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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 x NZW)F1 mice. Furthermore, we observed that ERα deletion in B cells was associated with decreased B cell activation in young, pre-autoimmune (NZB x 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.
Lupus is a systemic autoimmune disease in which patients lose immunologic tolerance and produce B and T cells reactive to nuclear antigens including double stranded DNA (dsDNA). Autoreactive B cells produce autoantibodies, and a subset of these autoantibodies, particularly the anti-dsDNA autoantibodies of the IgG isotype, have the potential to form immune complexes. Immunoglobulin-containing immune complexes that are deposited in tissues cause an inflammatory immune response through activation of complement and Fcγ receptors, which results in tissue damage . Although lupus patients display abnormalities in multiple immune cell lineages, including B cells, T cells, dendritic cells and macrophages [2–9], lupus is considered to be a primarily B cell driven disease. Consistent with this premise, when lupus-prone mice are rendered unable to produce mature B cells, lupus is completely ameliorated . However, when lupus-prone mice are genetically engineered to produce mature B cells that are unable secrete antibody, but are otherwise completely functional, lupus still developed, albeit in an attenuated manner . Altogether, these data suggest that B cells contribute to lupus pathogenesis through both antibody-dependent and antibody-independent mechanisms.
Like most autoimmune diseases, lupus shows a significant sex bias; approximately 90% of lupus patients are female. Furthermore, lupus is primarily a disease that affects young and middle aged women. Female sex hormones, particularly estrogens, have long been thought to promote lupus and to be responsible for the profound sex bias in this disease. Indeed, exposure to both endogenous and exogenous estrogens is a risk factor for developing lupus [12, 13]. Cells of the immune system, including B cells, express the cellular receptors for estrogens, estrogen receptor α (ERα) and estrogen receptor β [14, 15].
Previously, we have shown that targeted disruption of the ERα gene attenuates the development of autoantibodies and lupus pathogenesis in female lupus-prone mice, whereas more modest effects are seen in male mice . These studies were conducted using the (NZB x NZW)F1 mouse model of lupus, in which the development of pathogenic dsDNA IgG autoantibodies and fatal glomerulonephritis shows a strong female sex bias. These data indicate that although estrogen signaling through ERα promotes lupus in both sexes, the ability of ERα signaling to enhance autoantibody production and lupus is more pronounced in females than in males. We have also observed that estrogens and ERα signaling are responsible for the sex bias that is seen in mice carrying the lupus susceptibility locus Sle1, which controls loss of tolerance to nuclear antigens and immune cell activation. Targeted disruption of ERα attenuates the ability of Sle1 to promote loss of tolerance, autoantibody production and B cell activation preferentially in females . However, in these studies, the cell type or types responsible for these effects could not be identified because ERα was knocked out in all cells. Although it is hypothesized that estrogens influence lupus via actions within the immune lineage, there is little concrete evidence to support this hypothesis.
A growing body of literature suggests that estrogen, acting via ERα, can exert powerful effects on B cells. For example, sustained administration of a high level of the naturally occurring estrogen 17β-estradiol (E2) allows high-affinity dsDNA-reactive B cells to escape mechanisms that maintain tolerance, including clonal deletion at the immature stage and anergy induction at the T2 stage . Furthermore, continuous exposure to high levels of E2, leads to upregulation of CD22 and SHP-1 in B cells, which likely results in decreased B cell receptor (BCR) signaling, thereby increasing the concentration of antigen required for tolerization of autoreactive B cells, and protecting autoreactive B cells from receptor-mediated apoptosis [19, 20]. Importantly, the ability of estrogens to induce CD22 and SHP-1 expression is dependent upon ERα, although it is not known which cell type or types must express ERα to elicit this effect. High levels of E2 also cause increased expression of anti-apoptotic Bcl-2 and the B cell survival factor BAFF [19, 20]. By decreasing the strength of BCR signaling and increasing the expression of pro-survival molecules, estrogens may enhance the survival of high-affinity dsDNA-reactive B cells. In addition to autoreactive B cells that arise as the result of VDJ recombination, autoreactive B cells can be generated by somatic hypermutation in the periphery. Estrogens promote somatic hypermutation by stimulating the expression of activation-induced deaminase [21, 22]. Although these data suggest that estrogens can promote the development of autoreactive B cells at multiple stages of development, it is not known to what extent these various actions are mediated by B cell-intrinsic actions of ERα.
We hypothesize that ERα signaling in B cells promotes lupus. To test this hypothesis, we have generated lupus-prone (NZB x NZW)F1 mice in which ERα was deleted specifically in the B cell compartment. To generate (NZB x NZW)F1 mice with B cell specific deletion of ERα, we used the CD19-Cre driver strain and a floxed allele of ERα. In (NZB x NZW)F1 mice of both sexes, deletion of ERα in B cells significantly attenuated autoantibody production and extended survival. However, in female mice, B cell specific deletion of ERα also significantly reduced B cell activation, suggesting that ERα may enhance lupus in females by acting in a B cell-intrinsic fashion to promote B cell activation and thereby stimulate the production of autoantibodies.
To produce NZB mice carrying the CD19-Cre knockin allele, B6.129P2(C)-Cd19 tm1(cre)Cgn/J female mice were purchased (The Jackson Laboratory, Bar Harbor, ME, USA) and crossed with NZB male mice. Genotyping for the CD19-Cre knockin allele was performed using primers that amplified the Cre gene (IMR1084 F: 5′-GCGGTCTGGCAGTAAAAACTATC-3′ and IMR1085 R: 5′-GTGAAACAGCATTGCTGTCACTT-3′). A pair of primers that amplified the IL-2 receptor gene (COO3IC F: 5′-CTAGGCCACAGAATTGAAAGATCT-3′ and COO4IC R 5′-GTAGGTGGAAATTCTAGCATCATCC-3′) were used as an internal positive control. Offspring carrying the CD19-Cre knockin allele (CD19-Cre or CD19Cre/+) were serially backcrossed to NZB mice for 5 generations using simple sequence length polymorphism (SSLP) marker assisted selection as we have described previously [16, 23]. Briefly, at each backcross generation, mice were genotyped using 111 polymorphic markers spanning the 19 murine autosomes (detailed in Table 1). PCR genotyping was performed using oligonucleotide primers specific for SSLPs between the B6 and NZB strains. Primer sequences were obtained from the Mouse Genome Informatics database (www.informatics.jax.org). By the N5 generation, an average of only 7% of SSLP markers remained heterozygous in the NZB.CD19Cre/+ mice whereas 93% were homozygous for NZB allele (data not shown). At the N5 generation, the genetic background of the incipient congenic NZB.CD19Cre/+ strain was further confirmed at the DartMouse Mouse Speed Congenic Core Facility at Dartmouth Medical School (Supplemental Figure 1). DartMouse uses the Illumina, Inc. (San Diego, CA, USA) GoldenGate Genotyping Assay to interrogate 1449 possible SNPs spread throughout the genome. The raw SNP data were analyzed using DartMouse’s SNaP-Map™ and Map-Synth™ software, to determine the SNP genotype, and thus strain of origin of SNP alleles, in each mouse. Analysis of 733 polymorphic SNPs indicated that the incipient congenic NZB.CD19Cre/+ strain was homozygous for NZB alleles at 96% of markers evaluated (Supplementary Figure 1). A considerable fraction of markers remaining heterozygous were on distal chromosome 7 and thus linked to the CD19-Cre knockin allele. All areas of heterozygosity had been previously identified by our SSLP genotyping, and none of these areas of residual heterozygosity co-localized with known lupus susceptibility loci. Based upon these analyses, the NZB.CD19-Cre strain was determined to be extensively backcrossed onto the NZB background.
Incipient congenic NZB.CD19Cre/+ mice were then crossed to congenic NZB.ERα+/− mice, which are NZB mice heterozygous for a targeted deletion of exon 2 of ERα [16, 24], thereby producing NZB mice heterozygous for both CD19-Cre and an ERα knockout allele. Genotyping for the exon 2 deletion of ERα was performed as described (www.jax.org/protocols). The common ERαEx2 F primer: 5′-TACGGCCAGTCGGGCATC-3′ (0.5 uM/rxn) and the ERαEx2wtR: 5′-GTAGAAGGCGGGAGGGCCGGTGTC-3′ (0.06 uM/rxn) or ERαEx2null R: 5′-GCTACTTCCATTTGTCACGTCC-3′ (2 uM/rxn) primers were used to produce 234 bp (intact exon 2) and ~300 bp (deleted exon 2) products, respectively.
NZW.ERαfl/fl mice, which are congenic NZW mice homozygous for an ERα allele in which exon 3 is flanked by loxP sites, were previously produced by backcrossing the floxed ERα allele from B6.ERαfl/fl mice obtained from Ken Korach  to the NZW background using marker assisted selection . Genotyping for the ERαfl allele was done with the primers N6delcK F: 5′-GACTCGCTACTGTGCCGTGTGC-3′ and N6del3 R 5′-CTTCCCTGGCATTACCACTTCTCCT-3′. The ERα+ allele produces a 275 bp product, while the ERαfl allele produces a 475 bp product . To produce experimental mice, NZB.CD19Cre/+; ERα+/− female mice were crossed with NZW.ERαfl/fl males.
All animals were housed under conditions of controlled humidity, temperature, and lighting in facilities accredited by the American Association for Accreditation of Laboratory Animal Care, operating in accordance with standards set by the Guide for the Care and Use of Laboratory Animals. Mice had ad libitum access to 7904 Teklad Irradiated S-2335 Mouse Breeder Diet (Harlan Teklad, Madison, WI, USA). All procedures involving live animals were approved by the University of Nebraska Medical Center Institutional Animal Care and Use Committee.
To evaluate the efficiency of cre-mediated ERα deletion in B cells, CD19+ cells were isolated from the bone marrow and spleen of 2–6 month old (NZB x NZW)F1 CD19Cre/+; ERαfl/−, CD19Cre/+; ERαfl/+, CD19+/+; ERαfl/+, and CD19+/+; ERαfl/− mice. Briefly, bone marrow and spleen cells were isolated in MACS buffer, passed through a 70 μm cell strainer to create a single cell suspension, and red blood cells lysed with ACK Lysing Buffer (Gibco, Waltham, MA, USA). Cells were labeled using CD19 Microbeads, and CD19+ cells were isolated using an autoMACS Pro as per manufacturer protocol (Miltenyi Biotec, Bergisch-Gladbach, Germany). After isolation, DNA was isolated from CD19+ cells using a DNeasy Kit as per manufacturer protocol for cultured cells (Qiagen, Venlo, Netherlands). Quantitative PCR was performed on DNA isolated from CD19+ cells as previously described . Briefly, a primer set was developed flanking the loxP sites around ERα Exon 3 (ERαDel F: 5′-TGGAATGAGACTTGTCTATCTTCG-3′ and ERαDel R: 5′-AACCAAGGAGAACAGAGAGACT-3′). These primers generate a 161 bp product from an ERαfl allele that has successfully undergone cre-mediated recombination, but no product is generated from the wild type or intact floxed ERα alleles. A primer set in the unaffected ERα exon 5 served as a positive control in a separate reaction (ERαEx5 F: 5′-GGAAGGCCGAAATGAAATGGG-3′ and ERαEx5 R: 5′-CCAACAAGGCACTGACCATC-3′).
Survival of animals was monitored over one year. Animals were euthanized by CO2 asphyxiation when they exhibited albuminuria of 4+ (>2000 mg/dl) or physical signs of distress. Albuminuria was initially measured monthly, and subsequently with increasing frequency after a positive measurement, using Albustix (Bayer Corporation, Elkhorn, IN, USA).
Kidneys were collected from mice upon sacrifice. Tissues were fixed in 10% formalin, processed, paraffin embedded, and sectioned. Kidney sections were stained with Periodic Acid Schiff (Sigma-Aldrich, St. Louis, MO, USA) and 100 glomeruli per kidney were evaluated as previously described . Kidney sections were stained with anti-mouse IgG to detect immune complexes and color developed with the DAB kit (Vector Laboratories, Burlingame, CA, USA). To quantify the amount of immune complex (IC) staining in glomeruli, the color density of ≤ 20 immune complex stained and unstained glomeruli was measured and averaged. A ratio of the two densities was calculated, and a higher number indicates more IC staining.
Serum was collected from mice at monthly intervals beginning at 2 months of age. Enzyme-Linked Immunosorbent Assay (ELISA) was used to measure serum antibody levels. Anti-dsDNA IgG ELISAs were done using Immulon 2 HB plates (Thermo Fisher Scientific, Waltham, MA, USA) coated with excess calf thymus dsDNA (Rockland Immunochemicals Inc, Limerick, PA, USA). Diluted serum samples were incubated on the plate, followed by anti-mouse IgG HRP conjugate, then TMB substrate, and Stop Solution (Alpha Diagnostic International, San Antonio, TX, USA) was added and plates read at 450 nm. Plates were washed with 0.05% Tween-20 in PBS. Quantitative measurements of autoantibody concentrations for experimental samples were obtained using a standard curve generated from serial dilution of a positive control that consisted of serum pooled from female (NZB × NZW)F1 mice with heavy albuminuria. Autoantibody data is expressed as arbitrary Units.
Total IgM, IgG1, IgG2a, IgG2b, and IgG3 ELISAs were done using Immulon 2 HB plates (Thermo Fisher Scientific) coated with 5 ng/ml capture antibody (Southern Biotech, Birmingham, AL, USA) and incubated overnight at 4°C. Briefly, wells were incubated in blocking solution, incubated with samples and standards, then incubated with isotype specific alkaline phosphatase-labeled antibody, after which, 1 mg/ml PNPP solution was applied for 20 minutes (Southern Biotech). Plates were read on a microplate reader at 415 nm. The concentrations of IgG1, IgG2a, IgG2b, and IgG3 were added together to calculate total IgG.
Anti-dsDNA Ig2a and IgG2b ELISAs were performed by applying standards and samples to dsDNA coated Immulon 2 HB plates (Thermo Fisher Scientific). Isotype specific alkaline phosphatase-labeled antibodies, and PNPP solution (Southern Biotech) were added and plates read as for isotype ELISAs.
To assess lymphocytes by flow cytometry, single cell suspensions were prepared and incubated with primary antibody for 30 minutes on ice. After staining for surface proteins, cells were incubated with Propidium Iodide (BD Biosciences, San Jose, CA, USA) for 10 minutes as a live/dead stain. After staining, cells were fixed with 0.6% formalin. The antibodies used were CD4-PE, CD5-PE, CD19-FITC, CD69-FITC, CD86-PE, B220-APC, CD93-BB515, CD279-APC, CXCR5-PECy7 (all BD Biosciences), IgM-FITC (Southern Biotech), IgD-APC-Cy7 (BioLegend, San Diego, CA, USA), CD21-eFlour450, and CD23-PE-Cy7 (eBioscience Inc., San Diego, CA, USA). Apoptosis was analyzed with Telford reagent. Flow cytometry was performed with a BD LSRII Flow Cytometer and analyzed with FACSDiva software (BD Biosciences, v.8.0).
Statistical analysis was done using SPSS software (IBM, v. 22). P≤0.05 was considered significant. Kaplan Meyer survival curves with log rank tests were used to evaluate survival, and T tests or ANOVA were used to analyze QPCR, ELISA, histology, and flow cytometry data.
To assess the impact of deleting ERα specifically in B cells in lupus-prone (NZB x NZW)F1 mice, NZB.CD19Cre/+; ERα+/− females were crossed with NZW.ERαfl/fl males to produce (NZB x NZW)F1 CD19Cre/+; ERαfl/−, CD19Cre/+; ERαfl/+, CD19+/+; ERαfl/+, and CD19+/+; ERαfl/− mice. In the CD19Cre/+; ERαfl/− mice, cre-mediated deletion of the floxed allele of ERα in CD19+ B cells should result in B cell specific ERα deficiency. These CD19Cre/+; ERαfl/− mice remain heterozygous for ERα in all other cell lineages. In this regard, it is important to note that we have previously demonstrated that heterozygosity for ERα does not impact the development of autoantibodies or lupus in (NZB x NZW)F1 mice .
On the 129 genetic background, the CD19-Cre knockin allele has a deletion efficiency of 75–80% in bone marrow pre-B cells and 90–95% in splenic B cells . To assess the efficiency of CD19-Cre mediated deletion of ERα on the (NZB x NZW)F1 genetic background, CD19+ cells from bone marrow and spleen were isolated from 2–6 month old mice, and DNA from these cells was analyzed for deletion efficiency. The efficiency of deletion was not different in mice at the various ages evaluated, suggesting that the relative abundance of ERα deficient B cells did not change significantly over time (data not shown). Likewise, no sex-related differences in deletion efficiency were observed (data not shown). As expected, no deletion was detected in samples from CD19+/+; ERαfl/+ or CD19+/+; ERαfl/− mice. However, to our surprise we found that in CD19Cre/+; ERαfl/+ mice there was an average deletion efficiency of only 27% in CD19+ bone marrow cells (Figure 1a). This poor deletion efficiency was not significantly different from that observed in CD19Cre/+; ERαfl/− mice, which had an average deletion efficiency of just 18%. CD19+ spleen cells from CD19Cre/+; ERαfl/+ mice had an average deletion efficiency of 53%, whereas those from CD19Cre/+; ERαfl/− mice had an average deletion efficiency of 54%. Our results affirm previous findings which showed that the efficiency of CD19-Cre mediated gene deletion is higher in splenic B cells than bone marrow B cells. However, deletion efficiency on the (NZB x NZW)F1 genetic background is significantly lower than that which has been previously reported on other strain backgrounds. Because there was no significant difference in the efficiency of cre-mediated deletion in CD19Cre/+; ERαfl/+ and CD19Cre/+; ERαfl/− mice, the low to moderate efficiency of deletion of the ERαfl allele in CD19Cre/+; ERαfl/− mice cannot be due to the resulting ERα−/− B cells being at a competitive disadvantage. Rather, we posit that the lupus-prone genetic background of (NZB x NZW)F1 mice itself is the reason that only a moderate level of ERα deletion was observed.
Cre recombinase can cleave at loxP-like sites throughout the mammalian genome, causing double strand DNA (dsDNA) breaks . dsDNA breaks are not effectively repaired in lupus patients [30, 31]. These unrepaired dsDNA breaks lead to cell death. This means that cells that express low levels of cre recombinase may have a survival advantage, and this could contribute to the decreased efficiency of cre-mediated deletion observed in (NZB x NZW)F1 mice. Our lab has previously observed inefficient Lck-Cre mediated deletion of ERα in T cells in (NZB x NZW)F1 mice, as well as an increase in T cell apoptosis caused by Lck-Cre . To determine if the CD19-Cre allele was associated with increased B cell apoptosis, we examined apoptosis in young female (NZB x NZW)F1 CD19+/+ and CD19Cre/+ mice. We found that mice carrying the CD19-Cre knockin allele had significantly more apoptotic CD19+ splenocytes than CD19+/+ mice (Figure 1b; p=0.003). However, in the bone marrow, no difference was observed in the frequency of apoptotic CD19+ cells in (NZB x NZW)F1 CD19+/+ and CD19Cre/+ mice.
Thus, on the (NZB x NZW)F1 genetic background, deletion of ERα in B cells was only moderately efficient, resulting in a B cell population in CD19Cre/+; ERαfl/− mice which was composed of a mixture of ERα+/− and ERα−/− cells. Because of the residual ERα in the B cell compartment of CD19Cre/+; ERαfl/− mice, we anticipated that these mice might show a more modest attenuation of lupus than that seen in our previous study with the global ERα knockout, in which all B cells were ERα deficient .
The CD19-Cre knockin allele by itself has the potential to impact the development of lupus in (NZB x NZW)F1 mice. Mice that carry the CD19-Cre knockin allele have significantly lower levels of CD19 expression on B cells compared to CD19+/+ littermates (representative image shown in Figure 1c). In the bone marrow of CD19-Cre mice, surface CD19 expression was ~65% of that in CD19+/+ mice, and in the spleen of CD19-Cre mice, surface CD19 expression was ~53% of that in CD19+/+ mice (Figure 1d; p=6.0x10−9, p=3.3x10−13). This decrease in CD19 on the cell membrane could impact the strength of BCR signaling and inhibit the negative selection of autoreactive B cells. The CD19-Cre knockin allele could also affect lupus through toxic off-target effects of cre recombinase expression. Debris from apoptotic cells is a source of autoantigen and accelerates the onset of lupus [32, 33]. Because of its potential to impact lupus, the effects of the CD19-Cre knockin allele alone were examined on disease development in (NZB x NZW)F1 mice.
Comparison of CD19+/+; ERαfl/+ and CD19Cre/+; ERαfl/+ (NZB x NZW)F1 mice revealed that the CD19-Cre knockin allele itself significantly accelerated mortality in female and male mice. Female CD19+/+; ERαfl/+ mice had a median survival time of 211 days, whereas CD19Cre/+; ERαfl/+ females had a median survival time of 171 days (Figure 2a; p=0.0003). The 211 day median lifespan of CD19+/+; ERαfl/+ (NZB x NZW)F1 females was similar to the 238 day median survival we have observed previously for ERα+/+ (NZB x NZW)F1 females . CD19-Cre also accelerated mortality in (NZB x NZW)F1 males. Male CD19+/+; ERαfl/+ mice had a median survival time of 301 days, whereas CD19Cre/+; ERαfl/+males had a median survival time of 257 days (Figure 2b; p=0.0002). The 301 day median survival of CD19+/+; ERαfl/+ (NZB x NZW)F1 males is similar to the 321 day median survival we observed previously in ERα+/+ (NZB x NZW)F1 males . Although we are unable to determine if the accelerated mortality in CD19-Cre (NZB x NZW)F1 mice is an effect of cre recombinase or reduced CD19 expression, it is clear that the CD19-Cre knockin allele significantly accelerated mortality in (NZB x NZW)F1 mice. Therefore, the development of lupus in mice with B cell specific deletion of ERα must be compared to control mice also carrying the CD19-Cre knockin allele.
Our lab has previously shown that global deletion of ERα attenuates the development of lupus and extends survival in both female and male (NZB x NZW)F1 mice . To determine the effect of B cell specific deletion of ERα (albeit incomplete deletion) on survival in lupus-prone mice, CD19Cre/+; ERαfl/+ and CD19Cre/+; ERαfl/− (NZB x NZW)F1 mice were monitored for up to one year. Female CD19Cre/+; ERαfl/+ control mice had a median survival time of 171 days, which was significantly shorter than that observed in CD19Cre/+; ERαfl/− mice, in which the median survival time was 239 days (Figure 2c; p=0.001). Although this difference in lifespan was statistically significant, the 68 day increase in median survival observed in female mice with ERα deletion in B cells was certainly less dramatic than the > 200 day difference in survival observed between ERα−/− and ER+/− female mice in our previous study using the global ERα knockout . This moderate extension of median lifespan in the CD19Cre/+; ERαfl/− mice was likely due, at least in part, to the fact that a significant proportion of B cells in CD19Cre/+; ERαfl/− mice retain ERα expression. It is also possible that ERα signaling in other cell types continued to promote lupus in these mice.
Male mice with B cell specific ERα deletion also experienced significantly delayed mortality. Male CD19Cre/+; ERαfl/+ control mice had a median lifespan of 257 days, which is significantly shorter that of CD19Cre/+; ERαfl/− males which had a median lifespan of 329 days (Figure 2d; p=0.050). The 72 day difference in the median survival of male mice in this study was similar to the 85 day difference in median survival observed between ERα+/− and ERα−/− male (NZB x NZW)F1 mice in our previous study using the global ERα knockout . Thus, deletion of ERα from a moderate percentage of B cells results in attenuated mortality in male as well as female (NZB x NZW)F1 mice.
The primary cause of mortality in (NZB x NZW)F1 mice is severe glomerulonephritis. To assess the impact of CD19-Cre and B cell specific deletion of ERα on the development of glomerulonephritis, kidneys were collected from mice at the time of sacrifice, and Periodic Acid Schiff stained sections were analyzed to determine the extent of glomerular damage. (NZB x NZW)F1 mice with the CD19-Cre knockin allele developed glomerulonephritis that was histologically indistinguishable from CD19+/+ mice (data not shown). This indicates that mice with the CD19-Cre knockin allele died from lupus-related causes, not because of a separate CD19-Cre related effect. In kidneys of almost all female and male CD19Cre/+; ERαfl/+ and CD19Cre/+; ERαfl/− mice, ≥ 50% of glomeruli showed evidence of wire loop lesions, matrix deposition, and/or hypercellularity, which is indicative of severe glomerulonephritis (Figure 3a). Thus, although deletion of ERα in approximately half of the B cells in CD19Cre/+; ERαfl/− mice was sufficient to extend the lifespan of these mice, the ultimate development of severe nephritis was not prevented (Figure 3b–e). Nevertheless, CD19Cre/+; ERαfl/− mice did reach this stage of disease at a more advanced age than their CD19Cre/+; ERαfl/+ counterparts. The impact of B cell specific deletion of ERα on renal immune complex deposition was also evaluated. Interestingly, we found that the abundance of glomerular immune complexes in CD19Cre/+; ERαfl/− female mice was less than that in CD19Cre/+; ERαfl/+ female mice, but this difference fell short of statistical significance (Figure 3f, p=0.12). B cell specific deletion of ERα had no impact on renal immune complex deposition in males. Therefore, although mice with B cell specific deletion of ERα survive significantly longer than control mice, both groups of mice ultimately developed immune complex mediated glomerulonephritis.
The production of high levels of anti-dsDNA autoantibodies is highly specific to lupus . In lupus patients, dsDNA autoantibodies correlate with disease activity and disease-associated damage [35, 36]. In (NZB x NZW)F1 mice, the levels of anti-dsDNA IgG antibodies in the serum increase significantly as mice age and lupus progresses . The production of pathogenic autoantibodies is promoted by ERα [16, 38]. To determine the impact of B cell specific deletion of ERα on autoantibody production, serum anti-dsDNA IgG levels were measured by ELISA. Serum levels of anti-dsDNA IgG were significantly higher in control female CD19Cre/+; ERαfl/+ mice at 5 months of age compared to female CD19Cre/+;ERαfl/− mice (Figure 4a; p=0.03). Similarly, male CD19Cre/+; ERαfl/+ control mice had significantly higher levels of anti-dsDNA IgG antibodies compared to CD19Cre/+; ERαfl/− males at 7 months of age (Figure 4b; p=0.02). The lower levels of pathogenic autoantibodies in mice with B cell specific deletion of ERα is consistent with the attenuated development of lupus and extended lifespan in CD19Cre/+; ERαfl/− mice. Furthermore, these observations are consistent with the hypothesis that ERα promotes lupus by stimulating the development of pathogenic autoantibodies in a B cell-intrinsic manner.
Different isotypes of anti-dsDNA IgG antibodies have different pathogenic potentials; dsDNA-reactive IgG2a antibodies are more likely to bind to laminin and collagen, two proteins found abundantly in the kidney, resulting in the in situ formation of immune complexes in the kidney and glomerulonephritis . Both IgG2a and IgG2b autoantibodies are particularly pathogenic because they activate the immune system through both complement and Fcγ receptors . Additionally, global knockout of ERα in (NZB x NZW)F1 females caused a significant decrease in anti-dsDNA IgG2a and anti-dsDNA IgG2b . To determine if these subclasses of anti-dsDNA IgG autoantibodies were affected by B cell-intrinsic actions of ERα, serum levels of anti-dsDNA IgG2a and anti-dsDNA IgG2b were measured by ELISA at 4 months of age in females and 7 months of age in males. Compared to age and sex matched CD19Cre/+; ERαfl/+ controls, the levels of anti-dsDNA IgG2a were significantly lower in both female and male CD19Cre/+; ERαfl/− mice (Figure 4c; p=0.04, p=0.02). Additionally, compared to CD19Cre/+; ERαfl/+ controls, male CD19Cre/+; ERαfl/−mice had lower serum levels of anti-dsDNA IgG2b antibodies (Figure 4d; p=0.002). However, B cell specific ERα deficiency had no impact on anti-dsDNA IgG2b levels in female mice (Figure 4d).
To evaluate the effect of B cell specific deletion of ERα on total antibody production, and to determine if the significant reduction in anti-dsDNA IgG autoantibodies in mice with B cell specific deletion of ERα was due to global changes in antibody production, we measured the total serum IgM, IgG1, IgG2a, IgG2b, and IgG3 levels by ELISA. Female CD19Cre/+; ERαfl/+ mice produced significantly more IgG3 than female CD19Cre/+; ERαfl/− mice (Figure 5a; p=0.005). No differences were observed in the level of IgM, total IgG, or any other IgG isotype in female CD19Cre/+; ERαfl/+ and CD19Cre/+; ERαfl/− mice (Figure 5a). In male mice, there were no genotype dependent differences in the production of IgM, total IgG, or any IgG isotype (Figure 5b). Therefore, the lower levels of anti-dsDNA IgG autoantibodies produced by mice with B cell specific deletion of ERα were not due to overall suppression of antibody production, but were specific to anti-dsDNA autoantibodies. Thus, the attenuation of lupus in mice with B cell specific deletion of ERα is likely due to the reduction in anti-dsDNA IgG autoantibodies in these mice and not the consequence of systemic immunosuppression.
Estrogens can potentially affect the development of B cells in the bone marrow. Specifically, it has been shown that E2 causes a reduction in the number of developing bone marrow B cells on a mixed C57BL/6/129 genetic background [40, 41]. There is some discrepancy in the literature regarding the impact of ERα knockout on B cell populations in the bone marrow; however, there is consensus that the effects of E2 on B cell populations are mainly mediated by signaling through ERα [40, 41]. It is not known if the effects of estrogens on B cell development in the bone marrow are B cell-intrinsic.
To evaluate the B cell-intrinsic effects of ERα on B cell populations in the bone marrow, cells from 3 month old pre-autoimmune (NZB x NZW)F1 mice were analyzed by flow cytometry. In female but not male mice, the CD19-Cre knockin allele was associated with a decrease in the percentage of pre-B cells and an increase in the percentage of immature B cells (Table 2). However, there were no changes in developing B cells in the bone marrow or spleen that could be attributed to ERα deletion in B cells in either female or male mice. These results indicate that in pre-autoimmune (NZB x NZW)F1 mice, deletion of ERα in B cells does not impact the relative abundance of bone marrow B cell populations.
The effect of global ERα knockout on B cell populations in the spleen has not previously been reported. We evaluated follicular and marginal zone B cell populations in 3 month old ERα−/− and ERα+/+ (NZB x NZW)F1s to provide a comparison for mice with B cell specific ERα deletion. The follicular B cell population was not affected by ERα genotype (Table 3). However, the population of marginal zone B cells was significantly increased in ERα−/− female mice (Table 3; p=0.00004). This was somewhat unexpected because the marginal zone B cell population is enriched for autoreactive B cells compared to follicular zone B cells, and ERα−/− female mice produce less autoantibodies than ERα+/+ mice [16, 42]. However, the marginal zone B cell population also increases significantly in high-estrogen conditions or upon treatment with an ERα specific agonist [18, 20. Female ERα−/− mice have significantly elevated serum E2 , and thus this may be the reason for the increase in marginal zone B cells in these mice.
The same analysis of splenic immune cell populations was performed in 3 month old (NZB x NZW)F1 CD19-Cre mice. The relative abundance of follicular and marginal zone B cells were similar in ERα+/+ and CD19+/+; ERαfl/+ mice indicating that the floxed allele of ERα had no impact on these populations. ERα deletion in B cells did not have a significant impact on the follicular or marginal zone B cell populations (Table 4). However, the CD19-Cre knockin allele did significantly impact these populations. The CD19-Cre knockin allele caused a significant increase in the proportion of follicular B cells and a significant decrease in the proportion of marginal zone B cells (Table 4; females p=0.002, 0.02, males p=0.08, p=0.0009, respectively). Several other lymphocyte populations were examined in the spleen; no significant differences were observed in transitional B cell or T follicular helper cell populations (Table 4). These results suggest that CD19-Cre reduces the relative abundance of marginal zone B cells in the spleen, but that deletion of ERα in B cells does not affect the relative abundance of splenic B cell populations. Thus, the attenuated lupus development in CD19Cre/+; ERαfl/− mice is not the result of a shift in the relative abundance of splenic B cell subsets.
Our previous studies suggest that ERα promotes lupus by enhancing immune cell activation . Therefore, the relative abundance of activated B and T cells in the spleen was also examined in these same groups of young (NZB x NZW)F1 mice. Female CD19Cre/+; ERαfl/+ mice had significantly more activated B cells than CD19Cre/+; ERαfl/− mice (Table 5; p=0.006). Interestingly, no such difference was observed in male mice. B cell specific deletion of ERα had no impact on the proportion of T cells that were activated in mice of either sex. This result suggests that ERα promotes B cell activation in female lupus-prone mice in a B cell-intrinsic manner. Furthermore, the enhanced B cell activation in young, pre-autoimmune CD19Cre/+; ERαfl/+ female mice suggests that the ability of ERα to promote B cell activation may underlie the ability of ERα to promote lupus.
To study the role of B cell-intrinsic actions of ERα on the development of lupus, we created (NZB x NZW)F1 mice with ERα deletion specifically in B cells. This was accomplished using an allele of ERα in which one exon is flanked by loxP sites in conjunction and the CD19-Cre knockin allele, which results in expression of cre recombinase in CD19+ B cells and thus B cell specific deletion of ERα. The cre-loxP system is widely used in mammalian systems to cause targeted deletion of DNA segments from the genome. Although this system is potentially very useful, experiments using the cre-loxP system can be complicated by the off target effects of cre recombinase [29, 44, 45].
In the present study, we found that the CD19-Cre knockin allele by itself significantly accelerated lupus development in (NZB x NZW)F1 mice. Cre recombinase can cleave endogenous pseudo-loxP sites that are found throughout the mouse genome, causing severe DNA damage [46, 47]. The accelerated development of lupus in CD19-Cre (NZB x NZW)F1 mice may be due, at least in part, to cre-induced DNA damage, leading to increased B cell apoptosis and increased nuclear antigen load. It is known that lupus-prone (NZB × NZW)F1 mice have impaired clearance of apoptotic debris [48, 49]. Thus, in young (NZB × NZW)F1 mice, enhanced B cell apoptosis would likely lead to the accumulation of apoptotic debris and an increase in the nuclear antigen load, which would, in turn, promote the earlier development of autoantibodies and thereby accelerate the development of lupus. Indeed, a recent study from our lab showed that the Lck-Cre allele significantly accelerated lupus in (NZB x NZW)F1 mice by causing increased apoptosis . In the present study, we observed a small but significant increase in apoptotic CD19+ splenocytes in CD19-Cre mice, and this increase could be sufficient to accelerate lupus. However, the CD19-Cre knockin allele not only causes cre recombinase expression in B cells, but because it is a knockin allele, it also interrupts the coding region of CD19, resulting in a null allele of CD19. To our knowledge, the effects of CD19 heterozygosity on B cell development, B cell function, and lupus pathogenesis have not been reported. It is known that homozygosity for the CD19 knockout does not impact the development of conventional B2 cells; however, CD19 deficiency does decrease the B cell response to antigens requiring T cell help . In (NZB x NZW)F1 mice, homozygosity for the CD19 knockout allele does accelerates the development of lupus . Thus, in our (NZB x NZW)F1 CD19Cre/+ mice, the accelerated development of lupus could be attributable to the decreased expression of CD19 and/or the expression of cre recombinase.
We also observed that the CD19-Cre allele modulated the relative abundance of B cell subsets in (NZB x NZW)F1 mice. In females, the CD19-Cre knockin allele lead to a decrease in pre-B cells and an increase in immature B cells in the bone marrow. In the spleen, we found that the CD19-Cre knockin allele increased the proportion of follicular B cells and decreased the proportion of marginal zone B cells in the spleen of both female and male (NZB x NZW)F1 mice. Because of these immunologic effects and the fact that the CD19-Cre allele accelerated the lupus phenotype in (NZB x NZW)F1 mice, it was imperative to use CD19Cre/+; ERαfl/+ mice as controls for the analysis of the CD19Cre/+; ERαfl/− mice that were generated to examine the impact of B cell specific deletion of ERα.
On the 129 genetic background, the CD19-Cre allele results in gene deletion in ~75–80% of bone marrow B cells and 90–95% of spleen B cells . However, in our study, the observed efficiency of CD19-Cre mediated deletion on the (NZB x NZW)F1 genetic background was much lower. Only ~20% of CD19+ bone marrow cells successfully deleted ERα whereas ~50% of CD19+ B cells deleted ERα. These results indicate that the B cells in CD19Cre/+; ERαfl/− mice were a mixed population of ERα+/− and ERα−/− cells. Therefore, the effects that we have observed in these studies are the result of loss of ERα signaling in only a portion of the B cell population.
Despite the fact that only ~50% of B cells successfully deleted ERα in CD19Cre/+; ERαfl/− (NZB x NZW)F1 mice, this reduction in ERα was sufficient to significantly attenuate lupus in these mice. Female and male CD19Cre/+; ERαfl/− mice survived significantly longer than their CD19Cre/+; ERαfl/+ counterparts. In terms of survival, the impact of deleting ERα in a fraction of B cells in male (NZB x NZW)F1 mice was roughly equivalent to the impact of global deletion of ERα deletion seen in our previous study . By contrast, in female (NZB x NZW)F1 mice, the increased survival in mice with ERα deletion in a fraction of B cells was not as dramatic as the increase in survival we have seen previously in mice with global deletion of ERα . There are several possible explanations for the observation that the impact of B cell specific ERα deletion on survival in (NZB XNZW)F1 female mice was less than that seen with global ERα deletion. One of the major factors likely to contribute to this difference is the fact that in CD19Cre/+; ERαfl/− mice, many, perhaps often the majority of B cells do not delete ERα; in these mice the efficiency of B cell specific deletion of ERα barely exceeds 50%. Thus, we postulate that the delay in the onset of lupus in CD19Cre/+; ERαfl/− female mice would have been greater if there had been deletion of ERα in a larger proportion of B cells. We also note that in female mice with a global ERα knockout, the hypothalamic-pituitary-gonadal axis is disrupted resulting in significant alterations in the serum levels of multiple hormones, including testosterone and prolactin, which could attenuate the development of lupus in ERα deficient (NZB x NZW)F1 females independently of the effects of ERα. Thus, it is possible that the more modest impact of B cell specific deletion of ERα on lupus development in (NZB x NZW)F1 females is a reflection of the fact that we have avoided disruption of the hypothalamic-pituitary-gonadal axis by using the cre-loxP system. Finally, in addition to B cells, other immune cells such as dendritic cells and T cells, which also express ERα, are also known to contribute to lupus. Thus, our observation that B cell specific deletion of ERα attenuates lupus in females to a lesser degree than global deletion of ERα may reflect the fact that ERα signaling in other immune cells also contributes significantly to lupus in females.
Higher levels of anti-dsDNA IgG autoantibodies are associated with increased disease severity in lupus-prone mice. In both female and male (NZB x NZW)F1 mice with B cell specific deletion of ERα, we observed significantly lower levels of pathogenic anti-dsDNA IgG antibodies as compared to sex and age matched control mice. Some isotypes of anti-dsDNA antibodies are particularly pathogenic, especially the IgG2a and IgG2b isotypes which cause immune activation through multiple pathways . CD19Cre/+; ERαfl/+ mice of both sexes had higher levels of anti-dsDNA IgG2a autoantibodies than sex and age matched CD19Cre/+; ERαfl/− mice. CD19Cre/+; ERαfl/+ male mice also had higher levels of anti-dsDNA IgG2b autoantibodies. By contrast, total serum levels of IgG and IgM antibodies were not different between CD19Cre/+; ERαfl/− and CD19Cre/+; ERαfl/+ mice, clearly indicating that B cell specific deletion of ERα has no impact on overall IgG levels. Altogether, these data support the idea that ERα signaling specifically promotes pathogenic autoantibody production in a B cell-intrinsic manner.
Global deletion of ERα has not been reported to affect the relative abundance of B cell populations in the bone marrow of non-autoimmune mice . However, there are no published reports in which the impact of global ERα deficiency on splenic B cell populations has been examined. Interestingly, in female (NZB x NZW)F1 mice homozygous for a global ERα knockout, we found that the marginal zone B cell population was significantly increased. By contrast, B cell specific deletion of ERα has no effect on the relative abundance of various subsets of splenic B cells. One possible explanation for this difference could be the relatively inefficient deletion of ERα in splenic B cells in (NZB x NZW)F1 mice. Alternatively, it is possible that the expansion of the marginal zone B cell compartment in female (NZB x NZW)F1 mice homozygous for the global ERα knockout allele could be the result of disruption of the hypothalamic-pituitary-gonadal axis, and the resulting high levels of serum estradiol in these mice. Indeed, an expansion of the marginal zone B cell compartment is also seen in transgenic mice expressing a DNA reactive heavy chain following continuous treatment with high levels of E2 [20, 42].
The deletion of ERα in B cells in (NZB x NZW)F1 mice did not affect T cell activation in the spleen. These observations suggest that ERα deficiency in B cells does not impair the ability of B cells to present antigen to T cells and thereby activate T cells. By contrast, ERα deletion in B cells did result in a significant decrease in the proportion of activated splenic B cells in female mice specifically. These observations are consistent with our previous finding that ERα deficiency attenuates B cell activation in female, but not male, mice carrying the lupus susceptibility locus Sle1 . However, the data presented here reveal that ERα acts in a B cell-intrinsic manner to promote B cell activation. Furthermore, this effect is seen in young, pre-autoimmune mice, which indicates that the ability of ERα to enhance B cell activation likely contributes to its ability to promote autoantibody production and the pathogenesis of lupus in females. Interestingly, ERα deficiency in B cells did not impact B cell activation in age matched male mice, suggesting that the enhanced B cell activation phenotype seen in female lupus-prone mice is dependent upon B cell-intrinsic actions of ERα. However, it is possible that ERα signaling might enhance B cell activation in older, pre-autoimmune male mice.
In conclusion, these studies show that loss of ERα signaling in a moderate proportion of B cells significantly attenuates lupus in (NZB x NZW)F1 mice. ERα deletion in B cells significantly decreased the production of autoantibodies, and prolonged survival in both female and male mice. These data indicate that ERα signaling in B cells specifically promotes lupus in both females and males. Of particular note, we observed a decrease in the proportion of activated B cells in young, pre-autoimmune female mice with B cell specific deletion of ERα, whereas no such effect was seen in males. These data indicate that ERα acts in a B cell-intrinsic manner to promote B cell activation specifically in female mice. The ability of ERα to stimulate B cell activation in females likely contributes to the enhanced development of autoantibodies and more robust lupus observed in female (NZB x NZW)F1 mice compared to male mice.
This work was supported by the National Institutes of Health R01 AI075167 (KAG) and a pre-doctoral research assistantship awarded by the University of Nebraska Medical Center (DET). We thank Kimberly Bynoté for performing the isotype ELISAs. We also thank Jenny Nuxoll for conducting genotyping during backcrossing. We gratefully acknowledge Victoria Smith, Samantha Wall, and Dr. Philip Hexley of the University of Nebraska Medical Center Flow Cytometry Research Facility, who contributed to the acquisition and analysis of flow cytometric data. The UNMC Flow Cytometry Research Facility is administrated through the Office of the Vice Chancellor for Research and supported by state funds from the Nebraska Research Initiative (NRI) and The Fred and Pamela Buffett Cancer Center’s National Cancer Institute Cancer Support Grant. Major instrumentation in this facility has been provided by the Office of the Vice Chancellor for Research, The University of Nebraska Foundation, the Nebraska Banker’s Fund, and by the NIH-NCRR Shared Instrument Program
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