Identification of Stage-Specific Gene Modulation during Early Thymocyte Development by Whole-Genome Profiling Analysis after Aryl Hydrocarbon Receptor Activation

Abstract

The aryl hydrocarbon receptor (AHR) is a basic helix-loophelix transcription factor, implicated as an important modulator of the immune system and of early thymocyte development. We have shown previously that AHR activation by the environmental contaminant and potent AHR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) leads to a significant decline in the percentage of S-phase cells in the CD3 Ϫ CD4 Ϫ CD8 Ϫ triple-negative stage (TN) 3 and TN4 T-cell committed thymocytes 9 to 12 h after exposure. In the more immature TN1-or TN2-stage cells, no effect on cell cycle was observed. To identify early molecular targets, which could provide insight into how the AHR acts as a modulator of thymocyte development and cell cycle regulation, we performed gene-profiling experiments using RNA isolated from four intrathymic progenitor populations in which the AHR was activated for 6 or 12 h. This microarray analysis of AHR activation identified 108 distinct gene probes that were significantly modulated in the TN1-4 thymocyte progenitor stages. Although most of the genes identified have specific AHR recognition sequences, only seven genes were altered exclusively in the two T-cell committed stages of early thymocyte development (TN3 and TN4) in which the decline of S-phase cells is seen. Moreover, all seven of these genes were reduced in expression, and five of the seven are associated with cell cycle regulatory processes. These seven genes are novel targets for modulation by the TCDD-activated AHR and may be involved in the observed cell-cycle arrest and suppression of early thymocyte development.

Aryl Hydrocarbon Receptor Activation in Hematopoietic Stem/Progenitor Cells Alters Cell Function and Pathway-Specific Gene Modulation Reflecting Changes in Cellular Trafficking and Migration

“…Only cell suspensions with viability Ͼ95% as measured by exclusion of trypan blue were used for further experimentation. All flow cytometric analyses and separations were performed on viable populations only, as measured by exclusion of the fluorescent dye 7-amino-actinomycin D (7AAD (Singh et al, 2010). To examine differentiation of LSK cells under conditions in vitro, LSK cells were obtained from mice treated with TCDD (30 g/kg, 7 days) or vehicle.…”

“…TCDD was obtained from Cambridge Isotopes (Andover, MA), and 6-g/ml aliquots were prepared as described previously (Laiosa et al, 2010). Either 30 g of TCDD in olive oil/kg b.wt.…”

“…1 and 2). A modified ability of HSCs and progenitors to move within the marrow niche may also explain the effects of AhR activation by TCDD on more differentiated cells [i.e., decreased thymic (Fine et al, 1990;Laiosa et al, 2010)] and altered B cell numbers (Thurmond et al, 2000). Trafficking events are dependent on the ability of HSCs to "sense" their microenvironment by cell-cell interactions and the recognition of soluble factors through cell surface proteins.…”

“…In addition, there is much data indicating that persistent activation of AhRs results in immunosuppression in different animal models (Stevens et al, 2009). AhR-dependent thymic atrophy, a hallmark of exposure to varying doses of TCDD, may be elicited by different mechanisms including 1) reduced seed-ing of the thymus by bone marrow (BM)-derived progenitors (Fine et al, 1990), 2) reduced stromal-mediated proliferation of thymocytes (Kremer et al, 1994;Frericks et al, 2007), and 3) a skewing of the thymic output through direct effects on developing thymocytes (Laiosa et al, 2010). In addition, AhR activation leads to 1) alterations of B lineage cells during gestation with possible consequences on autoimmunity (Mustafa et al, 2008), 2) decreased numbers of immature B cells (Murante and Gasiewicz, 2000), and 3) altered functional activity of early hematopoietic progenitors (Murante and Gasiewicz, 2000;Sakai et al, 2003;Singh et al, 2009).…”

Consequences of TCDD treatment on intra-hepatic lymphocytes during liver regeneration

“…Exposure to TCDD has also been shown to suppress in vivo hepatocyte proliferation in a rodent model of liver regeneration induced by 70% partial hepatectomy (PH) (Bauman et al, 1995;Mitchell et al, 2006), but no overt changes in p27Kip1 expression were detected in the regenerating liver of TCDD-treated mice. Interestingly, observations in other cell types indicate that TCDD treatment can elicit a cell cycle arrest independently of changes in p27Kip1 gene expression (Laiosa et al, 2010). Taken together, these findings raise the possibility that the mechanisms by which TCDD elicits a G 1 arrest in the regenerating liver may be distinct from those mechanisms identified in vitro.…”