Defining the Molecular Basis of MicroRNA-146A Risk Allele Effects in Systemic Lupus Erythematosus

Va Eastern Colorado Health Care System, Aurora CO

Investigators

Abstract

Systemic Lupus Erythematosus (SLE) is caused by a combination of genetic and environmental factors; however, the mechanisms by which these factors mediate disease risk remain incompletely defined. We recently elucidated a putative molecular mechanism for one of the few SLE risk loci that confers risk across all ancestral population groups examined. We fine-mapped the putative casual variant, rs2431697 and applied genome- editing approaches to demonstrate that this locus encodes an enhancer of MIR146A gene expression. Initial data suggested that this enhancer mediates SLE risk through its effects on type I interferon signaling, a pathway known to be dysregulated in SLE. However, follow up data reveal that despite in vitro effects on type I interferon production in myeloid cells, this risk variant does not impact serum type I Interferon activity in vivo in several large SLE cohorts. Interim observations suggest a plausible mechanism. First, rs2431697 is an expression quantitative trait locus eQTL for MIR146A in B cells. Second, the transcription factor, BHLHE40, exhibits allele- dependent physical interaction with the SLE risk variant in B cells. Third, our data highlight a role for both genes in B cell anergy, the usual mechanism of autoreactive B-cell tolerance to self-antigens. Together, these observations prompt our hypothesis. We hypothesize that the SLE risk enhancer at the MIR146A locus confers risk in SLE by modulating the development of B cell anergy. This hypothesis links the genetics of SLE with ongoing work by Dr. Harley during his rheumatology fellowship to develop novel tools to understand human B- cell anergy. Thus, we propose the following Aims. Aim 1) define the cellular locus of MIR146A dysregulation in vivo. To do this we will define whether the molecular correlates of our hypothesized model are present in diverse immune cell populations from healthy individuals and those with SLE analyzed immediately ex vivo or whether alternative models better explain how variation at this locus impacts SLE risk. We will apply a combination of RNA-Flow cytometry and spectral flow cytometric peripheral blood immunophenotyping with genetic analysis to determine whether the molecular correlates of our hypothesized model are present in B-cells ex vivo as predicted. Aim 2) [define the functional relationship between BHLHE40 and MIR146A in B cell anergy. We will determine whether MIR146A expression regulates autoreactive B-cell tolerance ex vivo in primary cells (Aim 2a), in vitro in cell lines (Aim 2b) and in vivo in mice (Aim 2c).] Aim 3) define whether global down regulation of microRNA metabolism occurs in anergic B cells. Experiments in T-cells indicate that most microRNAs undergo decay downstream of acute T-cell antigen receptor stimulus. Since chronic B-cell antigen receptor signaling is a major mechanism of autoreactive B-cell tolerance, global microRNA decay may enforce tolerance. To interpret our MIR146A results in this broader context, we must determine whether this is true. Doing so will also advance training goals for Dr. Harley’s career. The concept that the MIR146A SLE risk enhancer might impact SLE risk through regulation of B cell anergy and the other aspects of our model are highly innovative and have never before been experimentally demonstrated. Results from this proposal would provide strong rationale and inform planned efforts to develop MIR146A directed therapies, offering the prospect of improving outcomes for patients with SLE. SLE is a disease that both more commonly and more severely afflicts individuals within several demographic groups (women, African American & Hispanic individuals) that are projected by the VetPop 2018 project to increase as a proportion of the VA population in the coming decades. Since current SLE therapies inappropriately balance immunosuppression and immunopathology, they result in substantial morbidity & mortality. By understanding the mechanisms of SLE genetic risk factors and developing therapeutic approaches informed by this mechanistic understanding, we aim to improve care for this growing population of Veterans.