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The circadian gene Arntl2 on distal mouse chromosome 6 controls thymocyte apoptosis

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Abstract

Nonobese diabetic (NOD) mice are a model for type 1 diabetes that displays defects in central immune tolerance, including impairment of thymocyte apoptosis and proliferation. Thymocyte apoptosis is decreased in NOD/Lt mice compared to nondiabetic C3H/HeJ and C57BL/6 mice. Analysis of a set of NOD.C3H and NOD.B6 congenic mouse strains for distal chromosome 6 localizes the phenotype to the 700 kb Idd6.3 interval. Idd6.3 contains the type 1 diabetes candidate gene aryl hydrocarbon receptor nuclear translocator-like 2 (Arntl2), encoding a circadian rhythm-related transcription factor. Newly generated Arntl2 −/− mouse strains reveal that inactivation of the B6 allele of Arntl2 is sufficient to both decrease thymocyte apoptosis and proliferation. When expressed from C3H or B6 alleles, ARNTL2 inhibits the transcription of interleukin 21 (Il21), a major player in the regulation of immune responses. IL-21 injection abolishes the B6 allele-mediated decrease of apoptosis and proliferation. Interestingly, IL-21 also leads to an increase in thymic proinflammatory Th17 helper cells. Our results identify Arntl2 as a gene controlling thymocyte apoptosis and proliferation along with Th17 development through the IL-21 pathway.

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Acknowledgments

We thank Pierre-Henri Commère, Corinne Veron, Chantal Bécourt, Gaelle Chauveau-Le Friec and Abokouo Zago for technical assistance, and Roberto Mallone for correcting the manuscript. The authors acknowledge the financial support of their work by Laboratoire d’Excellence Revive (Investissement d’Avenir; ANR-10-LABX-73), European Foundation for the Study of Diabetes (EFSD)/Juvenile Diabetes Research Foundation (JDRF)/Novo Nordisk Programme, Domaine d’intérêt majeur (DIM): Cardiovasculaire-Obésité-Rein-Diabète (CORDDIM) and by recurrent funding from the Centre national de la recherche scientifique (CNRS), Institut national de la santé et de la recherche médicale (INSERM) and Institut Pasteur.

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Authors and Affiliations

  1. Mouse Molecular Genetics Laboratory, CNRS URA 2578, Institut Pasteur, 25 rue du Docteur Roux, 75015, Paris, France

    Basile Lebailly, Philip Avner & Ute Christine Rogner

  2. University Pierre and Marie Curie, Cellule Pasteur UPMC, 25 rue du Docteur Roux, 75015, Paris, France

    Basile Lebailly

  3. Mouse Genetics Engineering Center, CNRS URA 2578, Institut Pasteur, 28 rue du Docteur Roux, 75015, Paris, France

    Francina Langa

  4. Inserm U1016 - CNRS UMR8104, Groupe Hospitalier Cochin, Université Paris Descartes, 123 boulevard Port-Royal, 75014, Paris, France

    Christian Boitard & Ute Christine Rogner

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  1. Basile Lebailly
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  2. Francina Langa
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  3. Christian Boitard
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  4. Philip Avner
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  5. Ute Christine Rogner
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Correspondence to Ute Christine Rogner.

Electronic supplementary material

Below is the link to the electronic supplementary material.

335_2016_9665_MOESM1_ESM.eps

Online Resource 1: Genetic configuration for distal chromosome 6 (GRCm38.p4) of the different mouse strains. C3H alleles are indicated by grey boxes, NOD alleles by white boxes, B6 alleles by black boxes. NOD.C3H congenic strains: 6.VIII, 6.VIIIa-c; Arntl2 -/- strains: NT28 and N.B62A-; Idd6 NOD.B6 congenic strains: N.B6, N.B6A-. Idds, markers and positions in Mb are shown to the left; +/+ = wildtype Arntl2, -/- mutated Arntl2. Supplementary material 1 (EPS 2020 kb)

335_2016_9665_MOESM2_ESM.eps

Online Resource 2: Examples of cytometric analyses. a) Selection of the lymphocytes (left panel), selection of the different types of lymphocytes in the thymus: DP = CD4+CD8+, DN = CD4-CD8- (middle panel) and selection of splenic CD4+ T cells (right panel); b) Characterization of apoptotic cells at early stage using annexin V (AnV) and propidium iodide (PI) in the thymus of mice from strain N.B6 (middle panel) and N.B6A2- (right panel); c) Characterization of the CD4+IL-21+ and CD4+IL-17+ cells in the thymus of mice from strain N.B6 (middle panel) and N.B6A2- (right panel) compared to an unstimulated sample (left panel). Supplementary material 2 (EPS 678 kb)

335_2016_9665_MOESM3_ESM.eps

Online Resource 3: Diabetes incidence a) spontaneously in the NOD (n=50), N.B6A2- (n=22) and N.B6 (n=27) strains. P-values: NOD versus N.B6A2- =0.012; NOD versus N.B6 <0.009; N.B6A2- versus N.B6 =0.947. b) Diabetes incidence after transfer to NOD/SCID mice of NOD (n=15), N.B6A2- (n=15) and N.B6 (n=15) CD25- splenocytes (5.5 x 106). P-values: NOD versus N.B6A2- =0.617; NOD versus N.B6 <0.0001; N.B6A2- versus N.B6 =0.001. (Roche, Mannheim, Germany). Animals were considered diabetic when their urine glucose level exceeded 250 mg/dL. Time-to-event distributions were calculated by Kaplan-Meier estimation and compared by log-rank tests during the period of observation. Supplementary material 3 (EPS 251 kb)

(P_values_numbers) Tables of P-Values for all strains. Supplementary material 4 (PDF 41 kb)

Supplementary material 5 (PDF 43 kb)

Supplementary material 6 (PDF 45 kb)

Supplementary material 7 (PDF 36 kb)

Supplementary material 8 (PDF 37 kb)

Supplementary material 9 (PDF 41 kb)

Supplementary material 10 (PDF 37 kb)

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Lebailly, B., Langa, F., Boitard, C. et al. The circadian gene Arntl2 on distal mouse chromosome 6 controls thymocyte apoptosis. Mamm Genome 28, 1–12 (2017). https://doi.org/10.1007/s00335-016-9665-4

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  • DOI: https://doi.org/10.1007/s00335-016-9665-4

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