Xist Deletion in B Cells Results in Systemic Lupus Erythematosus Phenotypes

Claudia Lovell¹, Nikhil Jiwrajka², Hayley Amerman³, Michael Cancro⁴ and Montserrat Anguera⁵, ¹Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, ²Hospital of the University of Pennsylvania, Philadelphia, PA, ³University of Pennsylvania School of Veterinary Medicine, Philadelphia, PA, ⁴Perelman School of Medicine, Philadelphia, PA, ⁵University of Pennsylvania, Philadelphia, PA

Meeting: ACR Convergence 2024

Keywords: Autoantibody(ies), B-Lymphocyte, Epigenetics, lupus-like disease, Non-coding RNA

Session Information

Date: Monday, November 18, 2024

Title: Abstracts: SLE – Etiology & Pathogenesis

Session Type: Abstract Session

Session Time: 3:00PM-4:30PM

Background/Purpose: Systemic lupus erythematosus (SLE) exhibits a strong female sex bias but the mechanisms underlying this bias are not well understood. Epidemiological studies demonstrate that SLE risk increases with the number of X chromosomes carried by an individual, suggesting important genetic contributions from the X chromosome. To ensure dosage compensation of X-linked genes relative to XY males, mammals with multiple X chromosomes utilize X-chromosome inactivation (XCI), a multi-layered epigenetic mechanism that is initiated and maintained by the long non-coding RNA Xist, which is expressed from and coats the inactive X chromosome. The X chromosome is enriched for genes with immunomodulatory functions that are relevant to B cell function. B cells from female SLE patients and mouse models of SLE exhibit mislocalization of Xist RNA and aberrant expression of X-linked genes, suggesting that impairment of XCI may contribute to disease. We sought to determine whether perturbed XCI maintenance in the B cell compartment contributes to SLE pathogenesis.

Methods: We generated female BALB/c mice with a B cell specific deletion of Xist (mb1^WT/creXist^fl/fl, “Xist cKO”). We measured serum levels of anti-dsDNA autoantibodies in a cohort of WT (n=109) and Xist cKO (n=66) mice. In a cohort of Xist cKO mice with high anti-dsDNA (n=3 “Xist cKO High”), Xist cKO mice with low anti-dsDNA (n=4), and WT controls (n=7) we measured sera for various autoantibodies, quantified splenic B cell subsets, and assessed kidney pathology. We also injected a cohort of WT (n=46) and Xist cKO mice (n=36) with pristane to induce SLE-like disease and measured anti-dsDNA autoantibodies, kidney pathology, and splenic B cells during disease. We performed RNAseq on activated B cells from Xist cKO mice with spontaneous and pristane-induced SLE.

Results: Some Xist cKO mice spontaneously developed elevated anti-dsDNA, anti-Smith U1-RNP, anti-MDA5, anti-PM/Scl-100, and anti-Ro/SS-A, as well as glomerular kidney pathology. These Xist cKO mice also had more activated B cell subsets including GL7+ activated B cells, class-switched B cells, age-associated B cells (ABCs), and plasma cells. Pristane-treated Xist cKO mice developed higher levels of anti-dsDNA, had slightly more severe glomerular kidney pathology, and had more activated B cell subsets compared to pristane-treated WT mice. Activated B cells from Xist cKO mice with spontaneous and pristane-induced SLE demonstrated upregulation of the X-linked TLR7 pathway member Tasl.

Conclusion: Our data demonstrate that perturbed XCI maintenance via Xist deletion in the B cell compartment results in the upregulation of X-linked Tasl, a contributor to abnormal immune signaling in SLE, along with activated B cell subset expansion, production of disease-specific autoantibodies, and glomerulonephritis. These findings suggest that disruption of XCI maintenance in B cells can both spontaneously confer and exacerbate SLE-like disease, thereby providing a novel pathogenic mechanism that simultaneously accounts for the strong female sex bias of SLE.

A. Breeding schematic used to generate mice with a B cell-specific Xist deletion. Xist 2lox mice with loxP sites flanking the first 3 exons of Xist were successively bred to mb1-cre mice for deletion in the B cell lineage staring with early proB cells, to generate mice with a homozygous Xist deletion (“Xist cKO/cKO”) or a heterozygous Xist deletion (“Xist +/cKO”) in the B cell compartment. B. Serum levels of anti-dsDNA autoantibodies from female BALB/c mice 12 months or older (n=39 female WT mice, n=27 female Xist cKO/cKO mice) measured by enzyme-linked immunosorbent assay (ELISA). Each circle represents an individual mouse. Grey bar bounded by dotted lines represents serum levels of anti-dsDNA in NZB/W (n=8) mice that spontaneously develop SLE-like disease. C. Mean serum anti-dsDNA autoantibody levels ± standard deviation (SD) in female Xist cKO/cKO mice with elevated anti-dsDNA autoantibodies (“Xist cKO High”, n=3), age-matched female Xist cKO/cKO mice with low anti-dsDNA autoantibodies (“Xist cKO Low”, n=4), and age-matched female WT controls (“WT”, n=7) as measured by ELISA. D. Mean number of anti-dsDNA producing cells per 1,000,000 splenocytes ± SD as measured by ELISPOT. E. Mean serum anti-Smith RNP autoantibody levels ± SD as measured by ELISA. F. Heatmap depicting individual sample-level data for those autoantibodies significantly enriched in Xist cKO High mice compared to WT mice or Xist cKO Low mice, as measured by an autoantigen microarray. Bold autoantibodies in black are either specific for or associated with non-SLE female biased autoimmune diseases. G. Mean total glomerular pathology ± SD. H. Mean total tubular pathology ± SD. I. Mean interstitial inflammation ± SD. J. Mean scores ± SD for individual features of glomerular pathology. K. Representative image of glomeruli from a WT mouse. L. Representative image of glomeruli from Xist cKO High mice. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 via ordinary one-way ANOVA with Tukey’s multiple comparisons test.

A-D. Cells were pre-gated on live single cells that are CD4-CD8-Gr_1-F4/80-. Quantification of splenic (A) GL7+ activated B cells (B220+CD19+IgM-IgD-CD138-GL7+), (B) class-switched B cells (B220+CD19+IgM-IgD-), (C) short-lived plasma cells (SLPCs; IgD-CD138+B220+) and long-lived plasma cells (LLPCs; IgD-CD138+B220-), and (D) age-associated B cells (ABCs; B220+CD19+CD11c+). Data are depicted as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 via ordinary one-way ANOVA with Tukey’s multiple comparisons test. E. Volcano plot depicting X-linked genes that are significantly differentially expressed (purple) between Xist cKO High and WT CD11c+ ABCs. F. Heatmap of differentially expressed X-linked genes between Xist cKO High and WT CD11c+ ABCs.

A. Mean serum anti-dsDNA autoantibody levels ± SEM in pristane-treated Xist cKO mice (n=32), pristane-treated WT mice (n=41), and PBS-treated WT mice (n=10) over 7 months. p < 0.05 by mixed effects model with the Geisser-Greenhouse correction (not assuming equal variability of differences). B. Mean serum anti-dsDNA autoantibody levels ± SD in pristane-treated Xist cKO mice (n=12), pristane-treated WT mice (n=18), and PBS-treated WT mice (n=3) at 9 months post-pristane injection. C. Mean total glomerular pathology score + SD. D-E. Cells were pre-gated on live, single CD4-CD8-Gr_1-F4/80- cells. Quantification of splenic (D) age-associated B cells (ABCs; B220+CD19+CD11c+) and (E) GL7+ activated B cells (B220+CD19+IgM-IgD-CD138-GL7+) after 4, 7, or 9 months of pristane. Data are depicted as mean + SD. * p < 0.05, ** p < 0.01, *** p < 0.001 via pairwise Mann-Whitney U test comparing pristane-treated Xist cKO to pristane-treated WT mice. F. Volcano plot depicting X-linked genes that are significantly differentially expressed (pink) in CD11c+ ABCs from pristane-treated Xist cKO vs. pristane-treated WT mice. G. Heatmap of differentially expressed X-linked genes in CD11c+ ABCs from pristane-treated mice. H. Volcano plot depicting X-linked genes that are significantly differentially expressed (pink) in GL7+ activated B cells from pristane-treated Xist cKO vs. pristane-treated WT mice. I. Heatmap of differentially expressed X-linked genes in GL7+ B cells from pristane-treated mice.

Disclosures: C. Lovell: None; N. Jiwrajka: None; H. Amerman: None; M. Cancro: None; M. Anguera: None.

To cite this abstract in AMA style:

Lovell C, Jiwrajka N, Amerman H, Cancro M, Anguera M. Xist Deletion in B Cells Results in Systemic Lupus Erythematosus Phenotypes [abstract]. Arthritis Rheumatol. 2024; 76 (suppl 9). https://acrabstracts.org/abstract/xist-deletion-in-b-cells-results-in-systemic-lupus-erythematosus-phenotypes/. Accessed .

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