Epidemiological studies provide very compelling evidence that prenatal and early postnatal environmental factors influence the risk of developing chronic diseases, such as diabetes, cardiovascular disease, and maybe even obesity (Gluckman and Hanson, 2004
; Jirtle and Skinner, 2007
; Rhind et al., 2003
). Although the evidence is not as extensive, environmental factors appear to influence the development and programming of the immune system as well (Dietert, 2005
; Holladay and Smialowicz, 2000
; Luebke et al., 2006
; Merlot et al., 2007
; Pinkerton and Joad, 2006
; Prescott, 2006
; Zhang et al., 2005
). Even subtle changes in immune function could result in poor vaccine efficacy and decreased resistance to infectious disease. Growing concern over emerging and re-emerging infectious diseases necessitates a better understanding of how environmental factors influence immunity and contribute to differential susceptibility to infectious diseases. Furthermore, imbalances in immune function can also enhance responsiveness to nonpathogenic antigens, as is the case in autoimmune disease and hypersensitivity reactions.
Among the many factors that contribute to the establishment of proper immunoregulatory balance, the AhR clearly plays an important role. Inappropriate or sustained AhR activation during fetal and neonatal development results in changes in the immune system that persist into adulthood. At higher doses of TCDD, thymic atrophy and bone marrow hypocellularity have been reported. However, at lower doses of TCDD, functional alterations are present in adult offspring in the absence of detectable alterations in the cellular composition of primary and secondary immune organs (Vorderstrasse et al., 2004
). This suggests AhR activation during development may reprogram the immune system such that certain responses are inappropriately elevated and other responses are dampened. Numerous epidemiological and animal studies have suggested that prenatal and early life environmental factors, including exposure to exogenous chemicals and maternal stress, influence states of health and disease later in life (Jirtle and Skinner, 2007
; Merlot et al., 2007
; Rhind et al., 2003
; Zhang et al., 2005
). Delineating the epigenetic mechanisms by which developmental programming occurs is an active area of study. In addition to determining how AhR modulates the programming of the immune system, it will be important to delineate whether exposure to TCDD, and other AhR ligands, during development skews programming due to interference with the function of endogenous AhR ligands, or via inappropriate alterations of AhR-responsive genes and signaling pathways. Interestingly, phenotypic characterization of transgenic mice with a constitutively active AhR suggests that alterations in immune parameters occur in the absence of TCDD (Andersson et al., 2003
; Tauchi et al., 2005
). Although this supposition awaits validation in the context of assessing specific immune responses in vivo,
it provides further evidence that the consequences of sustained AhR activation during the development of the immune system extend beyond toxicity caused by dioxin exposure.
The observation that the dose of TCDD administered to pregnant mice did not cause long-lasting changes their responses to influenza virus and OVA challenge strongly suggests that the persistence of functional changes following developmental exposure stems from reprogramming rather than residual TCDD. In support of this idea, it is important to recall that only a very small amount (≤ 0.5%) of the maternal dose is transferred to the fetus (Birnbaum, 1986
; Weber and Birnbaum, 1985
), and that hepatic levels of TCDD in 5-week-old offspring exposed gestationally and via lactation were 75% lower than levels in the fetus (Nau et al., 1986
). Thus, even when exposure continues via lactation, TCDD levels in adult offspring are substantially lower than levels detected in the fetus. In our exposure paradigm, where the maximum cumulative dose to the dam was 4 μg/kg, the amount of TCDD remaining in adult offspring is likely well below levels that are immunomodulatory. In fact, we have previously shown that adverse effects of TCDD on the immune response to influenza virus are dose-dependent (Vorderstrasse et al., 2003
). Administration of a single dose of 5 μg TCDD/kg modulated IFN-γ levels, T cell expansion in the lymph node, and the number of CD8+
T cells and neutrophils in the lung. However, at the lowest dose administered (1 μg/kg) these endpoints were unaffected. Given that we show here that TCDD causes long-lasting changes in developmentally exposed animals versus transient immune modulation in pregnant adult animals that were exposed acutely to the same chemical, it seems very likely that the developing immune system is more sensitive to modulation by AhR activation. This conclusion is consistent with the general thinking that the developing immune system is more sensitive to perturbation by environmental factors, and that adverse effects of a chemical are more persistent when exposure occurs during development rather than during adulthood. What remains to be determined is whether AhR activation modulates the mature and developing immune systems through the same or different molecular mechanisms.
A step-wise understanding of when different leukocyte subpopulations and their direct progenitors arise is an active area of study. Current information indicates that different leukocyte lineages begin to form at different times. Cells of the myeloid lineage are first detected during mid-gestation, whereas T cells and a thymus are not detected until late gestation, and lymphocyte maturation continues into early postnatal life (Dzierzak and Medvinsky, 1995
; Zhu and Emerson, 2002
). This suggests that factors that influence neutrophils may be sensitive to AhR-mediated programming during an earlier window of time than T cells. Our data support this idea. The representative functional responses examined herein suggest subtle but distinct differences in the critical periods during development that are the most sensitive to long-lasting perturbation by AhR activation. Impairment in the expansion of virus-specific CD8+
T cells was apparent when AhR was activated in late gestation and lactation and solely during lactation, suggesting that events that occur after parturition may be key regulatory targets of CD8+
T cell function. These events may include the generation of lymphoid lineage precursors and the selection of T cells in the thymus. In contrast, neutrophil recruitment and increased IFN-γ levels in the lung only showed significant treatment effects when AhR activation was initiated during gestation and continued via lactation. Thus, perturbation of these responses requires sustained AhR activation during an earlier period of time and lasting for a longer amount of time, and suggests that events in the AGM or fetal liver and spleen may be sensitive targets of AhR. One variable not fully accounted for in the present study is the slight variation in the total dose administered to the dam. For offspring exposed during gestation and lactation, gestation only and lactation only, the total cumulative dose to the dam was the same (4 μg/kg). However, for offspring exposed only during late gestation/lactation, the total dose administered to the dam was 2 μg/kg. Regardless of these subtle differences, the most striking effect of developmental exposure was observed in offspring of dams treated throughout gestation and 2 days after parturition.
Although AhR activation during development causes long-lasting changes in innate and adaptive responses to infection, these effects likely result from distinct AhR-mediated events within and extrinsic to bone marrow–derived cells. We show here that AhR activation during development modulates programming of bone marrow–derived cells directly, such that CD8+
T cells demonstrate decreased responsiveness to antigen even when transferred to recipients that have not been given exogenous AhR agonists. This does not prove TCDD acts directly within T cells, but indicates that this altered functional capacity is inherent in bone marrow–derived cells. In contrast, increased IFN-γ levels in the lung are probably not inherent to hematopoietic cells. This is a surprising result and suggests that AhR-regulated pathways extrinsic to bone marrow–derived cells play a pivotal role in enhancing IFN-γ levels in the infected lung. This is consistent with findings in adult mice acutely exposed to TCDD, in which AhR-mediated events extrinsic to hematopoietic cells direct enhanced IFN-γ production by phagocytic cells in the lung (Neff-LaFord et al., 2007
). Because the majority of IFN-γ+ cells in the lung are myeloid cells (primarily neutrophils), it is tempting to speculate that increased IFN-γ is simply due to the increased recruitment of neutrophils to the lung. However, the answer may not be that simple. Although the amount of IFN-γ in BAL fluid was enhanced in TCDD-exposed chimeras reconstituted with naïve CD45.1+
bone marrow cells, there was not a corresponding increase in the number of neutrophils. Thus, increased IFN-γ levels likely reflect changes in extrahematopoietic pathways that regulate IFN-γ production.
This dichotomy between AhR activation affecting functions of cells in the innate and adaptive arms of the immune system via distinct pathways is consistent with recent reports in adult mice exposed acutely to TCDD. Using a combination of adoptive transfer and bone marrow chimeras, it is clear that AhR-mediated modulation of T cell responses is dependent upon the presence of AhR within bone marrow–derived cells, whereas AhR-mediated increases in IFN-γ production by neutrophils and macrophages are not (Funatake et al., 2005
; Kerkvliet et al., 2002
; Lawrence et al., 2006b
; Neff-LaFord et al., 2007
). Similarly, the idea that the AhR modulates the immune system through a combination of direct effects in bone marrow–derived cells and indirect action in extra-hematopoietic tissues is supported by mechanistic studies of TCDD-induced thymic atrophy and bone marrow hypocellularity. Thymic atrophy caused by TCDD has been shown to require AhR in hematopoietic cells, but not in thymic stromal tissues (Staples et al., 1998
). Direct affects on thymocytes are further supported by studies using a T-cell specific lck
-conditional knockout of ARNT, which showed that mice that do not express ARNT in their T cells do not exhibit thymic involution following TCDD exposure (Laiosa et al., 2003
; Tomita et al., 2003
). However, there is also evidence that thymic stromal cells contribute to TCDD-induced thymic atrophy (Camacho et al., 2005
; Greenlee et al., 1985
; Kremer et al., 1994
; Riecke et al., 2003
). Similarly, effects of AhR on hematopoietic cells in the bone marrow likely reflect direct and indirect mechanisms. HSC from mice that were acutely treated with TCDD (40 μg/kg) were less able to reconstitute bone marrow of irradiated recipients (Sakai et al., 2003
). These effects were not observed at lower doses of TCDD, and were not observed in AhR-deficient mice, suggesting a direct, AhR-mediated effect on hematopoietic cells, at least at high doses of TCDD. However, AhR-dependent events in bone marrow stromal cells contribute to suppressed differentiation of B cell precursors, suggesting that, at least for the B cell compartment, AhR-mediated events in stromal cells also play a role (Allan et al., 2003
; Wyman et al., 2002
; Yamaguchi et al., 1997
). When taken together, these findings suggest that AhR-mediated alterations in primary immune organs (thymus and bone marrow) reflect a composite of changes in regulatory pathways within and extrinsic to bone marrow–derived cells.
The new findings reported here have important implications with regard to the design and interpretation of studies aimed at delineating how AhR contributes to immunoregulatory balance. For instance, in order to comprehend the mechanism at a more detailed level, we need to better understand how extra-hematopoietic cells modulate the magnitude and nature of the response of leukocytes to antigens. More broadly, this study reiterates the point made by others (Dietert, 2005
; Holsapple et al., 2004
) that a full understanding of how environmental factors in-play during development cause long-lasting and often subtle changes in immune function, we will need to conduct studies in which functional endpoints form a critical component.