title: Detection and function of anti-cytokine autoantibody in respiratory disease
Creators: name: Almeshleb, Mohammed Talib Mohammed
Publisher: name: University of Glasgow
Year: 2019
Subjects: subject: anti-cytokine, autoantibody, asthma, COPD.

description: Background: Cytokines play an important and variable role in respiratory diseases, including Chronic Obstructive Pulmonary Disease (COPD) and asthma. Some cytokines can induce airway inflammation, allergy and asthma and thereby contribute to the pathogenesis of respiratory disease by different mechanisms. Anti-cytokine autoantibodies have been detected in several infectious, inflammatory and autoimmune disorders, and may have a protective role in the pathogenesis of these diseases. However, the presence and function of serum anticytokine autoantibodies in COPD and asthma is unknown. Furthermore, the use of neutralising antibody against cytokines is a powerful approach in drug development to block cytokine-mediated pathogenic effects in disease. Hypothesis: My hypothesis is that anti-cytokine autoantibodies are produced naturally in respiratory disorders (Asthma and COPD) and that these may be differentially associated with the severity and pathogenesis of these diseases, depending on the nature of the cytokine and the associated autoantibody. Aims: To address this hypothesis I set out the following aims: 1) To investigate whether COPD and asthma patients develop anti-cytokines autoantibodies, 2) To study their association with the severity, clinical phenotypes and biomarkers of COPD and asthma (Chapter 3 and 4), 3) To study the immunological features and functions of anti-IL-33 autoantibodies in asthma (Chapter 4), and 5) To investigate how anti-IL-33 autoantibodies are developed in asthma patients (Chapter 5). Methods: To test this hypothesis and to detect anti-cytokine autoantibodies in asthma and COPD serum, I firstly established the Luciferase Immunoprecipitation System (LIPS) assay for the detection of autoantibody against 14 cytokines in clinical serum samples (Chapter 3). I addressed the aims using different experimental protocols including neutralisation assays to investigate the function of anti-IL-33 autoantibody in asthma samples (Chapter 4). Several methods, including in vitro immunisation, flow cytometry and antibody gene cloning were used to understand the development and function of the anti-IL-33 autoantibodies in asthma (Chapter 5). Results: a) I successfully established and optimised the luciferase immunoprecipitation assay system (LIPS) for the expression of 14 luciferase-fused Th1, Th2, Th17 and Treg cytokines in Cos-1 cells. Using LIPS, several anti-cytokine autoantibodies against IL-1α, IL-8, IFNγ, IL-10, IL-37, TGFβ were detected at significantly high titres for the first time in COPD serum samples. The anti-cytokine titres do not correlate with the concentration of the corresponding cytokines in serum and in lung fluid from COPD patients, and they do not correlate with disease severity nor are they predominant in any of the COPD phenotypes studied (Chapter 3). The function of these serum anti-cytokine autoantibodies in COPD remains unresolved. b) For the first time, serum anti-IL-33 and anti-IL-5 autoantibodies were detected in significantly raised titres in asthma patients, mainly in those with mild asthma. These anti-IL-33 autoantibodies had neutralising activity and could block IL-33 mediated effects in vitro. Furthermore, the anti-IL33 autoantibodies are predominantly of the IgG isotype and their titre correlated negatively with the serum concentration of IL-33, IL-4, IL-5 and IL-25 which are key cytokines involved with the pathogenesis of asthma. The anti-IL-33 titre also correlated negatively with the serum total IgE concentration in asthma patients (Chapter 4). c) Several key experimental strategies and methods were established to understand the development and function of the anti-IL-33 autoantibodies in asthma including (i) in vitro immunisation to understand how the anti-IL33 autoantibodies were generated, (ii) flow cytometry strategies to identify the IL-33-specific B cell subset in asthma, and (iii) strategies were established for the IgG V-region cloning of the anti-IL-33 autoantibody from asthma patients (Chapter 5). Conclusion and prospects: the data presented demonstrated that the LIPS assay is a sensitive, specific and high-throughput method for screening autoantibodies, including anti-cytokine autoantibody in asthma and COPD. My results also demonstrated that patients with asthma and COPD have developed natural anti-cytokine autoantibodies and the antibody profiles are different between the two disorders, further confirming their difference in pathology and immune mechanism. The anti-IL-33 autoantibodies in asthma patients have neutralising activity and may be associated with disease severity. However, the functions of anti-cytokine autoantibodies in COPD are still largely unknown. The prospects from this work include to reveal their development and function in clinical asthma and COPD, identify the cytokine specific B and T cells and also cloning and bulk synthesis of natural human anti-IL-33 with therapeutic potential.