Estrogen receptor alpha promotes lupus in (NZB×NZW)F1 mice in a B cell intrinsic manner

Abstract

Lupus is a systemic autoimmune disease characterized by the production of autoreactive antibodies against nuclear antigens. Women are disproportionately affected by lupus, and this sex bias is thought to be due, in large part, to the ability of estrogens to promote lupus pathogenesis. Previously, we have shown that global deletion of estrogen receptor alpha (ERα) significantly attenuated loss of tolerance, immune cell activation, autoantibody production, and the development of lupus nephritis. Here we show that targeted deletion of ERα specifically in B cells retards production of pathogenic autoantibodies and the development of nephritis in lupus-prone (NZB×NZW)F1 mice. Furthermore, we observed that ERα deletion in B cells was associated with decreased B cell activation in young, pre-autoimmune (NZB×NZW)F1 females. Altogether, these data suggest that ERα acts in a B cell-intrinsic manner to control B cell activation, autoantibody production, and lupus nephritis.

Keywords: B cell; Estrogen receptor alpha; Immune cell activation; Immunologic tolerance; Lupus.

Figure 1. CD19-Cre increased apoptosis and decreased CD19 expression in (NZB x NZW)F1 mice

The efficiency of ERα deletion in B cells was measured in (NZB x NZW)F1 mice. (a) There was moderate ERα deletion in CD19⁺ bone marrow and spleen cells in both CD19^Cre/+; ERα^fl/+ and CD19^Cre/+; ERα^fl/− mice (bone marrow n= 8–15; spleen n= 8–16). (b) The frequency of apoptotic cells in CD19⁺ splenocytes was increased in female mice carrying the CD19-Cre knockin allele (p=0.003, bone marrow and spleen n= 5–6). (c) The fluorescence intensity of CD19 staining on B cells was lower in mice carrying the CD19-Cre knockin allele. (d) Mean fluorescence intensity of CD19 staining was consistently and significantly reduced in both bone marrow and spleen B cells of CD19-Cre mice (bone marrow p=6.0x10⁻⁹, spleen p=3.3x10⁻¹³; bone marrow and spleen n= 9–13).

Figure 2. Both the CD19-Cre knockin allele and B cell specific deletion of ERα impacted the survival of (NZB x NZW)F1 mice

Survival was monitored for up to one year. (a) Female and (b) male CD19^+/+; ERα^fl/+ mice had significantly longer median survival compared to CD19^Cre/+; ERα^fl/+ mice (females p=0.0003, n= 62–73 ; males p=0.0002, n= 51–63). Compared to CD19^Cre/+; ERα^fl/+ control mice, CD19^Cre/+; ERα^fl/− (c) female and (d) male mice had significantly longer median survival (females p=0.001, n= 22–73; males p=0.050, n= 25–63).

Figure 3. Deletion of ERα from B cells did not prevent the development of glomerulonephritis

(a) The majority of female and male CD19^Cre/+; ERα^fl/+ and CD19^Cre/+; ERα^fl/− mice developed severe glomerulonephritis by the end of the survival study (females n= 9–17; males n= 7–11). Representative histological sections from (b) female CD19^Cre/+; ERα^fl/+, (c) female CD19^Cre/+; ERα^fl/−, (d) male CD19^Cre/+; ERα^fl/+, and (e) male CD19^Cre/+; ERα^fl/− mice showed abnormalities characteristic of glomerulonephritis. (f) Semi-quantitative analysis of immune complex staining showed immune complex deposition occurred equally in CD19^Cre/+; ERα^fl/+ and CD19^Cre/+; ERα^fl/− mice of both sexes (females p=0.12, n=8–16; males n= 8–12).

Figure 4. ERα deletion in B cells reduced levels of pathogenic autoantibodies

(a) Female and (b) male CD19^Cre/+; ERα^fl/+ mice produced significantly more anti-dsDNA IgG than CD19^Cre/+; ERα^fl/− mice (females p=0.03, n=7–11; males p=0.02, n= 5–10). (c) More anti-dsDNA IgG_2a was produced by both female and male CD19^Cre/+; ERα^fl/+ mice than CD19^Cre/+; ERα^fl/− mice (females p=0.04, n= 7–13; males p=0.02, n= 5–12). (d) Male CD19^Cre/+; ERα^fl/+ mice produced more anti-dsDNA IgG_2b than CD19^Cre/+; ERα^fl/− males (females n= 7–13; males p=0.002, n= 5–12).

Figure 5. B cell specifc deletion of ERα did not impact total IgM or IgG

There was no difference in total IgM, total IgG, or IgG₁, IgG_2a, or IgG_2b in (a) female and (b) male mice (females n= 7–15; males n=6–13). Female CD19^Cre/+; ERα^fl/+ mice produced more total IgG₃ than CD19^Cre/+; ERα^fl/− controls (p=0.005).