Inflammation in Nonalcoholic Steatohepatitis

R Christopher Harmon; Dina G Tiniakos; Curtis K Argo

Disclosures

Expert Rev Gastroenterol Hepatol. 2011;5(2):189-200. 

In This Article

Histological Evaluation of NAFLD

Liver

Adult NAFLD The histological lesions that characterize noncirrhotic NAFLD in the adult are accentuated in zone 3 (perivenular) of the hepatic acinus. The most important include triglyceride accumulation within hepatocytes (i.e., steatosis), acinar (lobular) inflammation and, in progressive cases, hepatocellular injury, in the form of ballooning, apoptosis or necrosis, and/or pericellular/perisinusoidal fibrosis. Mallory–Denk bodies (MDBs), megamitochondria, mild iron deposition and glycogenated (vacuolated) nuclei in periportal hepatocytes are also described in NAFLD.[87,88]

The triad of steatosis, inflammation and hepatocyte ballooning is currently considered diagnostic of NASH (Figure 1), while the presence of fibrosis is not required for the diagnosis, as it is not required for the histological confirmation of other chronic liver diseases.[87,88] Liver biopsy is generally accepted as the best method to diagnose NASH despite recognized limitations, including sampling error and a small, but real, risk to patients.[87,88] Necroinflammatory activity in NASH may be graded as mild, moderate or severe by semiquantification of steatosis, acinar and portal inflammation and hepatocellular ballooning.[89]

Figure 1.

Nonalcoholic steatohepatitis with moderate activity in a middle-aged man with unexplained hypertransaminasemia. Low magnification (×100) demonstrates macrovesicular steatosis with acinar zone 3 accentuation (arrowhead), acinar inflammation (white arrows) and mild/moderate portal chronic inflammation. On high magnification (×400, insert), hepatocellular ballooning (black arrow) and a focus of mixed inflammation (white arrow) is noted in zone 3.
PT: Portal tract, THV: Terminal hepatic venule.

With progression of NASH, additional portal/periportal fibrosis may be evident, followed in some cases by bridging fibrosis with central–central and central–portal fibrous septa. Cirrhosis may be the end result of this process, with corresponding reduction of perisinusoidal fibrosis and other features of active NASH, including steatosis, ballooning and inflammation, probably giving rise to the numerous cases of 'cryptogenic' cirrhosis.[87–90] HCC and liver failure are known consequences of NASH-related cirrhosis.[31]

Longitudinal studies in biopsy-proven NAFLD have demonstrated that steatohepatitis is more commonly found in patients who progress to cirrhosis and/or die from liver disease, while 'simple' or 'bland' steatosis usually has a benign course.[12,91–94] However, it should be noted that in most of these studies it is not clear if 'simple steatosis' refers to steatosis only or steatosis with inflammation, since different authors have used different criteria. A more accurate and homogeneous terminology for 'simple/bland steatosis' is clearly needed to correctly assess the effect of acinar and/or portal inflammation per se – that is, without hepatocellular injury or fibrosis – in the natural history of NAFLD.[95]

Pediatric NAFLD In pediatric NAFLD, inflammatory changes are frequently portal-based and zone 3 predominance of diagnostic histologic lesions is uncommon. Portal-based fibrosis, more extensive steatosis, infrequent occurrence of hepatocellular ballooning and MDBs are also distinctive features of childhood NAFLD. The portal accentuation pattern (borderline zone 1 pattern), previously referred to as type 2 pediatric NASH, is observed in many pediatric NAFLD cases, while definite NASH resembling adult NASH with zone 3 injury, previously known as type 1 pediatric NASH, is less common.[96–98] Overlap cases with features of both histological patterns are common in pediatric NAFLD.[98] In the largest study to date evaluating 137 children with NAFLD, mild or moderate acinar and/or mild portal inflammation was present in most patients, while inflammation scores were higher in patients with significant fibrosis.[98] Portal fibrosis is common in pediatric NAFLD, and may evolve to periportal fibrosis and bridging fibrosis. Cirrhosis is observed in up to 3% of the cases.[99]

Histology of Inflammation in NAFLD Inflammation is a central histologic feature in NAFLD and it may involve the acini, the portal tracts or both. The presence of acinar inflammation is essential for the histological diagnosis of NASH,[88] but recent studies demonstrate that portal inflammation may also be important for the progression of NAFLD.[95,100] Moderate/severe acinar and moderate portal inflammation are associated with the presence of definite NASH in the largest cohort of NAFLD patients studied to date from the NASH Clinical Research Network (NASH-CRN).[101] As previously mentioned, a systematic review of longitudinal studies has demonstrated that inflammation is an independent predictor of advanced fibrosis in NASH patients,[40] highlighting the significance of this histological feature in the pathogenesis of progressive NAFLD.

Acinar Inflammation Acinar inflammation in NAFLD is usually mild and frequently consists of a mixed, acute and chronic, inflammatory-cell infiltrate composed of lymphocytes (mainly CD3+ T cells), plasmacytes, monocytes, macrophages and neutrophils within sinusoids. Eosinophils are occasionally present, mainly in relation to lipogranulomas (see later).[87,89,90] In steatohepatitis, the inflammatory cells may also be seen close to injured hepatocytes. Satellitosis, a lesion referring to ballooned hepatocytes containing MDBs surrounded by neutrophils, may rarely be seen in NASH, while it is a more frequent finding in steatohepatitis of alcoholic etiology.[102] It is proposed that MDBs are chemotactic for neutrophils, thus contributing to satellitosis.[103] Acinar inflammation is evaluated by measuring the number of necroinflammatory foci per 20× optical field.[89,104]

Lipogranulomas, consisting of chronic inflammatory cells, Kupffer cells and rare eosinophils surrounding a steatotic hepatocyte or a large lipid droplet, are frequently seen in NAFLD. Lipogranulomas are found near terminal hepatic venules, scattered in the acinus or in portal tracts. Usually they are small in size, but when large may be associated with fibrosis.[105,106] Microgranulomas, consisting of clusters of Kupffer cells, and single, periodic acid-Schiff-diastase-positive pigmented Kupffer cells may also be noted scattered in the acini, possibly indicating prior necroinflammatory activity. Kupffer cell aggregation in steatohepatitis usually occurs in zone 3, while in steatosis and normal liver, Kupffer cells are seen diffusely in the acini.[107] The number of microgranulomas[108] or CD68+ Kupffer cells[109] has been correlated with necroinflammatory activity, NASH diagnosis and stage of fibrosis in NAFLD. These findings support the significant role of Kupffer cells in the pathogenesis and progression of NAFLD by regulating hepatic triglyceride storage,[110] mediating the inflammatory response,[111] contributing to hepatocyte injury[112] and initiating fibrosis through TNF-α and TGF-β secretion.[88] On the other hand, data from experimental animal models indicate that macrophages are also important for fibrosis regression as they can also degrade extracellular matrix proteins and exert anti-inflammatory actions.[113]

Portal Inflammation Portal inflammation in adult NAFLD is usually of chronic type and is frequently mild. When the chronic inflammatory infiltrate is marked and/or the portal inflammation is disproportionate to the acinar inflammation, the suspicion of a superimposed chronic liver disease, such as chronic viral hepatitis C, should be raised.[106] The presence of neutrophils in portal and periportal regions raises the suspicion of alcoholic or cholestatic liver disease. In untreated NAFLD patients, increased (more than mild) portal inflammation has been proposed as a marker of advanced disease[95,100] and has been associated with steatosis severity, presence of hepatocyte ballooning[100] and diagnosis of definite NASH.[114] In treated NASH, portal lymphocytic inflammation is considered indicative of disease resolution, irrespective of the type of therapy.[115,116]

Immunopathology of Inflammation in NAFLD Recent studies have demonstrated the importance of innate immunity in the pathogenesis of NASH.[117] Most of the cells in the acinar or portal chronic inflammatory cell infiltrate are lymphocytes, and immunohistochemistry helps to highlight the effector cells of the innate immune response. Their number varies depending on the severity of necroinflammatory injury.[118] Natural killer (NK) T cells are regulatory T lymphocytes that are preactivated in situ by endogenous glycolipids, and are therefore considered to be innate immune effectors. NKT cells are present in normal liver and are relatively depleted in steatosis.[119] Initial evidence points towards their fourfold increase in NASH-related cirrhosis, but there are no data on their numbers in early-stage NASH.[120] Further work is needed in order to characterize the relationship between NK and NKT cell populations in NASH and to evaluate the effect of different NKT subsets on disease outcome.

The adaptive immune response in NAFLD involves CD4+ T-helper cells, and although NASH is not classically considered a Th1-polarized disease, recent data suggest that its pathogenesis may be influenced by an imbalance between a relative excess of proinflammatory Th1 cytokines (i.e., IFN-γ) and a deficiency in anti-inflammatory IL-4 and IL-10 cytokines.[117] Emerging evidence also points towards a functional role of Th17-mediated T-cell responses in the pathogenesis of NASH.[119] Th17 cells are a recently described subset of CD4+ T-helper cells producing the cytokine IL-17. IL-17 can induce the expression of neutrophil-attracting chemokines in epithelial and endothelial cells, but it can also by itself mobilize and activate neutrophils.[121] In fatty liver, the characteristic perivenular infiltration of both neutrophils and lymphocytes suggests enhanced recruitment via their two major receptors CXCR1 and CXCR2.[122]

Adipose Tissue & Muscle

Cross-sectional studies of NAFLD patients have highlighted the importance of visceral adipose tissue in the pathogenesis of steatohepatitis, and have demonstrated its direct association with increased liver inflammation and fibrosis.[123] Adipose tissue in obese individuals is characterized by adipocyte hypertrophy, increased angiogenesis, immune cell infiltration and extracellular matrix overproduction.[124] Interestingly, adipocyte hypertrophy has been recently associated with common variants in PNPLA3 known to confer susceptibility to NAFLD.[125]

Increased total numbers of macrophages and 'M1' or 'classically activated' macrophages are present in the adipose tissue of obese insulin-resistant individuals, and these are thought to be the primary source of circulating inflammatory molecules. Adipose tissue macrophages may also inhibit adipocyte differentiation, leading to altered adipokine secretion and ectopic storage of fat in non-adipose tissues, including liver and skeletal muscle.[126]

Intramyocellular fat content, measured by magnetic resonance spectroscopy, is increased in severely obese individuals, not only in skeletal,[127] but also in cardiac muscle.[128] Experimental studies have recently demonstrated that lipid overload does not induce significant 'lipotoxic' effects in skeletal muscle, as measured with markers of apoptosis, autophagy and proteolysis.[129] By contrast, myocardial steatosis may cause alterations in myocardial metabolism and efficiency in nondiabetic obese patients with NAFLD.[130]

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