2105 Pathophysiology of Asthma PDF

Asthma Dr Neil S Holden  Objectives After this lecture you should be able to: Describe the immunopathology of asthma. This will include defining asthma. Describing the different variants of asthma. Describe the Immunopathology of asthma. In...

Asthma Dr Neil S Holden  Objectives After this lecture you should be able to: Describe the immunopathology of asthma. This will include defining asthma. Describing the different variants of asthma. Describe the Immunopathology of asthma. Including the immune cells & mediators involved. Describe the anti-inflammatory mechanisms of corticosteroids & how combination therapy works.  What is Asthma? The word asthma is a noun (ασθμα) derived from the Greek verb aazein (αάζειν) meaning short drawn breath or panting The earliest appearance of asthma that still survives today is in Homer’s The Iliad The term asthma is referenced in book XV, line 10: "He saw Hector lying on the plain, his companions sitting round him. Hector was gagging painfully, dazed and vomiting blood." Asthma It was also used in the writings of the school of Hippocrates of Kos (460-370 BC) to describe any form of breathlessness  Asthma the Timeline 460-370 BC 1698 AD 1892 AD 1960 AD Hippocrates of Kos Sir John Foyer Sir William Osler Dunnill et al Breathlessness Described “asthma” Noted bronchial Described full extent Many different Classified asthma as asthma special form of airway lung diseases “periodic” and of inflammation of inflammation in “continued” the small bronchioles patients who died of acute asthma Since 1960 the definition for asthma has continually evolved – Inflammation has become an accepted feature of even mild asthma in the absence of symptoms Historically just about all of the definitions of asthma have focused on defining asthma purely through the symptoms.  Asthma the Definition(s) British Thoracic Society 1993 “…..a common and chronic inflammatory condition of the airways, whose cause is not completely understood. As a result of inflammation, the airways are hyper-responsive and they narrow easily in response to a wide range of stimuli. This may result in coughing, wheezing, chest tightness, and shortness of breath; these symptoms are often worse at night. Narrowing of the airways is usually reversible, but in some patients with chronic asthma the inflammation may lead to irreversible obstruction of airflow. Characteristic pathological features include the presence in the airways of inflammatory cells, plasma exudation, oedema, hypertrophy of smooth muscle, mucus plugging, and shedding of the epithelium. These changes may be present even in patients with mild asthma when they have few symptoms” Global Initiative for Asthma (GINA) 2006 “A chronic inflammatory disorder of the airways in which many cells and cellular elements play a role. The chronic inflammation is associated with airway hyperresponsiveness that leads to recurrent episodes of wheezing, breathlessness, chest tightness and coughing, particularly at night or in the early morning. These episodes are usually associated with widespread, but variable airflow obstruction within the lung that is often reversible either spontaneously or with treatment” Global Initiative for Asthma (GINA) 2015 “Asthma is a heterogeneous disease, usually characterized 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”  Asthma Stratification One of the major definitions of asthma is that it is a heterogeneous disease This means many different forms (phenotypes) of asthma exist. One method used to stratify asthma is the severity of the disease  Asthma Stratification Other methods include to classify asthma by the phenotypic features. Many phenotypes have been identified. Some of the most common include: Allergic asthma: most easily recognized asthma phenotype. Often commences in childhood. Associated with a past and/or family history of allergic disease such as eczema, allergic rhinitis, or food or drug allergy. Induced sputum of these patients before treatment often reveals eosinophilic airway inflammation. Patients usually respond well to inhaled corticosteroid (ICS) treatment. Non-allergic asthma: some adults have asthma not associated with allergy. The cellular profile of the sputum of these patients may be neutrophilic, eosinophilic or contain only a few inflammatory cells (paucigranulocytic). Patients often respond less well to ICS. Late-onset asthma: some adults, particularly women, 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 to corticosteroid treatment. Asthma with fixed airflow limitation: some patients with long-standing asthma develop fixed airflow limitation that is thought to be due to airway wall remodelling. Asthma with obesity: some obese patients with asthma have prominent respiratory symptoms and little eosinophilic airway inflammation.  Asthma Prevalence Because of the lack of a clear definition and almost certain under- reporting of milder forms of asthma, it is likely that we underestimate the prevalence of asthma in the world  Asthma Burden One of the most common chronic diseases, with an estimated 300 million affected worldwide Has become more common in both children and adults around the world in recent decades Rate of asthma increases as communities adopt western lifestyles and become urbanised; projected increase in urban population (from 45% to 59% in 2025) likely to significantly increase the number of asthmatics worldwide in the future (estimated additional 100 million by 2025) It is estimated that asthma accounts for about 1 in every 250 deaths worldwide. Many of the deaths are preventable, being due to suboptimal long-term medical care and delay in obtaining help Normal Lung Physiology Asthmatic Lung Physiology  Immunology – a quick refresher Lines of Defence 1st 2nd 3rd Barriers The Immune System Physical Chemical Innate Adaptive  The innate immune system Cells of the innate immune system Cells of the adaptive immune system Antigen presenting cell (APC)  Inflammation aids the killing frenzy Symptoms of inflammation have been recognised for a long time medically Calor: heat Rubor: redness Turgor: swelling Dolor: pain Blood vessels dilate, leading to local swelling and the accumulation of components of the immune system.  Inflammation in different forms of asthma Severe Refractory Mild/Moderate Asthma Asthma Eosinophils ++ Neutrophils+ Macrophages+ Macrophages Immune Cells CD4+ T-Lymphocytes (Th2) CD4+ T-Lymphocytes (Th2) Mast cells CD8+ T-Lymphocytes (Tc1) Eotaxin IL-8 Key Mediators IL-4, IL-5 IL-13 IL-5, IL-13 Nitric Oxide +++ Nitric Oxide ++ Oxidative Stress + +++ Proximal airways Site of Disease Proximal airways Peripheral airways Fragile Epithelium Consequences Mucous metaplasia Thickened basement membrane Smaller bronchodilator Large bronchodilator response response Response to therapy Good response to Reduced/no response to corticosteroids corticosteroids Cascades in allergic/atopic asthma  Activators of asthma exacerbations Rhinovirus (RV) Respiratory syncytial virus (RSV) Viruses Human metapneumovirus (HMV) Influenza virus Mycoplasma pneumoniae Bacteria Chlamydia pneumoniae Virus Fungi Tree, Weed and grass pollen Allergen Indoor allergens House dust mite, Cockroach Animal exposures Occupational Rhinovirus Enterovirus Chemical exposures 16,00 3,00 Coronavirus Airway pollutants 2,17 39 Influenza virus Irritants Parainfluenza virus Cigarette smoke 5,17 Respiratory Syncytial virus 8,67 Metapneumovirus Aspirin Adenovirus Other Exercise 3,17 9,50 8,33 Bocavirus Cold air Bacteria 5,00 Immunopathology  The Epithelial cell The Epithelium plays a crucial role in asthma Pathogenesis. 1. It is the first point of contact of allergens, pollution and other irritants, and more importantly a number of pathogens including respiratory viruses. 2. But importantly the epithelium is also the first point of contact for all the inhaled therapies patients take The Epithelial cilia beat in a synchronised pattern. This helps clear the mucus and “sweep” the allergens/irritants out of the lung  The Epithelium The asthmatic epithelium undergoes a number of structural changes 1. The beating of the cilia often become desynchronised, coupled with goblet cell hyperplasia and mucus gland hypertrophy causes an accumulation of airway mucus 2. The epithelial cells can lose their ciliated physiology or slough off leading to local lung remodelling 3. The epithelium is a source of a large number of pro-inflammatory cytokines/chemokines and other inflammatory mediators  The Airway Smooth Muscle 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 numerous GPCRs which control the contraction and relaxation Bronchoconstriction The Asthmatic Airway Smooth Muscle Enhanced Contraction Impaired Relaxation ASM Function ASM Airway Structure Inflammation Cytokines/Chemokines Increased ASM mass Cell Adhesion Airway wall thickening Cellular Infiltrate/Oedema Increased Mucus Increased IgE  The Mast/Basophil cell 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-leukotrienes and Prostaglandins  The Mast/Basophil cell Input Output Antigen specific/ Adaptive Histamine Antigen + IgE or IgG Heparin Substance P C3a, C5a Cytokines IL-4, IL-13 Proteases E.coli FimH IL-5, IL-6, TNFα Tryptases, tPA Endothelin Chymases, Cathepsin G Kit Ligand Gelatinases A & B NGF Growth Factors Carboxypeptidase A ATP bFGF, VEGF, NGF Dipeptidylpeptidase I Adenosine Chemokines IL-8, MIP-1α IL-4 IL-5 Eicosanoids Antigen-independent/ PGD2, LTC4 innate  Histamine Four Histamine receptors all G-protein coupled receptors but linked to various different Gα subunits The H1 receptor is linked to Gqα The H2 receptor is linked to Gsα The H3 and H4 receptors are linked to Giα  Leukotrienes Leukotrienes are formed from 5-Lipoxygenase (5-LO) arachidonic acid uses FLAP to convert arachidonic acid into 5- The precursor form LTA4 can either hydroperoxyeicosatetrae form LTB4 or the cysteinyl noic acid (5-HPETE), leukotrienes (LTC4, LTD4, and LTE4) which spontaneously Cysteinyl leukotrienes (CysLT) can reduces to 5- bind to CysLT1 receptor on goblet hydroxyeicosatetraenoic cells, airway smooth muscle and acid (5-HETE). The eosinophils ultimately causing enzyme 5-LO acts again inflammation and on 5-HETE to convert it bronchoconstriction into leukotriene A4 (LTA4), Leukotriene receptor antagonists bind to and block the CysLTR1 receptor.  Signalling via Gqα e.g. Histamine Leukotrienes Ligand PIP2 PLCβ G G & Ca2+ Gq α Calmodulin ↑ MLCK Ca2+ Ca2+ Ca2+ ↓ MLCP Ca2+ Ca2+ Ca2+ Ca2+ Ca2+ Ca2+ Bronchoconstriction Ca2+ Ca2+Ca2+ Ca2+ Ca2+ Ca2+ Ca2+ 2+ Ca2+ Ca Ca2+ SR Ca2+ Ca2+  The T-lymphocyte T-lymphocyte cells come in a number of “flavours” Thymus Millions of thymocytes with different TCRs Regulatory/supressor cells “dampen and control Treg the immune response Regulatory/ TC suppressor Cytotoxic cells “kill” pathogens and pathogen- TH Cytotoxic infected cells Helper The Helper T-cells co-ordinate many different Lymphoid organ immune responses through production of numerous different cytokines The T helper lymphocyte  The Th2 imbalance in asthma Healthy individuals Balance = Health High Th2 = Allergy/ Asthma have a either a Th1 balance of Th1 and Th1 Th2 Th2 cells, or have a Th2 Th1 biased response The Th2 imbalance observed in allergic asthma led to the “hygiene” hypothesis More recently this hypothesis has now evolved into the counter-regulation hypothesis, which takes into account the effect of infection on development of Treg cells Th2 cytokines  Eosinophils can work in gangs Eosinophils evolved in the main to help destroy parasites Eosinophils attacking a schistosome larva: it’s too big…but if it is coated with complement, eosinophils can recognise it and collectively kill it The Eosinophil  The Eosinophil in Asthma During the late asthmatic reaction eosinophils infiltrate the airways and degranulate causing inflammation The number of eosinophils both in the airways and within the blood is closely correlated to the severity of the disease in eosinophilic asthmatics  Eosinophil degranulation products Eosinophil cationic protein (ECP) - Also known as Ribonuclease 3 Eosinophil-derived Neurotoxin (EDN) – aka Ribonuclease 2 or RNaseA - Both of these evolved to attack RNA in pathogens such as viruses, but also both have cytotoxic effects which are independent of their RNase activity Major basic protein (MBP) – aka Proteoglycan 2 (PGR2) - Multiple pro-inflammatory effects, is a strong helminthotoxin, but also damages cells (epithelial) and causes bronchospasm Eosinophil peroxidase - Catalyses the conversion of hydrogen peroxide into hypohalous acids e.g. hypochlorus acid (HOCl)which is a powerful oxidising agent which causes damage to pathogens, but when released inappropriately damages the lung tissue H2O2 + Cl- → HOCl- + H2O  The Alveolar Macrophage Macrophages, are the main “professional phagocytes” in the body. They recognize and remove senescent, 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 pathogen free. An Electron- A macrophage micrograph of ingesting 5 red a macrophage blood cells  The Asthmatic Macrophage – a double edged sword Mediator Stimulus Effect Anti-inflammatory Greater in asthmatics, upregulated Suppression of inflammatory IL-10 by corticosteroids and LPS response Greater in asthmatics, upregulated IL-12 Neutralize Th2 response by LPS Immunosuppression (inhibition of Nitric Oxide Greater in asthmatics cytokine production) Pro-inflammatory Up-regulated by mast cell IL-17 mediators and down-regulated by AHR and airway inflammation IL-10 Reactive oxygen intermediates IgG immune complexes, IFNγ, (ROI): superoxide anion (O2−), Antimicrobial defense and chronic platelet-derived growth factor hydrogen peroxide (H2O2), and lung injury (PDGF), GM-CSF, TNFα, LTB4 hydroxyl radical (OH−) Pro-inflammatory cytokines: IgE Inflammation TNFα, IL-1β, IL-8, MCP-1, MIP-1α Inflammation and modulation of Products of arachidonic pathway IgE smooth muscle tone TNFα IgE/amplified by IFNγ Inflammation Activated B cells differentiate into antibody- secreting effectors. Resting B cell: Has membrane-bound 1 μm Effector B cell (plasma cell): antibodies that constitute Massive increase in ER allows secretion the B cell receptor of ~ 5000 antibodies per second Each individual plasma cell secretes an antibody specific for the antigen that elicited the immune response  Antibodies Mature B cells can undergo isotype 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 the order of the exons is μ-δ-γ3-γ1-γ2-γ4-ε-α1-α2  The B-lymphocyte in asthma Th2 cytokines (particularly IL-4 and IL-13) promotes the isotype 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 crosslinking results in degranulation Asthmatics/atopics can have up to 10x the concentration of circulating IgE than non- asthmatics/non-atopics http://img.medscape.com/boards/user/11e55398/ige%20asthma%20severity.png http://image.slidesharecdn.com/rhinitis-150226115535-conversion-gate01/95/rhinitisbronchial-asthma-and-immunotherapy-56-638.jpg?cb=1424973468  Severe Neutrophilic Asthma 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 However eosinophilic and neutrophilic asthma are not mutually exclusive Pathogens Overview - Immunopathology Eotaxin Numerous Cytokines & Chemokines Treating inflammation  Corticosteroids/glucocorticoids 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 and 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, Sjögren's syndrome, gout and others The Hypothalamic–pituitary–adrenal (HPA) axis Negative Hypothalamus Feedback CRH Corticotropin Releasing Anterior Hormone Pituitary ACTH AdrenoCorticoTropic Hormone Adrenal Cortex Cortisol Metabolism Immune supression  A Brief history of corticosteroids 1849 1948 September 30th 1949 1981 1988 2000 https://www.mims.com/resources/drugs/Malaysia/pic/Pulmicort%20inhaler%20200%20mcg_pufffd6da827-54d7-420f-9f28-9faa0009f93a.GIF Cortisone Thomas Addison Edward Calvin Kendall Guy’s Hospital London The Mayo Clinic 1955 1959 1970s Budesonide approved in Budesonide Turbuhaler Symbicort Combination Scandinavia introduced therapy introduced Schering and Upjohn Dexamethasone Inhaled BDP  Common side effects of corticosteroids Local vs Systemic Systemic side effects Adrenal SuppressionLocal side effects – suppression of the HPA axis Growth suppression – a concern for juvenile asthmatics Dysphonia – vocal cord impairment Bruising – thinning of the skin particularly in the elderly Oropharyngeal Candidiasis – Yeast infection Osteoporosis – effects on bone formation and resorption Cataracts & Glaucoma – an increased statistical risk has been Cough identified Pneumonia – mostly in– COPD Metabolic abnormalities e.g. possible effects on diabetes patients Psychiatric disturbances – various effects have been reported  Nuclear Hormone Receptors Ligands of NHR include glucocorticoids, Hormones (e.g. Estrogen, Testosterone), Vitamins (A & D). There are a number of orphan receptors which may bind a number of different metabolic intermediates File:Nuclear Receptor Structure.png  The Glucocorticoid receptor signalling pathway Glucocorticoid Cytoplasm FKBP51 FKBP51 Hsp90 GR FKBP51 Hsp90 Hsp70 GR Hsp90 GR Hsp70 Hsp70 FKBP51 GR Hsp90 Nucleus Hsp70 Pol II GRE Transactivation p65 p50 κB RE Transrepression  Transactivation Vs Transrepression Old hypothesis: The anti-inflammatory effects of corticosteroids are due to the transrespression of pro-inflammatory transcription factors such as NF-κB and AP1. The side effects associated with corticosteroid use are due to transactivation of metabolic genes. GR Inhibition of pro-inflammatory Stimulation of metabolic genes e.g. cytokines chemokines e.g. IL-1β, IL-6, tyrosine aminotransferase, serine IL-8, TNFα etc dehydrogenase, phosphoenol pyruvate carboxykinase GR p65 Pol II p50 GR GR κB RE Transrepression GRE Transactivation Anti-inflammatory Side Effects  Negative glucocorticoid response elements Hypothesis: Genes contain negative glucocorticoid response elements which overlap essential transcriptional machinery binding sites, and thus inhibit transcriptional activation. y TBP x GR GR TATAAA RE nGRE Osteocalcin – nGRE overlaps the TATA box POMC – GR multimers bind to nGRE close to TATA and transcriptional start site Glycoprotein hormone α submunit – nGRE overlaps the CREB response element However Genomic analysis has shown that only a few genes contain nGREs Those gene that do possess nGREs are typically NOT pro-inflammatory Thus nGREs cannot explain the anti-inflammatory effects of corticosteroids  The HDAC hypothesis Acetylation of Histone tails increases gene transcription Deacetylation decreases gene transcription  HDAC2 and the transrepression effects of corticosteroids Corticosteroids cause recruitment of HDAC2 to the promoters of inflammatory genes Evidence for corticosteroid inducible genes as anti-inflammatory mediators GR p65 p50 κB RE Transrepression Anti-inflammatory  Evidence for corticosteroid inducible genes as anti-inflammatory mediators Microarray RGS2 profiling indicates that corticosteroids can induce hundreds of genes many with clear anti- inflammatory potential Corticosteroids and redundancy Do anti-inflammatory genes require “classical” transactivation? Corticosteroid Insensitivity vs Resistance 60  What causes steroid insensitivity? In vitro Pro-inflammatory cytokines, such as IL-1β, TNFα, and a combination of TNFα & IFNγ Oxidative stress such as H2O2 and cigarette smoke extract Viruses and virus mimetics, including Rhinovirus (HRV-16) and poly IC What causes steroid insensitivity? Factors important in the disease state? Smoking Infection The balance of inflammatory cells in the lung?  What are the molecular mechanisms? The up-regulation of the dominant negative Glucocorticoid Receptor β The inhibition of HDAC2 The down-regulation of glucocorticoid-dependent transcription e.g. by MIF Defective glucocorticoid receptor binding and translocation Change in the balance of lymphocytes e.g. Reduction of regulatory T cells Multidrug resistance efflux pumps e.g. P-glycoprotein Enhancing corticosteroid potency and efficacy – LABAs and PDE inhibitors Observation by Greening et al Greening et al demonstrated that addition of a glucocorticoid to a long acting β2- adrenoceptor agonist (LABA) results in superior asthma control than increasing the dose of glucocorticoid alone BDP = Beclomethasone dipropionate PEF = Peak Expiratory flow A number of subsequent studies have confirmed this in both asthma and COPD, and have shown beneficial effects on symptom control, exacerbation number and mortality And now Symbicort and Advair are widely used. Author | 00 Month Year 64 A. P. Greening et al 1994 The Lancet, 344, (8917), 219-224 Enhancing corticosteroid potency and efficacy – LABAs GRE GRE Luciferase gene 40 Control GRE Reporter Activity 30 + Salmeterol 30.7 (fold induction) Increased 20 2.6-fold efficacy 10 11.9 ~9.3-fold 0 -10 -9 -8 -7 log [Fluticasone (M)] 1 nM 9.3 nM Increased potency M. Kaur et al. 2008 Mol Pharmacol Jan;73(1):203-14 Enhancing corticosteroid potency and efficacy – LABAs Form + + Bude - + - + RGS2 GAPDH  Take home message Corticosteroid biology is complex !! We are still working to understand the mechanisms that underlie their clinical actions However it is probably because of this complexity that corticosteroids are so effective Separating out the side effects of corticosteroids from the anti-inflammatory effects is not as simple as scientists originally thought it would be However lower doses of corticosteroids may be used when we combine them with other therapies