Critical role of IRF-5 in regulation of B-cell differentiation

Abstract

IFN-regulatory factor 5 (IRF-5), a member of the IRF family, is a transcription factor that has a key role in the induction of the antiviral and inflammatory response. When compared with C57BL/6 mice, Irf5(-/-) mice show higher susceptibility to viral infection and decreased serum levels of type I IFN and the inflammatory cytokines IL-6 and TNF-alpha. Here, we demonstrate that IRF-5 is involved in B-cell maturation and the stimulation of Blimp-1 expression. The Irf5(-/-) mice develop an age-related splenomegaly, associated with a dramatic accumulation of CD19(+)B220(-) B cells and a disruption of normal splenic architecture. Splenic B cells from Irf5(-/-) mice also exhibited a decreased level of plasma cells. The CD19(+) Irf5(-/-) B cells show a defect in Toll-like receptor (TLR) 7- and TLR9-induced IL-6 production, and the aged Irf5(-/-) mice have decreased serum levels of natural antibodies; however, the antigen-specific IgG1 primary response was already dependent in IRF-5 in young mice, although the IgM response was not. Analysis of the profile of transcription factors associated with plasma cell differentiation shows down-regulation of Blimp-1 expression, a master regulator of plasma cell differentiation, which can be reconstituted with ectopic IRF-5. IRF-5 stimulates transcription of the Prdm1 gene encoding Blimp-1 and binds to the IRF site in the Prdm1 promoter. Collectively, these results reveal that the age-related splenomegaly in Irf5(-/-) mice is associated with an accumulation of CD19(+)B220(-) B cells with impaired functions and show the role of IRF-5 in the direct regulation of the plasma cell commitment factor Blimp-1 and in B-cell terminal differentiation.

Fig. 1.

Splenomegaly and alteration of splenic architecture in aging Irf5^−/− mice. (A) Spleens (arrows) and lymph nodes (arrowheads) of C57BL/6 mice and Irf5^−/− mice (10 months old). (B) Spleens of C57BL/6 mice and Irf5^−/− mice (10 months old) were fixed in 3.7% formaldehyde, sectioned, and stained with H&E. White pulp (WP), red pulp (RP), and marginal zones (MZ) are marked.

Fig. 2.

Analysis of B-cell subtypes in spleens of C57BL/6 and Irf5^−/− mice at 2, 7, and 14 months (mo) of age. Total splenocytes from C57BL/6 (Top) and Irf5^−/− (Bottom) mice aged 2, 7, and 14 mo were stained with CD19 and B220 (A) or B220 and CD138 (B), respectively, and analyzed by flow cytometry. CD19⁺ B cells and CD138⁺ plasma cells were gated on live lymphocytes by forward and side scatter.

Fig. 3.

Antibody response to T-cell-dependent and -independent antigens in Irf5^−/− and C57BL/6 mice. Groups of five Irf5^−/− and C57BL/6 female mice (8 weeks old) were injected i.p. with 4-Hydroxy-3-nitrophenyl (NP); Keyhole Limpet Hemocyanin (KLH) NP-KLH/alum (1:1, 100 μg) or NP-LPS (20 μg) and bled 7 and 14 days later. Antigen-specific IgG1 and IgM were determined by ELISA, as described in Materials and Methods. Values indicate mean titers for groups of five animals, and the error bars indicate the SEM. The P values are given at the top of the respective figures. *p ≤ 0.05 **p ≤ 0.005

Fig. 4.

Irf5^−/− B cells show decreased expression of Blimp-1, which can be rescued by ectopic IRF-5. (A) RNA was isolated from purified splenic B cells of C57BL/6 and Irf5^−/− mice, and the relative levels of Blimp-1,BCL6,IRF-4,PAX5, and IRF-8 expression were determined by semiquantitative RT-PCR. The intensity of analyzed bands was normalized to β-actin levels. (B) Expression of IRF-5 and IRF-4 in purified splenic B cells of C57BL/6 and Irf5^−/− mice was determined by Western blot analysis. (C) Purified splenic B cells from C57BL/6 and Irf5^−/− mice were stimulated with LPS (100 μg/mL) for 3 days and then infected with retrovirus expressing the IRF-5 gene. RNA was isolated from infected cells 3 days postinfection, and the relative levels of Blimp-1 transcripts in transduced and untransduced Irf5^−/− cells and controls (C57BL/6 B cells) were detected by real-time RT-PCR.