The BXH2 mutation in IRF8 differentially impairs dendritic cell subset development in the mouse

Abstract

Among dendritic cell (DC) subsets, CD8alpha(+) DCs and plasmacytoid DCs (pDCs) produce high levels of IL12 and type I interferons (IFNs), respectively, and confer early innate immunity. Development of CD8alpha(+) DCs and pDCs requires the interferon regulatory factor 8 (IRF8). Recently, a spontaneous point mutation was identified in the Irf8/Icsbp gene in the BXH2 mouse, which exhibits an immunodeficient phenotype similar to the IRF8 knockout (KO) mouse. We show that this mutation, designated IRF8(R294C), abolishes the development of CD8alpha(+) DCs without impairing pDC development, and eliminates production of IL12p40, while retaining that of type I IFNs. Electrophoretic mobility shift and chromatin immunoprecipitation assays indicated that IRF8(R294C) failed to interact with partner transcription factors and did not bind certain promoters that require partner interactions. Together, this work indicates that IRF8-partner interactions play different roles in CD8alpha(+) DCs and pDCs, revealing a mechanistic separation that underlies development of these DC subsets.

Figure 1

Analysis of DC subsets in IRF8^R294C mice. (A) CD11c⁺ cells in spleen were analyzed for expression of the indicated DC subset markers by flow cytometry. (B) CD11c⁺ gated cells were tested for the pDC and IKDC markers, CD122 and PDCA1. (C) Total CD11c⁺ DCs per spleen. Values represent the average of 3 mice (± SD). (D) The percentages of 4 DC subsets in IRF8^WT and IRF8^R294C spleens. Values represent the average of 3 spleens (± SD). (E) BMDCs from IRF8^WT and IRF8^R294C mice grown in Flt3L were tested for B220 and CD24 as markers for pDCs and CD8α⁺ DCs, respectively. (F) BMDCs from IRF8^WT and IRF8^R294C mice were stimulated with CpG oligomer DNA D19 (1 μg/mL), LPS (100 ng/mL), or Poly IC (100 μg/mL) for 24 hours, and expression of CD8α was tested by flow cytometry. Numbers on plots are percentages of total cells.

Figure 2

Impaired cytokine induction and partner interactions in IRF8^R294C DCs. (A) BMDCs from IRF8^WT and IRF8^R294C mice were stimulated with indicated TLR ligands. IL12p40 and IFNα transcripts (measured 6 hours after stimulation) and proteins (measured 24 hours after stimulation) were measured by quantitative polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. Error bars represent SD. (B) BM cells from IRF8 KO mice were transduced with pMSCV vectors for IRF8^WT or IRF8^R294C, and expression of B220 and CD8α (24 hours after CpG stimulation) was detected by flow cytometry. (C) EMSA analysis: in vitro–transcribed and – translated proteins from control pcDNA, IRF8^WT, IRF8^R294C, and IRF8^R289E vectors (lanes 1–4) were mixed with the indicated in vitro–transcribed and – translated partner proteins and ³²p-labeled ISRE (for IRF2) and EICE (for PU.1 and SpiB). Asterisks indicates IRF8-partner complexes. Specificity of mobility shifts was verified by adding excess unlabeled probes (lane 5), which removed the shifted band or by adding antibodies for IRF8 (lane 6) or partner proteins (lane 7), which “supershifted” the band mobility. Lane 8 contained partner proteins without IRF8, which produced no shifted band. (D) Cystatin C transcript expression was tested for IRF8^WT and IRF8^R294C BMDCs (left) by quantitative reverse-transcription (RT)–PCR. ChIP analysis was performed for the Csc3 promoter for binding of IRF8 or PU.1 in above DCs. Normal rabbit IgG was used as a control. Data represent the average of 3 determinations (± SD). (E) Diagram of IRF8^R294C-directed DC development. The mutation abolishes the development of CD8α⁺ DCs, without affecting that of pDCs. The mutation results in increased CD4⁺ DCs and pDCs. The impaired ability to interact with partner proteins may partly account for the differential effect of this mutation on DC subset development.