Neil (Asthma immunopathology)

Question 1

What is the definition of asthma?

Answer 1

“Asthma is a heterogeneous disease, usually characterised by chronic airway inflammation. It is defined by the history of respiratory symptoms such as wheeze, shortness of breath, chest tightness and cough that vary over time and in intensity, together with variable expiratory airflow limitation” (GINA 2015)

Question 2

What does asthma being a ‘heterogeneous’ disease mean?

Answer 2

That many different forms (phenotypes) of asthma exist. One method used to stratify asthma is the severity of the disease- intermittent, mild, moderate, and severe.

Question 3

Asthma stratification by phenotype

Answer 3

Common phenotypes include
- Allergic asthma
- Non-allergic asthma
- Late-onset asthma
- Asthma with fixed airflow limitations
- Asthma with obesity

Question 4

Allergic asthma

Answer 4

Most easily recognised asthma phenotype. Often commences in childhood. Associated with past/family history and other atopic conditions e.g. eczema. Can be induced by exposure to allergen. Induced sputum before treatment reveals eosinophilic airway inflammation. Patients usually respond well to inhaled corticosteroid treatment

Question 5

Non-allergic asthma

Answer 5

Some adults have asthma not associated with allergy. Set off by environmental stress e.g. cold weather. The cellular profile of the sputum may be neutrophilic, eosinophilic or contain only a few inflammatory cells. Patients often respond less well to inhaled corticosteroid treatment

Question 6

Late-onset asthma

Answer 6

Some adults (particularly women around peri-menopausal age) present with asthma for the first time in adult life. These patients tend to be non-allergic, and often require higher doses of ICS or are relatively refractory (stubborn) to corticosteroid treatment

Question 7

Asthmas with fixed airflow limitations

Answer 7

Some patients with long standing asthma develop fixed limitation that is through to be due to airway wall remodelling. Constantly wheezy- airway structure.

Question 8

Asthma with obesity

Answer 8

Some obese patients with asthma have prominent respiratory symptoms with little eosinophilic airway inflammation

Question 9

Epidemiology of asthma

Answer 9

One of the most common chronic diseases with at least 300 million affected worldwide.
Has become more common in both children and adults in recent decades.
Rate of asthma increases as communities adopt western lifestyles and become urbanised.
Estimated that asthma accounts for 1 in every 250 deaths worldwide. Many are preventable being due to suboptimal long term medical care and delay in obtaining help

Question 10

Asthmatic lung physiology

Answer 10

More mucus in asthmatic airway.
Airways have decreased volume compared to non-asthmatic airways.
In asthma attack the smooth muscle is heavily constricted.
(Asthma is mostly a disease of the upper airways whereas COPD is further down)

Question 11

Lines of defence

Answer 11

1st- Physical/chemical barriers
2nd- Innate immune response
3rd- Adaptive immune response

Question 12

Cells of the innate immune system

Answer 12

Mast cell
Macrophage
Natural killer cell
Basophil
Eosinophil
Neutrophil
Complement protein
Dendritic cell

Question 13

Cells of the adaptive immune system

Answer 13

B cell
T cell
- CD4+ T cell
- CD8+ T cell
Antigen presenting cell (APC)
Antibodies

Question 14

What immune cells are involved in mild and moderate asthma?

Answer 14

Eosinophils
Macrophages
CD4+ T-Lymphocytes (Th2)
Mast cells

Question 15

What immune cells are involved in severe refractory asthma?

Answer 15

Neutrophils
Macrophages
CD4+ T-Lymphocytes (Th2)
CD8+ T-Lymphocytes (Tc1)

Question 16

Cascades in allergic/atopic asthma

Answer 16

Something stimulates the attack (pathogen/allergens/irritant) -> Mast cells -> bronchoconstriction/inflammation -> reversible airflow limitation

Something stimulates the attack (pathogen/allergens/irritant) -> Epithelia cells -> Eosinophil/CD4+ T-Lymphocyte -> bronchoconstriction/inflammation -> reversible airflow limitation

Mast cell stimulation is an immediate reaction whereas epithelial stimulation takes a while. This means that symptoms also happen 4-8 hours after the attack as the epithelial cells are activating the pathway.

Question 17

Activators of asthma exacerbations

Answer 17

Viruses
- Rhinovirus (RV)
- Respiratory syncytial virus (RSV)
- Human matapneumovirus (HMV)
- Influenza virus
Bacteria
- Mycoplasma pneumoniae
- Chlamydia pneumoniae
Allergen
- Fungi
- Tree, weed, grass pollen
- Indoor allergens
- House dust mite
Occupational
- Animal exposures
- Chemical exposures
Irritants
- Airway pollutants
- Cigarette smoke
Other
- Aspirin
- Exercise
- Cold air

Question 18

The epithelial cell

Answer 18

The epithelium plays an important role in asthma pathogenesis.
It is the first point of contact of allergens, pollution and other irritants, and pathogens including respiratory viruses.
It is also the first point of contact for all inhaled therapies
The epithelial cilia beat in a synchronised pattern which helps clear the mucus and ‘sweep’ the allergens out of the lung

Question 19

The epithelium

Answer 19

The beating of the cilia often become desynchronised, coupled with goblet cell hyperplasia and mucus gland hypertrophy causes an accumulation of airway mucus
The epithelial cells can lose their ciliated physiology or slough off leading to local lung remodelling
The epithelium is a source of a large number of pro-inflammatory cytokines/chemokines and other inflammatory mediators

Question 20

The airway smooth muscle (ASM)

Answer 20

Smooth muscle fibres surround the larger airways throughout the lung. Evolved to prevent inhaling noxious gases. The airway smooth muscle cell contains numerous receptors including GPCRs which control the contraction and relaxation

Question 21

How is ASM different in an asthmatic?

Answer 21

ASM function- enhanced contraction, impaired relaxation
Airway inflammation- cytokines/chemokines, cell adhesion, cellular infiltration, increased mucus, increased IgE
ASM structure- increased ASM mass, airway wall thickening

Question 22

What is hypertrophy and hyperplasia?

Answer 22

Hypertrophy- increase in the volume of organ or tissue due to the enlargement of its component cells
Hyperplasia- increased number of cells but cells remain same size

Question 23

Mast/basophil cell

Answer 23

Embedded within the smooth muscle fibres are a large number of mast cells. Mast cell degranulation causes bronchoconstriction. Mast cell products which cause bronchoconstriction and inflammation include histamine, cysteinyl-leukotrines and prostaglandins

Question 24

Histamine

Answer 24

Four histamine receptors all GPCRs but linked to various different G-alpha subunits
H1 receptor is linked to Gq-alpha
H2 receptor is linked to Gs-alpha
H3 and H$ receptors are linked to Gi-alpha

Question 25

Leukotrines

Answer 25

Leukotrienes are formed from arachidonic acid.
The precursor form LTA4 can either for LTB4 or the cysteinyl leukotrienes (LTC4, LTD4, and LTE4).
Cysteinyl leukotrienes can bind to cysLT1 receptor on goblet cells, airway smooth muscle and eosinophils ultimately causing inflammation and bronchoconstriction.
Leukotriene receptor antagonists bind to and block the cysLTR1 receptor.
(Montelukast is an example of a cysteinyl leukotriene receptor agonist)

Question 26

T-lymphocyte

Answer 26

T-lymphocyte cells come in a number of ‘flavours’.
The thymus produces millions of thymocytes with different TCRs (T cell receptors).
Regulatory/suppressor cells dampen and control the immune response.
Cytotoxic cells kill pathogens and pathogen infected cells.
Helper T cells coordinate many different immune responses through production of numerous different cytokines

Question 27

T helper lymphocytes

Answer 27

Th1
- Cell mediated immunity and inflammation
- Intracellular pathogens
- Autoimmunity
- Inflammation
- (Major pathogen)

Th2
- Antibody mediated immunity
- Extracellular parasites e.g. tapeworms, helminths
- Asthma, allergy
- Th2 cytokines produce lots of IL-5 which activate lots of eosinophils which move into the lungs

Treg
- Immune tolerance
- Lymphocyte homeostasis
- Regulation of immune responses

Th17
- Extracellular bacteria
Fungi
Autoimmunity

Question 28

Th2 imbalance in asthma

Answer 28

Healthy individuals have either a balance of Th1 and Th2 cells or have an Th1 biased response.

Factors favouring Th1 phenotype
- presence of older siblings
- early exposure to day care
- tuberculosis, measles, or Hep A infection
- rural environment

Factors favouring the Th2 phenotype
- widespread use of antibiotics
- western lifestyle
- urban environment
- diet
- sensitisation to house-dust mites

The Th2 imbalance observed in allergic asthma led to the hygiene hypothesis.
More recently this hypothesis evolved into the counter-regulation hypothesis which takes into account the effect of infection on development of Treg cells.

Question 29

Eosinophils

Answer 29

They work in gangs.
Involved in the early innate response against parasites.
Contain
- Chemoattractant receptors
- Receptors for lips mediators
- Adhesion receptors
- Cytokine and growth factor receptors
- Lipid body contents
- Fc receptors
Specific granule contents
- PRRs

Question 30

Eosinophil in asthma

Answer 30

During the late asthmatic reaction eosinophils infiltrate the airways and degranulate causing inflammation.
The number of eosinophils both in the airways and pithing the blood is closely correlated to the severity of the disease in eosinophilic asthmatics.

Question 31

Eosinophil degranulation products

Answer 31

Eosinophil cationic protein (ECP)
- evolved to attack RNA in pathogens such as viruses
- have cytotoxic effects which are independent of their RNAse activity

Major basic protein (MBP)
- multiple pro-inflammatory effects
- a strongg helminthotoxin
- damages epithelial cells and causes bronchospasm

Eosinophil peroxidase
- cats;yses the conversion of hydrogen peroxide into hypohalous acids which damages pathogens but also the lung tissue

Question 32

Alveolar macrophage

Answer 32

Macrophages are the main professional phagocytes in the body.
They recognise and remove senescent (aging), dead, and damaged cells in many tissues and are able to ingest large microorganisms such as protozoa.
Within the alveolar sacs there are resident macrophage which help keep the LRT (lower respiratory tract) pathogen free.

Question 33

Asthmatic macrophage

Answer 33

Anti-inflammatory mediators and effect
- IL-10 (suppression of inflammatory response)
- IL-12 (neutralise Th2 response)
- Nitric Oxide (immunosuppression, inhibition of cytokine production)

Pro-inflammatory mediators and effect
- IL-17 (airway hyperresponsiveness and airway inflammation)
- Reactive oxygen intermediates (antimicrobial defence and chronic lung injury)
- Pro-inflammatory cytokines (inflammation)
- Products of arachidonic (fatty acid) pathway (inflammation and modulation of smooth muscle tone)
- TNFalpha (inflammation)

In asthmatics pro-inflammatory is more prominent that anti-inflammatory.

Question 34

B cells

Answer 34

Activated B cells differentiate into antibody-secreting effectors.
Resting B cell- has membrane bound antibodies that constitute the B cell receptor
Effector B cell (plasma cell)- massive increase in ER allows secretion of about 5000 antibodies per second.
Each individual plasma cell secretes and antibody specific for the antigen that elicited the immune response.

Question 35

Antibodies

Answer 35

Mature B cells can undergo isotope switching, only affects the constant region not the variable regions, the antibodies produced will react against identical antigens. 2 daughter cells can produce 2 different antibodies against the same antigen.
Isotype switching involves recombination of the chromosome, where exons encoding the heavy chain are removed.

Question 36

B lymphocyte

Answer 36

Th2 cytokines promote the isotope switch of B cells to produce IgE.
IgE is strongly associated with asthma and allergy with high affinity receptors found on both mast cells and eosinophils cross linking results in degranulation.
Asthmatics/atopics can have up to 7 times the concentration of circulating IgE than non-asthmatics/non-atopics

Question 37

Severe neutrophilic asthma

Answer 37

generally speaking, mild/moderate asthmatics airways have an infiltrate of eosinophils.
Severe asthmatics who don’t respond to corticosteroids are often found to have an infiltrate of neutrophils.
(Corticosteroids stimulate apoptosis in eosinophils but increase lifetime of neutrophils).
However eosinophilic and neutrophilic asthma are not mutually exclusive.

Question 38

Corticosteroids/ glucocorticoids

Answer 38

Corticosteroids provide the cornerstone anti-inflammatory therapy of all but the mildest cases of asthma.

They are equally well tolerated in both asthmatic children and adults and impact both the morbidity ad mortality of asthma.

Corticosteroids are in general ineffective in COPD patients, having no effect on lung function, disease progression or mortality. But they do have some effect on exacerbation frequency in severe disease and this accounts for their use.

Other autoimmune/inflammatory disorders treated by corticosteroids include allergic rhinitis, rheumatoid arthritis, lupus, Sjogren’s syndrome and gout.

Question 39

The Hypothalamic-pituitary-adrenal (HPA) axis

Answer 39

Negative feedback loop
Hypothalamus releases CRH (Aorticotropin releasing hormone) which acts on the anterior pituitary which releases ACTH (AdrenoCorticoTropic hormone) which acts on the adrenal cortex which releases cortisol which surpasses the immune response

Question 40

Side effects of corticosteroids

Answer 40

Local
- Dysphonia- vocal cord impairment
- Oropharyngeal candidiasis- yeast infection
- Cough
- Pneumonia- mostly in COPD patients

Systemic
- Adrenal suppression- suppression of the HPA axis
- Growth suppression- a concern for juvenile asthmatics
- Bruising- thinning of the skin particularly in the elderly
- Osteoporosis- effects on bone formation and reabsorption
- Cataracts and glaucoma- an increased statistical risk has been identified
- Metabolic abnormalities- possible effects on diabates
Psychiatric disturbances- various effects have been reported

Question 41

Nuclear Hormone Receptors

Answer 41

Ligands of NHR include glucocorticoids, hormones (e.g. oestrogen, testosterone) and vitamins (A&D).
there are a number of orphan receptors which may bind a number of different metabolic intermediates

Question 42

The glucocorticoid receptor signalling pathway

Answer 42

Transactivation- switch on gene for expression
Transrepression- switch off gene for expression

Question 43

Transactivation vs transrepression

Answer 43

Old hypothesis: the anti-inflammatory effects of corticosteroids are due to the transrepression of pro-inflammatory transcription factors. The side effects associated with corticosteroid us are due to transactivation of metabolic genes. Can’t have one without the other.

Question 44

Negative glucocorticoid response elements (nGREs)

Answer 44

Hypothesis: genes contain negative glucocorticoid elements which overlap essential transcriptional machinery binding sites and therefore inhibit transcriptional activation.
However genomic analysis has shown that only a few genes contain nGREs. Those genes that do possess nGREs are typically not pro-inflammatory. therefore nGREs cannot explain the anti-inflammatory effects of corticosteroids.

Question 45

The HDAC hypothesis

Answer 45

Acetylation of histone tails increases gene transcription.
Deacetylation decreases gene transcription.

Question 46

What causes steroid insensitivity?

Answer 46

Pro-inflammatory cytokines such as IL-1beta, TNFalpha.
Oxidative stress such as H2O2 and cigarette smoke extract.
Viruses and virus mimetic, including Rhinovirus (HRV-16) and poly IC.

Factors important in the disease state?
Smoking
Infection
The balance of inflammatory cells in the lung.
Patients who smoke or have frequent viral lung infections do not respond as well to corticosteroids.

Question 47

What are the molecular mechanisms?

Answer 47

The up-regulation of the dominant negative glucocorticoid receptor beta.
The inhibition of HDAC2.
Th down regulation of glucocorticoid-dependant transcription
Defective glucocorticoid receptor binding and translocation.
Change in the balance of lymphocytes e.g. reduction of regulatory cells.
Multidrug resistance efflux pumps

Question 48

Enhancing corticosteroid potency and efficacy

Answer 48

Studies have shown that addition of a glucocorticoid to a long acting beta2-adrenoreceptor agonist (LABA) results in superior asthma control than increasing the dose of glucocorticosteroid alone. This has beneficial effects on symptom control, exacerbation number and mortality.