A role for the aryl hydrocarbon receptor in hematopoietic precursors: Consequences to the knockout phenotype and 2,3,7,8-tetrachlorodibenzo-p-dioxin toxicity

The aryl hydrocarbon receptor (AhR), the mediator of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxicity, is a bHLH-PAS transcription factor responsible for the regulation of a growing list of genes. Among endpoints is immunotoxicity, a hall mark of which is thymic atrophy. Concomitant with decreased thymus size is a decrease in prothymocytes, the source population for thymocytes. Recently, Lin - Sca-1+ cKit+ (LSK) bone marrow cells, a population that includes hematopoietic stem cells (HSC), were identified as TCDD targets. In vitro studies using AhR-WT and AhR-KO LSK cells suggested differential abilities for proliferation. Furthermore, AhR-KO mice have a 2-fold elevation in immature B-cells. In order to understand the toxicity of TCDD to hematopoiesis, it is important to understand the role of the AhR in these processes. Studies in this thesis were designed to test the hypothesis that the AhR regulates HSC and progenitor proliferation. In addition, it was hypothesized that environmental toxicants acting through the AhR regulate hematopoietic cell proliferation to shift the numbers of these cells. Three animal models were employed for these experiments: (1) young adult AhR-KO mice, (2) young adult AhR-WT mice exposed to TCDD, and (3) fetal AhR-WT mice exposed to TCDD in utero. Methods used include immunophenotyping and colony forming assays to measure numbers of progenitors, dual DNA/RNA staining to measure cell cycle, and in vitro culture systems and in vivo BrdU incorporation to assess proliferation. The chemotherapeutic agent 5-fluorouracil was used to measure the hematopoietic stress response in AhR-KO mice. Numbers of both phenotypically- and functionally-defined hematopoietic stem and progenitor cells were altered in AhR-KO mice or following TCDD exposure to juvenile or fetal mice. In the juvenile mouse models, these increases and decreases were transient, time-of-day specific, and related to changes in the circadian rhythms governing these cells. AhR-KO primary cultures and in vivo BrdU incorporations demonstrated an increased proliferative ability, while TCDD-treated cultures demonstrated a reciprocal decrease in proliferative ability. Overall, these data demonstrate the need for a functional AhR in order to maintain normal proliferation rates and numbers of murine hematopoietic precursors.