Aryl hydrocarbon receptor is required for optimal B‐cell proliferation

Abstract The aryl hydrocarbon receptor (AhR), a transcription factor known for mediating xenobiotic toxicity, is expressed in B cells, which are known targets for environmental pollutants. However, it is unclear what the physiological functions of AhR in B cells are. We show here that expression of Ahr in B cells is up‐regulated upon B‐cell receptor (BCR) engagement and IL‐4 treatment. Addition of a natural ligand of AhR, FICZ, induces AhR translocation to the nucleus and transcription of the AhR target gene Cyp1a1, showing that the AhR pathway is functional in B cells. AhR‐deficient (Ahr −/−) B cells proliferate less than AhR‐sufficient (Ahr +/+) cells following in vitro BCR stimulation and in vivo adoptive transfer models confirmed that Ahr −/− B cells are outcompeted by Ahr +/+ cells. Transcriptome comparison of AhR‐deficient and AhR‐sufficient B cells identified cyclin O (Ccno), a direct target of AhR, as a top candidate affected by AhR deficiency.

A qPCR analysis of Ahr expression in splenic B220 + and plasma cell (PC) subsets and bone marrow PC subset sorted from C57Bl/6 mice. Ahr expression was normalized to Hprt1. n = 2 independent experiments; mean AE range. B qPCR analysis of Ahr expression in T H 17 and splenic B-cell subsets sorted from Il17Cre R26R eYFP mice. Ahr expression was normalized to Hprt1. n = 1 experiment; mean. C qPCR analysis of Ahr expression in splenic B-cell subsets sorted from C57Bl/6 mice. Ahr expression was normalized to Hprt1. n = 2 independent experiments; mean AE range. D qPCR analysis of Ahr expression in splenic CD19 + cells isolated from C57Bl/6 mice and cultured for 6 h as indicated. Ahr expression was normalized to Hprt1; Ahr expression was normalized among groups to medium without BI605906 (medium À). n = 2 independent experiments; mean AE range. FC: fold change. E Western blot analysis of whole protein extract from splenic CD19 + cells isolated from C57Bl/6 mice and cultured for 6 h as indicated. Values above the blots indicate AhR protein quantification obtained by densitometry, normalized to b-actin and compared to the sample treated with a-IgM without BI605906. Representative data of n = 2 independent experiments. F Western blot analysis of whole protein extract from splenic CD19 + cells isolated from C57Bl/6 mice and cultured for 60 min as indicated. Values above the picture indicate IjBa protein quantification obtained by densitometry, normalized to b-actin and compared to the sample treated with medium without BI605906. Representative data of n = 2 independent experiments.   Figure EV2. The Ahr fl/fl R26R eYFP allele combined with the mb1 Cre system allows B cell-specific Ahr deletion and eYFP expression.

A
Breeding strategy to generate B cell-specific Ahr À/À mice, all carrying Cre recombinase. Ahr fl/À mb1 Cre+ mice lack Ahr in B cells. Ahr fl/+ mb1 Cre+ mice are Ahr +/À in B cells. Cre activity is reported via eYFP expression. B, C qPCR analysis of Ahr expression in the indicated cell subsets sorted from bone marrow (B) and spleen (C) of non-immune Ahr fl/+ mb1 Cre+ and Ahr fl/À mb1 Cre+ mice.
Ahr expression was normalized to Hprt1. Sorting strategy from bone marrow is depicted in the dot plot shown in (B). n = 3 independent experiments; mean AE SEM. D, E Flow cytometry analysis of eYFP expression in bone marrow (D) and spleen (E) from non-immune Ahr fl/+ mb1 Cre+ mice. Cells were gated as indicated above the dot plots. Representative data of n = 3 independent experiments.   Figure EV3. B cell-specific Ahr deficiency does not cause overt alterations in steady-state B-cell immunity.

A
Flow cytometry analysis of distribution of B-cell subsets sorted from spleen, bone marrow (BM), peritoneal cavity (PeC) and Peyer's patches (PP) of eight-week-old male non-immune Ahr fl/+ mb1 Cre+ (black) and Ahr fl/À mb1 Cre+ (white) mice. n = 3 mice per group; mean AE SEM; unpaired two-tailed t-test. B-G ELISA quantification of indicated antibody isotypes in the serum of 8-week-old male and female non-immune Ahr fl/+ mb1 Cre+ (black) and Ahr fl/À mb1 Cre+ (white) mice. Line indicates mean value; unpaired two-tailed t-test.
The Ahr fl/+ mb1 Cre+ (black) and Ahr fl/À mb1 Cre+ (white) mice immunized i.g. with 2.5 lg/mouse cholera toxin (Ctx). Representative data of n = 3 independent experiments. Line indicates mean value. I-L ELISA quantification at d14 post-immunization of serum (I, K) and faecal (J, L) anti-Ctx IgA antibodies from male (I, J) and female (K, L) Ahr fl/+ mb1 Cre+ (black) and Ahr fl/À mb1 Cre+ (white) mice immunized i.g. with 2.5 lg/mouse cholera toxin. Representative data of n = 3 independent experiments. Line indicates mean value.  Figure EV5. AhR deficiency does not affect the affinity maturation process in vivo.
The EMBO Journal ª 2016 The Authors