Non-alcoholic fatty liver disease (NAFLD) includes a variety of histological conditions (ranging from liver steatosis and steatohepatitis, to fibrosis and hepatocarcinoma) that are characterized by an increased fat content within the liver. The accumulation/deposition of fat within the liver is essential for diagnosis of NAFLD and might be associated with alterations in the hepatic and systemic inflammatory state. Although it is still unclear if each histological entity represents a different disease or rather steps of the same disease, inflammatory processes in NAFLD might influence its pathophysiology and prognosis. In particular, non-alcoholic steatohepatitis (the most inflamed condition in NAFLDs, which more frequently evolves towards chronic and serious liver diseases) is characterized by a marked activation of inflammatory cells and the upregulation of several soluble inflammatory mediators. Among several mediators, cytokines and chemokines might play a pivotal active role in NAFLD and are considered as potential therapeutic targets. In this review, we will update evidence from both basic research and clinical studies on the potential role of cytokines and chemokines in the pathophysiology of NAFLD.

In the last decade, there has been a remarkable scientific effort to improve our understanding of the pathogenesis, diagnosis, and treatment of non-alcoholic fatty liver disease (NAFLD). Clinical studies revealed dramatically high prevalence of NAFLD worldwide[1,2]. Worrying data on the prevalence of NAFLD in children and adolescents was also revealed[3]. Importantly, in American adolescents followed in the National Health and Nutrition Examination Survey between 1999 and 2004, serum elevation of hepatic enzymes [i.e., alanine aminotransferase (ALT)] was observed in 6% to 11% of subjects (depending of ethnicity)[4]. Furthermore, serum ALT increase was positively associated with waist circumference and insulin resistance, suggesting that NAFLD might be considered as the hepatic manifestation of other epidemic diseases, such as metabolic syndrome and obesity[1,2]. In fact, in obese children and adolescents, NAFLD affects about 20% to 74%, indicating that this disease might start early during life, providing more time for its deleterious evolution[5-7]. However, we believe that NAFLD is limited to patients suffering from obesity, metabolic syndrome, or other fat-related diseases. Although NAFLD has been described as an increased hepatic accumulation of fat (steatosis), a recent scientific consensus defined it as a complex spectrum of diseases, ranging from asymptomatic steatosis with possible aminotransferase alterations to non-alcoholic steato-hepatitis (NASH), cirrhosis, and also hepatocellular carcinoma[8-10]. Whether these conditions are different stages of a common progressive disease or should be considered as different entities, is still an open question. Thus, additional pathophysiological studies on improved animal models are needed to clarify this issue. Indeed, NAFLD is often underestimated, under diagnosed, and not treated in the current medical practice; therefore, its pathophysiological history is at risk of remaining a mystery for several years.

At present, the most suitable area for improving our knowledge of the pathophysiology of NAFLD is represented by the chronic inflammation that underlies all NAFLD entities/stages[11]. Soluble cytokines and chemokines, regulating inflammatory cell function and survival, could be considered as very promising candidates. On the other hand, hormonal axes, adipocytokines, and growth factors have also received attention from NAFLD scientists. In the following paragraphs, we focus on cytokines and chemokines, updating evidence of their role in NAFLD pathophysiology, both in human (Table 1) and animal studies.

Cytokines are soluble molecules that are involved in intercellular communication and are produced by a wide variety of cells in the body, including most types of liver cells[12]. They comprise several subfamilies, including interferons, interleukins, tumor necrosis factors (TNF), transforming growth factors (TGF), colony-stimulating factors, and chemokines. Cytokines mediate several fundamental biological processes, including body growth, adiposity, lactation, hematopoiesis, as well as inflammation and immunity. However, they are also implicated in various pathologies, such as atherosclerosis, rheumatoid arthritis, systemic lupus erythematosus, psoriasis, as well as NAFLD[13-16].

Under physiological conditions, constitutive cytokine generation is absent or minimal in the liver. Nevertheless, pathological stimuli like lipid accumulation induces hepatic cells to produce these inflammatory molecules. Cytokines might play an active role in the development and the potential progression of NAFLD through stimulation of hepatic inflammation, cell necrosis and apoptosis, and induction of fibrosis. Nevertheless, they are also essential for liver regeneration following injury[16]. Evidence on the pathophysiological role of cytokine in NAFLD is reported and discussed below.

TNF-α

TNF-α is an inflammatory mediator secreted by several inflammatory cell types, including monocyte/macrophages, neutrophils, and T-cells, but also by many other tissues, such as the endothelium, adipose tissue, or neuronal tissue. In the liver, TNF-α is secreted directly by hepatocytes and Kupffer cells or indirectly by abdominal fat[17]. Several studies have shown that TNF-α is a key factor in the development of NAFLD and NASH in both humans and animals. Hotamisligil et al[18] showed for the first time a relationship between TNF-α expression and insulin resistance in NASH. The authors stated that adipose tissue represents an important source of obesity-induced inflammation, notably by the expression of TNF-α, which can induce inflammation and insulin resistance. Indeed, in several rodent models of obesity, TNF-α expression in adipose tissue was upregulated as compared to controls[18]. Accordingly with these study, obese mice lacking TNF-α showed an improved insulin sensibility[19]. Recently, mice treated with the anti-TNF-α drug thalidomide showed some improvements in the hepatic alterations mediated by a high-fat diet[20]. Moreover, the use of anti-TNF-α antibodies in an experimental model of NASH decreased inflammation, necrosis, and fibrosis in rats[21].

Although TNF-α inhibition in animal models of NAFLD presents encouraging therapeutic perspectives, in humans, the role of this cytokine remains controversial. In patients, TNF-α levels were shown to be higher in obese than in lean individuals, and were correlated with insulin resistance[22,23]. Moreover, a positive correlation was observed between the degree of liver fibrosis and circulating TNF-α levels in patients with NASH[24]. Another study showed increased TNF-α expression in the liver and adipose tissue in NASH patients with significant fibrosis in comparison with those with a slight or nonexistent fibrosis[25]. More recently, Hui et al[26] strengthened these results, showing increased TNF-α levels in subjects with steatohepatitis as compared to controls. The potential involvement of TNF-α in NAFLD pathophysiology was recently suggested by genetic studies on its polymorphisms[27,28]. Moreover, treatment with pentoxifylline (a molecule inhibiting TNF-α) decreased the serum levels of aminotransferases and displayed hepatic beneficial effects in patients with NASH[29].

Nevertheless, the involvement of TNF-α in insulin resistance and NAFLD is questionable. Some studies did not show any correlation between insulin resistance and TNF-α levels[30,31], whereas two clinical studies, using an antagonist and an anti-TNF-α antibody, did not show any improvements in insulin sensitivity[32,33]. Moreover, in a recent study, Lucero et al[34] did not observe any difference in circulating levels of TNF-α between patients with NAFLD as compared to controls without NAFLD.

TGF-β

TGF-β is a cytokine/growth factor with immunosuppressive, anti-inflammatory, and pro-fibrotic properties[35]. In the liver, TGF-β1 is the most abundant isoform, and it is secreted by immune cells, stellate cells, and epithelial cells[36]. TGF-β1 plays a pivotal role in hepatic fibrosis by mediating the activation of stellate cells and their production of extracellular matrix proteins[37-39]. Indeed, Kupffer and stellate cells produce TGF-β1, which induces the transformation of resting stellate cells to myofibroblasts[40]. In experimental models of hepatic fibrosis induced by CCl₄ or schistosomiasis, expression of TGF-β1 is upregulated[41-43]. Moreover, in patients with liver fibrosis, the expression of TGF-β1 mRNA is increased[40,44,45]. Stärkel et al[46] showed that the upregulation of TGF-β1 is an early molecular step in the progressive fibrotic steatohepatitis. A study by Hasegawa and co-workers showed that TGF-β1 levels were increased in patients with NASH as compared to hepatic steatosis. Thus, the measurement of serum levels of TGF-β1 might be useful to distinguish NASH patients in the spectrum of NAFLD[47]. Moreover, polymorphisms that induce high angiotensinogen and TGF-β1 are associated with advanced hepatic fibrosis in obese patients with NAFLD[48].

Chemokines (chemotactic cytokines) are small heparin-binding proteins known to induce mainly leukocyte trafficking, growth, and activation in inflammatory sites[71,72]. Many cell types, including endothelial cells, smooth muscle cells, leukocytes, hepatocytes, and stellate cells, can secrete them. Approximately 50 currently identified chemokines are classified in four subfamilies (C, CC, CXC, CX₃C) according to their structural arrangement of N-terminal conserved cysteine residues. Chemokines need to bind to their coupled seven transmembrane protein G coupled receptors on target cells to induce cellular changes. Chemokines and their receptors have been implicated in multiple inflammatory diseases, such as atherosclerosis, multiple sclerosis, psoriasis, and insulin resistance[73]. Expression of several chemokines and chemokine receptors has been shown to be upregulated in the livers of obese patients with severe steatosis and NASH[74]. Inflammatory processes are crucial in the potential progression of NAFLD; therefore, chemokines might also play a pivotal role in NAFLD pathophysiology[75].

The take-home message of the present update on the inflammatory pathophysiology of NAFLD do not recommend any optimistic insights for the near future. Much research remains to be done to clarify the pathophysiology of NAFLD and to identify selective targets for treatment. The involvement of cytokines and chemokines and their receptors in the pathogenesis of NAFLD is only partially understood. Although the first studies attempting therapeutic strategies targeting the chemokine system have been recently published, we believe that scientific interest in NAFLD should be increased. In particular, effort is required to improve the consideration of NAFLD as a dangerous condition that should not be underestimated or by-passed. Another crucial aspect is represented by the identification of common cytokines and chemokines between NAFLD and metabolic or cardiovascular diseases. The most promising mediators (such as TNF-α, CCL2 and CCL5) also require more selective inhibitory drugs to safely improve NAFLD, limiting the potential risk of deleterious immune-suppression.