and hepatocellular carcinoma (4, 5). Currently, there is no approved drug regimen to treat NASH (5, 6), as a consequence of the lack of treatment and the growing global obesity epidemic, the prevalence of NAFLD is increasing and NASH-related cirrhosis is a major cause of liver transplantation in the United States (7).

The association between NAFLD, obesity, and type 2 diabetes favors a chronic low-grade inflammatory state, which predisposes to the development of other comorbidities, such as dyslipidemia, systemic arterial hypertension, atherosclerosis, and acute myocardium infarction (8, 9). In addition, inflammation is a hallmark of NAFLD progression characterized by activation of resident liver cells, recruitment of circulating inflammatory cells, and upregulation of several soluble inflammatory mediators (10, 11). Pro-inflammatory cytokines, such as interleukin (IL)-12, IL-6, IL-1β, and tumor necrosis factor (TNF) might play a fundamental role in the onset and progression of NAFLD promoting insulin resistance, oxidative stress, hepatic inflammation, cell necrosis, apoptosis, and liver fibrosis (12, 13). On the other hand, the anti-inflammatory cytokine IL-10 is essential to regulate these processes and to stimulate liver regeneration after injury (14–16).

The gold standard for diagnosis and staging of NAFLD is liver biopsy, which allows us to evaluate several histopathological parameters (5, 17). However, it is an invasive, painful and expensive procedure, which presents some risk to the patient and can lead to sampling errors (17, 18). Considering all these points, non-invasive biological methods have been emerging as a new perspective on NAFLD diagnosis and also the monitoring of disease progression. Cytokines may act as potential noninvasive biomarkers in NAFLD progression, however, further investigations are needed (11, 19, 20). There is insufficient knowledge about how cytokines are involved in the immunopathogenesis of NAFLD and can act as biomarkers in the progression and prognosis of the disease. Most of these studies are developed in animal models and few report associations between these mediators and clinical parameters like were presented in this study (21, 22). The current study was designed to investigate the potential association of plasma cytokines levels with clinical and biochemical parameters in NAFL and NASH patients and to evaluate their possible role as biomarkers in the immunopathogenesis and progression of NAFLD.

NAFLD has emerged as the most prevalent chronic liver disease all over the world (2) and recent studies suggest that women are at higher risk of NAFLD than men, mainly in the post-menopause stage (30, 31). However, its immunopathology remains unclear, being necessary a non-invasive biomarker that could accurately determine disease severity and prognosis to replace the need for liver biopsy (22, 32, 33). Soluble inflammatory mediators, such as pro-inflammatory cytokines might act promoting and modulating crucial processes during onset and evolution of NAFLD and liver regeneration following injury, being targets of novel therapies and studies about non-invasive biomarkers in the last years (16, 34–36). Despite that, the role of the immune response in patients with NAFLD, in particular, the relationship between the levels of inflammatory/regulatory cytokines and the course of liver damage is still unclear. Our data showed that IL-6, IL-12, and TNF associated with insufficient IL-10 modulation might favor liver inflammation, leading to development of metabolic disturbances and increasing the chances of liver damage associated with fibrosis during the progression of NAFLD.

According to the ‘two-hits’ hypothesis, insulin resistance, oxidative stress, and immune cell activation are candidate elements of the pathogenic transition from simple steatosis to steatohepatitis (37). Proinflammatory and regulatory cytokines, such as IL-6, IL-12, TNF, and IL-10 are believed to play a central role in these processes. The role of IL-6 in NAFLD is closely associated with obesity and insulin resistance (38). Some studies have shown that IL-6 plasma concentrations may be positively correlated with systemic insulin resistance, IL-6 liver expression, hepatic inflammation, and fibrosis degree in NAFLD patients (39–41). In contrast, it also has been demonstrated that IL-10 protects skeletal muscle, adipose, and hepatic tissues from insulin resistance mediated by action of IL -6 (16, 42). We observed that NASH patients showed higher levels of IL-6 associated with lower IL-10/IL-6 ratio when compared to controls, suggesting insufficient anti-inflammatory compensation in this group. On the other hand, NASH patients lose the positive correlation between IL-6 and IL-10, while assuming a positive correlation between IL-6, IL-12, and TNF, implying that IL-10 could modulate IL-6 activity in control individuals and NAFL patients, but not in NASH group.

In this context, an overproduction of proinflammatory cytokines, such as TNF and IL-6 connected to a defect in the function or production of anti-inflammatory cytokine IL-10 could increase nitric oxide and lipid peroxidation, enhancing steatosis severity (43) as observed in histological founds of NASH group. This proinflammatory microenvironment could also favor worse glycemic control and higher prevalence of diabetic patients, as observed in the NASH group (86,7%), leading to the development of comorbidities related to metabolic syndrome.

TNF has a dual effect on the physiology of the liver due to its ability to induce both hepatocyte cell death and hepatocyte proliferation (44, 45). Circulating TNF levels have been reported to be higher in patients with NASH than in patients with steatosis and control individuals (39, 40). Besides, we did not find any statistically significant in TNF levels between the study groups, TNF levels were significantly correlated with steatosis degree (CAP), liver stiffness (Kpa), and NAFLD liver fibrosis score, suggesting a chronic liver injury and inflammation in progression. It has also been demonstrated that TNF production may be important to the first event in liver injury (45), triggering the production of other cytokines, such as IL-12, which is responsible for recruiting inflammatory cells, destroying hepatocytes and initiating a healing response, including liver fibrogenesis (46).

Our results identified a relationship between these inflammatory mediators, biochemical profiles, and disease severity in NAFLD patients. We observed that IL-10 and IL-12 were negatively correlated with the glycemic profile in NAFL group. It suggests that, in the initial phase of the disease, IL-10 and IL-12 could be related to insulin resistance (1° hit). Conversely, in the NASH group, IL-12 was positively correlated with NAFLD fibrosis score, while IL-10 did not demonstrate any correlations in this group, indicating that these patients lose IL-10 anti-inflammatory modulation and IL-12 may be related to the disease progression.

Contiguous to this, we observed that IL-12 was positively correlated with the discreet increase in direct bilirubin in NAFL group, which has been inversely associated with NAFLD prevalence independent of the number of metabolic risk factors (47, 48), suggesting that it might be a protective biomarker for NAFLD due to its antioxidant and cytoprotective effects. Despite borderline levels of direct bilirubin found in NASH group, the same correlation was not observed, indicating that, in this case, direct bilirubin was not able to promote a similar effect.

Another topic that involves IL-6 deserves to be discussed. The importance of this cytokine in inflammatory responses and in a worse prognosis during NAFLD is well established in hepatology. Our data supported this theory when we observed higher levels of IL-6 in the NASH group, accompanied by higher percentages of high-producers of this cytokine in NASH compared to NAFL and the control groups. However, our findings did not demonstrate significant correlations of IL-6 with clinical parameters that assess liver function: elastography and liver fibrosis scores. This data suggests that IL-6 seems to play an important role in liver immunopathology, but it does not seem to be a good soluble biomarker. The absence of these correlations suggests that the IL-6 does not effectively reflects liver function as plasmatic marker, and cannot replace or be associated with a biopsy to assess NAFLD progression clinically.

Despite that, IL-12 and TNF showed a positive correlation with liver damage parameters (such as elastography and liver fibrosis scores), and negative correlations with clinical variables that assess early metabolic disorders. Indeed, IL-12 and TNF did not demonstrated significant plasmatic changes like IL-6 in the studied groups. However, through these correlations, these two cytokines have a potential to be characterized as plasma biomarkers, especially IL-12 by highest percentage of high producers in NASH group. It is also important to quote the sample size limitation in our study may have influenced this observation. Although preliminary, these data are very relevant to direct potential markers to be explored in larger populations in new studies.

Finally, we propose that cytokines might act in promoting and modulating crucial processes during the onset and evolution of NAFLD. We conclude that NAFL patients exhibited a balance between pro-inflammatory (IL-1β, IL-6, IL-12, and TNF) and regulatory (IL-10) cytokines concentrations, which were associated with the beginning of metabolic alterations in NAFLD context. However, an exacerbated production of inflammatory cytokines followed by insufficient IL-10 modulation might favor the liver inflammation, contributing to the appearance of the metabolic disturbances and increasing the chances of liver damage associated with fibrosis development. Therefore, plasma concentration analysis of these molecules and its association with clinical parameters may be used in the future as new strategy to monitoring NAFLD progression and as prognostic biomarkers for NASH.

The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.

The studies involving human participants were reviewed and approved by Ethics Committee of the Federal University of Minas Gerais (CAAE 56184716.3.0000.5149 and CAAE 67583317.3.0000.5149). The patients/participants provided their written informed consent to participate in this study.

Conceived and designed the experiments: JG. Performed the experiments: TM-C, MB-B, RM, and BS. Analyzed the data: TM-C, NM, BP, and JG. Contributed reagents/materials/analysis tools: JG and WD. Select and lead clinical management of patients: CC, TF, and LF. Histopathological analysis: PV. Wrote the paper: TM-C. Critical revision of the manuscript for important intellectual content: JG, NM, and WD. All authors contributed to the article and approved the submitted version.

This work was supported by grants from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brazil - #439942/2018), Fundação de Amparo á Pesquisa do Estado de Minas Gerais (FAPEMIG, Brazil - #PPM-00233-17), Pró-Reitoria de Pesquisa da Universidade Federal de Minas Gerais-PRPq, Brazil, and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). The funders had no role in study design, data collection, and analysis, decision to publish, or preparation of the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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