Virginia Research Day 2021

EGR2 differentially regulates plasma cell differentiation and function in normal C57BL/6 mice and autoimmune-prone B6/lpr mice Rujuan Dai 1 , Bettina Heid 1 , Zhuang Wang 1 , Christopher M. Reilly 1,2 , S. Ansar Ahmed 1 1 Department of Biomedical Sciences and Pathobiology, VMCVM, Virginia Tech, Blacksburg, VA24060 2 Edward Via College of Osteopathic Medicine (VCOM), Blacksburg, VA24060 Results Introduction/Background

Abstract ID: BF-4

The major phenomenon of autoimmune disease such as lupus is the production of autoantibodies that target self-organs. High affinity IgG autoantibodies are thought to arise through GC responses, which is important for the differentiation of autoantibody-secreting plasma cells (PCs). Major transcription factors that regulate the critical developmental checkpoint during GC response and PC differentiation have been identified 1 . Early growth response protein 2 ( EGR2 ) is a zinc finger transcription factor that belongs to the early growth response ( Egr ) gene family. Recent studies have shown that EGR2 plays an important role in T cell immunity and autoimmunity 2-5 . The B6 mice with EGR2 deficiency in T lymphocytes developed T - cell driven, lupus-like autoimmune disease with increased production of inflammatory cytokines and anti-dsDNA autoantibodies 3,4 . Further, it has been shown that in B6 mice, EGR2 depletion promoted GC response and GCB cell development, which contribute to elevated serum levels of autoantibodies and enhanced humoral response in EGR2 -/- B6 4,5 . Nevertheless, we have recently reported that EGR2 was upregulated in both human and murine lupus cells and that inhibition of EGR2 in vitro with EGR2 siRNA suppressed IFN  production in activated MRL- lpr splenic CD4 + T cells 6 . This data suggested that immune regulatory role of EGR2 could be context dependent, which varies at normal physiological and different pathological conditions. To investigate further the role of EGR2 in an autoimmune state, we derived a conditional EGR2 knockout mouse model in autoimmune-prone background (CD2-CreEGR2 -/- B6- lpr, also called EGR2 -/- B6- lpr herein). The aim of this study is to elucidate the differential role of EGR2 in autoimmune verse normal state with a focus on its role in B cell development and function.

Figure 1: Depletion of EGR2 increased spleen weight in both B6 and B6/ lpr mice. (A) Validation of EGR2 depletion in both T and B cells of EGR2 -/- B6- lpr mice (marked with blue). (B- D) EGR2 depletion increased spleen weight, but not absolute spleen cell number in B6-l pr mice. (E-G) EGR2 depletion increased spleen weight and absolute cell number in B6 mice. Graphs showed means  SD (n  7). Unpaired student t test; *, p <0.05; ** p <0.01.

Figure 4: EGR2 depletion promoted GCB cells in both B6/ lpr and B6 mice. (A) Flow cytometry analysis GCB cells in splenocytes EGR2 -/- B6- lpr and control mice. (B-E) Summary graphs showed the percentage of CD19 + and CD19 + GL7 + IgD - GCB cells in the spleens of EGR2 -/- B6- lpr (B&C) and EGR2 -/- B6 (D&E) mice and their controls. Graphs showed means  SD (n  6). Unpaired student t test; *, p <0.05 and ***, p <0.001.

Research hypothesis

Figure 5: EGR2 depletion suppressed the differentiation of plasmablasts (PBs) and plasma cells (PCs) in B6/ lpr, but not in normal B6 mice. (A) Representative Flow plots of CD19 + CD138 + PBs , CD19 - CD138 + PCs, CD93 - long lived and CD93+ short lived PCs in splenocytes of EGR2 -/- B6- lpr and control mice. (B-F) Summary graphs show the percentage of PBs, PCs and LLPCs in the spleens of EGR2 -/- B6- lpr (B-D) and EGR2 -/- B6 (E and F) mice and their respective controls. Graphs showed means  SD (n  6). Unpaired student t test; *, p <0.05 and ***, p <0.001.

The transcription factor, EGR2, functions aberrantly in autoimmune state when compared to the normal state. Specifically, In B6 mice, EGR2 suppresses both geminal center B cells (GCB) and plasma cells (PCs). In B6-lpr mice, EGR2 suppresses GCB, but promotes PCs, potentially via differentially regulating the key genes involved in PC differentiation.

Figure 2: Depletion of EGR2 increased serum levels of IFN , IL-10, IL-2 in B6/ lpr and B6 mice. Graphs showed the serum serum levels of cytokine IFN , IL-6, IL-10, TNF , IL- 2 and IL-17 in 6-month-old EGR2 -/- B6- lpr (A-F), 9-10-month-old EGR2 -/- B6 mice (G-L) and their age-matched controls. Graphs showed means  SD (n  8). Unpaired student t test; *, p <0.05; ** p <0.01.

Material and methods

Animals: EGR2 fl/fl mice in B6 background were kindly provided by Dr. Warren J. Leonard at NIH/NHLBI. EGR2 fl/fl mice were bred with B6/ lpr , and then CD2-CreB6-lpr mice to generate EGR2 -/- B6-lpr with EGR2 deficiency in T and B lymphocytes . The CD2-CreB6-lpr strain was developed by crossbreeding hiCD2-CRE (JAX) with B6- /pr ., EGR2 fl/fl mice were bred with hiCD2-CRE to obtain EGR2 -/- B6 mice. The EGR2 -/- B6-lpr and control EGR2 fl/fl B6- lpr were euthanized at 6 months-of age; and EGR2 -/- B6 and control EGR2 fl/fl B6 mice were euthanize at 9-10 months-of age for the experimental analysis. Both male and female mice were included in the study. Flow cytometry: The splenocytes and bone marrow cells were stained with different cell surface markers ( CD19, B220, CD43, IgM, IgD, GL7, CD138, CD93) to determine B cell development and specific B cell subsets. For intracellular stain of EGR2, the splenocytes were stained with surface marker, then fixed and permeabilized, followed by intracellular antigen stain with anti-EGR2. The stained cells were washed and visualized using a FACS Aria flow cytometer (BD Biosciences). The flow data were analyzed with FlowJo version 10 software. ELISA: The serum levels of inflammatory were measured by the Quanterix SP-X multiplex Chemiluminescent Assay kit. Serum levels of total IgG, IgM and IgG1 and IgG2a subtypes were measured with ELISA kits from Thermo Fisher scientific. Serum anti-dsDNA levels were detected by following a standard lab procedure as previously reported 7 . Statistics: The Graphs were generated with Prism software. All the values were reported as means ± SD. Unpaired student t tests were performed to assess the statistical significance between knockout and control mice.

Summary ❖ Conditional depletion of EGR2 promoted splenomegaly and cytokine IFN , IL-10 and IL-2 production in both B6 and B6/lpr mice. ❖ EGR2 depletion reduced serum levels of anti-dsDNA autoantibody, total IgG, IgM, and IgG1 and IgG2a in B6/ lpr mice. In contrast, EGR2 depletion increased anti-dsDNA autoantibody in B6 mice ❖ EGR2 depletion promoted GCB cells in both B6/ lpr and B6 mice, but suppressed the differentiation of PCs, particularly long-lived PCs in B6/ lpr mice. ❖ EGR2 depletion has no obvious effect on the early B cell development in bone marrow of B6/ lpr mice. Figure 6: EGR2 depletion had no obvious effect on the early B cell development in bone marrow of B6/lpr mice. ( A-E) Summary graphs show the percentage of pre-proB, Pro-B, pre-B, immature B, and mature B cells in bone marrow of EGR2 -/- B6- lpr and control mice . Graphs showed means  SD (n  6).

Figure 3: EGR2 depletion reduces anti-dsDNA autoantibody in B6/ lpr mice, but increase serum anti-dsDNA in B6 mice. Graphs showed serum serum levels of anti- dsDNA, IgG, IgM, and IgG subtypes in EGR2 -/- B6- lpr (A-E) , EGR2 -/- B6 mice ( F-J) and their age-matched controls. Graphs showed means  SD (n  8). Unpaired student t test; *, p <0.05; ** p <0.01.

References

5. Morita K, et al. Proc Natl Acad Sci U S A. 2016;113(50):E8131-E8140. 6. Dai R, et al. BMC Immunol. 2020;21(1):41. 7. Dai R, et al. PLoS ONE. 2010;5(12):e14302

1. Nutt SL, et al. Nat Rev Immunol. 2015;15(3):160-171. 2. Zheng Y, et al. J Exp Med. 2012;209(12):2157-2163. 3. Zhu B, et al. J Exp Med. 2008;205(10):2295-2307. 4. Okamura T, et al. Nat Commun. 2015;6:6329

Thanks for the support from VCOM-VMCVM One Health Research Seed Grants.

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