Abstract
Background Signaling through Toll-Like Receptor 9 (TLR9) is a central event in the activation of normal B cells and in the production of pathogenic autoantibodies in diseases such as systemic lupus erythematosus (SLE). We sought evidence for direct effects of repository corticotropin injection (RCI; H.P. Acthar Gel), an FDA-approved treatment for selected cases of SLE, on isolated human B lymphocytes activated in vitro by engagement of TLR9 and the B cell receptor.
Methods CD19 +B cells from healthy volunteers (n=3) were activated in vitro with the TLR9 ligand ODN 2395 and anti-IgM. RCI was added to cultures at concentrations over a 20-fold range (controls received equal volumes of placebo gel). Messenger RNA was isolated from cells after one day in culture and cDNA libraries were prepared for multiplexed high-throughput sequencing on an Illumina HiSeq 2500. Analyses of RNA-Seq reads utilized Illumina CASAVA pipeline Version 1.8. RUVSeq R package v3.1 and edgeR were used to identify differentially expressed genes in paired comparisons between unstimulated and TLR9/anti-IgM activated cells and between activated cells treated with RCI and placebo.
Results Treatment of B cells in steroid-free medium with ODN 2395/anti-IgM resulted in significant, reproducible induction of 162 distinct mRNAs (mean induction=8.87±0.95 fold; range=2.5–118.3-fold) and suppression of 80 mRNAs (mean suppression to 21.8%±0.8% of baseline; range=6% to 39% of baseline) at 24 hours. RCI treatment resulted in significant, reproducible suppression of 14 of the ODN 2395/IgM-induced mRNAs (mean suppression to 23.6%±3.1% of stimulated value; range 9.9% to 41.2%). The RCI-suppressed mRNAs included two key regulators of class switch recombination, AICDA and BATF. RCI treatment also resulted in significant, reproducible induction of 5 of the ODN 2395/IgM-suppressed mRNAs (mean induction by RCI=7.65±2.34 fold; range=4.7 to 16.9-fold). The RCI-induced mRNAs included SLAM family member Ly9 (SLAMF3), a cell surface receptor capable of inhibiting autoantibody responses. No ODN 2395/IgM-induced mRNAs were further increased after RCI treatment and no ODN 2395/IgM-suppressed mRNAs were further reduced after RCI treatment (p=0.0002; Fishers exact test).
Conclusions RCI treatment of human B cells cultured under steroid-free conditions and activated with TLR9 agonist ligand and B cell receptor stimulation resulted in reversal of elements of the early mRNA activation response, including two key regulators of somatic hypermutation, AICDA and BATF. Whether these effects are related to clinical actions of RCI in SLE will be of interest for future investigation.
Funding Source(s): Mallinckrodt Pharmaceuticals
