Module 3.3: Sterile inflammation & liver injury Flashcards
Explain why Alcoholic Hepatitis is relevant
- 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
Descibe the histology in alc hep
- Ballooning
- inflammatory infiltrate
- Steatosis
- Mallory Denk bodies
- Mega mitochondria
- Fibrosis
- Bile plugging
Describe how the alc hep is diagnosed
- Indistinguishable whether a patient has alcoholic hepatitis of NAFLD through histology
- <3 months jaundice
- Serum bilirubin >80mmol/L
- Alcohol consumption >10 units/day (men), >7 units/day (women)
- <2 months abstinence
- AST < 500; ALT <300
- Maddrey’s discriminant function ≥32
Give examples to the prognostic scores used in alc hep
- Scores of baseline liver function
• Maddrey’s discriminant function
• Model for End-stage Liver Disease (MELD)
• Glasgow Alcoholic Hepatitis Score - Histological scores
• Alcoholic hepatitis histology score - Scores of dynamic liver function
• Lille model
• early change in bilirubin level
Explain why we are unsure about the liver’s exact mechanism of response to alcohol
- 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
Describe the immune response to alcohol
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
Describe the findings that lead to the belief that bacterial overgrowth leads to hepatic inflammation in alcoholic hepatitis
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
give the main causes of death in alc hep within 90 days
hepatic failure - 41%
infection - 29%
renal - 8%
multi organ failure - 7%
Describe the role of infection in Alc hep
- 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.
Describe the role of monocytes in alcoholic hepatitis
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
Describe treatment associated defects in alc hep
- 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
Functions of Macrophages
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
What are Kuppfer cells
The principle macrophage population in the liver
• Filter blood coming through portal circulation to remove particles that pose a threat
How do macrophages work in immune response?
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
Describe how macrophages are phenotyped
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
Describe the role of myeloid cells in cancer
- 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
Compare Tumour associated macrophages and myeloid derived suppressor cells
- 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)
Therapeutic potential of TAMs
- 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
TAMs in HCC
- 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
TAMs in pancreatic adenocarcinoma
- 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
TAMs in gastric carcinoma
- TAMs are associated with poor prognosis and reduced survival
- TAMs are more abundant in tumours that have undergone treatment with some form of chemotherapy
TAMs in oesophageal carcinoma
- 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
Causes of chronic hepatitis
Chronic viral
autoimmune
drug induced
idiopathic
Clinical features of autoimmune hepatitis
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
Disease associations of AIH
- 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
Evidence for Autoimmune Pathogenesis of AIH
- 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
Pathophysiology of AIH
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
Possible reasons for abnormal HLA II presentation on hepatocytes in AIH
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
Autoantibodies in AIH
• 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
Classification of AIH
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
Overlap Syndromes in AIH and their significance
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
Histological features in AIH
- 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
Role of Liver Biopsy in AIH
• 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?
Tx in AIH
- Steroids
* Azathioprine
Define hepatic steatosis
presence of vesicles of fat, predominantly triglycerides, accumulating within hepatocytes (presence of fat in USS because you cannot biopsy the whole population)
Define Steatohepatitis
presence of hepatocellular inflammation in conjunction with steatosis
Define NAFLD
presence of hepatic steatosis as part of the metabolic syndrome of obesity, insulin resistance/ type 2 diabetes, dyslipidaemia and hypertension
Define Non-alcoholic steatohepatitis (NASH)
steatohepatitis associated with NAFLD.
Cannot be certain of this unless you have had a biopsy showing fat in liver
primary Causes of Fatty Liver
Associated with metabolic syndrome
o (Abdominal) Obesity
o T2DM
o Dyslidaemia
secondary Causes of Fatty Liver
o Alcohol
o Drugs (steroids, Amiodarone, HAART, MTX, tamoxifen)
o Hepatitis C infection (HBV?)
o Parenteral nutrition
Describe the histology of NASH
• 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
Describe the scoring systems that can be used for NASH
Kleiner/Brunt/ISHAK
Describe the epidemiology of NAFLD
- 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
Describe the causes of Morbidity and Mortality in NASH
(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
Describe the pathogenesis of steatosis
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.
Explain the gut-liver axis
• 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.
Immune Response in NAFLD
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
Danger Signals and Alarmins in NASH
• 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.
Inflammasome in NASH
• 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)
TLRs in NASH
- 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)
Complement in NASH
- 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
Monocytes and Macrophages in NASH
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.
Dendritic Cells in NASH
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.
T-Cells in NASH
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.
Non-Invasive Techniques for the Assessment of Hepatic Inflammation/Fibrosis in NAFLD
• 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
Commonest cause of ALF
paracetamol overdose
Types of Immune mediated drug hepatotoxicity
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
Classification I of DILI
- Hepatocellular (R>5)
- Mixed (R=2-5)
- Cholestatic (R<2)
R = (ALT level / ALT ULN) / (AP level / AP ULN)
Classification II of DILI
- 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
Flucloxacillin in Idiosyncratic Hepatotoxicity
- 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
- 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
HLA Associated Causes of Idiosyncratic Hepatotoxicity
HLA-DRB1 - Amoxicillin-clavulanate
Pathophysiology of DILI-HLA Associations: Theories
- 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)
- 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)
- 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)
- 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
inter-individual susceptibility in Paracetamol Hepatotoxicity
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
Mechanism of Liver Injury in Paracetamol
see images p127
- Excess paracetamol enters hepatocyte
- Glucuronidation and sulphonation pathways are saturated
- Increase in metabolism via the cytochrome P450 enzymes to reactive metabolite NAPQI
- Increased conjugation with glutathione eventually depletes levels of cellular glutathione
- Accumulation of reactive NAPQI (toxic metabolite that puts cells in stress)
- Binding of NAPQI to DNA and cellular proteins particularly to mitochondria
- Mitochondrial toxicity they lose the ability to produce ATP
- MPT: mitochondrial permeability transition allows solute flux, resulting in mitochondrial swelling and rupture
- Commencement of apoptosis (release of pro-apoptotic factors)
- Catastrophic energy failure leads to cell membrane compromise and necrosis
- 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
- Cell death by necrosis
- Release of DAMPs
- Local production of proinflammatory cytokines and chemokines by Kupffer cells
- 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 - Secondary immune mediated tissue damage
- Systemic inflammation driven by cytokines
- 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)
what are Immune Checkpoints
• 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
Roles Immune Checkpoint Inhibitors and the problem with them
- 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)
Treatment of Immune related adverse events IrAEs
- 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
Summarise the general mechanism of AIH
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
Epidemiology of AIH
Prevalence ranges from 11-36 cases per 100,000 of Type 1
Female > Male (4:1 in Type 1; 10:1 in Type 2)
presentation of AIH
• 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
Compare types of AIH
—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
Diagnostic criteria of Definite AIH
(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
Diagnostic criteria of Probable AIH
(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
International autoimmune hepatitis group revised diagnostic scoring system
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
Histology of AIH
- 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
Genetics of AIH
• 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)
Disease Triggers in AIH
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
Immune Mechanisms in AIH
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.
Role of PD-1 in impaired Immunoregulation in AIH
• 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
Role of Tregs in impaired Immunoregulation in AIH
- 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
Role of TIM3/GALECTIN-9 in impaired Immunoregulation in AIH
• 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
Findings in early trials for Tx of AIH
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
Summary of Immune Mechanisms of AIH
- 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
Describe the process of Fibrosis
- 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
What are the key concepts in the pathogenesis of fibrosis?
- Increase in extracellular matrix (ECM)
- Hepatic stellate cells and ECM producing cell populations
- Coagulation system in the pathogenesis of liver fibrosis
- Reversibility of fibrosis
Extracellular matrix in fibrosis pathogenesis
• 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
Hepatic Stellate Cell (HSC) in fibrosis pathogenesis
• 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?
Coagulation cascade in fibrosis pathogenesis
• 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
Reversibility of Fibrosis
• 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
Degradation of ECM
• 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
Degradation of ECM via MMPs
• 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.
Anti-fibrotic Therapy
• 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?
Roles of Monocytes and Macrophages in the liver
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
ALF vs ACLF
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.
Define ALF
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
Define ACLF
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
SIRS
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
CARS
- 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
INITIATION OF INFLAMMATION IN ALF AND ACLF
- 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
Pattern-Recognition Receptors (PRRs)
- 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.
Role of monocytes and macrophages in human liver failure
- 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
IMMUNOSUPPRESSION IN ALF AND ACLF
- ↓ 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
Characteristics of monocytes/macrophages in human liver failure
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
Immunotherapeutic strategies to treat liver failure
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.
