Module 3.3: Sterile inflammation & liver injury

Question 1

Explain why Alcoholic Hepatitis is relevant

Answer 1

• Median age of death: 37
• Bleak prognosis over 10 years
  o High early mortality
  o Sandahl 2011
• Affects younger people – ethical question regarding liver transplant
• Death to trial ratio extremely high. ETOh score 250x HepB, 24x PBC

Question 2

Descibe the histology in alc hep

Answer 2

• Ballooning
• inflammatory infiltrate
• Steatosis
• Mallory Denk bodies
• Mega mitochondria
• Fibrosis
• Bile plugging

Question 3

Describe how the alc hep is diagnosed

Answer 3

• Indistinguishable whether a patient has alcoholic hepatitis of NAFLD through histology

1. <3 months jaundice
2. Serum bilirubin >80mmol/L
3. Alcohol consumption >10 units/day (men), >7 units/day (women)
4. <2 months abstinence
5. AST < 500; ALT <300
6. Maddrey’s discriminant function ≥32

Question 4

Give examples to the prognostic scores used in alc hep

Answer 4

1. Scores of baseline liver function
   • Maddrey’s discriminant function
   • Model for End-stage Liver Disease (MELD)
   • Glasgow Alcoholic Hepatitis Score
2. Histological scores
   • Alcoholic hepatitis histology score
3. Scores of dynamic liver function
   • Lille model
   • early change in bilirubin level

Question 5

Explain why we are unsure about the liver’s exact mechanism of response to alcohol

Answer 5

• We can’t easily sample it
  o No transplants for ALD pts so no access to tissue
• Can’t be replicated in mice
  o Mice don’t drink alcohol
  o Tube-feeding alcohol not comparable to human consumption of alcohol
  o Mice have great enzymes to break down alcohol so they don’t get ALD, The most they get is mild damage/mild rise in ALT

Question 6

Describe the immune response to alcohol

Answer 6

LUCEY 2009

• The gut microbiota is different in patients who drink alcohol
• Alcohol causes overgrowth of pathogens
• Alcohol causes bacterial translocation through gut into the portal vein

o Predominance of G-ve bacteria

• The LPS on the pathogens activate macrophages in the liver leading to cytokine release
  o IL-1, IL-6, TNFa, PDGF, TGFb, IL-10, IL-8

NOTE that the mechanism of ethanol generates reactive oxgen species which leads to its own mechanism of tissue damage

• Fat cannot be oxidised properly  fat build-up in the liver
• Acetaldehyde (metabolic product of alcohol) creates new antigens that can be recognised by B-cells
  o Further immune injury due to B-cells attacking the liver

Question 7

Describe the findings that lead to the belief that bacterial overgrowth leads to hepatic inflammation in alcoholic hepatitis

Answer 7

Keshavarzian 2010

CHRONOLOGY is important

Urine lactulose is a marker of the permeability of the gut wall.

The initial increase –> breakdown of gut barrier

Serum endotoxin –> LPS –> indicator of bacterial content in bloodstream

After the gut barrier breaks down, bacteria in blood goes

Then the amount of fat in liver goes up suggesting that bacterial translocation may contribute to this

Liver MPO –> indicator of macrophage activity

INFLAMMATION IN THE LIVER

Human AH faeces transplanted to mice generates liver injury - LLOPIS 2016

• Gave faeces from alcoholic hepatitis patients to mice
• Mice who were resistant to alcoholic liver disease developed a form of liver injury, which could be reversed by faeces from patients who used a lot of alcohol but did NOT develop hepatitis
  o Suggests the presence of something in the faeces which drives the liver injury

Question 8

give the main causes of death in alc hep within 90 days

Answer 8

hepatic failure - 41%
infection - 29%
renal - 8%
multi organ failure - 7%

Question 9

Describe the role of infection in Alc hep

Answer 9

• Louvet et al. (Gastroenterology 2009)
  o 60 day survival without infection = 77%
  o Survival dropped by 50% in the case of infection while on steroids

o Mookerjee 2007
This study demonstrated that neutrophils have impaired phagocytosis

They have a higher resting oxidative burst activity

This higher resting oxidative burst suggests that neutrophils may be pre-activated in AH but, notably, the oxidative killing burst response to E coli is preserved in AH patients compared to normal controls.

So although phagocytosis of bacteria is impaired, the ability of neutrophils to kill the bacteria is preserved.

Question 10

Describe the role of monocytes in alcoholic hepatitis

Answer 10

Increase in number of circulating monocytes in AH compared to healthy pts and non-alcoholic cirrhotic patients

When you gave monocytes to AH patients, the amount of LPS generated increased
o Thought to be related to ethanol itself
o Still didn’t explain why AH pts got more infections

Oxidative Burst response to E. Coli was then tested using flow cytometry

A roaring oxidative killing burst to E coli in normal controls compared to a markedly attenuated response in AH patients in the panel below.

Phagocytosis was ok but there wasn’t enough oxidative species to kill it

NAPDH oxidase enzyme was defective  responsible for the defect
- Makes alcoholic hepatitis similar to Chronic Granulomatous Disease (seen in children)
- Susceptibility to opportunistic infections
- In chronic granulomatous disease, this can be treated with IFNg
o Injected subcutaneously
- This rationale used in AH to give pts IFNg
o Didn’t work in AH
- Potentially due to diminished levels of STAT1 and increased levels of SOCS-1
• Reducing the amount of oxidative burst

Question 11

Describe treatment associated defects in alc hep

Answer 11

-	STOPAH TRIAL
o	STeroids 
o	Or
o	Pentoxifylline for
o	Alcoholic
o	Hepatitis

o Showed:
no diference in mortality in steroid and non-steroid trearment in the long run

borderline significant reduction in mortality in the first 28 days BUT these people died later in the trial anyway

• PREDNISOLONE decreased the Lille score BUT doubled the infection rate

It WASN’T that the steroid was dampening the oxidative burst

No difference in oxidative burst function between steroid and non-steroid patient

Thought to involve inflammatory cytokines

TNFa blocking was attempted to see if liver injury could be reduced by reducing inflammatory cytokines

• Both Infliximab and Etanercept trialled

o Naveau 2004, Botticer 2008

o Both lead to INCREASED mortality and trials stopped

——> DUE TO INFECTION

Question 12

Functions of Macrophages

Answer 12

primary function is in clearance of pathogens and of tissue debris
• Can take up damaged cells, apoptotic cells
• Therefore involved in tissue homeostasis
• Allow removal of damaged cells without instigaging an immune response

• Effective antigen presenters
• DCs are the best APCs
• Macrophages aren’t as good, but they are good
• Also able to mediate antibody dependent cell-mediated cytotoxicity (ADCC)
• Key role, therefore, in the initiation and resolution of the inflammatory response

Question 13

What are Kuppfer cells

Answer 13

The principle macrophage population in the liver

• Filter blood coming through portal circulation to remove particles that pose a threat

Question 14

How do macrophages work in immune response?

Answer 14

When macrophage phagocytes a foreign body, it presents a fragment of foreign protein as an antigen on a MHC II molecule to be recognised by T-helper cells

This may lead to

• complement activation
• antibody dependent cellular phagocytosis
• direct signalling
• Antibody dependent cell mediated cytotoxicity

Question 15

Describe how macrophages are phenotyped

Answer 15

Macrophages are derived from circulating monocyte populations

They can have both pro- and anti-inflammatory actions, split into ‘M1’ and ‘M2’ macrophages respectively

M1
• Perform enhanced cytotoxicity

M2
• Resolution of immune response

These phenotypes are determined by the local microenvironment

Macrophages can display cytokine signatures and functionality typical of both subsets simultaneously, particularly in the tumour microenvironment

Most macrophages are now known to be replenished locally from embryonic tissue resident precursors

Tumour associated myeloid cells are primarily derived from the circulation

Question 16

Describe the role of myeloid cells in cancer

Answer 16

• Myeloid cells are the most abundant immune cell population within solid tumours (up to 50%)
• Their presence is associated with poor prognosis and impaired responses to therapy
• They (primarily) display a ‘resolution’ phenotype – secrete anti-inflammatory cytokines and promote tumour growth and development
• This alteration is tumour derived

o Can come in from peripheral circulation
o In cancer
 Local macrophages may be involved
 MDSCs are a by-product that feeds into the tumour associated macrophage cycle

Question 17

Compare Tumour associated macrophages and myeloid derived suppressor cells

Answer 17

• Both derived from monocyte precursors
• Share common surface markers and functionality
• MDSCs are more immature and heterogeneous – they are derived from bone marrow and splenic precursors by altered myelopoeisis in disease
• TAMs are principally derived from circulating monocytes; attracted to sites of tumour and altered by the microenvironment therein
• They overlap in function considerably
• Roles in the tumour microenvironment
  o Suppression of innate and adaptive immune mechanisms
  o Secretion of growth factors and those that support angiogenesis
  o Promotion of tumour cell motility and metastasis
  o Promotion of tumour cell ‘stemness’ (youngness)

Question 18

Therapeutic potential of TAMs

Answer 18

• Deplete the tumour associated macrophage population
• Will also remove ‘beneficial’ tumoricidal macrophages
• Skew them towards more of an M1 phenotype
• Target them via the M2 specific macrophage mannose receptor (CD206)
• Use anti-CD40 antibodies with existing chemotherapy
• Prevent them being recruited to the tumour in the first place
• CSF1R blockade in pancreatic tumours in mice
• CCL2-CCR2 axis blockade (prostate cancer; carlumab)
• Block the CD47 ‘don’t eat me’ signal on tumour cells to activate resident macrophages
• Multiple tumours including HCC cell lines have demonstrated CD47 positivity

Question 19

TAMs in HCC

Answer 19

• CD47 blockade is effective in improving macrophage function (mouse model)
• CSF1R blockade has shown promising effects in patients with HCC
• Possible role for other efferocytotic signaling pathways
• MerTK, Axl and Tyro3

Question 20

TAMs in pancreatic adenocarcinoma

Answer 20

• Play a key role in disease progression and response to chemotherapy
• Gemcitabine activates the tumoricidal actions of macrophages in vitro
• In patients, a high density of TAMs was associated with a positive response to gemcitabine chemotherapy in vivo
• This was irrespective of T-cell density within tumours

Question 21

TAMs in gastric carcinoma

Answer 21

• TAMs are associated with poor prognosis and reduced survival
• TAMs are more abundant in tumours that have undergone treatment with some form of chemotherapy

Question 22

TAMs in oesophageal carcinoma

Answer 22

• TAMs are primarily associated with disease progression and poor prognosis in squamous cell carcinoma
• A recent study identified that TAM mediated production of CXCL8 was able to induce increased motility and therefore metastatic potential of squamous cell cancer cells in vitro
• CXCL8 acts via the AKT and ERK pathways to stimulate motility

Question 23

Causes of chronic hepatitis

Answer 23

Chronic viral
autoimmune
drug induced
idiopathic

Question 24

Clinical features of autoimmune hepatitis

Answer 24

usually present with evidence of moderate-severe hepatitis

elevated ALT and AST

normal-marginally elevated alkaline phosphatase + GGT

Sometimes present with jaundice, fever and RUQ

Occasionally systemic symptoms such as arthralgias, myalgias, polyserositis, thrombocytopenia
- May be due to other autoimmune diseases

usually occurs in women (70%) between 15-40

• Termed “lupoid hepatitis” BUT patients with SLE do not have an increased incidence of AIH

Question 25

Disease associations of AIH

Answer 25

• Haematological complications (autoimmune haemolytic anaemia)
• Gastrointestinal complications (IBD)
• Proliferating glomerulonephritis
• Fibrosing alveolitis
• Pericarditis and myocarditis
• Endocrine complications (Graves disease)
• Rheumatological complications (RA)
• Febrile panniculitis
• Lichen planus
• Uveitis

Question 26

Evidence for Autoimmune Pathogenesis of AIH

Answer 26

• Infiltrate composed predominantly of cytotoxic T cells + plasma cells (characteristic of autoimmunity)
• Circulating autoantibodies
• Association with hypergammaglobulinaemia + presence of rheumatoid factor
• Association with other autoimmune diseases
• Response to steroid and immunosuppressive therapy

Question 27

Pathophysiology of AIH

Answer 27

Abnormal display HLA II antigens on the surface of hepatocytes –> facilitates presentation of normal liver cell membrane constituents to APC (remember: liver does not express much MHC I and II)

• These activated cells, in turn, stimulate the clonal expansion of autoantigen-sensitised cytotoxic T lymphocytes
• Cytotoxic T lymphocytes infiltrate liver tissue  release cytokines –> destroy liver cells
• HLA DR3+ more likely than other patients to have aggressive disease, which is less responsive to medical therapy (younger presentation)
• HLA-DR4+ more likely to develop extrahepatic manifestations of their disease

Question 28

Possible reasons for abnormal HLA II presentation on hepatocytes in AIH

Answer 28

o Genetic factors

Complement allele: C4AQO

HLA haplotypes: B8, B14, DR3, DR4, Dw3

C4A gene associated with development of AIH in younger patients

o Viral infections

HAV, HBV, EBV – by triggering an inflammatory response (and production of IFN which triggers MHC II display)

o Drugs (interferon, melatonin, nitrofurantoin)

The asialoglycoprotein receptor and the cytochrome mono-oxygenase P450 2D6 are proposed as the triggering autoantigens

Question 29

Autoantibodies in AIH

Answer 29

• Antinuclear antibody (ANA)

• Anti-smooth muscle antibody (ASMA)
typical of AIH (remember these are antibodies against actin)

• Anti-liver kidney microsomal antibody (LKM1)
• Antibodies against soluble liver antigen (anti SLA) directed at cytokeratins types 8 and 18 (hepatocyte cytokeratins)
• Antibodies to liver-specific asialogylcoprotein receptor or hepatic lectin

• Antimitochondrial antibody (AMA)
Possible to find in AIH but more common in PBC

• Antiphospholipid antibodies

Question 30

Classification of AIH

Answer 30

Always considered chronic even though the clinical presentation may be acute

Classified according to auto-antibody profile:

o Type 1: ANA +/- SMA (most common, adults)

o Type 2: LKM (associated in children, more difficult to treat)

o Type 3: ASLA

Question 31

Overlap Syndromes in AIH and their significance

Answer 31

Patients with disease that overlaps with PBC may have detectable AMA (usually in low titre) + histological findings of bile duct injury + presence of hepatic copper

These patients may improve with steroid therapy

But this isn’t ideal as PBC is associated with osteoporotic

However, because the overlap cannot be accounted for with individual drugs, steroid therapy is the mainstay treatment at the moment

Patients with disease that overlaps with PSC usually have concurrent IBD + liver biopsy findings reveal bile duct injury

Findings from cholangiograms are abnormal

Such patients have mixed hepatocellular and cholestatic liver chemistries and typically are resistant to steroid therapy

Question 32

Histological features in AIH

Answer 32

• Active disease with prominent plasma cells
• Hepatocytes rosettes – rounded clusters of hepatocytes
• Confluent perivenular necrosis – zone 3 necrosis, particularly found in drug-induced
• Lymphoid follicles – strongly associated with HCV
• Giant cells
• Canalicular cholestasis uncommon
• Emperipolesis –> thought to be the most diagnostic feature of AIH but rarely ever seen

Question 33

Role of Liver Biopsy in AIH

Answer 33

• Establishing diagnosis
o Histologically active disease without: (i.e. exclude other disease)
 Biliary lesions
 Well-defined granulomas
 Features suggesting different aetiology

• Monitoring the response to treatment
o LFTs are often normalised over time in AIH
o But histology presents a progressing disease – must not rely on serum evidence alone

GRADE = how active is the disease?
STAGE = how advanced is the fibrosis?

Question 34

Tx in AIH

Answer 34

• Steroids

* Azathioprine

Question 35

Define hepatic steatosis

Answer 35

presence of vesicles of fat, predominantly triglycerides, accumulating within hepatocytes (presence of fat in USS because you cannot biopsy the whole population)

Question 36

Define Steatohepatitis

Answer 36

presence of hepatocellular inflammation in conjunction with steatosis

Question 37

Define NAFLD

Answer 37

presence of hepatic steatosis as part of the metabolic syndrome of obesity, insulin resistance/ type 2 diabetes, dyslipidaemia and hypertension

Question 38

Define Non-alcoholic steatohepatitis (NASH)

Answer 38

steatohepatitis associated with NAFLD.

Cannot be certain of this unless you have had a biopsy showing fat in liver

Question 39

primary Causes of Fatty Liver

Answer 39

Associated with metabolic syndrome

o (Abdominal) Obesity
o T2DM
o Dyslidaemia

Question 40

secondary Causes of Fatty Liver

Answer 40

o Alcohol
o Drugs (steroids, Amiodarone, HAART, MTX, tamoxifen)
o Hepatitis C infection (HBV?)
o Parenteral nutrition

Question 41

Describe the histology of NASH

Answer 41

• Steatosis

• Ballooning of hepatocytes (see right)
o Appearance of hepatocytes undergoing apoptosis-like cell death

• Lobular inflammatory infiltrate
o Presence of immune cells such as lymphocytes and monocytes but also neutrophils (often in a lobular distribution

• Fibrosis
o Net deposition of a disordered ECM

Question 42

Describe the scoring systems that can be used for NASH

Answer 42

Kleiner/Brunt/ISHAK

Question 43

Describe the epidemiology of NAFLD

Answer 43

• Reported worldwide
• Increasingly recognised as the leading cause of liver dysfunction in the non-alcoholic, viral hepatitis negative population of the USA and UK

• NAFLD/NASH is a “Complex Disease Trait” where genetic and environmental factors interact to determine disease progression

• 49% of morbidly obese patietns with NAFLD progress to NASH
• 43-55% of patients with NAFLD + raised ALT progress to NASH
• Cirrhosis develops in 15-25% of patients with NASH

Question 44

Describe the causes of Morbidity and Mortality in NASH

Answer 44

(Ratzium EASL 2010, Targher NEJM 2010)

• Liver-related
o Decompensated liver disease (sepsis, variceal haemorrhage, encephalopathy)
o HCC
o Liver transplantation

•	Extra-hepatic
o	Insulin resistance 
o	Diabetes
o	HTN
o	CVD

• Direct ink between extra-hepatic morbidity/mortality is related to amount of fat in the liver
o i.e. it is thought that liver steatosis drives extra-hepatic complications

Question 45

Describe the pathogenesis of steatosis

Answer 45

Complex disease - genetic and environmental

Increased prevalence of NAFLD is strongly associated with the increasing prevalence of obesity

Development and progression of NAFLD are the result of a complex interplay between different organs and cell types.

expansion of visceral adipose tissue and gut-derived endotoxins are key factors in NAFLD and its progression to fibrosis

During obesity, adipocyte size may dramatically increase

This hypertrophy promotes the rupture of adipocytes leading to a local inflammatory phenotype marked by the recruitment and activation:

o of immune cells, such as macrophages and T cells, and
o by production of adipose tissue pro-inflammatory molecules (i.e., adipocytokines) that are released into circulation and can reach target organs, including the liver and skeletal muscle.

This aberrant activation of the immune response triggers harmful inflammation, which: (Alisi 2017)
o Impairs the ability of insulin to inhibit free fatty acids (FFA) release causing their accumulation in the liver and consequent lipotoxicity
o Induces hepatic insulin resistance, and drives the low-grade inflammatory pattern of NASH and, later, liver fibrosis.

Question 46

Explain the gut-liver axis

Answer 46

• Gut is now emerging as initiator of the events that contribute to obesity-associated systemic inflammation (Sica, 2012)

obesity prone subjects exhibit alterations in gut microbiota balance (dysbiosis).

major compound of gut bacteria, lipopolysaccharide (LPS), plays a key role in hepatic inflammation and in macrophage polarization during NAFLD.

In NAFLD subjects, LPS does not remain confined in the intestinal lumen but reaches the liver when the colonic mucosa immune function is impaired by gut dysbiosis (Mehal, 2013)

• Toll-like receptor 4 (TLR-4) on the plasma membrane of liver-resident cells recognizes LPS as a ligand that prompts receptor dimerization and activation of a signalling cascade.

the LPS/TLR-4 cascade causes the production of classical inflammatory cytokines, including TNF-𝛼, IL-1𝛽, and IL-6, exacerbating the hepatic inflammatory state and promoting fibrogenesis.

Question 47

Immune Response in NAFLD

Answer 47

the liver capillary system is lined with manifold different specialized cells such as the sinusoidal endothelial cells, Kupffer cells and liver dendritic cells.

these cells are capable of antigen presentation, as well as cytokine and chemokine production, and are therefore key players in initiating and shaping liver immune responses.

liver harbours ~80% of all body macrophages and is also patrolled by myeloid cells (such as blood monocytes), which scan the liver vasculature and eventually infiltrate into the liver.

Under steady-state conditions, monocyte-derived cells can develop into liver dendritic cells (DCs) or monocyte-derived macrophages (MoMFs), but do not contribute to the pool of Kupffer cells, in the liver.

Natural killer T (NKT) cells are a specialized subset of T cells that are particularly enriched in the liver compared with other organs and regulate immune responses in the context of autoimmunity, cancer and microbial infection.

Several mechanisms exist to trigger immune reactions in the liver:

o	Danger signals and alarmins
o	TLRs
o	Inflammasome
o	Complement 
o	Monocytes and macrophages
o	Dendritic cells
o	T-cells

Question 48

Danger Signals and Alarmins in NASH

Answer 48

• Danger signals or so-called danger-associated molecular patterns (DAMPs) are target structures, which, in comparison to pathogen-associated molecular patterns (PAMPs) derived from microorganisms, originate from the body and are released or induced upon damage to the tissue.

o Such signals or mediators are also termed ‘alarmins’ to express that these factors alert the immune system.

• In NASH, accumulation of cholesterol activation of liver macrophages and hepatic stellate cells  promoting the development of steatohepatitis and liver fibrosis.

Question 49

Inflammasome in NASH

Answer 49

• Inflammasomes are intracellular multiprotein complexes that sense intracellular danger signals.

• Inflammasome activation  a pro-inflammatory response commonly associated with caspase-1 activation followed by secretion of activated IL-1β and IL-18.
o The inflammasome complex most commonly activated in various liver diseases is NLRP3, which senses ATP81 release following necrotic cell death.

• The inflammasome complexes mainly respond to passive signals accompanying infections or cellular stress, targeting either factors released during necrotic cell death or harmful structures such as crystals (uric acid, cholesterol, β–amyloid, asbestos)

Question 50

TLRs in NASH

Answer 50

• TLRs are pattern-recognition receptors detecting PAMPs such as bacterial peptidoglycans, which are normally found upon microbial infection (especially TLR-4)
• However, due to the unique situation in the GI tract, it is also necessary to distinguish between noninvasive gut microbiota and pathogenic infection, triggering immune responses only in the context of threatening disease.
• One concept regarding altered microbial patterns in liver disease has received great attention lately: the influence of changes in the gut microbiota in various diseases (such as excessive alcohol intake, fatty liver disease)

Question 51

Complement in NASH

Answer 51

• The complement system is one of the first lines of defence against microbial infections, ultimately leading to the formation of cytolytic membrane attack complexes (MACs) similar to perforin pores formed by cytotoxic CD8+ T cells.
• In patients with NASH, activation of the complement cascade could be observed to the point of C9–MAC deposition, a mechanism also described in animal models of alcoholic liver disease, describing the functional contribution of C1q, C3 and C5 in liver inflammation and steatosis development

Question 52

Monocytes and Macrophages in NASH

Answer 52

Liver macrophages are a key component of liver inflammation and have remarkable functional diversity, being involved in upkeep of homeostasis, acute and chronic inflammation and regression of liver disease.

Traditionally, macrophages were categorized by their terminal differentiation stage: either pro-inflammatory (M1), wound-healing (M2) or immunosuppressive (MREG). However, extensive transcriptome analyses for human monocyte-derived macrophages in response to various stimuli revealed a spectrum of macrophage activation states by far extending the current M1 versus M2 polarization.

• In NASH:

Macrophages critically influence inflammation and insulin resistance in metabolic disorders. Individuals who are morbidly obese exhibit similar expression patterns of macrophage-related activation markers, which are closely related to the severity of liver steatosis and fibrosis.

Kupffer cells have an essential role in liver fibrosis in mouse models of NASH, propagating hepatic inflammation via TNF and leukocyte recruitment via ICAM-1 and VCAM-1.

In mice, macrophage activation during NASH is related to accumulation of toxic lipids such as ceramide.

Question 53

Dendritic Cells in NASH

Answer 53

DCs are commonly located at the portal tracts in healthy liver. Upon inflammation, they form periportal lymphoid structures, which serve as a priming site for liver- infiltrating T cells.

In metabolic liver disease, DCs have multiple, partially opposing functions. Overall, they are important immunoregulatory cells, as DC depletion exacerbates liver inflammation, liver damage and fibrosis development.

Interestingly, it is known that lipids and lipid products can impair antigen presentation by DCs, but the lipid- dependent functional alterations in hepatic DCs remain to be clarified.

Question 54

T-Cells in NASH

Answer 54

Similar to macrophages, hepatic T cells show remarkable heterogeneity regarding their diverse immunological profiles, enabling them to perform multiple pro-inflammatory and anti-inflammatory functions in liver disease.

The liver constitutively interacts with T cells roaming the sinusoidal vasculature, inducing imminent activation shown by expression of markers such as CD69 and CD25 and, ultimately, induction of CD45RO memory T cells.

The liver also harbours a substantial amount of non-classic T cells such as γδ T cells.

• In NASH:

T cells are involved in the pathogenesis of NASH, although this is not a typical antigen-driven entity such as infectious liver diseases.

The propagation of NASH seems to be closely related to the presence of TH17 cells. Development of steatosis in hepatocytes could be triggered by IL-17 in vitro, which in turn secrete IL-6, a mediator of TH17 induction acting together with IL-1β and retinoic acid, leading to a self-amplifying inflammatory T-cell response.

Question 55

Non-Invasive Techniques for the Assessment of Hepatic Inflammation/Fibrosis in NAFLD

Answer 55

• Imaging
o Ultrasound (first-line)
o Transient elastography (fibroscan)
o Magnetic resonance imaging and spectroscopy

• Blood markers
o Aminotransferases, AST:ALT ratio
o Serum panel markers, ELF
o Composite algorithms, NAFLD fibrosis score

• None yet can reliably distinguish NAFLD from NASH

Question 56

Commonest cause of ALF

Answer 56

paracetamol overdose

Question 57

Types of Immune mediated drug hepatotoxicity

Answer 57

o Intrinsic:

dose-dependent, predictable reactions that directly damage hepatocytes

o Idiosyncratic:

happen in a minority of patients, with variable time of onset and no obvious relationship to drug dose (but need to have a minimum threshold present – hence some degree of dose response), and they are not reproducible, unpredictable

 Idiosyncratic, non-allergic toxicity (isoniazid, tacrine) –> don’t necessarily have allergic evidence

 Idiosyncratic, allergic toxicity (halothane, flucloxacillin) –> eosinophilia, evidence of allergic response, most severe: Steven-Johnson’s syndrome

Question 58

Classification I of DILI

Answer 58

• Hepatocellular (R>5)
• Mixed (R=2-5)
• Cholestatic (R<2)

R = (ALT level / ALT ULN) / (AP level / AP ULN)

Question 59

Classification II of DILI

Answer 59

• Diagnosis: Roussel-Uclaf Causality Assessment Method (RUCAM) diagnostic score.

• Used in clinical trials predominantly  difficult to prove that a specific drug caused LFT derangement

• Different stages:
o Latency – how soon after drug introduced did LFT derangement occur?
o Course after de-challenge – if you withdraw drug, do LFTs return to baseline?
o Risk factors
o Co-medication – any other medication that patient is taking that is known to cause DILI?
o Exclusion of other aetiologies – e.g. viral hepatitis
o Data on drug hepatotoxicity – is the drug introduced known to cause DILI?
o Re-challenge – if drug re-introduced does LFT derangement recur? (rarely done due to risk of provoking more severe reaction)

• Balance of probability –> thought process is used in clinical decision making but not score in this way

Question 60

Flucloxacillin in Idiosyncratic Hepatotoxicity

Answer 60

• Penicillin antibiotics
• Widely used to treat soft tissue infections due to excellent staphylococcal cover
• Flucloxacillin and its metabolites are NOT directly toxic to the liver
• Idiosyncratic, cholestatic DILI that affects 8.5/100,000 users

1. GWAS that was done showing that HLA-B*5701 genotype is a major determinant of DILI due to flucloxacillin
   o Shown to confer 100x higher risk

Question 61

HLA Associated Causes of Idiosyncratic Hepatotoxicity

Answer 61

HLA-DRB1 - Amoxicillin-clavulanate

Question 62

Pathophysiology of DILI-HLA Associations: Theories

Answer 62

1. Hapten hypothesis – drug or its reactive metabolite binds covalently to an endogenous peptide to produce an antigenic ‘hapten-carrier complex’. Drug specific immune responses are then triggered when the hapten carrier complex interacts with the peptide binding groove of a specific HLA. (A on image)
2. Pharmacological interaction (p-i) – pharmacological interaction with immune receptors. Drug or metabolite binds directly to HLA and/or the T cell receptor. (eg. ximelagatran and HLA-DRB1*0701) (B on image)
3. Altered peptide repertoire model (eg minocycline and HLA-B*3502) suggests that drugs may transiently bind to the peptide binding cleft of the MHC, causing a conformational shift and therefore changing the repertoire of endogenous peptides that bind to it (C on image)
4. Altered T cell repertoire. Drug or reactive metabolites bind to and alter conformation of TCR (D on image)  same as the one above but specific to TCR

Question 63

inter-individual susceptibility in Paracetamol Hepatotoxicity

Answer 63

Paracetamol overdose is the commonest cause of acute liver failure in the UK and USA.

Accounts for 39-57% of cases

Commonest indication for super-urgent liver transplantation.

Drug mentioned as classic intrinsic DILI  true to some extent, one can poison someone’s liver with enough paracetamol hence dose dependent.

Gregory et al – Paper showed that the dose of paracetamol does not correlate with outcome of ALF in patients

Watkins et al – Paper showed that standard dose of paracetamol can indeed cause some liver injury (demonstrated by elevation in LFTs) even in healthy volunteers

Question 64

Mechanism of Liver Injury in Paracetamol

Answer 64

see images p127

1. Excess paracetamol enters hepatocyte
2. Glucuronidation and sulphonation pathways are saturated
3. Increase in metabolism via the cytochrome P450 enzymes to reactive metabolite NAPQI
4. Increased conjugation with glutathione eventually depletes levels of cellular glutathione
5. Accumulation of reactive NAPQI (toxic metabolite that puts cells in stress)
6. Binding of NAPQI to DNA and cellular proteins  particularly to mitochondria
7. Mitochondrial toxicity  they lose the ability to produce ATP
8. MPT: mitochondrial permeability transition  allows solute flux, resulting in mitochondrial swelling and rupture
9. Commencement of apoptosis (release of pro-apoptotic factors)
10. Catastrophic energy failure leads to cell membrane compromise and necrosis
11. Spillage of cell contents  leading to cell death by necrosis (this is zonal, starts at the centre of lobule near vein where most of NAPQI accumulates due to presence of higher levels of CYP2E1 + lower oxygen tension)

Possamai et al, J Hepatol, 2014

1. Cell death by necrosis
2. Release of DAMPs
3. Local production of proinflammatory cytokines and chemokines by Kupffer cells
4. Influx of innate immune effector cells (neutrophils and monocytes) into areas of necrosis, start to phagocytose debris
   o Neutrophils thought to cause more injury when recruited by damage to adjacent hepatocytes that were previously not necrosed
5. Secondary immune mediated tissue damage
6. Systemic inflammation driven by cytokines
7. Later anti-inflammatory mediators overspill and dampen immune responses (driven by monocyte maturation to macrophage M2 phenotype  however this overspill can dampen immune responses elsewhere around the body leaving these patients prone to sepsis + MOF)

Question 65

what are Immune Checkpoints

Answer 65

• Balance between stimulation and inhibition
• Determines the fate of T-cells – anergy v clonal expansion
• Immune checkpoints are inhibitory signals that limit T cell activation and prevent autoimmunity
o The ‘Brakes’ – hold the immune system in check, prevent autoimmunity and allow for tolerance
o Part of peripheral tolerance

CTLA-4
• Exclusively expressed on T cells – inducible on conventional T cells and constitutive on Tregs
• Affects the amplitude of the initial T cell response to activation
• Counteracts the co-stimulatory effects of CD28 (see A on image)

PD-1/PD-L1
• Limits the activation of T cells in peripheral tissues during an inflammatory response
• Reduces autoimmunity
• Is an important immune resistance mechanism in the tumour microenvironment by which cancer cells resist immune-mediated killing.
• Also highly expressed on Tregs

Question 66

Roles Immune Checkpoint Inhibitors and the problem with them

Answer 66

• Immune checkpoint inhibitors are effective new anti-cancer therapies
• These are not specific to a tumour but instead focus on “removing the brakes” of the immune system to allow it to detect the tumour and clear it
• Problem: increases autoimmunity (imbalance = leading to hepatotoxicity mimicking AIH)

o Can occur in any organ (but focus on liver)
o Typically detected 8-12 weeks after commencing therapy –> associated with increased in ALT/AST
o Associated with elevation in transaminases and other LFTS
o Variety of histopathological presentations seen (typically see interface hepatitis and nuclear cell infiltrates)
o Theory: require a second hit e.g. infection or another drug reaction to cause this adverse event
o Treated dependent on severity  drug withdrawal, rechallenge if mild hepatotoxicity, immune suppression (azathiopeinw/MMF)

Question 67

Treatment of Immune related adverse events IrAEs

Answer 67

• Drug withdrawal
• Rechallenge considered depending on severity of reaction (contraindicated in very severe liver injury but if mild liver injury with good cancer outcomes then may be restarted)
• Hepatitis treated as AIH
• Steroid
• Immunosuppression – MMF or Azathioprine

Question 68

Summarise the general mechanism of AIH

Answer 68

Occurs in genetically susceptible individuals with an environmental trigger that leads to loss of immune tolerance to liver autoantigens

Activated autoreactive CD4+ helper T-cell

recruitment of monocytes, cytotoxic T and NK cells

differentiation of B cells into plasma cells producing Igs

impairments in T-regs and control mechanisms

Question 69

Epidemiology of AIH

Answer 69

Prevalence ranges from 11-36 cases per 100,000 of Type 1

Female > Male (4:1 in Type 1; 10:1 in Type 2)

Question 70

presentation of AIH

Answer 70

• Presentation is often non-specific – nausea, vomiting weight loss, amenorrhoea, abdominal pain, itching

25% are asymptomatic and detected by routine blood tests

Rarely AIH can present as acute liver failure

• 1/3 of patients have histological evidence of cirrhosis at presentation.

o Without treatment 40% mortality within 6 months, and 10% 10 year survival

o With treatment, 10 year survival 80-93%

o 20% of patients have or will develop another AI disease
o 40% have a FH of AI disease

Question 71

Compare types of AIH

Answer 71

—Autoantibodies—
- Type 1
ANA (Usually homogenous, occasionally speckled)
Anti-SMA (Smooth muscle autoantibodies are directed against components of the cytoskeleton including actin, tubulin, vimentin and skeleton)
Anti-actin
Anti-soluble liver antigen or anti-liver pancreas antigen

• Type 2
  Anti—LKM type1 (Anti-LKM-1 antibodies stain the proximal renal tubules (A) and hepatocellular cytoplasm (B) Pattern is sometimes confused with AMA, which stain distal tubules and gastric parietal cells, but not liver)
  Anti-liver cytosol antibody type1
  Anti-LKM type 3

—Geographical variation—
both worldwide

• –Age at presentation—
• Type 1 - all ages
• Type 2 - Childhood and young adulthood
• –Female:Male—
• 1 - 1:4
• 2 - 1:10
• – Clinical phenotype —
• 1 - variable
• 2 -generally severe
• – Histopath features at presentation —
• 1 - Range from mild to cirrhosis
• 2 - Generally advanced with inflammation and cirrhosis common
• – Treatment failure —
• 1 - Rare
• 2 - Common
• – Relapse —
• 1 - Variable
• 2- Common
• – Long-term maintenance —
• 1 - variable
• 2 - almost universal

Question 72

Diagnostic criteria of Definite AIH

Answer 72

(M. Henegan et al Lancet 2013)

Liver histology

• Interface hepatitis of mod or severe activity +/- lobular hepatitis or bridging necrosis
• No biliary lesions, granulomas or changes suggestive of other aetiology

Laboratory features

• Any serum aminotransferase abnormality esp if ALP normal
• Normal α1-antitrypsin, copper & caeruloplasmin

Serum immunoglobulins
- Globulin, γ-globulin or IgG >1.5 x ULN

Serum autoantibodies

• ANA, Anti-SMA, or anti-LKM type1 antibodies at > 1:80 titre
• No AMA

Viral Markers
- Negative markers for Hepatitis A,B and C

Other exposures

• Alcohol <25g/day
• No recent use of hepatotoxic drugs

Question 73

Diagnostic criteria of Probable AIH

Answer 73

(M. Henegan et al Lancet 2013)

Liver histology
- Same as for definite AIH

Laboratory features

• Same
• (abnormal copper levels can be included if Wilson’s excluded by other means)

Serum immunoglobulins
- Any increase in globulin, γ-globulin or IgG above ULN

Serum autoantibodies
- Same but titres of >1:40

Viral Markers
- Same

Other exposures

• Alcohol <50g/day
• No recent use of hepatotoxins

Question 74

International autoimmune hepatitis group revised diagnostic scoring system

Answer 74

Alvarez F et al J Hepatol 1999
•	Pre-treatment score >15: definite AIH
•	10-15: probable AIH
•	Post-treatment score >17 definite AIH
•	12-17 probable AIH

Question 75

Histology of AIH

Answer 75

• Periportal “interface hepatitis” with an immune infiltrate that interrupts the limiting plate.
• Infiltrate is composed of lymphocytes, plasma cells and macrophages.
• T lymphocytes predominate and most are CD4+ - ie of the T helper/inducer phenotype a minority are CD8+ cytotoxic T lymphocytes
• NK cells, macrophages, B cells and plasma cells are also present

Question 76

Genetics of AIH

Answer 76

•	The strongest associations are within the HLA-DRB1 locus. Alleles encoding  confer susceptibility in European & N American populations to AIH-1
o	DR3 (DRB1*0301)
o	DR4 (DRB1*0401) 

•	Susceptibility to and severity of AIH-2 has been linked to alleles encoding
o	DR3 (DRB1*0301)
o	DR7 (DRB1*0701)
o	DQ2 (DQB1*0201)

Question 77

Disease Triggers in AIH

Answer 77

Molecular mimicry

• Theory which suggests immune responses to exogenous pathogens cross react with similar self components
• Autoimmunity is triggered by infection with a pathogen

• In AIH-2 the target of anti-LKM-1 antibodies is CYP450 2D6.
o The HCV polyprotein shares high amino-acid homology.
o Other potential viral trigger include HBV, CMV, HSV… Unknown virus?

• Other environmental triggers have been identified such as drugs including nitrofurantoin, minocycline, statins, diclofenac, propylthiouracil

Question 78

Immune Mechanisms in AIH

Answer 78

A number of steps need to occur before one loses tolerance to self-antigens (summarised below: R. Liberal et al J Autoimm 2013)

• First necessary step: failure of central tolerance of negative selection i.e. T-cell that is “auto-reactive” to self-antigens escapes thymus
o Insufficient to induce autoimmune disease on its own

• Second necessary step: failure of peripheral tolerance i.e. T-cell co-stimulation occurs with no T-cell inhibition (balance of costimulation and coinhibition)
• Third necessary step: cytokine environment is needed for activated T-cell to develop Th1/Th2 phenotype

o Important: Th1 cell is involved in pro-inflammatory reactions leading to increase in IFN-gamma which increases the MHC class presentation of liver cells

 So liver cells present more self-peptides to immune cells further driving local immune response

Th2 cell  involved in proliferation of B-cells  produce autoantibodies that are detected in patient sera

1) INITIATION: Liver damage is initiated by the presentation of a self-antigenic peptide within a major histocompatability molecule (MHC) by professional antigen presenting cells (APCs). The presence of appropriate costimulation alongside exposure to various cytokines drives the differentiation of uncommitted CD4 helper T-cells (Th0).
2) IL6 and IL1b lead to differentiation into pathogenic Th17 cells that secrete the proinflammatory cytokine IL17. Th17 cells promote hepatocyte secretion of IL6, which in turn further enhances Th17 development.

3) Exposure to IL12 leads to the differentiation of Th1 cells secreting IFNg, which induces monocyte (MF) differentiation, activates cytotoxic CD8
T-cells and promotes NK cell killing. IFNg also increases MHC class I and induces class II expression by hepatocytes, further exacerbating inflammation. 

4) Exposure to IL4 leads to Th2 differentiation. Th2 cells secrete IL13, IL4 and IL10 that enable B cell maturation into plasma cells with the consequent production of autoantibodies. Autoantibodies are in turn involved in antibody-mediated cellular cytotoxicity and complement activation.

Question 79

Role of PD-1 in impaired Immunoregulation in AIH

Answer 79

• PD-1 and its ligands play an important role in downregulating the immune system by preventing the activation of T-cells  this reduces autoimmunity and promotes self-tolerance

• Matsumoto K et al Journal of Gastroenterology and Hepatology 2013: found anti-PD1 antibodies in AIH patients
o These individuals essentially form their own “immune checkpoint inhibitors”
o Provides evidence that side effect profile of immune checkpoint inhibitors do indeed produce an AIH-like phenotype

Question 80

Role of Tregs in impaired Immunoregulation in AIH

Answer 80

• Tregs from controls or AIH patients added to CD4+CD25- cells and proliferation assessed.
• Tregs from controls reduced the T cell count by 57%, but those from AIH only reduced T-cell count by 34% (p=0.02)  i.e. small proportion of T-reg cells found in individuals suffering from AIH, but also function of T-Reg cells = reduced

Summary of T-Reg Cells  Longhi MS et al J Hepatol 2004, Ferri S et al Hepatology 2010
• Regulatory T cells (CD4+ CD25hi) in AIH:
o are numerically reduced in adult and paediatric AIH.
o express lower levels of FOXP3 than those of HCs.
o have impaired inhibitory activity
o NKT cells in AIH are also quantitatively and qualitatively impaired

HOWEVER: LATER REPORT FOUND NO QUANTITATIVE OR QUALITATIVE T-REG IMPAIRMENT IN AIH WHICH DIRECTLY CONFLICTS WITH PREVIOUS REPORTS AND HAS CAUSED SOME CONTROVERSY. IT HAS BEEN SUGGESTED THAT T-REG IMPAIRMENT MAY BE LOCALISED TO THE INFLAMMED LIVER AND AS A RESULT OF THE INFLAMMATORY MICROENVIRONMENT - Peiseler M et al Hepatology 2012
• This remains a controversial area and further research is required

Question 81

Role of TIM3/GALECTIN-9 in impaired Immunoregulation in AIH

Answer 81

• Murine studies have shown that galectin-9 (Gal9), a member of the galectin family expressed by T-regs, inhibits T helper 1 (Th1) effector immune responses after binding to the T cell immunoglobulin and mucin domain 3 (Tim-3), its receptor on CD4 effector cells
• R. Liberal et al Hepatology 2012: human studies
o In AIH TRegs showed decreased expression of Gal-9
o In AIH effector CD4 T cells showed decreased expression of Tim-3
o Tregs treated with Gal-9 knockdown RNAis were less effective at suppressing T cell proliferation

Question 82

Findings in early trials for Tx of AIH

Answer 82

Sherlock et al, Q J Med, 1971
• Randomised patients to 22+27 patients into either corticosteroid and control groups (respectively) suffering from AIH
o Mortality in control group = 56% compared to 14% in corticosteroid group
o Trial stopped early due to preliminary results

R. Liveral et al J Autoimmun 2013 – Steroid Based Regimens
• Induction = steroids, maintenance therapy = azathioprine (and other alternative treatments if intolerant to azathioprine listed on the right) + steroid weaned
• Remember: side effect profile of steroids  osteoporosis particularly important in a female preponderant population

Question 83

Summary of Immune Mechanisms of AIH

Answer 83

• The overall process in AIH is a loss of tolerance to liver autoantigens.
• Failure of central tolerance. Autoreactive T cells are not destroyed in the thymus by negative selection/clonal deletion. (this is common and not sufficient to cause AI disease)
• Presentation within MHC II of self antigen to Th0 cell by a professional antigen presenting cell with co-stimulation/ absence of inhibition. Role of PD-1 and CTLA4. Failure of peripheral tolerance (clonal anergy) allows autoreactive Th cell to form
• Differentiation of uncommitted self-reactive Th0 cell.
• Propagation of immune-response with recruitment of cytotoxic T cells, NK cells and monocytes that can induce hepatocyte death.
• IFNγ stimulates MHC I and II expression by hepatocytes, worsening damage.
• B cell maturation to plasma cells, production of autoantibodies and antibody-mediated cytotoxicity
• Failure of Treg control possibly due to quantitative and qualitative defect? Possibly due to decreased susceptibility of effector T cells to suppression? Possibly due to impaired Gal-9/Tim-3 signalling

Question 84

Describe the process of Fibrosis

Answer 84

• Wound healing response in which damaged regions are encapsulated by extracellular matrix or scar tissue
• It is a dynamic and complex process

despite the complexities of the pathways, they all ultimately lead to one common pathway via hepatic stellate cell activation
 This common pathway needs to be targeted

• Recruitment and activation of platelets, inflammatory cells, hepatic stellate cells and other extra-cellular matrix producing cells  it is a paradigm for other tissue fibrosis models (lung, renal, skin)
• Develops in almost all patients with chronic liver injury

• Hepatic scar composition similar regardless of type of injury but the distribution differs (see below):
o Portal-portal – Cholestatic, PSC, PBC
o Portal-central (bridging) – Viral hepatitis
o Central-central – Budd-Chiari, heart failure
o Peri-cellular – Alcohol, NASH

• The rate of fibrosis depends on cause of liver disease and host factors (e.g. other diseases: fatty liver disease, alcohol, diabetes)
• Usually takes months to years of on-going injury

Question 85

What are the key concepts in the pathogenesis of fibrosis?

Answer 85

• Increase in extracellular matrix (ECM)
• Hepatic stellate cells and ECM producing cell populations
• Coagulation system in the pathogenesis of liver fibrosis
• Reversibility of fibrosis

Question 86

Extracellular matrix in fibrosis pathogenesis

Answer 86

• Macromolecules which provide the scaffolding of the normal liver and also act as transducers of extracellular signals

•	Types:
o	Collagens: I, III, V, XI (predominate)
o	Non-collagen glycoproteins
o	Matrix bound growth factors
o	Proteoglycans
o	Matricellular proteins

• Fibrotic liver
o There is a change in the ECM
o 3-10X increase in collagen content
o Increase in glycoproteins, proteoglycans, glycosaminoglycans in ECM – change in normal constituent of ECM
 The change in constituents affects hepatocyte, stellate cell function and impairs transport of solutes
o Positive feedback to further amplify fibrosis: (vicious cycle)
 Integrins provoke HSC activation
 Release of fibrogenic/proliferative growth factors
 Increased liver stiffness/collagen IV  HSC activation via TGFB1

• ECM producing cell populations
o Hepatic stellate cells – KEY
o Sinusoidal endothelial cells – important in early fibrosis
o Portal fibroblasts – cholestatic disease/ischaemia
o Bone marrow recruitment of mesenchymal cells1

Question 87

Hepatic Stellate Cell (HSC) in fibrosis pathogenesis

Answer 87

• Primary source of ECM
• Located in subendothelial space of Disse
• In normal liver = storage site for retinoids
• Activation – common pathway to hepatic fibrosis
o Transition from quiescent Vit A rich cell to proliferating fibrogenic cell (myofibroblast)
o Diminution of VitA droplet
o Proliferation/activation occurs in regions of most injury
o Leads to accumulation of scar, which leads to loss of hepatocyte function

• There are two steps to its activation:
o Initiation
 Renders HSC sensitive to cytokines/stimuli
 Via stimuli from neighbouring cells:
• TGFb from endothelial and Kupffer cells
• PDGF from platelets
• Fas and TRAIL from hepatocyte apoptosis

 

o	Perpetuation – Key State
	Maintaining activated phenotype and increase accumulation of ECM
	Major changes after during perpetuation:
•	Fibrogenesis
•	Proliferation
•	Contractility (Dhar et al 2012)
•	Matrix degradation
•	Chemotaxis
•	Retinoid loss
•	White blood cell chemoattraction

• Fibrogenesis
o Increased ECM (type 1 collagen, fibronectin, proteoglycan)
o Stimulated by TGFb (but also retinoids, angiotensin II, TNF, CTGF, Fe)

• Proliferation
o Mitosis induced by PDGF (Kupffer cells)

• Chemotaxis
o Migration towards chemoattractants

• White blood cell chemo-attraction
o Amplifies inflammatory response

• Contractility
o Impedes blood flow in fibrotic livers, contributing to portal HT
o So portal HT is caused by both the mechanical element of disrupted architecture AND by the dynamic element of stellate cell contractility
o Stellate inactivation may increase prognosis in portal HT

• Matrix degradation via matrix proteases
• Retinoid loss: ? Role?

Question 88

Coagulation cascade in fibrosis pathogenesis

Answer 88

• The activation of the pathways lead to thrombin production
o This normally occurs via the extrinsic pathway (other pathways include: intrinsic and common)
• 3 bodies of evidence suggest a role of coagulation in liver fibrosis:

• Parenchymal extinction – Wanless et al 1996
o Microthrombi deposition from small veins of the liver, leading to venous obstruction
o The obstruction would cause oedema  necrosis
o The necrotic tissue would be replaced by fibrous tissue

• Prothrombin tendencies and acceleration of HCV fibrosis – Wright et al 2003
o HCV patients are classified as FAST or SLOW fibrosers according to stage and duration of infection
o The greatest interest has centred on the Factor V leiden mutation – mutation that leads to prothrombotic tendency
o There was increased OR of FAST fibrosis in HCV infected carriers of the FvL mutation
o N.B: further validated by teams in France and Greece
 Papatheodoridis et al, Gut, 2003 – looked at other prothrombotic tendencies e.g. Protein C deficiency and showed that host genetics that predispose to fast thrombotic factors can indeed increase the extent of live fibrosis in chronic viral hepatitis

• Thrombin and FXa mediated PAR ligation leading to HSC activation – Duplantier et al, Gut, 2004
o Animals were given carbon tetrachloride for the induction of fibrosis
o They were then given drugs (experimental antithrombin agents) and found that they had less fibrosis than control but also reduced activity of HSC
o Blocking thrombin may lead to beneficial effect

• Thrombin mediated stellate cell activation – Martinelli et al 2007
o The most likely mechanism by which a pro-thrombotic state influences hepatic fibrogenesis is via the PAR receptor
o PAR-1 expressed on surface of stellate cells
o Cleavage of PAR-1 by thrombin leads to stellate cell activation and deposition of tissue fibrosis

• FXa mediated stellate cell activation – Dhar et al 2012
o FXa also activates HSC via PAR1 and 2

Question 89

Reversibility of Fibrosis

Answer 89

• Human and animal models both prove degree of reversibility
• Wanless 1999
o Lamivudine in HepB  improved histopathology of fibrosis in viral hepatitis patients (i.e. if you take away the cause of fibrosis, there is a chance of recovery)
• Fibrosis is a balance between ECM degradation and production

Question 90

Degradation of ECM

Answer 90

• Bad:
o Early degeneration of ECM of normal matrix by matrix proteases
o Replacement by scar tissue = pathologic

• Good
o Resorption of excess matrix in fibrotic patients = resolution
o Potential opportunity to reverse dysfunction and portal HT

Question 91

Degradation of ECM via MMPs

Answer 91

• Metalloproteinases (MMPs) have a critical role in ECM degradation
• Family of calcium dependent enzymes
• Degrade collagens and non-collagenous substrates
• 5 subtypes
• Activated by cleavage via MT1-MMP or plasmin
• Inhibited by TIMPs (tissue inhibitors of metalloproteinases)
o TIMP1/2 produced by stellate cells and are anti-apoptotic for them
• Balance between MMPs and TIMPs that will dictate how effective the degradation process is
Pathologic phase
• Degradation of the normal matrix
• MMP-2 and MMP-9 are the main MMPs involved in the collagen degradation
• Stromelysin-1 cause proteoglycan and glycoprotein degradation

Progressive fibrosis phase
• Failure to degrade scar matrix
• MMP-1: main protease in scar degradation
• However, marked increase in TIMP-1/2 = net decrease in protease activity of MMP-1

Resolution phase
• There is decreased TIMP expression  increased expression of MMPs leading to reversal of fibrosis
• HSC either revert to inactive form or undergo cell death (NK/Kupffer cell mediated apoptosis)

This process of MMP and TIMPs can also be manipulated therapeutically in order to ameliorate fibrosis.

Question 92

Anti-fibrotic Therapy

Answer 92

• FIRST – withdraw primary injury
o Stop drinking
o Lose weight
o Treat viral hepatitis
• However, since a common pathway of fibrosis is present – there is a role for anti-fibrotic therapy whereby we may not only prevent fibrosis but result in resolution of pre-existing fibrosis
• There is no licensed agent yet
• BUT
o Many agents have been unsuccessfully trialled
o Many more currently being trialled: warfarin, rapamycin

Potential Targets
• Any step in the mechanism of activation of fibrosis potential target
• Developing an anti-fibrotic treatment may not only prevent fibrosis, but result in resolution of pre-existing fibrosis

Anticoagulation
• If a prothrombotic state may accelerate fibrosis, an anti-thrombotic state may slow down hepatic fibrosis
• Animal studies
o Suggest therapeutic potential
o Provide new insight into pathogenesis of hepatic fibrosis
o Heparin to rats – Abe
 Heparin to rats decreased in areas of fibrosis even in additional CCl4 insult

• However heparin has problems – injection everyday is not convenient, also has risks for hepatitis and osteoporosis
•
• Warfarin
o Decrease in amount of fibrosis in mouse again

• FXa inhibition – Rivaroxaban – Dhar et al
o Significant reduction in FXa, causing decreased fibrosis
o Warfarin affects both thrombin and FXa, but FXa inhibition causes direct effect

• Warfarin anticoagulation for fibrosis in HCV – Dhar and Thursz et al
o HCV post transplantation – 20/30% patients can progress to fibrosis within 5y, maybe due to interaction with immunosuppression

• Rapamycin
o In vitro: decreased proliferation of HSC, Collagen I, TGFb
o In vivo: reduction fibrosis in mouse models
o Can we use this post transplantation for HCV?

Question 93

Roles of Monocytes and Macrophages in the liver

Answer 93

ontogeny, tissue microenvironment and stress signals shape macrophage responses
 tissue development, homeostasis and inflammation
 host defense and clearance of apoptotic cells
 biological plasticity & great functional diversity
 contribute to a variety of inflammatory pathologies

• In peripheral blood, circulating monocytes represent ∼5–10% of peripheral blood white blood cells (WBCs)
• Highly heterogenic population
• circulation monocytes are recruited to tissues where they can differentiate into dendritic cells or tissue macrophages
• In the adult liver, macrophages may originate from either a haematopoiesis-independent, self-renewing population derived from embryonic yolk sac cells (Kupffer cells) or from the recruitment of bone marrow-derived circulating monocytes
• The liver macrophage pool consists of:
  o liver-resident Kupffer Cells (KCs) which are embryonically derived and have self-renewal capacity
  o bone marrow derived monocytes which can differentiate into monocyte-derived macrophages (MoMFs)
   tissue-destructive and pro-restorative/tissue-repair roles
   initiation and progression of liver diseases
• hepatic inflammation  systemic inflammation  immunosuppression
  o e.g. ALF and ACLF

Question 94

ALF vs ACLF

Answer 94

p154

• ALF:
  o hepatocellular loss of a magnitude and at a rate which exceeds the liver’s regenerative capacity
  o hepatocyte death results in synthetic “loss of function” and provokes a robust innate immune response
• ALCF:
  o presence of CLD and occurrence of acute hepatic dysfunction with synthetic failure that progresses to cause extrahepatic organ failure
  o EASL-CLIF definition: CLD must be cirrhosis

Background liver
Normal vs chronic liver disease +- cirrhosis

Demographics:
mean age 36 vs 56
female vs male

Causes:

• ALF: paracetamol, DILI, acute viral hepatitis, ischaemia, pregnancy, autoimmune
• ACLF:
• — CLD: alcohol, chronic viral hep, NASH
• — other precipitant: GI bleed, viral reactivation, etc.

Clinical features:

• ALF: coaguloapthy, jaundice, hepatic encephelopathy, high incidence of SIRS, extrahepatic organ failure, susceptibility to infection
• ACLF: coagulopathy, jaundice, extrahepatic organ failure, high sincidence of hepatic encephelopathy, SIRF, infection

Infection susceptibility:

• Bacterial: 35-40% vs 37% at diagnosis, 66% by 4 weeks
• Fungal: 11.2% vs 2.5-3%

Infection onset:
Late (> 5days) vs early OR late

Mortality
40 % vs 40-80% hospital mortality

In both ALF and ACLF, infections are key drivers or life-threatening complicating factors in these syndromes. Discounting the etiological hepatotropic viruses, bacterial infections are the primary microbial clinical issue in liver failure.

Question 95

Define ALF

Answer 95

Acute liver failure (ALF) is a rare condition in which coagulopathy, jaundice, and hepatic encephalopathy arise in the context of an acute hepatic injury and the absence of chronic liver disease (CLD); sub-categorizations of ALF have been proposed which use the interval between the development of jaundice and emergence of hepatic encephalopathy

Question 96

Define ACLF

Answer 96

ACLF is a syndrome in patients with chronic liver disease with or without previously diagnosed cirrhosis which is characterized by acute hepatic decompensation resulting in liver failure (jaundice + prolonged international normalized ratio) and one or more extrahepatic organ failure that is associated with increased mortality within a period of 28 days and up to 3 months from onset

Question 97

SIRS

Answer 97

o constellation of clinical signs, suggestive of immune activation and inflammation
o pro-inflammatory (e.g. TNF-α, IL-1β, IL-6) cytokines secreted in the liver
o associated with extra-hepatic organ dysfunction and adverse outcome in patients
o It is defined by the presence of any two or more of: fever or hypothermia, tachycardia, tachyopnoea, and white cell response, either leukocytosis, leucopaenia or >10% immature neutrophils

 modulatory effects on immune cell function (e.g. monocytes/macrophages)
 immune dysregulation  defective immune responses to microbial cues

Question 98

CARS

Answer 98

• Compensatory Anti-inflammatory Response Syndrome (CARS)
  o anti-inflammatory (e.g. IL-10, SLPI) cytokines/mediators secreted in the liver
  o counter-regulatory, homeostatic mechanism  prevent overwhelming inflammation
  o circulating anti-inflammatory cytokine levels: predict poor patient outcome

 modulatory effects on immune cell function (e.g. monocytes/macrophages)
 immune dysregulation  defective immune responses to microbial cues

Question 99

INITIATION OF INFLAMMATION IN ALF AND ACLF

Answer 99

• Pathogen-Associated Molecular Patterns (PAMPs)
  o mostly derived from the gut
  o lipopolysaccharide (LPS), flagellin, viral RNA
• Damage-Associated Molecular Patterns (DAMPs)
• mostly derived from damaged hepatocytes
• HMGB-1, S100A8/9, IL-1α, IL-33, ATP, mitochondrial DNA, histone-associated DNA, purines, heat-shock proteins and bile acids

Question 100

Pattern-Recognition Receptors (PRRs)

Answer 100

• expressed on monocytes and liver macrophages
• toll-like receptors (e.g. TLR 3, 4, 9)
• purinergic receptors (P2X7)
• receptor for advanced glycation end-products (RAGE)

After acetaminophen overdose, oxidative stress and direct mitochondrial damage are induced in hepatocytes, which consequently release DAMPs that can be recognized by KCs. In turn, activated KCs secrete pro-inflammatory cytokines (e.g., TNF-α), reactive oxygen species (ROS), and chemokines (e.g., CCL2) that amplify the pro-inflammatory signal and increase the recruitment of bone-marrow derived cells into the liver, mainly neutrophils and monocytes, thereby enhancing the inflammatory process. The sustained KC-released cytokines can also recruit other inflammatory cell subsets, such as eosinophils, DCs, and T cells.

Question 101

Role of monocytes and macrophages in human liver failure

Answer 101

• Monocytes and macrophages in the immunopathology of acute and acute-on-chronic liver failure. (Left)
• Different causes lead to development of acute (bottom) and acute-on-chronic (top) liver failure.
• A major component of the immunopathology of both syndromes is liver inflammation initiated by release of various DAMPs and DAMPs/PAMPs, respectively. (Right)
• During these syndromes, there is a reciprocal interaction of the immune responses between the liver and systemic circulation throughout the different phases.
  o Initiation phase: Kupffer cells become activated after recognition of PAMPs/DAMPs and initiate a pro-inflammatory response.
  o Propagation phase: Bone-marrow derived monocytes are recruited to the liver and differentiate into inflammatory macrophages, expanding the macrophage pool and promoting tissue destruction.
   During the propagation phase, innate immune activation is self-perpetuating with recruitment of effector cells driving further cytokine and chemokine production; their release to systemic circulation provokes SIRS.
• These macrophage-derived mediators contribute to vascular endothelial dysfunction and microcirculatory disturbances, resulting in extra-hepatic organ dysfunction.

see pg 156

Question 102

IMMUNOSUPPRESSION IN ALF AND ACLF

Answer 102

• ↓ monocyte HLA-DR expression
• ↑ plasma IL-4, IL-6, IL-10 levels
• HLA-DR & IL-10 levels: correlation with disease severity, organ failure & patient outcome
• circulating monocytes
  o ↓ numbers, ↑ CCR2 expression
• ↑ plasma and hepatic CCL2 levels
  o associated with monocytopenia and disease severity
• hepatic immune cell infiltrate:
  o CD68+ > MPO+ > CD3+

-	expanded hepatic macrophage population
o	recruitment (MAC387+) vs local proliferation (Ki67+)

• phagocytosing hepatic macrophages
• anti-inflammatory hepatic micro-environment
  o ↑ IL-10, TGF-β, favours resolution of inflammation

Question 103

Characteristics of monocytes/macrophages in human liver failure

Answer 103

Characteristics of human pro-restorative monocytes and macrophages in acute liver failure syndromes. The schematic summarizes the phenotypic and functional characteristics of (Left) steady-state inflammatory and (Right) pro-restorative monocytes and macrophages described in acute liver failure syndromes, which can arise in response to micro-environmental cues (IL-10, SLPI, PGE2)

In parallel to SIRS, a CARS develops that is due to release of anti-inflammatory mediators from the liver. Resolution/tissue-repair phase: In response to anti-inflammatory cytokines/mediators and efferocytosis of apoptotic cells, macrophages undergo functional reprogramming toward a pro-restorative phenotype, favoring resolution, and tissue recovery. “Spill over” of anti-inflammatory mediators from the liver to systemic circulation enhances CARS and causes monocyte functional reprogramming toward a pro-restorative phenotype, eventually leading to relative immunosuppression that predisposes susceptibility to infectious complications

Question 104

Immunotherapeutic strategies to treat liver failure

Answer 104

 pathways regulating macrophage recruitment (chemokines), responses to injurious/infectious insults (PRRs, DAMPs, PAMPs) and differentiation/polarization are well-conserved between mice & humans
 drugs for liver disease patients (e.g., albumin, glucocorticoids, NAC)  immune-modulatory effects
 high scavenging capacity of liver macrophages  preferential targeting using drug carrier materials
 animal models are not fully representative of the mechanistic spectrum of human diseases
 mice bear few immunological differences
 greater heterogeneity in patients compared to inbred mouse strains
 macrophage heterogeneity and their context-specific functions are better understood in the mouse

Liver macrophages are central to the pathogenesis of ALF and ACLF driving the initiation, propagation, and resolution of injury and related inflammation. Therefore, they are an attractive target for developing new therapeutic approaches to treat such conditions.