Week 9 Flashcards
Pathogens have choices where to live
Viruses are obligate intracellular pathogens
Bacteria, some fungi and parasites have a ‘choice’ or a preferred environment either inside or outside the cell
Intracellular bacteria
Can be found free in the cytoplasms or restricted to vacuoles/vesicles
Which cells do pathogens infect
Bacteria can infect immune and not immune cells
Eg epithelial cells
Eg macrophages (professional phagocytes) therefore they can hide in the cells meant to kill them
Survival inside phagocytes
Advantage:
-intracellular environment provides a continuous supply of nutrients to the pathogen
-pathogen shielded from extracellular immune system eg complement, antibodies, drugs (eg some antibodies)
Disadvantage:
-need to overcome innate anti microbial killing mechanisms of the phagocyte
What can be done about a pathogen that can survive inside a phagocyte
Intracellular
Cytoplasmic: NK cells, cytotoxic cells protective immunity
Vesicular: T cell and NK cell dependent macrophage activation
Phagosome processing pathway for MHC class II
Macrophage
Pathogen ingested by phagocytosis
Early phagosome
Late phagosome
Lysosome (contains hydrolytic enzymes, cationic peptides, proteases) fuses with phagosome to form phagolysosome
Microbe digested
MHC class II loading compartment
Degraded non-peptide material released,
Mtb peptide/MHC presented on surface
Phagosomal living intracellular pathogens
Phagosomal pathogens block processing pathway at an early stage on phagosome development
Eg mycobacterium tuberculosis Mtb;
Replicates in the early phagosome
So avoids contact with lysosome contents
Involves surface Mtb glycolipid lipoarabinomannan LAM
Cytosolic living intracellular pathogens
Eg listeria monocytogenes
Escapes into cytosol from early phagosome
Replicates in cytosol
So avoids contact with lysosome contents
Intracellular microbial pathogens are killed by a combination of innate processes
Acidification: pH=~3.5-4.0, bacteriostatic or bactericidal
Toxic oxygen derived products: superoxide O2-, hydrogen peroxide H2O2, singlet oxygen 1O2. Hydroxyl radical .OH, hypohalite OCl-
Toxic nitrogen oxides: nitric oxide NO
Antimicrobial peptides: defensins and cationic proteins
Enzymes: lysozyme- dissolves cell walls of some gram-positive bacteria. Acid hydrolases- further digest bacteria
Competitors: lactoferrin (bind Fe) and vitamin B12-binding protein
CD4 Th1 immunity
Activated Th1 cell:
IFN-gamma and CD40 ligand: activates macrophage to destroy engulfed bacteria
Fas ligand or LT-alpha: kills chronically infected cells, releasing bacteria to be destroyed by fresh macrophages
IL-2: induces T cell proliferation, increasing numbers of effector cells
IL-3 and GM-CSF: induces macrophage differentiation in the bone marrow
TNF-alpha and LT-beta: activates endothelium to induce macrophage binding and exit from blood vessel at site of infection
CCL2: causes macrophage to accumulate at site of infection
Priming Th1 cells
CD4 Th1 cells are primed by dendritic cells (antigen presenting cells) in LN/spleen through presenting peptides through MHC class II
Important for vesicle dwelling pathogens
Primed Th1 cells recognise infected cells through peptide presentation on MHC class II
Start of response: antigen receptor binding and Co stimulation of T cell by dendritic cell, proliferation and differentiation of T cell to acquire effector function. Lymph nodes or spleen
End function response: LN or spleen, liver, lungs, skin. Th1 cell recognises complex of bacterial peptides with MHC class II and activates the macrophage
Primed Th1 cells activate macrophages
Th1 driven macrophage activation enhances macrophage killing
No Th1 cells= no bacterial clearance
Not having T cells is bad
Activation of macrophages makes them more able to recognise pathogen and more able to kill pathogen
Th1 activated macrophages
Increase expression of:
-MHC class I and class II
-CD40
-CD80/CD86 amplification
Fas expression (apoptosis)
increase TNF and TNFR promotes autocrine nitric oxide production and apoptosis
Lysosome numbers
Reactive O2 intermediates (antimicrobial)
Factors that promote phagolysosome maturation (acidification and fusion)
CD8 cytotoxic T cell killing through MHC class I
Important for killing of cytosolic resident bacteria
Antigen in the infected cell is presented through MHC class I to CD8 T cells
CD8 T cells kill infected cells that by recognising foreign peptides through MHCI
Cytotoxic T cell recognises a specific peptide from the bacterium (eg listeria) presented through MHC class I and the T cell provokes cell death
Neighbouring uninflected cells not killed
Independent of where the infecting bacterium resides within the cell it can still be identified and killed
Mycobacterium tuberculosis
Mtb- an intracellular pathogen lives in vacuoles
Acid fast (gram +ve) bacilli
Slow growing: 12-24 hours replication
Complex lipid envelope that affects longevity, drug permeability, host resistance
Mtb host avoidance
Inhibits 3 key innate killing mechanisms used by phagocytes:
-reactive oxygen radical generation
-reactive nitrogen radical generation
-phagosome fusion with lysosomes
Mtb expresses:
Superoxide dismutase SOD- an enzyme that catalyses the breakdown of superoxide O2- to hydrogen peroxide
Catalase: an enzyme that catalyses the breakdown of hydrogen peroxide to water
Mtb actively block phagosome-lysosome fusion allowing them to survive and replicate within macrophages. Associated with the actions of Mtb cell wall glycolipid (lipoarabinomannan). M.tuberculosis have an unusually complex cell wall covered by a thick waxy coat
Tuberculosis disease clinical symptoms
Breathlessness
Chest pains
Loss of appetite
Coughing up blood
Tiredness
Persistent cough
Loss of weight
Night sweats
The persistence of mycobacteria in the host
Usually immunity develops- bacteria restricted to granulomas
T cells surround epithelioid cell, multi-nucleated giant cells, with mycobacteria in
Activation macrophages
Th1 cells enhance macrophage function through their activation
After activation phago-lysosome fusion is more efficient
Intracellular Mtb then killed
We are not very good at killing Tb:
-3-4 antibiotic treatments required 6-12 months, multidrug resistance increasing
Granulomas help ‘quarantine’ infection
Granulomas
Sometimes they collapse and become caseous
Centre of Granuloma becomes caseous- mass of dead macrophages (semi solid pus)
Anaerobic, low pH, cytokines (IFN gamma, LT and TNF a), macrophage, mycobacteria
Sometimes granulomas can leak and this enables bacteria to spread
Granulomas grows until erodes into: air pocket, blood vessel ,another Granuloma
If bacteria gets in the lungs they can be transmitted to others through sputum
-coughing: spread infection to other areas of lung, transmits infection to new hosts via infected sputum
Preventing TB
Bacillus calmette-Guerin vaccine BCG
Vaccine is 70-80% effective against most severe forms of TB eg TB meningitis
Less good at preventing respiratory disease
The vaccine is developed from mycobacterium bovis which is commonly found in cows
Live vaccine so shouldn’t be given to patients with immunodeficiency or who are on immunosuppression
Many factors influence susceptibility to TB infection (and other intracellular bacteria)
HIV
Malnutrition
Close living
Stress
Diabetes
Alcohol/drug abuse
Immunosuppresion (steroids anti-TNF for RA)
Genetic predisposition
Susceptible patients:
-CD4+ T cel defects (SCID)
-Th1 cytokine or receptor defects IFN gamma/ IL12/23 defects
Interferon -gamma is one of the critical protective factors, patients with partial defects in IFN gamma production or in IFNgamma R fucntion are highly susceptible to poorly pathogenic mycobacterial infections
TNFa is also critical as treatment of latent TB infected patients with anti-TFNa therapy for rheumatoid arthritis can develop post primary tuberculosis due to disease reactivation
BCG becomes disseminated and causes disease when live vaccine not controlled
A typical course of acute infection- viral titres
Rapid viral replication once infection established
Viraemia controlled within a day
Rapid resolution of viraemia once neutralising antibody kicks in
The prodromal response to infection
Most common symptoms of viral infection are due to the general effects of innate immune responses
The common cold
Rhinoviruses (~100):
-majority >50% of common colds
-ssRNA virus of the picornaviridae family
Adenoviruses (~60):
-second most common source of respiratory infections
-dsDNA virus of the adenoviridae family
Clinically almost indistinguishable- acute viral infections stimulate the host response in a similar way with similar results
Viral recognition
Innate immune responses target common motifs used by many viruses
Pathogen associated molecular patterns and pattern recognition receptors (PAMPs/PRRs)
Remember toll like receptors but also RIG-I-like receptors and nod-like receptors
Recognise viruses surface molecules or RNA/DNA
Interferon: interfere with viral infection
Large family of cytokines
Type I-interferon a and b- key antiviral cytokines:
-IFN-a produced by plasmacytoid dendritic cells and monocytes
-IFN-b produced by many cell types
-IFN-a can be used to treat hepatitis B and C infections while IFN-b can be used to treat multiple sclerosis
Type II-interferon gamma:
-activated by IL-12
-important for Th1 immunity
Type III:
-less well understood but role in anti-fungal/viral immunity
Interferon stimulated gene: ISG
ISG is a gene whose expression is stimulated by interferons
Viruses trigger the type I interferon system-> transcription of 100s of ISGs
The products of these ISGs have numerous antiviral effector functions interfering with all steps in a virus life cycle- entry, uncoating, transcription, translation, assembly, egress
MX1, PKR, OAS1- ‘classical’ ISGs
Potent antiviral activity
More recently discovered ISGs find a gradient of antiviral activity- dont always need the full arsenal to complete a job
Viral inhibition by interferons/ISGs
Inhibition of viral protein synthesis
Degradation of viral RNA through activation of an RNAse enzymes
Inhibition of viral gene expression and virion assembly
Upregulate MHC class I
Increases p53 activity (tumour suppressor gene) which kills virus infected cells by promoting apoptosis
Protein kinase R (PKR)
- Infected cell produces: interferons
- Interferons activate PKR
- Phosphorylation of translation initiation factor elF2a (eukaryotic initiation factor 2 alpha)
- Phosphorylated elF-2a forms an inactive complex which blocks translation and protein synthesis
-also able to mediate NF-kB and cellular activity
-activate tumour suppressor PP2A which regulates cell cycle and metabolism
-PKR able to induce cellular apoptosis
Medicines that act like interferons to reduce translation and therefore viral protein synthesis
Thiazolide class of antivirals/parasitics
Viruses have evolved to turn IFN cascade off
Primary immunodeficiency
TLR3 pathways
NEMO and NFkB pathways
Dysregulation of innate intracellular DNA sensing
Failure leads to auto-inflammatory and auto-immune disease (eg SLE)
STING:
-STING-stimulator of interferon genes
-STING has a key role in stimulating interferon production
-STING-associated vasculopathy with onset in infancy (SAVI)-Dysregulated inflammatory response
MHC-I loading,
The key method for viral recognition by CD8 T cells
Cross presentation of antigen
MHC class I induces T cell proliferation and activation
Engagement of peptide loaded MHC class I induces proliferation of peptide-specific cytotoxic T lymphocytes CTL
CTLs recognise peptide expressed on MHC class I
All nucleated cells have MHC class I molecules on cell surface
MHC class I displays peptides derived from any proteins being synthesised by cell
Viruses can interfere with MHC class I
Virus infected cells often have a “MHCI low” phenotype
Without MHC class I, CTLs cant ‘see’
How cytotoxic T cells kill
Enzymes- perforin, granzymes and granulysin
-enter cytoplasm of target cell and triggers caspase cascade which leads to apoptosis
Receptor interaction through FAS (effector cell) and FAS ligand (T cell)
The MHC class I low phenotype triggers NK cell killing
MCH class I on normal cells is recognised by inhibitory receptors that inhibit signals from activating receptors
NK cell does not kill the normal cell
‘Altered’ or absent MHC class I cannot stimulate a negative signal . The NK cell is triggered by signals from activating receptors
Activated Nk cell releases granule contents inducing apoptosis in the target cell
Directly neutralising antibodies
Mechanisms of action:
-block virus/receptor interaction
-block endocytosis
-block release into cytoplasm
-aggregate virus
Indirectly neutralising antibodies- ADCC antibody-dependent cellular cytotoxicity
Antibodies bound to virus can:
-induce classic complement cascade
-bind to Fc(gamma)RIII (CD16) receptors on NK cells- activation
1. Antibodies bind antigens on the surface of target cells
2. NK cell CD16 Fc receptors recognise cell-bound antibodies
3. Cross linking of CD16 triggers degranulation into a lytic synapse
4. Tumour cells die by apoptosis
Protective mechanisms of non-neutralising antiviral antibodies
ADCC- apoptosis
ADCP- virolysis and antigen presentation
ADCML- virolysis or cytolysis
Epitope unmasking- enhanced neutralisation
Intracellular neutralisation -enhanced MHCI presentation
When ADCC goes wrong: antibody dependent enhancement of infection
ADEI occurs when non-neutralising antiviral proteins facilitate virus entry into host cells, leading to increased infectivity in the cells
Some viruses have no easy route into cell
After first infection antibodies are produced but these are non-neutralising
On secondary infection the Fc domain of these non neutralising antibodies bind Fc receptor on cells and virus loads up the antigen binding region and through this gets access to the cell
Dengue is the prototype for ADEI
Dengue virus uses CD16 (Fc(gamma)RIII) as its entry receptor
1. Dengue has 4 serotypes, each antigenically distinct
2. In primary infection, dengue replicates in dendritic cells, conventional prodromal acute (painful) flu-like illness, 7-10 days ~1% mortality
3.infection generates neutralising Abs to infecting serotype
4. On a second exposure with a new serotype, non neutralising Abs bind dengue and initiate ADCC by NK cells by binding to Fc(gamma)RIII
5. Enhanced antibody mediated virus delivery to target cells
6. Dengue haemorrhagic fever ~10-50% mortality
An important consideration for vaccination
Poor antigenicity of any one strain could lead to ADEI
Hypersensitivity
Reactions of the immune system that are detrimental to the host
-normal immune response triggered and persistent in absence of true pathogen
-inappropriately exaggerated immune response
Gel and Coombs described 4 different hypersensitivity reactions
-type I IgE mediated reaction is ‘allergy’
Coombs and Gel classification of hypersensitivity mechanisms
Type I:
-IgE mediated mast cell degranulation and activated eosinophils
-anaphylaxis, venom/drug/food allergy
Allergy IgE
Type II:
-self IgG antibodies bind antigens on the patients own cell surfaces and directly cause damage
-autoimmune haemolytic anaemia
Cytotoxic IgG
Type III:
-immune complex between circulating antigen and IgG causes damage
-‘Arthu’s reactions
-glomerulonephritis, vasculitis
-immune complex disease
Type IV:
-cellular immune response mediated by T cells not by antibody
-contact dermatitis, Mantoux test
-delayed hypersensitivity, usually 24-72 hours after contact
Clinically useful to know type
Type I:
-immediate- within minutes of exposure
-patient usually tells you what they are allergic to
-life threatening
Type IV:
->4 hours, often a day or 2 after antigen exposure
-more difficult to find the allergen
-nasty but not life threatening
Contact dermatitis- Type IV
Specific, repeatable immune reaction mediated by T cells eg nickel hypersensitivity
Symptoms occurs 24-72 hours after exposure to the causative agent
Patch testing may be useful
-mark redness, patient comes back after 2 days if redness settled-irritant response. If not settled- type IV reaction -hypersensitivity reaction
Other reactions
Irritant response (burn/ chemical)
-compared to type IV improves when irritant removed
Food tolerance
-mechanism not well understood
Side effect;
-predictable reaction to a substance- “off target effect”
Important to get mechanism and diagnosis right as there are major implications on management
We need a clear message of what is allergy and what is something else
Different types of hypersensitivity/other reactions which have different risks attached
Lots of things that are mislabelled as allergy
-people who are not at risk of severe reaction may be inappropriately concerned
-people who are at risk of severe reaction may not take that risk seriously
Confuses the clinical message
Developing type I allergy
Allergen exposure
— step 1: sensitisation
Specific IgE production
—IgE undergoes class switching
Re exposure to allergen
—step 2 allergic reaction, memory response
IgE
Very low concentrations in blood compared with the other immunoglobulin classes
Class switching to IgE requires Th2 cytokines IL4 and or IL13
Extra constant region domain allows binding of allergy effector cells via Fc(epsilon)R1
Type I allergy
Antigen/allergen- the thing you’re allergic too
IgE antibody class that can stimulate allergy cells through Fab region
The high affinity receptor on the mast cell that cross links the IgE/allergen complex. FceR1
Mast cell, eosinophils, basophils
The effector cells that releases mediators that cause the allergic symptoms
Allergy effector cells
All deprived from haematopoetic stem cells in the bone marrow
All express FceRs
High granular (preformed mediators stored in granules and ready to respond)
Mast cells:
-concentrated in the tissues
-sentinels of immune system
-role in innate immune response as well
(Depends on how triggered eg IgE v TLR)
Basophils:
-circulating not in tissues
-migrate to lymph node and help prime for allergic responses (IgE class switching)
Eosinophils
Circulate in blood and migrate into tissues
Haematopoiesis in the bone marrow stimulated by IL-5 from Th2 cells
-clinically important as we block IL-5 in severe eosinophilic asthma
Attracted to tissues through chemokines (eotaxins)
Kills through lysosomal granules containing toxic mediators
Major role in anti-helminth immunity but also in allergic disease
Mast cell mediators
Very powerful substances with profound physiological reactions
So most are very short acting- minutes so not easy to measure
Tryptase lasts about 8 hours so is clinically useful to help diagnose presence of anaphylaxis
-part of NICE guidelines but not always release
If high then drops - sign of mast cell degranulation
Mast cell mediators together cause the symptoms of allergy
Histamine
Kallikrein
Cytokines (TNFa)
Tryptase
Prostaglandin
Leukotrienes
Serotonin
Vasodilation
Vascular permeability
Heart rate, cardiac contraction
Glandular secretion
Bronchoconstriction
Irritates nerve endings
—hypotension and tachycardia
Mast cells release mediators according to their trigger:
-eg IgE mediated release different to toll like receptors
-called degranulation
Anaphylaxis
Severe/life threatening allergic reaction
-medical emergency
Dramatic fall in blood pressure and high heart rate
-chest pain
Severe airway constriction
-tight chest, wheeze, swelling of throat, hoarseness, difficulty breathing
Collapse, loss of consciousness
-anxiety, headaches, dizziness, impending sense of doom
Skin
-hive, angioedema
GI symptoms
-diarrhoea, vomiting, nausea, abdominal cramps
Treatment
-intramuscular adrenaline: as lots symptoms mediated through beta-adrenergic system
The type I IgE mediated allergy spectrum
Hay fever, allergic asthma, atopic dermatitis: local symptoms rarely could cause anaphylaxis
Food allergy
Drug allergy
Latex allergy
Venom allergy —— can cause much more serious reactions
Type 1 allergy tests
Serum specific IgE
Skin prick testing- 15-20mins if not there then not allergic
-utilise IgE mediated immunology (a very specific reaction)
-if the hypersensitivity is not IgE mediated- these are the wrong tests
-don’t diagnose ‘allergy’ but do diagnose sensitisation
The allergic march- being atopic
Once allergic to one thing then more likely to be allergic to others
Allergy- the human cost
Allergy impacts on:
-Quol
-concentration
-performance- work/school/exams
-socialisation
-diet
-sleeping/snoring
-anxiety
Why do we get allergy
Genes:
-polymorphism (IL4R, FceR1)
-filaggrin
Environment:
-early life exposure and how exposed to allergens
-hygiene
-house dust mite
-infections RSV
-antibiotics/paracetamol
-smoking
-air pollution
Hygiene hypothesis
The lack of exposure to germs early in life skew our immune system away from a Th1 towards a Th2 phenotype
It is well recognised allergic disease is associated with a Th2 phenotype and the cytokines associated
T cells for allergy
IL-4, IL-5, IL-13
- Th2
— IL-4, IL-9: mast cell activation
—IL-4, IL-13: IgE class switching
—IL-3, IL-5: eosinophil activation and recruitment
Evidence for the hygiene hypothesis
More allergic disease in the western world
German unification
First child more likely to be allergic
Farm children less likely to be allergic (endotoxin/LPS exposure)
Killing of parasites
Evolutionary purpose of IgE mediated responses
Helminth recognised by mast cells:
-first line defence
-near body surfaces
-increase lymph and blood flow (angio-oedema)
-recruit other effectors
Parasite killing
Too large for phagocytes
Anti-helminth immunity triggered by IgE recognising and binding
Eosinophils bind to IgE via FceR1
Kill helminth by:
-toxic lysosomal granules:
— major basic protein MBP, ribonucleases
—reactive oxygen and nitrogen free radicals
Eradication
IL13 from effector cells causes goblet cell hyperplasia and mucus production
Also contraction of smooth muscle cells (gut)
Worm expulsion
Anaphylaxis and bronchoconstriction
Hypersensitivity
Reactions of the immune system that are detrimental to the host
Allergy
A specific immune mechanism against an allergen- IgE
Sensitisation
The immune system recognises an allergen but does not necessarily induce systemic symptoms
Atopy or atopic
Predisposition to allergic disease
Anaphylaxis
A severe life threatening, generalised or systemic IgE mediated reaction
Anaphylactoid
Systemic reaction not IgE mediated, can be severe and life threatening
