Inflammation induced tissue damage

Cytokine-driven pathways to hepatocellular cancer

Lymphotoxins (LTα and LTβ) are members of the tumor necrosis factor (TNF) superfamily. Under physiological conditions LTs are expressed by activated T-, B-, NK- and lymphoid tissue inducer cells and are crucial for organogenesis and maintenance of lymphoid tissues. Whereas LTβ contains a transmembrane domain, LTα is soluble. Consequently, LT can exist as membrane bound heterotrimers (LTα1β2 or LTα2β1) interacting with LTβR or as soluble secreted homotrimers (LTα3) triggering TNF receptor 1, 2 (TNFR1, TNFR2) and the herpes virus entry mediator receptor (HVEM).

A causal relationship between chronic hepatitis, hepatocellular damage, fibrosis and carcinogenesis is well established. Various etiologies, including chronic alcohol consumption, chronic drug abuse, autoimmune disorders, toxins (e.g. aflatoxin B) or infections with hepatotropic viruses (e.g. HBV, HCV) can lead to chronic hepatitis, liver fibrosis and cirrhosis. HBV- and HCV-infections are by far the most common cause of chronic hepatitis in humans. Chronic HBV- and HCV-infections are frequently associated with HCC, the most prevalent primary human liver cancer, and except for HBV-infections, liver cirrhosis precedes HCC in most cases. The exact mechanisms driving hepatitis-induced liver cancer remain elusive. Among others, aberrant expression of cytotoxic cytokines is thought to be critically involved.

We currently investigate the involvement of various cytokines including LTs, LIGHT and other TNF superfamily members in the development of inflammation induced liver cancer in humans with chronic hepatitis B or C and appropriate mouse models for inflammation induced liver carcinogenesis.

Figure 1:
mRNA expression of TNF-superfamily members in viral (HBV, HCV induced) and non-viral liver diseases. Analysis of hepatic LTa, LTb, LTbR, LIGHT, TNFR1, and TNFa transcription by real-time PCR. Healthy individuals (Ctrl; n=15), patients chronically infected with HBV (n=19), HCV (n=49), affected by HCC (n=30) or suffering from various non-virus related liver disorders were investigated. Non-virus related liver diseases with hepatitis include alcoholic steatohepatitis (ASH; n=13), cholestasis (CH; n=3), primary biliary cirrhosis/ autoimmune cholangitis (PBC; n=5), end stage liver cirrhosis due to alcoholic liver disease (CIR; n=8), a1-antitrypsin deficiency (a1AT; n=1) and focal liver fibrosis (FLF; n=2). Non-virus related liver diseases without hepatitis include steatosis (ST; n=5), hemochromatosis/siderosis (HE; n=3), and Wilson’s disease (WD; n=1). Focal nodular hyperplasia (FNH; n=8) was investigated as a benign primary liver tumor. Diseases such as a1AT (●), FLF (▲), HE/SID (♦), and WD (Δ) are listed under “other liver diseases” (OLD). Horizontal bars represent the average mRNA expression level. The y-axis describes the DDCT values on a log2 scale. *, **, *** indicate statistical significance: * = p≤0.05; ** = p<0.001; *** = p<0.0001.

Investigating the cellular and molecular mechanisms of hepatitis induced hepatocarcinogenesis

Among others, deregulation of cytotoxic or inflammatory cytokines was linked to the development of chronic inflammation induced carcinogenesis.

We have recently described a strong upregulation of particular pro-inflammatory cytokines in humans suffering from chronic hepatitis B or C (HBV; HCV) or HCV/HBV induced liver cancer.

Figure 2: Development of chronic inflammation and HCC in mice expressing LTa and LTb under the control of the Albumin promoter. (A) Immunohistochemical analysis of representative 9 month-old C57BL/6 and AlbLTab livers. B220+ stained B-cells, CD3+ T-cells, F4/80+ macrophages, Kupffer cells and A6+ oval cells (scale bar: 150mm). Ki67+ proliferating hepatocytes (arrow heads) and inflammatory cells are indicated (scale bar: 50mm). (C) Macroscopy of C57BL/6 (left panel) and AlbLTab livers at the age of 12 (middle panel) and 18 months (right panel). White arrows indicate tumor nodules. White arrowhead indicates a liver lobe completely affected by HCC. Scale bar size is indicated. (C) Histological analysis of livers derived from C57BL/6 and AlbLTab mice at the age of 12 months. Dashed line depicts the HCC border. Collagen IV staining highlights the broadening of the liver cell cords and loss of collagen IV networks indicative of HCC in AlbLTab mice (scale bar: 200mm). High numbers of Ki67+ proliferating hepatocytes (arrowheads) are only found in AlbLTab HCC (right column; scale bar: 100mm). From Haybaeck et al., 2009, Cancer CELL.

Consequently, we have established a mouse model for inflammation-induced carcinogenesis (chronic hepatitis-induced hepatocellular carcinoma) by overexpressing pro-inflammatory cytokines specifically on hepatocytes.

Currently, we are studying the contribution of various cell types as well as different cytokines/chemokines to the inflammatory process and to liver carcinogenesis. For this reason, mice were intercrossed and analyzed at several time-points. Moreover, we analyze transcriptional changes and chromosomal aberrations within liver tumors.

Figure 3: aCGH analysis of AlbLTab HCC. The q-arm of each chromosome is shown and chromosome numbers are indicated. Black ellipses on the top of each q-arm represent the centromere. Dark horizontal bars within the symbolised chromosomes represent G bands. Chromosomal deletions are indicated in blue, amplifications in red (see methods for details). (A) HCC of individual AlbLTab mice were hybridized against liver tissue of age matched C57BL/6 mice and analyzed by aCGH analysis. Columns next to each chromosome represent individual HCC (1; 2; 3) with numerous chromosomal aberrations on the q-arm of various autosomes. No common pattern of chromosomal aberrations could be detected. (B) aCGH analysis of six representative HCC (1, 2, 3, 4, 5 and 6) taken from different lobes of the same AlbLTab liver. From Haybaeck et al., 2009, Cancer CELL.

Studying the impact of cytokines/chemokines on metastasis

We are studying the impact of cytokines/chemokines on metastasis in a model of cancer cell metastasis in the lung.

In this project we apply various knockout and transgenic mouse models and analyse tumor cell infiltration into the lung at different time-points.

Techniques involve e. g. real-time PCR, protein analysis (ELISA, Western Blot, immunohistochemistry), FACS for various immune cell types, serum analysis, cell culture and mouse transgenesis.

The role of inflammatory cytokines in the control of HCV replication

In this project we investigate the molecular mechanisms how cytokine signalling controls the replication of non-infectious HCV amplicons or infectious HCV/HBV by modulating cytokine signaling pathways (e.g. application agonists or antagonists; siRNA knockdown experiments of ligand and receptors).

We determine whether the replication of HCV amplicons in human hepatocytes (primary hepatocytes, transformed and non-transformed hepatocyte cell lines) depends solely on cyotkine signalling itself by specifically blocking cytokine receptor signalling on hepatocytes. Vice versa we will also try to enhance HCV amplicon replication by inducing cytokine receptor signalling with appropriate agonists.

If this leads to inhibition of viral replication we will specifically block downstream signalling components of the various investigated downstream pathways.

Chronic pancreatitis and pancreatic cancer

Chronic pancreatitis is long-standing inflammation of the pancreas that results in irreversible deterioration of pancreatic structure and function due to organ fibrosis, induration, duct obstruction, atrophy and, consequently, loss of endocrine and exocrine function. Hallmarks of the disease are severe abdominal pain, elevated pancreatic enzyme (amylase, lipase) levels. Chronic pancreatitis is predominantly caused by alcohol abuse, smoking or by other toxic substances and also genetic causes have become more common. In advanced cases the disease may result in pancreatic exocrine insufficiency and/or diabetes. Furthermore, chronic pancreatitis is a well-described risk factor for pancreatic adenocarcinoma, especially in cases of hereditary chronic pancreatitis.

The destruction of acinar cells in the exocrine pancreas leads to infiltration of mononuclear cells, neutrophils and macrophages and to the replacement of the parenchyma by fibrous tissue. It is still not fully understood which process triggers and maintains the inflammatory response underlying chronic pancreatitis.

In various organs, chronic inflammation generates a microenvironment that contains different types of leukocyte, such as normal tissue macrophages, tumor-associated macrophages, dendritic cells, neutrophils, mast cells and T cells. A plethora of cellular mediators, including cytokines, chemokines, and enzymes, complement this microenvironment.

Therefore, we analyzed human pancreatic tissue from patients with chronic pancreatitis and pancreatic cancer to investigate which cytokines and chemokines are playing an important role in inflammatory environment and cancer.
Based on this study we have generated a transgenic mouse model to determine whether the over expression of inflammatory cytokines within the pancreas is sufficient to induce inflammatory infiltrates and, if so, whether that inflammatory infiltrate is a sufficient condition to lead to diabetes and/or cancer.

Chronic myositis

Chronic myositis "Myositis" defines illnesses that involve chronic, or persistent, muscle inflammation. Myositis refers to several different illnesses, including polymyositis, dermatomyositis, and inclusion body myositis.  In most cases chronic muscle inflammation results in muscle weakness,  lack of contractability - leading to movement disablities, degeneration of muscle fibres, inflammation of peripheral nerves as well as muscle swelling and muscle pain. Myositis can affect many parts of the body. Sometimes the joints, heart, lungs, intestines, and skin can become inflamed. 

Many such conditions are considered likely to be caused by autoimmune conditions, rather than directly due to infection, although autoimmune conditions can be activated or exacerbated by infections. Myositis is also a documented side effect of the lipid-lowering drugs statins and fibrates.

We have generated various models of muscle inflammation (e.g. in skeletal muscle; heart) and currently apply those together with analysis of human specimens (e.g. cryo-frozen human muscle tissue) to better understand the mechanisms driving chronic inflammation induced tissue damage in the muscle.

Chronic inflammation in the CNS

To investigate the effects of chronic inflammation in the central nervous system (CNS) - we have generated transgenic mice with chronic inflammation in the CNS. We currently investigate the effects as well as molecular and cellular mechanisms that drive this inflammatory phenotype and examine its effects on myelin integrity, autoimmunity and CNS tissue destruction.