An Investigation Into the Role of IgA and Its Fc Receptor (FcαRI) in Activation of Pro-Inflammatory Signaling and Inhibition Thereof The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters Citation Getchell, Kristen. 2019. An Investigation Into the Role of IgA and Its Fc Receptor (FcαRI) in Activation of Pro-Inflammatory Signaling and Inhibition Thereof. Master's thesis, Harvard Extension School. Citable link http://nrs.harvard.edu/urn-3:HUL.InstRepos:42004156 Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of- use#LAA An Investigation into the Role of IgA and its Fc Receptor (FcαRI) in Activation of Pro- inflammatory Signaling and Inhibition Thereof Kristen Getchell A Thesis in the Field of Biotechnology for the Degree of Master of Liberal Arts in Extension Studies Harvard University May 2019 Copyright 2019 Kristen Getchell Abstract Autoimmune diseases are defined as conditions in which the immune system mistakenly attacks the person’s tissues or organs. This often leads to inflammation, tissue destruction, and organ damage. In many cases, unknown precursors trigger autoimmune diseases, which presents a difficult therapeutic problem. Although some therapeutics are effective, many are not across all patients highlighting an unmet medical need. Some non-responder patients with autoimmune diseases have increased levels of immunoglobulin A (IgA). IgA’s sole activating Fc receptor, FcRI, activates the Immunoreceptor Tyrosine Activation Motif (ITAM) which triggers downstream pro- inflammatory pathways. These pathways are only activated by polymeric IgA. Scientists showed that inhibiting FcRI with a blocking antibody prevents this activation; however, there is limited understanding of how polymeric IgA’s structure influences activation of inflammatory pathways. Furthermore, a crosstalk between IgA and IgG receptors has been reported. These studies sought to develop in vitro methods to facilitate further investigation into IgA’s activating pro-inflammatory mechanisms. A method was designed to create synthetic immune complex memetics of IgA and IgG, which were used to probe the activation of pro-inflammatory pathways in the U937 monocytic cell line. A comprehensive gene panel of pro-inflammatory pathways associated receptors and cytokines was developed and used to measure via qPCR the downstream impact of IgA- and IgG-immune complex mimetics. IgA-immune complexes induced an increase in expression of IL8, FCAR1, CXCL2, TNFA, and IL1B. IgG-immune complexes also induced a pro-inflammatory signal, which had a unique signature in comparison to IgA- immune complex activation. Immune complex activation could be inhibited with a FcRI receptor blocking antibodies; thus, reversing the pro-inflammatory signal and suggesting this signal is mediated by FcRI. Therefore, these studies outline a method development process that resulted in a reproducible assay that can be used to analyze IgA- and IgG- immune complex activation and subsequent inhibition with blocking FcαRI antibodies by measuring gene expression changes in U937 cells. These methods can therefore be useful for screening of therapeutic candidates designed to inhibit IgA and IgG mediated pro-inflammatory signals and further understanding of structural properties of immune complexes necessary to induce cellular activation. Acknowledgments This thesis would not have been possible without the support of my colleagues at Momenta Pharmaceuticals, Inc. Carlos Bosques, my thesis director, for his mentorship and knowledge. Jay Duffner for his experimental design help and assistance in developing the idea for this research. Naveen Bhatnagar for his help with conducting the size exclusion chromatography experiments. John Scheck for his help to conduct endotoxin removal of the immunoglobulins. And lastly, for the Momenta Research Team, for their thought-provoking discussions surrounding this research throughout this past year. v Table of Contents Acknowledgments................................................................................................................v List of Tables.................................................................................................................... viii List of Figures...................................................................................................................... x Chapter I Introduction ..........................................................................................................1 IgA Structure and Function ..........................................................................4 IgA Deficiency .............................................................................................6 IgA Deposition .............................................................................................7 The IgA Fc Receptor: FcαRI .......................................................................8 IgA and FcαRI: Anti- and Pro-inflammatory Mechanisms .........................8 IgA and Autoimmunity ..............................................................................11 Chapter II Materials and Methods .....................................................................................14 Cell Culture of U937 Cells ........................................................................14 Endotoxin Removal, Biotinylation, and Quantification of IgA and IgG ...15 Size Exclusion Chromatography Analysis of IgA- and IgG-Immune Complexes..................................................................................................16 IgA- and IgG-immune Complex Activation in U937 Cells .......................17 Inhibition of IgA and IgG-Immune Complex Activation by Antibodies ..17 RNA Extraction and Quantitation ..............................................................18 Quantitative Reverse Transcription Polymerase Chain Reaction ..............19 Chapter III Results .............................................................................................................21 Endotoxin Analysis ....................................................................................21 vi Preparation and Characterization of IgA- and IgG-Immune Complexes via SEC Analysis .............................................................................................22 Selection of Genes to be Measured by qPCR to Monitor After Cellular Activation ...................................................................................................26 Optimization of Immune Complex Driven Cellular Stimulation of U937 Cells ...........................................................................................................28 Varying Concentrations of IgA-immune Complexes to Induce Activation ....................................................................................................................31 Varying Concentrations of IgG-immune Complexes to Induce Activation ....................................................................................................................32 Inhibition of IgA- and IgG-immune complex Activation with Blocking Antibodies ..................................................................................................33 Chapter IV Discussion .......................................................................................................38 Appendix........................................................................................................................... .42 Additional Figures .............................................................................................................42 References........................................................................................................................ ..92 vii List of Tables Table 1. Primer List for Measuring Gene Expression in Pro-Inflammatory Pathways. ....42 Table 2. Gene Array for Measuring Gene Expression Changes in Pro-Inflammatory Pathways with qPCR..........................................................................................................43 Table 3. Statistical analysis (p values) of all genes measured for expression and the effects of cell culture media at 30 minutes incubation with IgA-immune complex mimetics…….. ...................................................................................................................50 Table 4. Statistical analysis (fold change log2) of all genes measured for expression and the effects of cell culture media at 30 minutes incubation with IgA-immune complex mimetics…….. ...................................................................................................................51 Table 5. Statistical analysis (p values) of all genes measured for expression and the effects of cell culture media at 60 minutes incubation with IgA-immune complex mimetics…….. ...................................................................................................................52 Table 6. Statistical analysis (fold change log2) of all genes measured for expression and the effects of cell culture media at 60 minutes incubation with IgA-immune complex mimetics…….. ...................................................................................................................53 Table 7. Statistical analysis (p value) of all genes measured for expression