Histopathologic and Transcriptomic Phenotypes of a Conditional RANKL Transgenic Mouse Thymus

Authors

Maria M Szwarc
Lan Hai
Vineet K Maurya
Kimal Rajapakshe
Dimuthu Perera
Michael M Ittmann
Qianxing Mo
Yong Lin
Matthew L Bettini
Cristian Coarfa
John P Lydon

Publication Date

12-1-2022

Journal

Cytokine

DOI

10.1016/j.cyto.2022.156022

PMID

36099756

PMCID

PMC10255830

PubMedCentral® Posted Date

6-9-2023

PubMedCentral® Full Text Version

Author MSS

Published Open-Access

yes

Keywords

Agglutinins, Animals, Cytokines, Doxycycline, Epithelial Cells, Forkhead Transcription Factors, Keratin-5, Ligands, Mice, Mice, Transgenic, NF-kappa B, Phenotype, RANK Ligand, Receptor Activator of Nuclear Factor-kappa B, Thymus Gland, Transcriptome, Thymus, Mouse, RANKL, Doxycycline, Cytokeratin 5, Cortex, Medulla, Transcriptome, RNA-seq

Abstract

Although conventional knockout and transgenic mouse models have significantly advanced our understanding of Receptor Activator of NF-κB Ligand (RANKL) signaling in intra-thymic crosstalk that establishes self-tolerance and later stages of lymphopoiesis, the unique advantages of conditional mouse transgenesis have yet to be explored. A main advantage of conditional transgenesis is the ability to express a transgene in a spatiotemporal restricted manner, enabling the induction (or de-induction) of transgene expression during predetermined stages of embryogenesis or during defined postnatal developmental or physiological states, such as puberty, adulthood, and pregnancy. Here, we describe the K5: RANKL bigenic mouse, in which transgene derived RANKL expression is induced by doxycycline and targeted to cytokeratin 5 positive medullary thymic epithelial cells (mTECs). Short-term doxycycline induction reveals that RANKL transgene expression is significantly induced in the thymic medulla and only in response to doxycycline. Prolonged doxycycline induction in the K5: RANKL bigenic results in a significantly enlarged thymus in which mTECs are hyperproliferative. Flow cytometry showed that there is a marked enrichment of CD4+ and CD8+ single positive thymocytes with a concomitant depletion of CD4+ CD8+ double positives. Furthermore, there is an increase in the number of FOXP3+ T regulatory (Treg) cells and Ulex Europaeus Agglutinin 1+ (UEA1+) mTECs. Transcriptomics revealed that a remarkable array of signals-cytokines, chemokines, growth factors, transcription factors, and morphogens-are governed by RANKL and drive in part the K5: RANKL thymic phenotype. Extended doxycycline administration to 6-weeks results in a K5: RANKL thymus that begins to display distinct histopathological features, such as medullary epithelial hyperplasia, extensive immune cell infiltration, and central tissue necrosis. As there are intense efforts to develop clinical approaches to restore thymic medullary function in the adult to treat immunopathological conditions in which immune cell function is compromised following cancer therapy or toxin exposure, an improved molecular understanding of RANKL's involvement in thymic medulla enlargement will be required. We believe the versatility of the conditional K5: RANKL mouse represents a tractable model system to assist in addressing this requirement as well as many other questions related to RANKL's role in thymic normal physiology and disease processes.

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