Almost a decade has passed since the approval of belimumab, an mAb directed against B lymphocyte stimulation and the first targeted therapy approved for systemic lupus erythematous (SLE) in over 50 y. Although well tolerated, the efficacy of belimumab remains limited and is not labeled for patients suffering from nephritis, the leading cause of patient mortality. We sought to explore alternative targets of autoreactive B lymphocytes through manipulation of affinity maturation. The BXSB/MpJ mouse, a well-established model of human SLE, develops elevated antinuclear Abs and immune complex–mediated nephritis along with other manifestations of SLE-like disease. To limit interfering with critical background genetics, we used CRISPR-Cas9 to disrupt activation-induced cytidine deaminase (AID; Aicda) directly in BXSB zygotes. Homozygous null mice demonstrated significantly prolonged survival compared with wild-type. Although mice continued to develop plasma cells, splenic follicular structure was restored, and renal pathology was reduced. Mice developed expanded germinal center B lymphocyte populations as in other models of AID deficiency as well as increased populations of CD73+ B lymphocytes. Treatment with the small molecule inhibitor of RAD51, 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid, resulted in minimal changes in disease markers in BXSB mice. The prolonged survival in AID-deficient BXSB mice appears attributed primarily to the reduced renal pathology, warranting further exploration, as current therapeutics targeting lupus nephritis are limited and, thus, in great demand.
Systemic lupus erythematosus (SLE) is a complex, B lymphocyte–driven autoimmune disease in which high levels of circulating autoreactive Abs exert toxic effects that lead to organ damage, most significantly severe nephritis. SLE has no effective cure, and current treatments are based on global immunosuppression, often insufficiently controlling symptoms coupled with significant side effects. Advances in therapeutics for SLE have been slow, with the notable exception of belimumab, an mAb targeting B lymphocytes, which became the first Food and Drug Administration–approved drug for SLE in decades (1–3). The success of belimumab establishes the clinical effectiveness of targeting pathogenic B lymphocytes in SLE; however, only ∼–30% of lupus patients benefited from this treatment in clinical trials (4) with no current labeling for patients with nephritis. This warrants further investigations into novel therapeutics targeting B lymphocytes for lupus patients.
A defining hallmark of SLE is the presence of copious amounts of antinuclear autoantibodies (ANA) generated by autoreactive B lymphocytes (5, 6). The accumulation of ANA and other autoantibodies results in the formation of immune complexes that activate downstream effector mechanisms, including the fixation of complement, activation of monocytes, and the abundant secretion of proinflammatory cytokines, most notably type 1 IFNs (7, 8). These events ultimately lead to multisystem organ erosion, in particular, renal failure, which is the leading cause of death among SLE patients (9). The majority of pathogenic autoantibodies in human patients with SLE are highly mutated and isotype switched from IgM and IgD to IgG (5, 6). This indicates the autoantibodies are produced by affinity-matured B lymphocytes that have undergone class-switch recombination (CSR) and somatic hypermutation. Both CSR and somatic hypermutation are driven by activation-induced cytidine deaminase (AID), an enzyme that initiates dsDNA breaks (DSB) predominantly in Ig genes (10–14). These DSB are then repaired via homologous recombination by the RAD51 protein family (15–20). Failure to repair DSB leads to cell apoptosis and loss of CSR, thus providing a potential approach for disrupting CSR and abrogating pathogenic autoantibody production (21).
AID is expressed principally in B lymphocytes residing within germinal centers (GCs) (http://www.immgen.org), and its expression is increased in the MRL/MpJ-Faslpr/J (MRL/lpr) mouse model of SLE (22). Previous studies show disrupting CSR through introduction of an Aicda null gene in MRL/lpr mice results in marked reduction in disease symptoms, including decreased nephritis and decreased levels of IgG autoantibodies, leading to increased survival (23). These mice also had high levels of autoreactive IgM that exerted a protective effect (24), suggesting that preservation of circulating IgM may be beneficial. However, when Aicda was knocked out in C57BL/6 mice carrying the lpr mutation, SLE-like disease was rapidly accelerated, driven by autoreactive IgM Abs (25). These conflicting studies raise the important question of whether IgM autoantibodies can substitute for their IgG counterparts in the development of SLE. The BXSB/MpJ mouse offers a model in which SLE-like disease develops through the effects of multiple SLE genetic loci and duplicated expression of Tlr7 (26, 27). This model exhibits a spontaneous, robust extrafollicular response with reduced marginal B lymphocyte numbers and copious plasma cell generation (28, 29). BXSB mice, with a disease pathogenesis distinct from MRL/lpr mice, provide a good model to resolve controversy regarding the role of AID in SLE development.
In this study, we used CRISPR-Cas gene–editing techniques to ablate AID expression directly in the BXSB background. This technique allowed for gene targeting with minimal disruption to background genetics critical for the study of complex traits. We found SLE-like disease diminished in BXSB mice lacking AID with significant improvements in lupus nephritis, a rebound in marginal zone B lymphocyte populations and a restoration of splenic and GC architecture. We then investigated the ability of an inhibitor RAD51, previously found successful in limiting autoimmune disease (30), to attenuate the progression of lupus-like disease in our mice.
Materials and Methods
Mice were maintained under specific pathogen-free conditions with the approval of the Institutional Animal Care and Use Committee of Virginia Polytechnic Institute and State University. BXSB/MpJ mice (henceforth referred to as “BXSB”) were provided by Dr. D. Roopenian (JR000740, available from The Jackson Laboratory). BXSB.Aicda−/− mice were generated using CRISPR-Cas 9 technology as described (30). Briefly, the following single-guide RNAs were used to target Aicda exon 1 or 2 respectively: 5′-gaaattaatacgactcactataggAGTCACGCTGGAGAC-CGATAgttttagagctagaaatagc-3′ or 5′-gaaattaatacgactcactataggACTTCTTTTGCTTC-ATCAGAgttttagagctagaaatagc-3′ (the uppercase letters being the complementary sequences to the targeted region). The resulting progeny were backcrossed to wild-type (wt) BXSB mice. N1 progeny were sequenced, and a founder with a 101-bp deletion on exon 1 was identified. This founder was backcrossed one additional time to BXSB to further reduce the chance of off-target effects with the resulting progeny then intercrossed to homozygosity. The line was maintained by brother–sister matings (referred to as BXSB.Aicda−/− in the text). Genotyping primers (Integrated DNA Technologies) used for Aicda were forward, 5′-TCACACAACAGCACTGAAGC-3′ and reverse, 5′-ACCCAAA-AGACCTGAGCAGA-3′. PCR products were run on a 1.5% agarose (Lonza) gel and imaged on ChemiDoc XRS+ (Bio-Rad Laboratories) using Image Lab Software. The band size for wt is 230 bp and for Aicda knockout is 129 bp. Ear pinna collected at the time of notching for identification was used for genotyping.
In vitro CSR assay
Splenocytes from 8- to 9-wk-old BXSB and BXSB.Aicda−/− male mice were treated with ammonium–chloride–potassium (ACK) lysis buffer and then incubated with biotin–CD43 (S7; BD Biosciences) for 30 min at 4°C, followed by Streptavidin MicroBeads (Miltenyi Biotec) for 15 min at 4°C. Cells were washed and passed through MACS LD Columns (Miltenyi Biotec) according to the manufacturer’s instructions. Enriched B lymphocytes were resuspended at 1 × 106/ml in X-VIVO (Lonza) supplemented with 2-ME (Sigma-Aldrich). Cells were then stimulated with IL-4 (50 ng/ml; PeproTech) and anti-CD40 (2 μg/ml, HM40-3; BD Biosciences) at 37°C (5% CO2) for 48 h. After 48 h, cells were restimulated with IL-4 (25 ng/ml) and anti-CD40 (1 μg/ml). At 96 h, cells were incubated with anti-IgG1 (A85-1; BD Biosciences) for 30 min at 4°C and collected on an Attune NxT Flow Cytometer (Thermo Fisher Scientific). Results were analyzed using FlowJo software (FlowJo).
Splenocytes were isolated from 12- to 15-wk-old BXSB and BXSB.Aicda−/− males and enriched for B lymphocytes as described in CSR assay. These B lymphocytes were resuspended at 1 × 106/ml in X-VIVO (Lonza) supplemented with 2-ME (Sigma-Aldrich). Cells were then stimulated with IL-4 (10 ng/ml; PeproTech) and anti-CD40 (0.1 μg/ml, HM40-3; BD Biosciences) at 37°C (5% CO2) for 72 h. Cells were harvested for RNA extraction using Quick-RNA MicroPrep Kit (catalog ZR1050; Zymo Research). cDNA was synthesized using High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). Primers for Aicda RT-PCR are 5′-CAGGGACGGCATGAGACCT-3′ and 5′-TCAGCCTTGCGGTCTTCACA-3′ and primers for Gapdh are 5′-GAGAAACCTGCCA AGTATGATGAC-3′ and 5′-TGATGGTATTCAAGAGAGTAGGGAG-3′ (30). PCR products were run on a 1.5% agarose (Lonza) gel and imaged on ChemiDoc XRS+ (Bio-Rad Laboratories) using Image Lab Software. The band size for Aicda is 302 bp and for Gapdh is 421 bp.
In vitro plasma cell generation
Splenocytes from 8-wk-old BXSB males were isolated to gain enriched B lymphocytes as described in CSR assay. Enriched B lymphocytes were stimulated with IL-4 (4 ng/ml; PeproTech), TGF-β (2 ng/ml; R&D Systems), anti-Igδ/dex (100 ng/ml; Fina Biosolutions) and retinoic acid (10 μM; Sigma-Aldrich) plus either anti-CD40 (2 μg/ml, HM40-3; BD Biosciences) for T cell–dependent response or LPS (5 μg/ml; Sigma-Aldrich) for T cell–independent response. After 66 h, cells were stained with Abs and analyzed using flow cytometry as described.
Splenocytes were treated with ACK lysis buffer. (For the 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid [DIDS] experiments, splenocytes were not lysed, because DIDS-exposed RBCs are refractory to lysis. RBC were excluded by gating for size in those experiments.) Splenocytes and femoral bone marrow were counted using a Nexcelom cell counter and resuspended at 2 × 107 cells/ml. Cells were then stained with fluorochrome-conjugated Abs for 30 min at 4°C, and propidium iodide (BioLegend) or 7-aminoactinomycin D (7-AAD) (BioLegend) was added before analyzing to differentiate live and dead cells. The following fluorochrome-conjugated Abs (clones) were used: B220 (RA3-6B2), CD4 (GK1.5), CD8 (53-6.7), CD21 (7E9), CD23 (B3B4), GL7 (GL7), CD73 (TY/11.8), CD138 (281-2), CD11b (M1/70), CXCR5 (L138D7), ICOS (C398.4A), and PD-1 (RMP1-30) (BioLegend) and FAS (Jo2) and IgG1 (A85-1) (BD Biosciences). Apoptosis was characterized using Apoptosis Kit (BioLegend) following the manufacturer’s instructions. All experiments were performed on an Attune NxT Flow Cytometer (Thermo Fisher Scientific), and data were analyzed using FlowJo software (FlowJo). All analyses were done after gating on single cells.
Antinuclear Abs in the sera were detected using an ANA Test Kit (Antibodies). Either FITC anti-mouse κ L chain (187.1; BD Biosciences) or FITC anti-mouse IgM (RMM-1; BioLegend) was used as secondary Ab. Slides were imaged with an Eclipse Ti microscope using NIS-Elements software (Nikon). Intensity was measure using ImageJ software (National Institutes of Health).
Urine albumin and creatinine assay
Free catch urine samples were collected from BXSB (n = 8) and BXSB.Aicda−/− (n = 6) males at 6 and 16 wk of age. Samples were stored at −20°C until processed using the Mouse Albumin ELISA Kit (Bethyl Laboratories) and Creatinine Colorimetric Assay Kit (catalog 500701; Cayman Chemical) per manufacturer’s instructions. The albumin-to-creatinine ratio was calculated by dividing albumin concentration in milligrams per deciliter by creatinine concentration in milligrams per deciliter.
Sera Ab ELISA
Different Ab isotypes were quantified by ELISA as described (28). Briefly, plates were incubated with appropriate coating Ab at 4°C overnight; diluted sera were added to the plate and incubated for 1 h at room temperature (RT), followed by detection Abs for 1 h at RT. Anti-dsDNA Abs were quantified as described (31). The plate was coated with calf thymus DNA (Sigma-Aldrich) at 4°C overnight. After blocking for 1 h at RT, diluted sera were added to the plate and incubated for 1 h at RT, followed by detection Abs for 1 h at RT. Detection Abs used in this study were either goat anti-mouse κ-chain (polyclonal; SouthernBiotech) or anti-mouse IgM (polyclonal; Bethyl Laboratories) conjugated to alkaline phosphatase. Plates were developed with 1-Step p-nitrophenyl phosphate disodium salt (Thermo Fisher Scientific) and read on an Infinite M200 PRO plate reader using Magellan 7.0 software (Tecan).
Tissues were embedded in OCT (Thermo Fisher Scientific), frozen on dry ice for 10 min, and stored at −80°C until processing. Frozen tissues were cut to 8 μm using a Microm HM550 Cryostat (Thermo Fisher Scientific). Slides were fixed with acetone (catalog A18-4; Fisher Chemical) at −20°C for 10 min and washed with PBS (Life Technologies) for three times. A PAP pen (MilliporeSigma) was used to circle the sections and blocking buffer (3% FBS, HyClone; Thermo Fisher Scientific) was added onto the slides and incubated in a moist chamber for over 1 h. Slides were washed with PBS for three times and incubated with fluorochrome-conjugated Abs against B220 (RA3-6B2), CD4 (RM4-5), CD73 (TY/11.8), and GL7 (GL7) (BioLegend) and κ-chain (187.1) (BD Biosciences) and C3c (polyclonal, Nordic-MUbio) at RT for 1 h. CD16/32 (93; BioLegend) was used for Fc Block prior to Ig labeling for 30 min at RT. DAPI was used for nuclear counterstaining. Slides were imaged using an LSM 880 Confocal or Zeiss Axio Observer (ZEISS) microscope.
Kidneys were fixed in 10% formaldehyde (Fisher Chemical) overnight and transferred to 70% ethanol (Decon Laboratories). Samples were sent to Histo-Scientific Research Laboratories for processing and H&E staining. The sections were analyzed in a blinded fashion and scored as described by a trained pathologist (32). Briefly, kidneys were scored for the severity of glomerular mesangial proliferation and immune complex deposition, tubular degeneration with protein casts, interstitial inflammation, and vasculitis.
ELISPOT for dsDNA-producing plasma cells was performed as described (33). Briefly, ELISPOT MultiScreen plates (catalog MSIPS4W10; MilliporeSigma) were precoated with 10 μg/ml methylated BSA (Sigma-Aldrich) at 37°C for 2 h, followed by 10 μg/ml calf thymus dsDNA (Sigma-Aldrich) or PBS (control group) at 4°C overnight. Femurs and spleen were collected from 12-wk-old BXSB (n = 5) and BXSB.Aicda−/− (n = 6) males. Bone marrow cells were flushed using syringes and resuspended at 5 × 106/ml in RPMI 1640 (Life Technologies) supplemented with 2 g/L sodium bicarbonate (Sigma-Aldrich), 25 mM HEPES (Sigma-Aldrich), 2 mM l-glutamine (Sigma-Aldrich), 1% penicillin–streptomycin (Sigma-Aldrich), and 10% FBS and 100 μM 2-ME (Sigma-Aldrich). Splenocytes were treated with ACK lysis buffer and resuspended at 2 × 106/ml in complete RPMI 1640. After blocking with complete RPMI 1640 RT for 2 h, cells were seeded and cultured at 37°C (5% CO2) overnight. After washing with PBS three times and PBS with Tween 20 three times, plates were incubated with either goat anti-mouse κ (polyclonal; SouthernBiotech) or anti-mouse IgM (polyclonal; Bethyl Laboratories) conjugated to alkaline phosphatase for 1 h. Spots were developed with nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (Thermo Fisher Scientific), and plates were read on an automated ELISPOT reader and analyzed by the software (AID Autoimmun Diagnostika). The number of anti-dsDNA–secreting cells was calculated by subtracting the number in the control group (no dsDNA coating) from the number in the experimental group.
DIDS (Tocris Bioscience) was injected i.p. at 50 mg/kg DIDS beginning at 6 wk of age in BXSB male mice. Mice were injected weekly for a total of eight injections. DIDS was reconstituted in 0.1 M potassium bicarbonate to a concentration of 0.1 M and further diluted for injection in sterile PBS. Control mice received 0.1 M potassium bicarbonate diluted in sterile PBS at the same injection interval.
In vitro DIDS treatment
Splenocytes from 7-wk-old BXSB males (n = 4) were stimulated with IL-4 (50 ng/ml; PeproTech) and anti-CD40 (0.2 μg/ml, HM40-3; BD Biosciences) in the presence of DIDS (Tocris Bioscience) (0, 50, 100, 150, and 300 μM) or potassium bicarbonate (Fisher Chemical) at 37°C (5% CO2) for 48 h. After 48 h, cells were restimulated with IL-4 (25 ng/ml) and anti-CD40 (0.1 μg/ml). At 96 h, cells were harvested and incubated with fluorochrome-conjugated Abs (clones) against B220 (RA3-6B2), CD80 (16-10A1), CD86 (GL-1), MHC class II (M5/114.15.2), CD11c (N418), F4/80 (BM8), CD4 (GK1.5), and CD8 (53-6.7, all from BioLegend) and IgG1 (A85-1; BD Biosciences) for 30 min at 4°C and collected on an Attune NxT Flow Cytometer (Thermo Fisher Scientific). Results were analyzed using FlowJo software (FlowJo). DIDS was reconstituted in 0.1 M potassium bicarbonate and added to the cells on day 0 of culture.
Statistical significance was determined by Mann–Whitney U test for two groups comparison in Prism (GraphPad). One-way ANOVA and two-way ANOVA were used for multiple groups comparison (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001). Log rank was used for survival assessment.
Disruption of AID prohibits CSR and retards lupus-like disease progression in BXSB mice
To determine the effect of affinity maturation on the pathogenesis of the lupus-like disease in the BXSB mouse, we employed CRISPR-Cas gene editing to target the Aicda gene directly in BXSB zygotes. Because of the complex background genetics of lupus-like disease, we chose this technology to preserve the background genetics of our model. Resulting PCR product sizes are shown in Fig. 1A. B lymphocytes were cultured under conditions to induce CSR (34). RT-PCR confirmed that lack of transcript produced in BXSB.Aicda−/− B lymphocytes under stimulatory conditions (Fig. 1B). Although wt BXSB B lymphocytes successfully produced a subset of cells expressing IgG1, the BXSB.Aicda−/− B lymphocytes remained unswitched (Fig. 1C). Sera Ig levels in BXSB.Aicda−/− mice reflect this deficiency as well, because these mice lack IgG isotypes and exhibit elevated IgM levels as seen previously in Aicda−/− mice (Fig. 1D (23, 30, 35). Male BXSB.Aicda−/− exhibited extended lifespans with 100% of mice surviving past 36 wk, whereas wt BXSB males averaged 15-wk survival (Fig. 1E).
Given that male BXSB.Aicda−/− mice survived significantly longer than their wt BXSB counterparts, we sought to phenotype disease progression in these two strains. BXSB lupus-like disease is characterized by elevated sera IgG (Fig. 1D), glomerular disease with IgG and C3 deposition and the presence of antinuclear autoantibodies (28, 36–38). Histologic renal pathology seen in BXSB.Aicda−/− mice was significantly less than in wt BXSB mice (Fig. 2A, Supplemental Fig. 1). The primary renal lesions in wt BXSB mice were membranoproliferative glomerulonephritis and tubular protein casts. Interstitial inflammation was predominantly minimal, and there was no evidence of vasculitis. As BXSB.Aicda−/− mice lack IgG, renal Ig deposition was evaluated using an all isotype (pan)-Ig detection Ab. The mean intensity of glomerular Ab deposition between the two strains did not significantly differ; however, staining appears to be confined to the mesangial regions in the Aicda−/− mice and extends to the glomerular loops in wt BXSB (Fig. 2C, 2D, Supplemental Fig. 2). Despite no measured differences in renal Ig staining, C3c deposition was significantly reduced in BXSB.Aicda−/− mice compared with wt BXSB mice (Fig. 2E, 2F).
To evaluate levels of ANA, a pan-Ig Ab was used with BXSB.Aicda−/− mice exhibiting reduced ANA staining intensity compared with wt BXSB mice (Fig. 3A). To assess levels of autoreactive IgM Abs, ANA intensity was measured using an IgM detection Ab, and no differences in labeling intensity were seen in the BXSB.Aicda−/− compared with wt BXSB mice (Fig. 3A). The predominant pattern seen with the IgM ANA was perinuclear localization, whereas the pan-Ig Ab showed principally nuclear staining. (Supplemental Fig. 3). Anti-dsDNA Abs were also quantitated using a pan-Ig Ab as well as an IgM Ab; however, no significant differences were observed with low levels detected in both strains (Fig. 3B). Anti-dsDNA ELISPOT showed no differences in bone marrow or splenic plasma cell numbers between the two strains (Fig. 3C, 3D).
Splenic cellularity as assessed using flow cytometry did not vary dramatically between the two strains. B and T lymphocyte percentages were unchanged (Fig. 4A). The marginal zone B lymphocyte compartment is known to be reduced in male BXSB mice (28, 38), and this reduction was at least partially restored in mice deficient in AID (Fig. 4B). Consistent with other Aicda−/− strains (30, 39), the GC B cell compartment was expanded in the BXSB.Aicda−/− mice (Fig. 4C); however, a corresponding increase in T follicular helper (Tfh) cells was not appreciated (Fig. 4D). The characteristic expansion of splenic monocytes (CD11b+) did not differ between the two strains (data not shown). Splenic architecture appears distorted in BXSB mice, with follicular and GC structure difficult to determine; however, AID deficiency resulted in restoration of this architecture with more discernable follicles and GCs (Fig. 4E).
The ability of BXSB B lymphocytes to mature into plasma cells remained unhampered in the absence of AID
Extrafollicular plasma cell generation is a hallmark of lupus-like disease in BXSB mice (28, 29). We hypothesized that inability to undergo CSR would inhibit plasma cell generation in this model. We found no significant differences in splenic or bone marrow CD138+ populations between wt BXSB and BXSB.Aicda−/− (Fig. 5A). To determine if this response was intrinsic to B lymphocytes, we performed in vitro stimulation for plasma cell generation. We found that both wt BXSB and BXSB.Aicda−/− B lymphocytes were able to differentiate into plasma cells under both T cell–independent and T cell–dependent culture conditions (Fig. 5B, 5C). As negative selection during B lymphocyte maturation is thought to lead to apoptosis in low-affinity B cells (40), we asked if BXSB.Aicda−/− B lymphocytes unable to undergo somatic hypermutation would experience an increase in cell death. We found no increase in apoptosis indicators in the AID-deficient BXSB B lymphocytes (Fig. 5D).
Memory B lymphocytes are expanded in AID-deficient BXSB mice
Previously, we have reported expansion of B memory cells in Aicda-deficient mice on the NOD background (30). These memory B cells were also expanded in BXSB.Aicda−/− mice, with the majority of GC B cells being CD73+ (Fig. 6A, 6B). A change in CD73 mean fluorescence intensity (MFI) on B lymphocytes was not appreciated (Fig. 6C). CD73 was also present on a large proportion of CD4+ lymphocytes and Tfh cells (Fig. 6D). No change in CD73 MFI was seen on these CD4+ T lymphocytes (Fig. 6E). These CD73+ cells were generally associated with the distorted follicular structures seen in wt BXSB, whereas BXSB.Aicda−/− mice showed localization of CD73 mainly in GC structures (Fig. 6F).
Disruption in repair of AID-induced DNA breaks resulted in minimal attenuation of lupus-like disease in BXSB mice
DSB are initiated by AID during the process of CSR. The RAD51 complex repairs these breaks, and inability to mend the DNA damage that occurs during this process results in cell death (34, 41). DIDS, a disulfonated trans-stilbene derivative, has previously been shown to bind to RAD51 and inhibit its function (42). In vitro and in vivo use of DIDS inhibits DNA break repair and leads to B cell death without evidence of off-target toxicity (21). We recently showed that use of this small molecule inhibitor significantly delayed the development of type 1 diabetes in NOD mice (30). We sought to investigate the effect of DIDS treatment in BXSB model of SLE. In vitro assessment of DIDS on B lymphocytes stimulated to undergo CSR showed a dosage-dependent loss of total B cells and IgG1+ cells (Supplemental Fig. 4A, 4B) without a corresponding decrease in T lymphocytes (Supplemental Fig. 4C). Macrophages and dendritic cells (DCs) also showed a dosage-dependent decrease in cell numbers (Supplemental Fig. 4D), and whereas B cell activation markers increased with dosage of the DIDS (Supplemental Fig. 4E–G), DC expression of CD80 and CD86 declined (Supplemental Fig. 4H–J). To assess if these results translated in vivo, BXSB mice were treated weekly, beginning at 6 wk of age with 50 mg/kg DIDS for eight injections. mice were euthanized 1 wk following the final injection. CD4+ T and B lymphocyte percentages in the spleen were decreased with treatment (Fig. 7A); however, no differences were seen in splenic B cell subsets (Fig. 7B). GC B cell percentage was unaffected, whereas the Tfh cell population trended toward reduction in the presence of DIDS (Fig. 7C, 7D). The percentage of splenic monocytes was significantly reduced with DIDS administration (Fig. 7E). ANA intensity remained consistent between the treated and control groups (Fig. 7F), whereas the circulating IgG levels over the course of the experiment trended toward reduction but did not reach significance (Fig. 7G). No change was seen in renal pathology scoring between the two groups (Fig. 7H).
The disruption of affinity maturation of B lymphocytes through ablation of AID in BXSB mice significantly prolonged survival with decreased renal damage and restoration of splenic architecture. Pathogenic glomerular changes were diminished, as was urine protein concentrations. Immune complexes were still present in the Aicda−/− mice; however, complement activation appeared absent. These results suggest that these IgM Abs lacking somatic hypermutation are not as adept at activating the inflammatory profile characteristic of lupus nephritis. It is generally accepted that IgG isotypes initiate IFN production fueling renal damage (43), and a potential explanation needing further exploration in this model is the inability of IgM to induce IFN production. Although hyper-IgM syndrome appears to exist in BXSB.Aicda−/− mice as in other AID-deficient models (23, 30, 35), antinuclear IgM Ab levels do not differ between wt BXSB and BXSB.Aicda−/− mice, suggesting no increase in autoreactivity. However, the pattern of Ig and IgM ANA binding is altered between the strains. BXSB.Aicda−/− sera presents a cytoplasmic, dense, fine-speckled pattern, whereas wt sera shows a predominately homogeneous nuclear pattern (https://www.anapatterns.org). This suggests altered Ag affinity that would be of interest to explore further.
Although the cellular phenotype of spleens by flow cytometry remained mostly consistent between the two strains, the organization of these cells within the spleen was noticeably altered. GC B cell percentages were increased as previously reported in mice with targeted AID deficiency (30, 35). A corresponding increase in Tfh cells was not observed, suggesting that the creation of GC B cells may not be accelerated, but perhaps their movement out of this compartment is hampered. A reduction in marginal zone B cells is a consistent change seen in BXSB mice, and abrogation of AID restored this population. This lack of detectable changes using traditional flow cytometry in a model with significantly prolonged survival cautions against relying on these markers as an indicator of disease improvement. Further exploration in situ revealed, despite little change in cellular percentages and numbers, splenic architecture was restored in the spleens of the knockout mice with discernable follicles and GCs readily visible. Previously, we reported that BXSB mice exhibit a robust extrafollicular response (36), which has also been appreciated in other lupus models (44, 45) as well as suspected in human patients (46). The return to more-normal architecture in the AID-deficient mice suggests a shift from an extrafollicular response to a GC response. However, no differences were seen in plasma cell generation, including anti-dsDNA Ab–producing plasma cells. BXSB historically has low production of anti-dsDNA Abs (47), and assay sensitivity may be clouding these results. The possibility exists that although follicular structure is distorted in the wt BXSB mice, the plasma cell–generating interactions between B and T cells are still occurring in a similar manner between the two models. Further studies are needed to investigate this possibility.
BXSB.Aicda−/− B lymphocytes expressed increased CD73, a memory B cell marker, which is similar to what we showed in the NOD.Aicda−/− model (30). In the NOD model, these CD73+ memory B cells exert a protective effect (30). The regulatory role of CD73+ memory B cells has yet to be tested in the BXSB model. The literature presents a model in which low-affinity GC B cells move into the memory compartment (48). These results support this model, as B lymphocytes unable to undergo affinity maturation would presumably remain as lower affinity, increasing the memory B cell compartment.
Given the reduced production of Igs and increase in the GC B cell compartment in BXSB.Aicda−/− mice, we anticipated the generation of plasma cells would be reduced. With diminished autoreactive Ab levels in these mice, we hypothesized that the Aicda−/− B lymphocytes would have limited survival because of low affinity and would not undergo plasma cell differentiation. We did not, however, find a statistically significant drop in the percentage of plasma cells. In fact, we saw that BXSB.Aicda−/− B cells were able to differentiate into plasma cells as readily as their wt counterparts in vitro and that these cells were not subject to early or late apoptosis. Further study into the generation of these cells and the reduction in pathogenic B lymphocyte development in this model are warranted.
Because disruption of the AID pathway led to significantly improved life span in BXSB mice, targeting this pathway therapeutically may prove beneficial. The small molecule inhibitor of RAD51, DIDS, blocks the ability of B cells to repair the DSB induced by AID, causing these cells to undergo early cell death (21). The point of intervention in the pathway is, therefore, different from targeted knockout of the Aicda gene. The NOD model of type 1 diabetes showed success with this therapy, and diabetes onset was greatly delayed (30). Although the NOD model was successful at a dosage of 10 mg/kg, BXSB mice were not responsive (data not shown), and a higher dosage of 50 mg/kg (21) was investigated. Although in vitro assays with DIDS successfully decreased B lymphocyte populations, we saw no improvement in vivo in our mice, namely glomerular pathology was not significantly altered. We did appreciate a decrease in splenic B lymphocytes with a trend toward reduced levels of Igs. In vitro stimulation revealed a toxic effect on DCs and macrophages of BXSB origin not previously reported, whereas the effect of decreasing B lymphocytes and sparing T lymphocytes was consistent with earlier studies (21). We suspect this unexpected effect contributed to the lack of efficacy seen; however, further exploration is needed. Although targeting with the DIDS molecule appeared unsuccessful in diminishing glomerular disease in our mice, we feel our success in genetically targeting of this pathway warrants further exploration for potential therapeutic development.
In conclusion, targeting B lymphocyte maturation in the BXSB model alleviated lupus-like nephritis and prolonged survival. One limitation of this work is that only a single founder was used to develop the BXSB.Aicda−/− line, and thus, off-target effects cannot be ruled out. The authors did perform an additional backcross to BXSB to limit this possibility when creating these mice. Our results are supported by previous work in the MRL model targeting this same molecule (23). Because these two mouse models of SLE vary greatly in disease pathogenesis, these similar findings indicate greater promise that this work could translate to human patients, particularly those with nephritis. Further explorations into ways to target this pathway for the treatment of SLE are warranted.
This work was supported by the National Institute of Diabetes and Digestive and Kidney Diseases of the National Institutes of Health under Award K08DK101735 and the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health under Award R03AR066787.
The online version of this article contains supplemental material.
Abbreviations used in this article:
activation-induced cytidine deaminase
mean fluorescence intensity
T follicular helper
The authors have no financial conflicts of interest.