Iron inhibits activation-induced cytidine deaminase enzymatic activity and modulates immunoglobulin class switch DNA recombination

Abstract

Immunoglobulin (Ig) class switch DNA recombination (CSR) and somatic hypermutation (SHM) are critical for the maturation of the antibody response. Activation-induced cytidine deaminase (AID) initiates CSR and SHM by deaminating deoxycytidines (dCs) in switch (S) and V(D)J region DNA, respectively, to generate deoxyuracils (dUs). Processing of dUs by uracil DNA glycosylase (UNG) yields abasic sites, which are excised by apurinic/apyrimidinic endonucleases, eventually generating double strand DNA breaks, the obligatory intermediates of CSR. Here, we found that the bivalent iron ion (Fe(2+), ferrous) suppressed CSR, leading to decreased number of switched B cells, decreased postrecombination Iμ-C(H) transcripts, and reduced titers of secreted class-switched IgG1, IgG3, and IgA antibodies, without alterations in critical CSR factors, such as AID, 14-3-3γ, or PTIP, or in general germline I(H)-S-C(H) transcription. Fe(2+) did not affect B cell proliferation or plasmacytoid differentiation. Rather, it inhibited AID-mediated dC deamination in a dose-dependent fashion. The inhibition of intrinsic AID enzymatic activity by Fe(2+) was specific, as shown by lack of inhibition of AID-mediated dC deamination by other bivalent metal ions, such as Zn(2+), Mn(2+), Mg(2+), or Ni(2+), and the inability of Fe(2+) to inhibit UNG-mediated dU excision. Overall, our findings have outlined a novel role of iron in modulating a B cell differentiation process that is critical to the generation of effective antibody responses to microbial pathogens and tumoral cells. They also suggest a possible role of iron in dampening AID-dependent autoimmunity and neoplastic transformation.

FIGURE 1.

Fe²⁺ suppresses CSR. A, surface expression of B220 and IgG3 (induced by LPS), IgG1 (induced by LPS plus IL-4), or IgA (induced by LPS, IL-4, TGFβ plus anti-δ mAb/dex) in the absence (nil, black) or presence of 80 μm FeCl₂ (plum) or FeSO₄ (teal). The number inside each rectangle indicates the percentage of B220⁺ cells that are switched to the indicated Ig isotypes. Ratios of the proportion of switched B cells in B cells stimulated in the presence of Fe²⁺ to that of B cells stimulated in the absence of Fe²⁺ are depicted in histograms (mean ± S.E. of data from three independent experiments). B, titers of IgG3, IgG1, or IgA in supernatants of B cells stimulated with LPS, LPS plus IL-4, or LPS, IL-4, TGFβ plus anti-δ mAb/dex in the absence (nil, black) or presence of 80 μm FeCl₂ (plum) or FeSO₄ (teal) (mean ± S.D. of data from three independent experiments). C, levels of postrecombination Iμ-Cγ3, Iμ-Cγ1, or Iμ-Cα transcripts in B cells stimulated with LPS, LPS plus IL-4, or LPS, IL-4, TGFβ plus anti-δ mAb/dex in the absence (nil, black) or presence of 80 μm FeCl₂ (plum) or FeSO₄ (teal). Data were normalized to the expression of Cd79b and are expressed as ratios of values in B cells stimulated in the presence of Fe²⁺ to those in B cells stimulated in the absence of Fe²⁺ (mean ± S.E. of data from three independent experiments). **, p < 0.05 (p values: t test).

FIGURE 2.

Fe²⁺ does not alter B cell surface Igμ expression, proliferation, or viability. A, surface expression of B220 and Igμ in B cells stimulated with LPS, LPS plus IL-4, or LPS, IL-4, TGFβ plus anti-δ mAb/dex in the absence (nil) or presence of 80 μm FeCl₂ or FeSO₄. The number inside each rectangle indicates the percentage of cells that are Igμ⁺. Data are representative of three independent experiments. B, proportion of surface IgG1⁺ B cells after stimulation of CFSE-labeled B cells with LPS plus IL-4 in the absence (nil) or presence of 80 μm FeCl₂ or FeSO₄. Data are representative of three independent experiments. C, proportion of IgG3⁺, IgG1⁺, or IgA⁺ B cells in each cell division after CFSE-labeled B cells were stimulated with LPS, LPS plus IL-4, or LPS, IL-4, TGFβ plus anti-δ mAb/dex in the absence (nil, black) or presence of 80 μm FeCl₂ (plum) or FeSO₄ (teal). Data are representative of three independent experiments. D, proportion of live (7-AAD⁻) B cells after stimulation with LPS, LPS plus IL-4, or LPS, IL-4, TGFβ plus anti-δ mAb/dex in the absence (nil, black) or presence of 80 μm FeCl₂ (plum) or FeSO₄ (teal). Data are expressed as ratios of values in B cells stimulated in the presence of Fe²⁺ to those in B cells stimulated in the absence of Fe²⁺ (mean ± S.E. of data from three independent experiments).

FIGURE 3.

Fe²⁺ does not inhibit plasmacytoid differentiation. A, proportion of CD138⁺ (syndecan-1⁺) plasmacytoid cells/plasmacytes after stimulation of B cells with LPS, LPS plus IL-4, or LPS, IL-4, TGFβ plus anti-δ mAb/dex in the absence (nil) or presence of 80 μm FeCl₂ or FeSO₄. B220 surface expression was down-regulated in B cells stimulated with LPS or LPS plus IL-4. Data are representative of three independent experiments. B, levels of Irf4, Prdm1, and Xbp1 transcripts in B cells stimulated with LPS, LPS plus IL-4, or LPS, IL-4, TGFβ plus anti-δ mAb/dex in the absence (nil, black) or presence of 80 μm FeCl₂ (plum) or FeSO₄ (teal). Data were normalized to the level of Cd79b and are expressed as ratios of values in B cells cultured with LPS, LPS plus IL-4, or LPS, IL-4, TGFβ plus anti-δ mAb/dex in the presence of Fe²⁺ to those in B cells cultured in the absence of Fe²⁺ (mean ± S.E. of data from three independent experiments).

FIGURE 4.

Fe²⁺ does not alter induction of Aicda, 14-3-3γ, or Paxip1, or germline I_H-S-C_H transcription in general. Levels of Aicda, 14-3-3γ, and Paxip1 transcripts in B cells were stimulated with LPS, LPS plus IL-4, or LPS, IL-4, TGFβ plus anti-δ mAb/dex in the absence (nil, black) or presence of 80 μm FeCl₂ (plum) or FeSO₄ (teal) as well as germline Iμ-Cμ and Iγ3-Cγ3, Iγ1-Cγ1 or Iα-Cα transcripts in those B cells. Data were normalized to the level of Cd79b and are expressed as ratios of values in B cells stimulated in the presence of Fe²⁺ to those in B cells stimulated in the absence of Fe²⁺ (mean ± S.E. of data from three independent experiments). **, p < 0.05 (p values: t test).

FIGURE 5.

Fe²⁺ inhibits AID-mediated dC DNA deamination. A, schematics of AID deamination assay. B, dose-dependent inhibition of AID mediated dC DNA deamination by FeCl₂. AID dC DNA deamination activity in the presence of different concentrations of FeCl₂ is indicated below each lane in the left panel and depicted by an inhibition curve in the right panel. C, AID dC DNA deamination was inhibited over different periods of the enzymatic reaction by different concentrations of FeCl₂, as indicated. AID dC DNA deamination activity is indicated below each lane. D, AID dC DNA deamination was inhibited over different periods of the enzymatic reaction by different concentrations of FeCl₂, as indicated, in the absence or presence of RNase A. AID dC DNA deamination activity is depicted by kinetic curves in the right panel. Data are representative of three independent experiments.

FIGURE 6.

Bivalent metal ion Zn²⁺, Mn²⁺, Mg²⁺, or Ni²⁺ does not affect AID-mediated dC DNA deamination. AID dC DNA deamination activity in the absence or presence of 10 or 20 μm FeCl₂, ZnCl₂, MnCl₂, MgCl₂, or NiCl₂ is indicated below each lane in the left panel and depicted by inhibition curves in the right panel. Data are representative of three independent experiments.

FIGURE 7.

Fe²⁺ does not alter UNG dU excision activity. A, schematics of UNG-mediated dU excision assay. B, omission of UNG yielded no cleavage products. C, UNG-mediated dU excision in the absence or presence of FeCl₂, ZnCl₂, MnCl₂, MgCl₂, or NiCl₂ is shown. Numbers below each lane indicate the percentage of the substrate showing dU excision. Data are representative of three independent experiments.