Leukocyte Beta-Catenin Expression Is Disturbed in Systemic Lupus Erythematosus

Abstract

Wnt/β-catenin signaling is relatively understudied in immunity and autoimmunity. β-catenin blocks inflammatory mediators and favors tolerogenic dendritic cell (DC) phenotypes. We show here that leukocytes from lupus-prone mice and SLE patients express diminished β-catenin transcriptional activity, particularly in myeloid cells, although other leukocytes revealed similar trends. Serum levels of DKK-1, an inhibitor under transcriptional control of Wnt/β-catenin, were also decreased in lupus-prone mice. Surprisingly, however, preemptive deletion of β-catenin from macrophages appears to have no effect on lupus development, even in mice with varying genetic loads for lupus. Although myeloid-specific loss of β-catenin does not seem to be important for lupus development, the potential role of this transcription factor in other leukocytes and renal cells remain to be elucidated.

Fig 1. B-catenin and its targets are decreased in lupus-prone Mrl-lpr mouse and human SLE splenocytes.

(A) Splenocytes from 6-month-old healthy B6 control and lupus-prone Mrl-lpr mice were analyzed by Western blot for total β-catenin, phosphorylation-inactivated (pSer33) β-catenin, negative feedback target Axin-2, and loading controls. Mrl-lpr splenocytes exhibit a loss of β-catenin (p = 0.0011) and Axin-2 (p = 0.0015) as quantified by densitometry using ImageJ. (B) Peripheral blood mononuclear cells (PBMCs) from SLE patients and healthy controls were analyzed by Western blot for total β-catenin, negative feedback target Axin-2, and loading controls. SLE PBMCs exhibit a loss of β-catenin (p = 0.0003) and Axin-2 (p<0.0001) as quantified by densitometry using ImageJ. All plotted data have been normalized against the GAPDH loading control.

Fig 2. Splenocytes from multiple, genetically diverse lupus-prone strains express lower levels of β-catenin target transcripts.

Splenocytes from 6-month and 13-month healthy B6 and lupus-prone Mrl-lpr, BWF1, and B6.Sle1.Sle3 mice were isolated and probed for Ctnnb1 and Axin2 transcripts by RT-PCR. Lupus-prone mice exhibit a loss of β-catenin-related transcription versus healthy age-matched B6 controls.

Fig 3. Serum DKK-1 is suppressed in lupus-prone mice over time.

Sera from young (6 week) and aged (6 month) B6 healthy control, B6.Sle1.Yaa lupus-prone, and BWF1 lupus-prone mice were collected and assayed by ELISA for DKK-1 and sFRP. (A) DKK-1, an inhibitor under transcriptional control of Wnt/β-catenin, did not rise over time in lupus-prone mice. (B) Serum sFRP, which by contrast is an epithelial cell-restricted Wnt/β-catenin transcription target, showed no such changes.

Fig 4. Lupus-prone CD11b+ splenocytes express reduced β-catenin.

Splenocytes were extracted from 8-month-old healthy B6, lupus-prone B6.Sle1, and lupus-prone B6.Sle1.Yaa mice and analyzed by flow cytometry for intracellular β-catenin (blue) versus isotype control staining (red). Representative plots shown. Pop3 represents total live cells. CD11b+ cell β-catenin was significantly lower in lupus-prone splenocytes versus healthy controls (by MFI ratios, p = 0.0476).

Fig 5. Lupus-prone F4/80-positive splenocytes express reduced β-catenin signaling.

Spleens from 4 month old healthy B6 and lupus-prone B6.Sle1.Sle3 mice were sectioned and stained for phosphorylated (i.e. inactivated) β-catenin protein (green) and F4/80 (blue). These inactivated β-catenin proteins co-localized with F4/80-positive splenocytes (i.e. macrophages).

Fig 6. LyzM-cre-mediated β-catenin knockout does not affect lupus pathogenesis.

B6 mice were bred to produce mice expressing the lupus-susceptibility locus Sle1, the lupus-accelerating locus Yaa (males only), and β-catenin-floxed loci in the presence or absence of the LyzM-cre locus. Mice expressing all of these loci are genetically lupus-prone and lack macrophage β-catenin expression. Mice expressing all but the LyzM-cre locus are sibling controls. Mice lacking macrophage β-catenin exhibit similar levels of anti-dsDNA IgG (A) but significantly lower levels of anti-dsDNA IgM (B). These mice did not exhibit significantly different markers of kidney function as measured by serum creatinine (C) and urine protein (D).