Virginia Research Day 2021

Selective inhibition of B Cell Lymphoma 6 (BCL6) D ecreases Germinal Center (G C) F ormation and R educes K idney Disease in L upus I nduced M ice Ashton Shiraz 1,3 , Eric Panther 3 , Samuel Dickerson 3 , Chandler Davis 2 , Christopher Reilly 1, 2, 3 1. Biomedical and Veterinary Sciences, Virginia-Maryland College of Veterinary Medicine, Virginia Tech, Blacksburg, VA. 2. Department of Cellular and Molecular Physiology, via Virginia College of Osteopathic Medicine. 3. Virginia Polytechnic Institute and State University, Virginia Tech, Blacksburg, VA.

Results

Introduction

Methods In vitro Studies: for the B cell activation, splenocytes were isolated and homogenized from a 26 weeks old NZB/W mice. Splenocytes were then stimulated with LPS (1µg/ml), IL4 (25ng/ml), Anti-CD40 (1µg/ml), and CpG-ODN (20µg/ml) as well as different doses of the BCL6-I drug were added to the cultured media. For cell cycle analysis and differentiation, splenocytes were isolated and homogenized from a 26 weeks old NZBWF1 mice. Splenocytes were then stimulated with, IL4 (1µg/ml) and Anti-CD40 (5µg/ml) and different doses of the BCL6-I drug were added to the cultured media (Data is not shown). In vivo studies: 20-week-old NZB/W mice were IP injected with different doses of the BCL6-I drug five days a week for 6 weeks. Proteinuria was measured each week by collecting urine from each group and assessed semi-quantitively via Uristix. dsDNA was measured by blood collection prior to imitation of treatment at 20 weeks-of-age and every 2 weeks until euthanasia. Immunofluorescence assay: IHC detection of GC in spleen tissue was determined. The sections were incubated with GL-7, IgD, and CD3e respectively. Statistical analysis: The significance of differences of the results was determined by One-way ANOVA a p < 0.05 was considered to be statistically significant. leads to clonal expansion, immunoglobulin somatic hypermutation, and class switch recombination leading to antibody affinity maturation (2). GC B cells represent the normal counterpart of most B-cell lymphomas, which are characterized by deregulated BCL6 expression or BCL6-mediated pathways (3). NZB/W female mice spontaneously develop autoimmunity that closely resembles human SLE. Hypothesis The selective B Cell Lymphoma 6 (BCL6) inhibitor decreases Germinal Center (GC) formation and reduces kidney disease in the NZB/W Mice Systemic lupus erythematosus (SLE) is a chronic, systemic autoimmune disease triggered by disruptions in the immune system. Both genetic and environmental factors contribute to disease progression (4). The serological hallmark of SLE is the production of autoantibodies, which target antigens located in the nuclear complex or the cell surface in the cytoplasm and are secreted by cells (1). B cell lymphoma 6 (BCL6) is a transcription repressor with many crucial roles in many immune cells and contributes to both innate and adaptive immune response. BCL6 functions as a main regulator of the germinal center (GC) B cell formation during hormonal responses. GCs emerge in the secondary lymphoid organs upon B cell activation, which

BCL6-I inhibits GC formation

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Summary

References • Selective BCL6-I decreases GC formation in lupus-prone mice. This was demonstrated both in vivo and in vitro . • Selective BCL6-I may be a potential therapeutic for the treatment of lupus nephrites. [1] Hatzi, K. and A. Melnick (2014). "Breaking bad in the germinal center: how deregulation of BCL6 contributes to lymphomagenesis." Trends Mol Med 20(6): 343-352. [2] Moulton, V. R., et al. (2014). "Pathogenesis of Human Systemic Lupus Erythematosus: A Cellular Perspective." Trends Mol Med 23(7): 615-635. [3] Huang, C. and A. Melnick (2015). "Mechanisms of action of BCL6 during germinal center B cell development." Sci China Life Sci 58(12): 1226-1232. [4] Salazar-Camarena, D.C., Palafox-Sánchez, C.A., Cruz, A. et al. Analysis of the receptor BCMA as a biomarker in systemic lupus erythematosus patients. Sci Rep 10, 6236 (2020).

(A, B) Spleen pictures from different treated groups to visualize the spleen size and the weight of spleens from each groups. (C) Proteinuria measurement of from 26-week-old NZB/W. (D-I) Flow cytometric analysis of different subsets of B cells in the spleen and Bone Marrow. (D) (E) Germinal centers of frozen 5-µm sections from spleen of 26-week-old control (Methyl Cellulose) and drug-treated mice were visualized by confocal microscopy. Slides were stained for GL-7 (green), IgD (red), and CD4 (blue) to detect germinal centers, B cells, and T cells, respectively. (F) Representative immunobiological staining of kidney section for IgG and C3. Data are shown as mean ± standard error of the mean (s.e.m) n = 5 mice for each group. Graphic analysis of mean fluorescent intensity (MFI) of IgG and C3 is also provided. Data are shown as mean ± standard error of the mean (s.e.m) n = 10

Acknowledgment

We thank the VMCVM flow cytometry facility, as well as, Virginia Tech confocal microscopy facility.

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