rnabioco/bcellmicro repository overview

⁠Defining whether global MicroRNA processing acts as a break on autoimmune diseases

PI: Isaac TW Harley, MD, PhD, MS

⁠Background

Immune mediated inflammatory diseases (IMIDs) are immune system disorders affecting one in four human beings. IMID etiologies remain largely idiopathic. Incompletely understood polygenic and environmental risk factors trigger disease development. Systemic Lupus Erythematosus (SLE) is a prototypical example. SLE is an autoimmune disease characterized both by multi-organ system failure and production of pathogenic antibodies that react with self-antigens. In patients with SLE, there is a defect in the normal silencing program when B lymphocytes (B cells) that produce autoantibodies develop. This contrasts with healthy persons, in whom B cells capable of producing autoantibodies develop. However, these autoreactive B cells are made inert through chronic B cell antigen receptor (BCR) stimulation. This silencing program is termed anergy. In persons with SLE, anergic B cells can become activated. However, in healthy persons anergic B cells remain in an inert state and are not activated upon restimulation.

Our recent work identified microRNA-146A as a risk factor for SLE [doi:10.1038/s41467-020-20460-1.]. A single nucleotide change that disrupts an enhancer of microRNA-146A is a risk factor for SLE, as well as two related diseases: rheumatoid arthritis and Sjögren’s syndrome. Our subsequent unpublished work has identified dysregulation of autoreactive B cell development as the likely mechanism of this genetic risk factor. We found that anergic B cells exhibit decreased microRNA-146A expression as compared to their antigen naïve counterparts both in the peripheral blood of humans and in the spleen of mice.

An important interpretive caveat of our data is that most microRNAs we have tested using quantitative PCR exhibit decreased expression in anergic as compared to naïve B cells. This is finding is consistent with an observation in a similar physiologic context in T lymphocytes (T cells). In T cells, acute T cell antigen receptor (TCR) stimulation leads to decreased expression of most microRNAs across the entire genome [doi: 10.1084/jem.20111717.]. This implicates microRNA processing as an important regulator of cell state during lymphocyte activation. Our data and the genome-wide decreased expression of most microRNAs following antigen receptor stimulation suggest two competing hypotheses. The first hypothesis is that microRNA-146A expression is specifically downregulated as part of the B cell anergy program and this is the likely mechanism of SLE risk. The second hypothesis is that microRNA-146A downregulation is not specific but is part of a broader silencing program involving microRNA processing that enforces B cell anergy.

⁠Specific Aims

Thus, we aim to:
Aim 1: Understand microRNA-146A expression in anergic B cells in the context of microRNA processing Aim 2: Develop a comprehensive atlas of microRNA expression and processing in anergic B cells
Aim 3: Identify new microRNAs that induce or prevent B cell anergy to target in IMID & SLE therapy

⁠Research Plan

We optimized a protocol to sort both anergic B cells (BND) and their naïve counterparts (MN) from peripheral blood without inducing BCR stimulation. We will sort these two populations from the peripheral blood of 12 healthy persons. We will then perform both RNA-Seq and small RNA-Seq to characterize mRNA, pri-miRNA, pre-miRNA and miRNA expression within these two populations. We will also perform Argonaute pull-down experiments to characterize components of the RNA-induced silencing complex (RISC). Integrative analysis of these three levels of data will define whether the hypothesized global change in microRNA expression distinguishes anergic cells from their non-anergic counterparts. It will also identify the level of differential regulation of microRNA expression. Having RBI informatics fellow support will ensure that we are able to make the most of this multilayered data set and advance our understanding of this disease-relevant aspect of fundamental RNA biology. In terms of feasibility, we completed a pilot study of 3 healthy persons and have obtained high quality RNA that will be sent for sequencing next week.

⁠Broader impact

We anticipate that this data set will be a resource for advancing fundamental understanding of RNA biology. Specifically, it will advance our understanding of the regulatory mechanisms whereby microRNA processing impacts B lymphocyte function. It will advance our understanding of this key step on the path to developing several autoimmune diseases. In aggregate, these diseases affect a large proportion of the population. Also, there is potential broader impact if we can identify new microRNAs that induce or prevent B cell anergy. If we are able to therapeutically manipulate B cell anergy modulating microRNAs, there are clear translational applications across multiple health and disease states. This is because the balance between antipathogen and anti-self immune responses impacts IMIDs, infectious disease, vaccine development and cancer immunotherapies.

⁠Data

Project directory: /beevol/home/riemondy/Projects/harley-RBI-2023-005

⁠2024-01-22

These data are from the bulk RNA-Seq pilot experiment from 3 healthy persons of sorted BND* and Mature Naïve** B cells. Low input ribo depleted library prep to determine quality of RNA-seq data from sorted B cells popualtions

raw data: /beevol/home/rbilab/data/harley/raw_data/20240122_LH00407_0013_A22HKT7LT3