All-or-none suppression of B cell terminal differentiation by environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin

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Abstract

Many environmental contaminants can disrupt the adaptive immune response. Exposure to the ubiquitous aryl hydrocarbon receptor (AhR) ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and other agonists suppresses the antibody response. The underlying pathway mechanism by which TCDD alters B cell function is not well understood. The present study investigated the mechanism of AhR-mediated pathways and mode of suppression by which TCDD perturbs terminal differentiation of B cells to plasma cells and thereby impairs antibody production. An integrated approach combining computational pathway modeling and in vitro assays with primary mouse B cells activated by lipopolysaccharide was employed. We demonstrated that suppression of the IgM response by TCDD occurs in an all-or-none (binary) rather than graded mode: i.e., it reduces the number of IgM-secreting cells in a concentration-dependent manner without affecting the IgM content in individual plasma cells. The mathematical model of the gene regulatory circuit underpinning B cell differentiation revealed that two previously identified AhR-regulated pathways, inhibition of signaling protein AP-1 and activation of transcription factor Bach2, could account for the all-or-none mode of suppression. Both pathways disrupt the operation of a bistable-switch circuit that contains transcription factors Bcl6, Prdm1, Pax5, and Bach2 and regulates B cell fate. The model further predicted that by transcriptionally activating Bach2, TCDD might delay B cell differentiation and increase the likelihood of isotype switching, thereby altering the antibody repertoire. In conclusion, the present study revealed the mode and specific pathway mechanisms by which the environmental immunosuppressant TCDD suppresses B cell differentiation.

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.

References (62)

  • S. Mangan et al.

    The coherent feedforward loop serves as a sign-sensitive delay element in transcription networks

    J. Mol. Biol.

    (2003)
  • F. Mora-Lopez et al.

    Human BSAP and BLIMP1 conform an autoregulatory feedback loop

    Blood

    (2007)
  • K. Ochiai et al.

    Plasmacytic transcription factor Blimp-1 is repressed by Bach2 in B cells

    J. Biol. Chem.

    (2006)
  • J.R. Pirone et al.

    Fluctuations in transcription factor binding can explain the graded and binary responses observed in inducible gene expression

    J. Theor. Biol.

    (2004)
  • F.M. Rossi et al.

    Transcriptional control: rheostat converted to on/off switch

    Mol. Cell

    (2000)
  • A. Schebesta et al.

    Transcription factor Pax5 activates the chromatin of key genes involved in B cell signaling, adhesion, migration, and immune function

    Immunity

    (2007)
  • A.L. Shaffer et al.

    Blimp-1 orchestrates plasma cell differentiation by extinguishing the mature B cell gene expression program

    Immunity

    (2002)
  • J. Suh et al.

    Aryl hydrocarbon receptor-dependent inhibition of AP-1 activity by 2,3,7,8-tetrachlorodibenzo-p-dioxin in activated B cells

    Toxicol. Appl. Pharmacol.

    (2002)
  • C.A. Turner et al.

    Blimp-1, a novel zinc finger-containing protein that can drive the maturation of B lymphocytes into immunoglobulin-secreting cells

    Cell

    (1994)
  • B.A. Vorderstrasse et al.

    Aryl hydrocarbon receptor-deficient mice generate normal immune responses to model antigens and are resistant to TCDD-induced immune suppression

    Toxicol. Appl. Pharmacol.

    (2001)
  • M. Watanabe-Matsui et al.

    Heme regulates B-cell differentiation, antibody class switch, and heme oxygenase-1 expression in B cells as a ligand of Bach2

    Blood

    (2011)
  • D. Adalsteinsson et al.

    Biochemical network stochastic simulator (BioNetS): software for stochastic modeling of biochemical networks

    BMC Bioinformatics

    (2004)
  • A. Barberis et al.

    A novel B-cell lineage-specific transcription factor present at early but not late stages of differentiation

    Genes Dev.

    (1990)
  • S. Bhattacharya et al.

    A bistable switch underlying B-cell differentiation and its disruption by the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin

    Toxicol. Sci.

    (2010)
  • S.R. Biggar et al.

    Cell signaling can direct either binary or graded transcriptional responses

    EMBO J.

    (2001)
  • C.J. Broccardo et al.

    Single cell analysis of switch-like induction of CYP1A1 in liver cell lines

    Toxicol. Sci.

    (2004)
  • R.B. Crawford et al.

    2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) alters the regulation and posttranslational modification of p27kip1 in lipopolysaccharide-activated B cells

    Toxicol. Sci.

    (2003)
  • K.N. De Abrew et al.

    An integrated genomic analysis of aryl hydrocarbon receptor-mediated inhibition of B-cell differentiation

    Toxicol. Sci.

    (2010)
  • B. Ermentrout

    Simulating, Analyzing, and Animating Dynamical Systems: A Guide to XPPAUT for Researchers and Students

    (2002)
  • D.T. Gillespie

    Exact stochastic simulation of coupled chemical reactions

    J. Phys. Chem.

    (1977)
  • C.Y. Huang et al.

    Ultrasensitivity in the mitogen-activated protein kinase cascade

    Proc. Natl. Acad. Sci. U.S.A.

    (1996)
  • Cited by (0)

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