Introduction

Mallory bodies (MBs), also known as Mallory-Denk bodies (MDBs), are cytoplasmic hyaline inclusions of hepatocytes, once thought to be specific for alcohol-related steatohepatitis (ASH). MDBs are also seen in other liver diseases, including metabolic dysfunction-associated steatohepatitis (MASH)—formerly known as nonalcoholic steatohepatitis (NASH),[1] cholestatic liver diseases, primary biliary cirrhosis (PBC), and hepatocellular carcinoma (HCC).[2] In 1911, Frank Burr Mallory discovered these histological findings while examining the hepatocytes of patients with alcoholic hepatitis. In 1975, Helmut Denk found the first animal model of MDBs by feeding mice griseofulvin, resulting in the renaming of these formations.[3] (See Image. Mallory Bodies.) As a result of transitioning from mouse models to applicability in humans, MDBs were found to primarily comprise keratins, chaperones, co-transporters, and other proteins. Broadly, they are categorized as classic, non-keratinous, rounded, and variants for clinicians grading patient biopsies. Histologically, these subtypes are specified when staging the disease process.

MDBs are formed by stress mechanisms leading to cell cycle dysregulation, which impairs the generation of normal hepatocytes and non-hepatic cells in the body. Since cell cycle dysregulation is a hallmark of the gastrointestinal (GI) diseases mentioned above, MDBs are clinically correlated. To an extent, the formation of MDBs is protective in inflamed tissue to limit further inflammation, leading to fibrosis, but several stressors may eventually accelerate the pathology. The learning activity below discusses the molecular basis of MDBs, the mechanisms by which they are formed, and evidence-based literature on clinical correlations. Typically, the interprofessional team may consist of primary care, gastroenterology, and pathology, leading to continued staging of disease progression. Students within these fields are equipped to identify the histological features of MDBs as they progress in training.

Causes

Hepatocytes

In general, MDB formation is known to present in liver diseases. Pertinent liver diseases include hepatitis B and C, alcoholic liver disease, metabolic function-associated steatotic liver disease (MASLD)—formerly known as nonalcoholic fatty liver disease (NAFLD),[1] hepatocellular carcinoma (HCC), focal nodular hyperplasia, Wilson disease, and copper toxicosis.[4] Pertinent gallbladder diseases include primary biliary cholangitis (PBC), chronic cholestasis, and cholangiocarcinoma. However, MDBs may also present following glucocorticoid therapy, intestinal bypass surgery for obesity, Weber-Christian disease, von Gierke disease, radiation pneumonitis, asbestosis, amiodarone, beta lipoproteinemia, porphyria cutanea tarda, antitrypsin deficiency, Indian childhood cirrhosis, perhexiline maleate hepatitis, cirrhosis, 2′3′-dideoxyinosine diethylaminoetheoxyhex-estriol–induced hepatitis, hepatic adenoma, sclerosing hyaline necrosis in Bloom syndrome, and congenital fibrosis.[5]

Non-Hepatic Cells

MDBs are rarely present in non-hepatic cells, but examples exist in asbestosis, renal cell carcinoma, and type 2 pneumocytes.[3] Clinical correlations discuss recent findings in lesser-known diseases detailed below.

Anatomical Pathology

P62 (a sequestosome), ubiquitin, and intermediate filament proteins keratins 8 and 18 (K8/18) are the significant elements that make up a Mallory body. In normal conditions, K8 and 18 are present in 1 to 1 ratio.[6] Protein misfolding, proteasome overload, a ratio of K8 greater than K18, and transamidation of K8 contribute to MDB formation.[7] K8 is insoluble and not easily amenable to degradation, resulting in MDBs. Overexpression of K18 inhibits MDB formation.[3] K8 is likelier to change the helical shape to cross-β sheets, resulting in MDB formation. Formations without K8 do not make MDB, thus linking cross-β sheets as necessary for MDB development.[8][9][10] A similar cross-linking is also seen in protein aggregation disorders such as prion disease and Alzheimer disease.

The results of recent studies continue to extrapolate the correlation of MDBs to other cell cycle markers, including stress-activated markers such as p16 and p21, suggesting a role in cellular senescence rather than just liver pathology.[11] Macrophages involved in the inflammatory process are transcriptionally heterogeneous.[12] Accordingly, aggregates such as MDBs are also implicated in activating nuclear factor-κB, though the downstream effects on inflammation are unknown.[13] The exact mechanism by which inflammation is upregulated is also unknown, though occurring at several stages of the cell regeneration process.

Clinical Pathology

Mallory-Denk bodies are ubiquitously found in alcohol-realted steatohepatitis (ASH), MASH, PBC, and HCC, among other GI disorders. Hepatotoxicity due to medications also causes MDBs; most recently, the use of amiodarone was directly linked.[14] The presence or absence of MDBs can determine a final diagnosis if the consultation of a pathologist is involved. Recently, MDBs were found in patients with COVID-19, suggesting a correlation to the pathological transformation of pneumocytes.[15] Formations appearing similar to MDBs were also seen in ovarian fibromas that underwent torsion.[16] The clinical correlations to conditions outside of gastroenterology are not yet diagnostic. Though MDBs indicate pathology, they do not typically inform the prognosis or influence the mortality rate. 

Cytoplasmic inclusion bodies, intracytoplasmic hyaline bodies (IHB), and MDBs can be visualized in HCC.[17] MDB was linked to the steatohepatitis variant of HCC, whereas intracytoplasmic hyaline bodies (IHBs) were not. However, the presence of IHBs correlates with shorter survival than MDBs.[18] MDBs are distinguishable from other cytoplasmic inclusions, such as IHBs. For example, MDBs consist of ubiquitin, p62, and keratin, whereas IHBs only consist of ubiquitin and p62. IHBs have intracytoplasmic hyaline bodies and are a morphological precursor to MDBs.[3][19]

Morphology

Mallory-Denk bodies are predominantly filamentous cytoplasmic inclusions, ranging from 3 to 24 nm versus 10 nm of intermediate filaments. 

The bodies are classified as:

• Type I: Parallels filaments
• Type II: Randomly oriented filaments occurring in the periphery
• Type III: Granular, amorphous, and occur around the center

MDBs occur in ballooned hepatocytes, but not all ballooned hepatocytes lead to MDBs; both features indicate active tissue inflammation. The formation occurs in stages:

1. Misfolded proteins (typically degraded by the proteasome pathway)
2. Young MDB in ballooned hepatocytes
3. Mature MDB

They are visible via hematoxylin-eosin stain; however, immunohistochemical staining of cytokeratin or ubiquitin is more sensitive and diagnostic. Pericellular fibrosis and neutrophils tend to surround the hepatocytes with MDB, causing satellitosis.[20] The distribution of MDB in the cells suggests different stages of their formation. For example, small cytoplasmic globular structures are early, large para-nuclear inclusions are mature, and those located in the periphery are considered old.[3] MDB are found in different liver zones and vary in location depending on the disease process. In PBC and Wilson disease, MDBs are seen in zone 1, whereas in ASH and MASLD, they are present in zone 3.[4]

Mechanisms

Before MDBs are formed, hepatocytes balloon, a response to oxidative stress, such as abnormal proteins (heat shock proteins) or fat, resulting in water accumulation in the hepatic cytoplasm.[4] Heat shock protein formation indicates cellular dysfunction.

Three mechanisms describe the formation:

1. The first mechanism is that epigenetics (acetylation, methylation, and ubiquitination of histones) contribute to genetic regulation during MDB formation. Hepatocytes have shown stored memory of previous inciting toxicity, leading to gene silencing.
2. The second pathway is the shift from the 26S proteasome to the immunoproteasome, though the exact mechanism of gene regulation is unknown. The 26S proteasome degrades intracellular (cytosolic, nuclear, and membrane) proteins.
   • The 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) agent induces MDB by changing 26s proteasome to immunoproteasome, accumulating protein. 
   • When chaperones (ie, heat shock protein 70) and proteins that refold misfolded proteins are uncontrolled, inclusion bodies such as MDB may form.
   • Toxins such as alcohol or DDC cause chaperones to become defective.
   • Misfolded proteins are targeted by ubiquitination, which signals the molecule via p62 for proteasomal and autophagic degradation.
3. The third is the chronic activation of the toll-like signaling receptors (TLR) that stimulate proinflammatory and cell growth pathways.
   • Interferon (IFR)-γ and tumor necrosis factor (TNF)-α stimulate TLR, which causes up-regulation of growth factors, resulting in the proliferation of MDB-forming cells.
   • Drugs can also create a shift to the formation of the immunoproteasome rather than the 26s proteasome.
   • DDC also stimulates TNF-α and IFN-γ expression, activating the TLR. Proinflammatory cytokines TNF-α and IFN-γ via TLR signaling cause up-regulation of the immunoproteasome and down-regulation of the 26s proteasome, resulting in undigested proteins and MDB formation.[3][7][21][22][23]

All 3 mechanisms describe different stress-induced points in the cell cycle, either at deoxyribonucleic acid replication, transcription, or translation. Depending on the inciting event, these mechanisms can occur simultaneously, sequentially, or separately.

Clinicopathologic Correlations

Liver Diseases

Mallory-Denk bodies as a marker for MASLD depend on the histological scoring system. Matteoni et al emphasized the formation of MDBs, while Kleiner et al, which is a more accepted scoring system, did not.[3] Kleiner's system emphasizes fibrosis, steatosis, inflammatory inflammation, and ballooning hepatocytes. Some recommend that the KRT8/K18 ratio is a biomarker for HCC.[6] In patients who use alcohol, a sensitive and specific test to detect MDB is Ub/immunostaining. Inclusions occur in the liver, muscle, and neural tissues, suggesting a common intracellular network of protein synthesis and degradation and responsiveness to stressors. MDBs can be considered a protective mechanism against cell injury or, rather, a step in the pathogenesis of liver damage.[4] The MDBs seem to reoccur within 2 to 3 days in animal models when alcohol use ceases after previous use of alcohol.[20]

Metabolic Dysfunction-Associated Steatohepatitis

Clinical findings of MASH (jaundice, leukocytosis, fever) may demonstrate classical histological findings of steatosis; however, few or no MDBs could be seen. MDBs are less developed in MASH, which tends to be less severe as a hepatic pathology. MASH in children often does not exhibit the formation of MDBs, suggesting that aging may play a role. 

Clinical Significance

Areas for Further Research

Abnormal protein folding is known to cause other diseases, including Alzheimer disease. A recent literature review demonstrated common ground between the mechanisms involved in Alzheimer disease and MDB formation. Due to the similarities, future studies should test betaine or S-adenosyl-L-methionine's (SAMe) effect on Alzheimer disease, as they have been shown to prevent MDB formation.[5] Beta sheets that are present in amyloid deposits are also present in MDB.[3]

In patients with alcoholic liver disease, about 70% to 75% have MDB. However, in patients with MASLD, MDB ranges from 7% to 90%.[4] The wide range of MDBs in MASLD is likely the result of not having the specific amount, which qualifies as excessive alcohol use, categorizing the patient as alcoholic rather than non-alcoholic. In severe alcoholic hepatitis, non-responders to corticosteroids had high histopathological findings of ballooning degeneration and MDB, suggesting that histopathological findings can identify those who may respond to corticosteroids.[24] Study results have shown that autophagy contributes to MDB degradation along with other intracellular compartments.[25][26] Fenofibrate, a fibric acid derivative that lowers cholesterol, prevents MDB formation by preventing disruption of the intermediate filament.[27]

Methyl donors like betaine and SAMe prevent MDB formation. SAMe prevents the demethylation of histones, which occurs with DDC. In contrast, betaine prevents MDB formation by preventing the changes in methionine metabolism, and betaine-homocysteine methyltransferase (BHMT) methionine increases from homocysteine.[7][21][28] TLR and p62 pathways are also prevented by betaine and SAMe.

These identified markers for MDBs are potential areas for novel therapeutic intervention at the primary, secondary, tertiary, and quaternary protein formation levels and in patients undergoing treatment for related conditions.[29][30] Though the results in animal studies do not fully apply to clinical medicine, specifically when the MDBs revert or recur, the implications may benefit clinicians treating patients with GI conditions.

Interprofessional Collaboration

Given the established process for identifying, differentiating, and diagnosing conditions based on the presence or absence of MDBs, further inclusion or exclusion criteria could refine the diagnostic process. Though the molecular basis is elucidated, several downstream effects of epigenetics, proteasome regulation, TLR signaling, and immunomodulation are unknown, though the aggregations resemble several protein-aggregating diseases. The role of MDBs in lesser-known reproductive or pulmonary diseases could be explored.