Virginia Research Day 2021

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Depletion of microRNA-183-96-182 cluster in lymphocytes suppresses anti-dsDNA autoantibody production and IgG deposition in kidney in C57BL/6-Fas lpr/lpr mice Zhuang Wang 1 , Bettina Heid 1 , JingJing Ren 1 , Michael Edwards 1 , Thomas Cecere 1 , Ran Lu 1 , Deena Khan 1 , David Kirsch 3 , Christopher M. Reilly 1,2 , Ansar Ahmed 1 , and Rujuan Dai 1 1 Department of Biomedical Sciences and Pathobiology, Virginia-Maryland College of Veterinary Medicine (VMCVM), Virginia Tech, Blacksburg, VA 24061, USA 2 Edward Via College of Osteopathic Medicine, Blacksburg, VA, USA 3 Department of Radiation Oncology, Duke University Medical Center, Durham, NC27710, USA Introduction / Abstract Results

Material & Methods The miR-183-96-182 (miR-183C) is a highly conserved miRNA cluster among species. Our previous work found a significant upregulation of miR-183C in the splenic cells of three different murine models of systemic lupus erythematosus (SLE). Current studies revealed that miR-183C miRNAs are critically involved in immunity and autoimmunity. In this study, we found that inhibition of miR-182 alone or miR-183C in vitro with antagomirs significantly reduced lupus-related inflammatory cytokine IFN-  and IL-6 in activated splenocytes from MRL or MRL/lpr mice. To further characterize the pathogenic role of miR-182 and miR-183C in lupus in vivo , we developed B6- lpr mice with conditional depletion of miR-182 or miR-183C in CD2+ lymphocyte. We found that depletion of either miR-182 or miR-183C in the lymphocytes of B6/lpr mice had no obvious effect on T and B cell development as similar percentage of CD4+. CD8+, CD19+, as well as Tregs, follicular helper T (T FH ), germinal center B (GCB), and plasma cells were observed in the miR-182 -/- and miR-183C -/- and their respective control. Importantly, we observed a significant reduction of serum anti- dsDNA autoantibodies in miR-183C-/- mice when compared to age-matched controls and the B6/lpr mice with miR-182 or miR-183C deficiency have significantly reduced IgG deposition in the kidneys. Meanwhile, there was reduced IFN production in ex vivo activated splenocytes from the knockout mice. Furthermore, we demonstrated that miR-182 and miR-183C regulated the inflammatory response in splenocytes via targeting forkhead box O1 (Foxo1). Together, our data suggest a potential therapeutic effect of targeting miR-183C in lupus. • Genetically lupus-prone MRL-lpr (MRL/MpJ-Fas lpr /J) and MRL (MRL/MpJ) were purchased and bred in house. miR-183C fl/fl and miR-182 fl/fl mice were kindly provided by Dr. David G. Kirsch, then crossbred with B6 (C57BL/6J), B6- lpr (B6.MRL-Fas lpr /J) mice, and then CD2-CreB6-lpr (developed in house by crossbreeding) to develop miR-183C -/- B6- lpr and miR-182 -/- B6- lpr mice. Only female mice were used in this study. • miR-183C -/- B6- lpr, miR-182 -/- B6- lpr and control miR-183C fl/fl B6- lpr, miR- 182 fl/fl B6- lpr were monitored and euthanized at 29-32 wks old for further experimental analysis. • The splenocytes were stimulated with Lipopolysaccharide (LPS, 500 ng/ml), anti- CD3 (5 g/ml plate coated) plus anti-CD28 (2 g/ml), Concanavalin A (Con A, 5 g/ml), PMA (50ng/ml)/ionomycin (1 g /ml) for designated time. The supernatants were collected to analyze cytokine production by ELISA. • Scrambled control and specific antagomirs against miR-183C miRNAs, Accell Foxo1 siRNA were delivered into splenocytes by serum free Accell siRNA delivery medium. • All the values in the graphs were given as means  SEM. Paired student t tests, unpaired Student t -tests, and one-way ANOVAwith Tukey- Kramer all pair's comparisons were performed for statistical comparison. *, **, and *** indicate p < 0.05, p <0.01, and p <0.001 respectively.

IgM

IgG

miR-183C -/-

miR-183C fl/fl

anti-dsDNA

0.0 0.2 0.4 0.6 0.8 OD380

p =0.054

Foxo1 -actin

100 400 Splenocytes (x10 6 ) 200 300

Foxo1 -actin

0.04

0.03

0.0 0.2 0.4 0.6 0.8 1.0 Spleen Weight(g)

0.02

Foxo1

20 mg/mL

10 mg/mL

0.0 0.5 1.0 1.5

0.01

Body Weight (g)

Figure 2: Depletion of miR183C reduces the production of serum anti-dsDNA in B6-lpr mice. All the mice were euthanized at 29-32 wks old. (A)The body weight, spleen weight, spleen/body weight ratio and representative spleen images from miR-183C -/- B6- lpr and control mice. (B,C&D) The serum anti-dsDNA, total IgG and total IgM production level assessed by ELISA. Graphs show means  SEM. Unpaired t test was performed. miR-183C fl/fl miR-183C -/- 0 20 wks 24 wks 28 wks End point miR-183 fl/fl miR-183 -/- miR-183 fl/fl miR-183 -/- miR-183C fl/fl miR-183C -/- miR-183C fl/fl miR-183C -/- 0.00 Spleen/Body weight ratio miR-183C fl/fl miR-183C -/- 20

Foxo1 -actin

Relative mRNA levels

NC siRNA

Foxo1 siRNA

miR-182 fl/fl

miR-182 -/-

IgG p =0.062

anti-dsDNA

p =0.082

0 300 Splenocytes (x10 6 ) 100 200

20 25 30 35 40 Body Weight (g)

0.02

0.0 0.2 0.4 0.6 0.8 OD380

p =0.076

0.6

0.4

0.01

mg/mL

0.2

Summary • Deliberately inhibition of miR-183C miRNA significantly reduces the production of lupus-related inflammatory cytokines ( IFN , IL-6) in activated splenocytes from both MRL and MRL-l pr mice. • Depletion of miR-183C, but not miR-182 alone in CD2 + lymphocytes significantly suppresses the development of anti-dsDNA autoantibodies. • Depletion of miR-183C or miR-182 alone reduces IgG deposition in kidney of B6- lpr mice. • Depletion of miR-183C has no notable changes on T and B lymphocytes differentiation and composition in the spleen. Figure 6:The miR-183C family regulates IFN expression through targeting Foxo1. Foxo1 expression was reduced in MRL-lpr mice (A) and antagomiR treatment of MRL mice splenocytes increases Foxo1 expression (B). Foxo1 protein production was higher in splenocytes of miR-182 -/- and miR-183C -/- mice compared with respective control (C&D). IFN production was higher in Foxo1 siRNA transfected splenocytes of miR-182 -/- and miR-183C -/- mice while IL-6 level remained unchanged(E&F). References 1. Xu S, et al. MicroRNA (miRNA) transcriptome of mouse retina and identification of a sensory organ-specific miRNA cluster. The Journal of biological chemistry. 2007; 282:25053-25066. 2. Stittrich AB, et al. The microRNA miR-182 is induced by IL-2 and promotes clonal expansion of activated helper T lymphocytes. Nature immunology.2010;11:1057-1062. 3. Pucella JN, et al. miR-182 is largely dispensable for adaptive immunity: lack of correlation between expression and function. J Immunol.2015;194:2635-2642. 4. Ichiyama K, et al. The MicroRNA-183-96-182 Cluster Promotes T Helper 17 Cell Pathogenicity by Negatively Regulating Transcription Factor Foxo1 Expression. Immunity.2016;44:1284-1298. 5. Muraleedharan CK, et al. The miR-183/96/182 Cluster Regulates Macrophage Functions in Response to Pseudomonas aeruginosa. Journal of innate immunity.2019:1-12. 6. Dai R, et al. Identification of a common lupus disease-associated microRNA expression pattern in three different murine models of lupus. PLoS ONE.2010;5:e14302. 7. Dai R, Lu R, Ahmed SA. The Upregulation of Genomic Imprinted DLK1-Dio3 miRNAs in Murine Lupus Is Associated with Global DNA Hypomethylation. PLoS One.2016;11:e0153509. • miR-183C family targets Foxo1 in mouse splenocytes. Inhibition of Foxo1 is able to increase the expression of IFN in activated splenocytes of miR- 183C -/- B6- lpr and miR-182 -/- B6- lpr mice. • Further study is required to understand the mechanism of miR-183C regulation of autoimmunity in B6- lpr lupus mice. Thanks for the support from Alliance for Lupus Research (ALR) and the Office of Research &Graduate studies (RGS) at VMCVM.

0.00

0.0

Spleen Weight(g)

miR-182 -/-

miR-182 fl/fl

miR-182 -/-

miR-182 fl/fl

Spleen/Body weight ratio

miR-182 fl/fl

28wks

20 wks

24 wks

End point

Figure 3: Depletion of miR182 has limited effect in B6-lpr mice . All the mice were euthanized at 29-32 wks old. (A)The body weight, spleen weight, spleen/body weight ratio and representative spleen images from miR-182 -/- B6- lpr and control mice. (B,C&D) The serum anti-dsDNA, total IgG and total IgM production level assessed by ELISA. Graphs show means  SEM. Unpaired t test was performed.

miR-182 fl/fl miR-182 -/- miR-183C fl/fl , miR-182 fl/fl B6-lpr mice were stimulated LPS, anti- CD3/CD28 for 24h, PMA / ionomycin for 6h. The IFN (A&C) and IL-6 (B&D) levels in supernatant were measured by ELISA. The graphs show means  SEM (n  6). Unpaired student t test was performed. Figure 4: Depletion of miR183C or miR-182 alone reduces IFN production in ex vivo stimulated splenocytes. Splenocytes from miR- 183C -/- , miR-182 -/- B6- lpr and control

IL-6

15000

10000

5000

IL-6 pg/ml

LPS

anti-CD3/CD28

PMA+ionomycin

miR-183C fl/fl

miR-183C fl/fl miR-183C -/-

miR-183C -/-

B: Con A Results

Percentage

miR-182 fl/fl miR-182 -/- fluorescent intensity was measured with ImageJ (A&C). Splenocytes were stained with different cell surface markers and analyzed by Flow Cytometry (B&D). The graphs show means  SEM (n  6). Unpaired student t test was performed. Figure 5: Conditional Depletion of miR183C or miR-182 alone reduces IgG deposition in kidney while have limited effect on immune cell development. Frozen kidney slides of miR-183C -/- , miR-182 -/- B6- lpr and control miR-183C fl/fl , miR-182 fl/fl B6-lpr mice were stained with IgG-FITC and

(% splenocytes)

A: LPS

CD8 + CD19 + DN T

CD4 +

miR-182 fl/fl

0 10 20 30 40 50 Percentage

miR-182 -/-

Figure 1: Inhibition of miR183C or miR-182 alone reduces inflammatory responses in splenocytes. The splenocytes from 14-15 wks old female MRL and MRL- lpr mice were treated with either antagomir-182 alone, or antagomir-mix3 (mix of 3 antagomirs for miR-183C miRNAs) for 24hrs, and then stimulated with LPS for 48hrs (A), Con A for 24hrs (B). The cytokine levels in supernatant were measured by ELISA. The graphs show means  SEM (n  6). Paired t test was performed.

(% splenocytes)

CD8 + CD19 + DN T

CD4 +

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