AID dysregulation in lupus-prone MRL/Fas(lpr/lpr) mice increases class switch DNA recombination and promotes interchromosomal c-Myc/IgH loci translocations: modulation by HoxC4

Abstract

Immunoglobulin gene somatic hypermutation (SHM) and class switch DNA recombination (CSR) play important roles in the generation of autoantibodies in systemic lupus erythematosus. Systemic lupus is characterized by the production of an array of pathogenic high-affinity mutated and class-switched, mainly IgG, antibodies to a variety of self-antigens, including nuclear components, such as dsDNA, histones, and chromatin. We previously found that MRL/Fas(lpr/lpr) mice, which develop a systemic autoimmune syndrome sharing many features with human lupus, display greatly upregulated CSR, particularly to IgG2a, in B cells of the spleen, lymph nodes, and Peyer's patches. In MRL/Fas(lpr/lpr) mice, the significant upregulation of CSR is associated with increased expression of activation-induced cytidine deaminase (AID), which is critical for CSR and SHM. We also found that HoxC4 directly activates the promoter of the AID gene to induce AID expression, CSR and SHM. Here, we show that in both lupus patients and lupus-prone MRL/Fas(lpr/lpr) mice, the expression of HoxC4 and AID is significantly upregulated. To further analyze the role of HoxC4 in lupus, we generated HoxC4(-/-) MRL/Fas(lpr/lpr) mice. In these mice, HoxC4-deficiency resulted in reduced AID expression, impaired CSR, and decreased serum anti-dsDNA IgG, particularly IgG2a, autoantibodies, which were associated with a reduction in IgG deposition in kidney glomeruli. In addition, consistent with our previous findings in MRL/Fas(lpr/lpr) mice that upregulated AID expression is associated with extensive DNA lesions, comprising deletions and insertions in the IgH locus, we found that c-Myc to IgH (c-Myc/IgH) translocations occur frequently in B cells of MRL/Fas(lpr/lpr) mice. The frequency of such translocations was significantly reduced in HoxC4(-/-) MRL/Fas(lpr/lpr) mice. These findings suggest that in lupus B cells, upregulation of HoxC4 plays a major role in dysregulation of AID expression, thereby increasing CSR and autoantibody production and promoting c-Myc/IgH translocations.

Figure 1

HOXC4/HoxC4 and AICDA/Aicda are upregulated in PBMCs from SLE patients, and in lymph nodes, Peyer’s patches and spleens of autoimmune MRL/Fas^lpr/lpr mice. Total RNA was prepared from (a) the PBMCs from 5 female SLE patients (L1, L2, L3, L4 and L5) and 5 age- and sex-matched healthy subjects (H1, H2, H3, H4 and H5), or from (b) the spleen, lymph nodes, Peyer’s patches, thymus and liver of non-immunized 8-week old MRL/Fas^lpr/lpr or healthy C57BL/6 mice. Expression of HOXC4/HoxC4 and AICDA/Aicda transcripts was analyzed by real-time qRT-PCR using SYBR green and normalized to GAPDH/Gapdh expression. Data are from 3 independent experiments (mean values ± SD).

Figure 2

HoxC4 deficiency impairs AID expression in MRL/Fas^lpr/lpr mice. (a) AID and β-actin proteins in lymph nodes of non-immunized 12-week old female HoxC4^+/+ MRL/Fas^lpr/lpr mice and HoxC4^−/− MRL/Fas^lpr/lpr littermates, as well as non-immunized 9-month old female NZB/NZW F1 and NZB mice were detected by immunoblotting. Data are representative of 2 independent experiments. (b) Total RNA was prepared from the spleen and lymph nodes of HoxC4^+/+ MRL/Fas^lpr/lpr and HoxC4^−/− MRL/Fas^lpr/lpr littermates. Aicda transcripts were measured by real-time qRT-PCR using SYBR green and normalized to Gapdh expression. Data are from 3 independent experiments (mean values ± SD).

Figure 3

HoxC4 deficiency decreases levels of serum IgG1 and IgG2a autoantibodies, IgG2a kidney deposition and glomerular damage in MRL/Fas^lpr/lpr mice. (a) Titers of circulating anti-dsDNA IgG1 and anti-dsDNA IgG2a in 5 pairs of 16-week-old HoxC4^+/+ MRL/Fas^lpr/lpr mice and HoxC4^−/− MRL/Fas^lpr/lpr littermates (each symbol represents an individual mouse) depicted as the number of dilutions needed to reach 50% of saturation binding (EC₅₀). P values, paired t-test. (b) Kidney sections from 3 HoxC4^+/+ MRL/Fas^lpr/lpr and 3 HoxC4^−/− MRL/Fas^lpr/lpr mice were stained with H&E (left panels) or with FITC-labeled anti-IgG1 or anti-IgG2a mAb (middle and right panels). Depicted are kidney sections from one representative pair of HoxC4^+/+ MRL/Fas^lpr/lpr and HoxC4^−/− MRL/Fas^lpr/lpr mice.

Figure 4

HoxC4 deficiency impairs CSR in MRL/Fas^lpr/lpr mice. (a) B220⁺ B cells from the spleen or lymph nodes of HoxC4^+/+ MRL/Fas^lpr/lpr and HoxC4^−/− MRL/Fas^lpr/lpr littermates were analyzed for surface IgG1, IgG2a, IgG3 and IgA expression by flow cytometry. (b) B220⁺PNA^hi GC B cells from the spleen or lymph nodes of HoxC4^+/+ MRL/Fas^lpr/lpr and HoxC4^−/− MRL/Fas^lpr/lpr littermates were analyzed for surface IgG1, IgG2a, IgG3 and IgA expression by flow cytometry.

Figure 5

HoxC4 deficiency does not alter germline I_H-C_H transcripts but results in lower expression of circle I_H-Cμ and post-recombination Iμ-C_H transcripts in MRL/Fas^lpr/lpr mice. Total RNA was prepared from spleens of non-immunized HoxC4^+/+ MRL/Fas^lpr/lpr and HoxC4^−/− MRL/Fas^lpr/lpr mice. Germline I_H-C_H transcripts (a), circle I_H-Cμ transcripts (b) and post-recombination Iμ-C_H transcripts (c) were measured by real-time qRT-PCR using SYBR green and normalized to Gapdh expression. Data are from 3 independent experiments (mean values ± SD).

Figure 6

c-Myc to IgH translocations are frequent events in B cells from MRL/Fas^lpr/lpr mice, are reduced in the absence of HoxC4, and eliminated in the absence of AID. Genomic DNA was prepared from spleen cells of (a)HoxC4^+/+Aicda^+/− MRL/Fas^lpr/lpr, HoxC4^+/+Aicda^−/− MRL/Fas^lpr/lpr mice, and (b)HoxC4^+/+Aicda^+/+ MRL/Fas^lpr/lpr and HoxC4^−/−Aicda^+/+ MRL/Fas^lpr/lpr mice. c-Myc/IgH translocations were detected by nested PCR using primers specific to the IgH and c-Myc locus, and further specified by Southern blotting with a [γ-³²P]-labeled c-Myc specific probe. Each individual PCR assay was performed using a DNA template corresponding to 5 × 10⁵ cells. The corresponding frequency of translocations per cell is indicated underneath each panel. The data are representative of independent experiments with 3 sets of mice.

Figure 7

Sequences of c-Myc/IgH translocation junctions. Fourteen c-Myc/IgH junction DNAs from spleen B cells of HoxC4^+/+Aicda^+/+ MRL/Fas^lpr/lpr mice were amplified, cloned and sequenced. Each sequence is compared with germline c-Myc (blue) and germline IgH (green) sequences. Microhomologies or insertions are underlined.