Date: Sunday, November 8, 2020
Session Type: Abstract Session
Session Time: 10:00AM-10:50AM
Background/Purpose: The mechanisms self-tolerance loss that lead to autoantibody-mediated autoimmune disease remain underdefined. The rapid reversibility of peripheral B-cell tolerance in murine models suggests that non-durable biochemical mechanisms predominate to enforce this state, termed anergy. However, transcriptomic differences between anergic B-cells and their naïve non-anergic counterparts have also been described in murine models. Current understanding holds that B-cell anergy is maintained through chronic antigen receptor stimulation. This suggests that a core transcriptional program might extend across B-cell anergy in autoantigen systems of differing specificity. Here, we sought to understand whether such a core transcriptional program exists and performed initial functional characterization of one implicated pathway.
Methods: Data used in this analysis was from a combination of publicly available microarray/RNA-Seq data and a previously unpublished RNA-Seq experiment. All experimental conditions compared anergic B-cells to their naïve non-anergic counterparts. Data were analyzed using the NetworkAnalyst platform. Following normalization and batch effect correction, meta-analysis comparing anergic to naïve non-anergic B cells was carried out using Fisher’s method with a threshold of P < 1E-8. Pathway analysis was performed using Enrichr. For confirmatory studies, murine splenocytes or human PBMC were stained with CD36 along with B-cell subpopulation markers. For functional experiments, Ramos cells were pre-treated with palmitoyl-carnitine and calcium flux was determined kinetically following B-cell receptor crosslinking.
Results: Meta-analysis revealed 185 differentially expressed genes with this stringent significance threshold between anergic and naïve non-anergic B cells. Pathway analysis indicated expected enrichment of antigen receptor signaling pathway gene expression in anergic B cells. Surprisingly, genes involved in several metabolic pathways were differentially expressed. Of these, Cd36 stood out as one of the genes with lowest relative expression in anergic B-cells. We focused on its role as a key regulator of several metabolic pathways. Indeed, flow cytometry confirmed decreased CD36 protein expression on anergic B cells in comparison to their naïve non-anergic counterparts within the wild type repertoire. Similar decreases were observed in BCR-transgenic mouse models of anergy and the peripheral blood anergic human B-cell compartment. Ramos cells pre-treated with palmitoyl-carnitine, a metabolic intermediate downstream of CD36 exhibited dose-dependent suppression of calcium influx upon BCR-crosslinking.
Conclusion: Taken together, our results define a core transcriptional program of murine B-cell anergy and highlight metabolic pathways in the maintenance of tolerance in autoreactive B-cells. These findings are likely to inform our understanding of the long-term effects of therapies targeting metabolic pathways on the maintenance of tolerance in autoreactive B-cells. Further defining these mechanisms will nominate novel potential therapeutic approaches for autoimmune diseases.
To cite this abstract in AMA style:Harley I, Crute B, Getahun A, Cambier J. Transcriptomic Meta-analysis Reveals a Core Transcriptional Program in Murine B Cell Anergy and Implicates Immunometabolic Regulation as a Central Pathway in Maintaining Non-responsiveness of Autoreactive B-cells in Both Mouse and Man [abstract]. Arthritis Rheumatol. 2020; 72 (suppl 10). https://acrabstracts.org/abstract/transcriptomic-meta-analysis-reveals-a-core-transcriptional-program-in-murine-b-cell-anergy-and-implicates-immunometabolic-regulation-as-a-central-pathway-in-maintaining-non-responsiveness-of-autoreac/. Accessed December 3, 2021.
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