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. 2008 Aug 21;3(8):e3033.
doi: 10.1371/journal.pone.0003033.

Activation-induced cytidine deaminase deficiency causes organ-specific autoimmune disease

Koji Hase  1 , Daisuke Takahashi, Masashi Ebisawa, Sayaka Kawano, Kikuji Itoh, Hiroshi Ohno
Affiliations

Activation-induced cytidine deaminase deficiency causes organ-specific autoimmune disease

Koji Hase et al. PLoS One. .

Abstract

Activation-induced cytidine deaminase (AID) expressed by germinal center B cells is a central regulator of somatic hypermutation (SHM) and class switch recombination (CSR). Humans with AID mutations develop not only the autosomal recessive form of hyper-IgM syndrome (HIGM2) associated with B cell hyperplasia, but also autoimmune disorders by unknown mechanisms. We report here that AID-/- mice spontaneously develop tertiary lymphoid organs (TLOs) in non-lymphoid tissues including the stomach at around 6 months of age. At a later stage, AID-/- mice develop a severe gastritis characterized by loss of gastric glands and epithelial hyperplasia. The disease development was not attenuated even under germ-free (GF) conditions. Gastric autoantigen -specific serum IgM was elevated in AID-/- mice, and the serum levels correlated with the gastritis pathological score. Adoptive transfer experiments suggest that autoimmune CD4+ T cells mediate gastritis development as terminal effector cells. These results suggest that abnormal B-cell expansion due to AID deficiency can drive B-cell autoimmunity, and in turn promote TLO formation, which ultimately leads to the propagation of organ-specific autoimmune effector CD4+ T cells. Thus, AID plays an important role in the containment of autoimmune diseases by negative regulation of autoreactive B cells.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. AID−/− mice spontaneously developed gastritis.
(A) Stomachs of AID+/+ and AID−/− mice were cut longitudinally to expose the gastric mucosae. Inside-out tissues were examined by stereomicroscopy. A high magnification view of the mucosal surfaces revealed the presence of a number of nodule-like structures in the AID−/− mouse stomach (arrows). A representative sample from each group is shown. (B) Gastric tissue sections were stained with H&E for histological examination, or with Alcian blue-hematoxylin (AB&H) for detection of mucin-producing cells (shown as turquoise). Scale bars: 100 µm. (C) Gastric tissues of AID−/− mice at different ages were analyzed for development of ectopic lymphoid follicles and mucosal hyperplasia. The diagnostic criteria are described in Supplemental Table S1 online. (D) Stomach weights of AID+/+ and AID−/− mice were measured and the values were normalized to the body weight of each mouse. (E) Kaplan-Meier survival curves of AID−/− (solid line, n = 18) and AID+/+ (dotted line, n = 19) mice revealed significant differences in survival (P<0.05).
Figure 2
Figure 2. TLOs are formed in various organs of AID−/− mice.
The tissue samples were obtained from 12-month-old AID−/− or AID+/+ mice, and were subjected to histological examination after H–E staining. Asterisks represent TLOs. Scale bars: 100 µm.
Figure 3
Figure 3. Massive infiltration of inflammatory cells is observed in the gastric tissue of AID−/− mice.
Immunofluorescence staining for immune cell-specific markers was performed using serial tissue sections prepared from two different gastric samples with ectopic lymphoid follicles (upper) or mucosal hyperplasia (lower). DAPI staining (left panels) was used to identify cell nuclei. Scale bars: 100 µm.
Figure 4
Figure 4. Gastric mucosa-infiltrating lymphocytes in AID−/− mice have an activated phenotype.
(A) Lymphocytes were prepared from gastric mucosae (GM; upper panels) of AID−/− mice with gastritis. Since lymphocyte infiltration into gastric tissues was negligible in AID+/+ mice (Figure 1B), splenocytes (SP; lower panels) from AID−/− mice with gastritis were used as a control. (B) CD3+CD4+ gastric and splenic lymphocytes were gated and further analyzed for memory and regulatory T-cell markers. (C) Gastric CD3+CD4+ cells enriched with a MACS column were treated with PMA and Golgi-stop for 6 h and then analyzed for production of the indicated cytokines by intracellular staining. (D) Concentrations of the indicated cytokines in culture supernatants of gastric CD4+ cells stimulated with anti-CD3 and anti-CD28 mAbs for 48 h were determined by CBA and ELISA.
Figure 5
Figure 5. Q-PCR analysis of cytokine mRNA expression in gastric tissue.
Gastric tissue samples were obtained from 12-month-old AID+/+ mice and AID−/− mice with or without gastritis symptoms. Data are means±S.D. of at least four different samples. *P<0.05; **P<0.01.
Figure 6
Figure 6. Gastric microflora are irrelevant to gastritis development in AID−/− mice.
AID−/− and AID+/+ mice were raised under germ-free (GF) or conventional (CV) conditions to examine whether the presence of gastric microflora affects the development of gastritis. (A) Histological examination of gastric tissues from AID−/− and AID+/+ mice maintained under GF condition. (B) Stomach weight and pathological score of gastritis were compared between AID−/− mice in GF and CV conditions. (C) Cytokine expression levels in gastric tissues of AID−/− and AID+/+ mice under GF and CV conditions were measured by Q-PCR. The mRNA expression level of each cytokine was normalized to that of GAPDH.
Figure 7
Figure 7. Gastric mucosa-specific antibodies are elevated in AID−/− mice.
(A) Serum samples were collected from AID+/+ and AID−/− mice maintained under SPF condition to measure total IgM, and gastric-mucosa- or H+/K+-ATPase-specific IgM levels by ELISA. After histological examination, individuals with a total pathological score of 2 or more were considered to be gastritis-positive. (B) Serum levels of anti-gastric mucosa antibodies positively correlated with total pathological score in AID−/− mice (r = 0.6825; P<0.05).
Figure 8
Figure 8. CD4+ T cells contribute to the development of gastritis in AID−/− mice.
Gastric mucosa-infiltrating CD4+ T cells or CD8+ T cells were isolated from 12 month old AID−/− mice and were adoptively transferred into BALB/cnu/nu mice. After 3 months, the stomachs of recipient mice were removed and subjected to the following experiments. (A) Gastric tissue sections were stained with H&E for histological examination. (B) Stomach weights were normalized to body weight of each mouse. (C) Gastric tissue sections were examined by histology and assigned a pathological score. (D) Immunofluorescence staining revealed a massive infiltration of CD4+ and Mac1+ cells into the stomachs of BALB/cnu/nu mice that received CD4+ T cells by adoptive transfer. (E) Gastric CD3+CD4+ cells enriched with a MACS column were treated with PMA and Golgi-stop for 6 hr and analyzed for cytokine production by intracellular staining. (F) CD4+ T cells recovered from gastric tissues of CD4+ T-cell-transferred BALB/cnu/nu mice were stimulated with anti-CD3 and anti-CD28 mAbs for 48 h. Cytokine concentrations in the culture supernatants were measured by CBA and ELISA. Scale bars: 100 µm.
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