*Translating the Adipose Tissue-liver axis in Metabolic dysfunction associated SteatoHepatitis: finding inflammatory therapeutic targets and biomarkers*

Metabolic dysfunction associated steatotic liver disease (MASLD) is a condition
where fat accumulates in the liver (steatosis) occurring in 25% of people
worldwide (1). In obesity, MASLD risk rises to 90% (2). In 7-30%, steatosis
progresses to an inflammatory state called metabolic dysfunction associated
steatohepatitis (MASH), increasing the risk for developing fibrosis, cirrhosis
and liver failure (1), necessitating liver transplantation (3). MASH patients
also develop more hepatocellular cancers, cardiovascular disease and type 2
diabetes. These cardiometabolic diseases account for the majority of global
morbidity and mortality (WHO, 2020). Developing liver inflammation is thus a
crucial step in disease progression and clinical outcomes.

There are two major clinical problems with MASH. First, diagnosis of MASH is
difficult. MASH is a very dynamic condition, requiring multiple times of
assessment. Although elevated liver enzymes indicate hepatocyte damage, ALT
levels have disappointing 50% sensitivity and 61% specificity for MASH and some
patients retain normal ALT levels during disease progression. The so-called
*MASH test*, combining demographic parameters like age, sex and BMI, with
several serum parameters, has a negative predictive value of 81% for MASH, but
very poor sensitivity (4). The gold standard, i.e. a liver needle biopsy, is
invasive and with a significant risk of complications. Hence, less than 50% of
endocrinologists and general practitioners refer patients at risk for a biopsy
(5). Thus, many MASH cases remain undiagnosed. Nevertheless, even after a
biopsy, diagnosis is not straightforward. Such needle biopsies are very small
while the liver often displays heterogeneity, risking sampling bias. Moreover,
the dichotomy of MASH vs non-MASH based on histological scoring systems fails
to capture borderline cases, and leaves subtle inflammatory changes in the
liver that may precede MASH unrecognized. As a result, the true nature of MASH
in humans remains elusive. Second, there are currently no treatments for MASH
(6). These limitations are least partially due to a knowledge gap about the
mechanisms of liver inflammation in MASH development in humans.

MASH is influenced by crosstalk with other tissues (7). Given the high
incidence of MASH in obesity, the AT-liver axis is particularly interesting
(8). Our previous work in experimental animals showed that AT expansion induces
neutrophil and monocyte recruitment to the liver from the circulation, causing
inflammation and liver damage, independent of other risk factors (9). These
data Illustrate the causal contribution of AT to hepatic inflammation and the
involvement of circulating immune cells herein. Other groups also showed a role
for circulating immune cells in hepatic inflammation using experimental mouse
models (10-12).

We hypothesize that circulating immune cells are key mediators in the AT-liver
axis, and are therefore a promising biomarker and target for treatment of MASH.
Unfortunately, almost no data are available on the contribution of immune cells
to MASH development in the human AT-liver axis. This knowledge gap hampers the
development of diagnostic and therapeutic strategies targeting inflammation in
MASH. Here, we propose to investigate the role of immune cells in the AT-liver
axis during MASH development in humans.

Primary Objective:
To identify the immunological changes and associated mechanisms in the AT-liver
axis in overweight and obesity-related NASH.

Secondary Objective(s):
- Develop novel biomarkers of NASH
- Discover novel therapeutic targets for NASH

This research will be an observational cross-sectional study with invasive
measurements. Overweight and obese patients undergoing abdominal (bariatric,
gallbladder and other) surgery will be recruited. Biopsies of liver,
subcutaneous AT and visceral AT will be collected for histological evaluation,
as well as experimental measurements of immune cells (flow cytometry), gene
expression (qPCR, RNAseq), and biochemical measurements. Based on the hepatic
biopsies, MAFLD/MASH will be diagnosed. In addition, blood is collected for
immunological characterization and for measuring circulating plasma proteins.
Participants will be divided into the following groups, including expected
incidence based on previous research (09T30):
- Overweight and obese individuals without MAFLD (20% of individuals)
- Overweight and obese individuals with steatosis only (60% of individuals)
- Overweight and obese individuals with MASH without diabetes type 2 (10% of
individuals)
- Overweight and obese individuals with MASH with diabetes type 2 (10% of
individuals)

These groups will be compared to a control group of lean individuals undergoing
abdominal surgery, in which the occurrence of MAFLD is not expected. In this
way, we can evaluate the immunological and inflammatory changes in the tissues
and circulation that are specific to MASH in a background of overweight and
obesity. In addition, using the control group, we can delineate which changes
are specific for overweight and obesity. Patients with MASH are stratified
based on the presence of type 2 diabetes, since it is known that type 2
diabetes status impacts hepatic inflammation (13).

The research may have limited adverse effects. Patients will not notice
anything from the removal of pieces of fat and liver tissue as this will occur
during surgery while they are under anesthesia. There will be no additional
pain or scarring from the removal of the fat and liver tissue, and the patient
will not need to stay in the hospital longer because of it. Any minor bleeding
that occurs due to the removal of the fat will be immediately stopped by the
surgeon. In total, we will take (2 tubes) 10 ml of blood. This amount poses no
problems for adults and will be done during anesthesia.

Given the steep rise in obesity, the rate of MASLD and MASH is expected to rise
to worrying levels, while there is no cure available. MASH is associated with
increased risk of liver complications, i.e. cirrhosis, liver failure and
hepatocarcinoma. Furthermore, MASH greatly elevates the risk of developing
extrahepatic complications, including cardiovascular disease. Although weight
loss often leads to improvement of this condition, attaining and sustaining
weight loss are notoriously hard to achieve and do not always cure the
condition. Given the high chance of developing both hepatic and extrahepatic
complications in NASH and fibrosis patients, alternative intervention
strategies are urgently warranted. In addition to the lack of approved
treatments, there are no trustworthy biomarkers for MASH. This project will
identify novel biomarkers based on immune cell measurements that can ultimately
lead to a better identification of those patients at risk of developing MASH.
This will lead to improved risk stratification and patient care.
Thus, the participants in this study will contribute to increasing our
understanding of inflammation development in MASH in potentially finding novel
biomarkers and treatments for the disease. On the other hand, the proposed
procedures imposes no or very little additional burden on the participants as
the blood and tissue samples will be collected during already scheduled
clinical procedures and visits.
Potentials risks include small bleedings due to taking the biopsy, these will
immediately be stopped by the surgeon