All-or-none suppression of B cell terminal differentiation by environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin
Highlights
► TCDD suppresses B cell differentiation stimulated by LPS in an all-or-none mode. ► TCDD reduces the fraction of IgM-secreting cells, not the IgM level in those cells. ► A mathematical model indicates deregulation of AP-1 and Bach2 by AhR is involved. ► Both pathways interfere with the bistable switch underlying B cell differentiation. ► Disruption of the bistable switch leads to all-or-none mode of suppression.
Introduction
Activation of the humoral immune response to microbial challenge is an integral component of adaptive immunity. Many environmental pollutants and pharmaceutical compounds can modulate this process (Dooley and Holsapple, 1988, Peden-Adams et al., 2008, Salazar et al., 2005). To understand the toxicity mechanisms of environmental immunotoxicants, it is crucial to examine how these chemicals perturb the signaling dynamics of the molecular pathways and circuits underlying the physiological processes in immune cells. Studying the relevant pathways as dynamic systems and how they are perturbed by exogenous agents is a necessary step toward mechanistically-based interpretation and prediction of dose responses and improved risk assessment for human immune health.
Halogenated aromatic hydrocarbons (HAHs) are a ubiquitous class of environmental immunosuppressants, the most potent of which is 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (Kerkvliet, 2002, Sulentic and Kaminski, 2011). It has been well established that mature B cells are a highly sensitive target of TCDD (Sulentic and Kaminski, 2011). By acting via the aryl hydrocarbon receptor (AhR), TCDD suppresses the terminal differentiation of B cells into antibody-secreting plasma cells (Sulentic et al., 1998, Tucker et al., 1986, Vorderstrasse et al., 2001). Although some of the molecular targets of AhR in B cells are known (De Abrew et al., 2011, Henseler et al., 2009, Suh et al., 2002), it is largely unclear how the transcriptional dynamics of the B cell-specific gene circuit is perturbed by these targeted AhR pathways. Differentiation of B cells to plasma cells is an all-or-none phenomenon in individual cells, which express either low or high levels of immunoglobulin, respectively. Consequently, two distinct modes of suppression of the antibody response by TCDD are plausible in theory (Fig. 1A). In an all-or-none (binary) mode, the fraction of B cells that differentiate into antibody-secreting plasma cells would be reduced as the TCDD concentration increases, while the amount of immunoglobulin produced and secreted per plasma cell is unaffected. In a graded mode of suppression, the amount of immunoglobulin produced and secreted per plasma cell would be reduced by TCDD in a concentration-dependent manner while the fraction of plasma cells formed is largely unaffected. Additionally, a hybrid mode exhibiting a mixture of binary and graded suppression is also possible. The binary mode of suppression has been suggested based on earlier studies using Jerne plaque or ELISpot assays (Lu et al., 2010, Schneider et al., 2008, Tucker et al., 1986). However, since neither of these assays provides quantitative measurement of the intracellular immunoglobulin content in individual cells, the exact mode by which TCDD suppresses the antibody response and the responsible AhR signaling mechanism remain to be determined.
Terminal differentiation of B cells into plasma cells is believed to be controlled by a bistable gene regulatory network, which ensures all-or-none and irreversible differentiation dynamics (Bhattacharya et al., 2010, Martinez et al., 2012, Muto et al., 2010). The putative bistable-switch circuit is formed by interconnected feedback loops (Fig. 1B) composed of multiple transcription factors (TFs) including B cell lymphoma 6 (Bcl6), PR domain zinc finger protein 1 (Prdm1), paired box 5 (Pax5), BTB and CNC homology 2 (Bach2), and others (Lin et al., 2002, Mora-Lopez et al., 2007, Ochiai et al., 2006, Sciammas and Davis, 2004, Shaffer et al., 2002, Shapiro-Shelef and Calame, 2005, Tunyaplin et al., 2004, Vasanwala et al., 2002). Numerous studies have demonstrated that mature B cells and plasma cells have mutually exclusive expression profiles of these TFs, suggesting that these two cell types represent the two attractor states of the bistable-switch circuit (Barberis et al., 1990, Cattoretti et al., 1995, Johnson et al., 2005, Muto et al., 1998, Ochiai et al., 2006, Turner et al., 1994). A number of signal transduction pathways activated by antigens and cytokines can converge onto the bistable circuit as inputs to trigger the switch, driving B cells to differentiate into plasma cells (Calame, 2008). For the bacterial endotoxin lipopolysaccharide (LPS), a common polyclonal activator, Toll-like receptor 4 (TLR4)-mediated activation of activator protein 1 (AP-1) appears to be the primary signaling pathway (Ohkubo et al., 2005, Vasanwala et al., 2002). Pax5, highly expressed in B cells to maintain their identity and subsequently repressed in plasma cells, can be viewed as a primary output of the bistable system, and along with other TFs such as Bach2, it represses the production of immunoglobulin molecules in B cells (Delogu et al., 2006, Muto et al., 1998).
From a dynamic system's perspective, it could be argued that if TCDD blocks the signal transduction pathways that trigger the bistable gene circuit or interfere with the operation of the circuit itself, it would make the switching of the circuit by an activator more difficult. B cells would then be less likely to differentiate into plasma cells. However, for cells that do manage to switch the circuit on in the presence of TCDD, immunoglobulin genes would still be expressed at full levels because of the all-or-none nature of a bistable switch. This would lead to a binary mode of suppression. Alternatively, if TCDD directly inhibits the transcription and/or secretion of immunoglobulin molecules without interfering with the bistable switching process per se, B cells would differentiate into plasma cells unobstructed, but with each plasma cell producing a smaller amount of immunoglobulin molecules. This would produce a graded mode of suppression. Since TCDD suppresses the plasma cell response by perturbing multiple components of the underlying molecular network (Fig. 1B) including AP-1, Bach2 and immunoglobulin heavy chain (IgH) (De Abrew et al., 2011, Henseler et al., 2009, Suh et al., 2002), the possibility for both binary and graded modes of suppression exists. By using an integrated approach of in vitro experimentation and computational modeling of pathway perturbation, we demonstrate here that TCDD suppresses B cell differentiation in an all-or-none fashion at the level of individual cells. Our modeling study indicates that this all-or-none mode of suppression likely results from perturbations of pathways that interfere with the bistable gene circuit regulating B cell fate.
Section snippets
Animals
Virus-free, female B6C3F1 mice (6 weeks of age) were purchased from Charles River (Portage, MI, USA). Mice were randomized, transferred to plastic cages containing bedding (five per cage), and quarantined for 1 week. Mice were given food and water ad libitum and not used until their body weight reached 17–20 g. All experiments were approved by the Michigan State University Institutional Animal Care & Use Committee (East Lansing, MI, USA).
Chemicals
TCDD was purchased from Accustandard (New Haven, CT, USA)
LPS-activated all-or-none B cell differentiation
Flow cytometry revealed that in the absence of LPS there was a single distinct B cell population (CD19high) expressing low-level intracellular IgM (IgMlow) among freshly isolated mouse splenocytes (Fig. 2A). For cells continuously cultured with 5 μg/ml LPS, a distinct, second CD19high population displaying high levels of intracellular IgM (IgMhigh) gradually emerged over time. Histograms of intracellular IgM fluorescence intensity gated on viable B cells clearly showed two distinct peaks,
Discussion
Cells can respond to external perturbations with diverse gene expression patterns, which may vary in either an all-or-none or graded fashion (Biggar and Crabtree, 2001, Joers et al., 2004, Louis and Becskei, 2002, Rossi et al., 2000). The all-or-none mode of protein expression may arise from mechanisms including discrete gene promoter dynamics, ultrasensitive switches, and bistability (Huang and Ferrell, 1996, Ozbudak et al., 2004, Pirone and Elston, 2004, Zhang et al., 2006). As an important
Competing interests
The authors have declared no conflicts of interest. This manuscript has been reviewed by the U. S. Environmental Protection Agency and approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the agency; nor does the mention of trade names or commercial products constitute endorsement or recommendation for use.
Authors' contributions
All authors participated in the design of the in silico and in vitro experiments, and approved the final manuscript. QZ and SB developed the in silico model and performed numerical simulations. DEK and RBC2 performed the in vitro experiments. QZ and NEK wrote the manuscript. SB, RBC3, RST, MEA, and NEK critically reviewed the manuscript.
Acknowledgments
We would like to thank the Superfund Research Program of the National Institute of Environmental Health Sciences (P42ES04911 to NEK) for supporting this work.
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