Date: Monday, October 22, 2018
Session Type: ACR Poster Session B
Session Time: 9:00AM-11:00AM
Background/Purpose: Common molecular and histological features of idiopathic inflammatory myopathies (myositis) include activation of the type 1 interferon (IFN) response and aberrant expression of major histocompatibility complex (MHC) classes I and II in myofibers. The IFN response correlates with myositis disease activity, and IFN is known to up-regulate MHC in muscle. MHC is directly involved in autoimmune attack by presenting self-antigens to T cells, and certain of its alleles (human leukocyte antigens (HLAs)) confer susceptibility to myositis. These molecules are not expressed in healthy myofibers, and over-expression of MHC class I in mouse muscle recapitulates many characteristics of myositis including inflammation, atrophy, and ER stress. Here we report the development of a series of cell-based assays for quantitative high throughput screening (qHTS) for small molecule inhibitors of IFN-stimulated MHC class I expression in muscle.
Methods: The primary screen involves immunofluorescence of HLA-ABC in immortalized human myoblasts stimulated with IFN-beta and treated with large and diverse chemical libraries containing approved, well-characterized, or novel compounds, and then analyzed by laser cytometry and high content imaging. Active molecules displaying concentration-response profiles for inhibition of HLA-ABC expression will be validated in primary myoblasts from myositis patients by both immunofluorescence and RT-qPCR of several HLA genes. In addition, we are developing a CRISPR/Cas9 genome-edited myoblast with reporter genes inserted into the endogenous HLA loci to measure its expression level.
Results: We have developed both high throughput RT-qPCR and immunofluorescence assays for HLA-ABC in immortalized human myoblasts, have measured concentration-response profiles for IFN-beta, and are currently screening chemical libraries. We sequenced the HLA loci of this cell line, designed guide RNAs, and are optimizing Cas9 transfections to create a reporter gene edited cell line.
Conclusion: These efforts will be the first application of qHTS technology with a chemical genomics approach to interrogating the IFN-MHC response in myositis muscle.
To cite this abstract in AMA style:Kinder T, Dranchak P, Inglese J. Quantitative High Throughput Screening of Small Molecules to Inhibit Interferon-Stimulated Major Histocompatibility Complex Class I in Myositis Muscle [abstract]. Arthritis Rheumatol. 2018; 70 (suppl 10). https://acrabstracts.org/abstract/quantitative-high-throughput-screening-of-small-molecules-to-inhibit-interferon-stimulated-major-histocompatibility-complex-class-i-in-myositis-muscle/. Accessed August 13, 2020.
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