2,3,7,8-Tetrachlorodibenzo-p-Dioxin Induces Transcriptional Activity of the Human Polymorphic hs1,2 Enhancer of the 3′Igh Regulatory Region

Abstract

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is an environmental toxicant known to inhibit antibody secretion and Ig expression. Inhibition of Ig expression may be partially mediated through repression of the 3’Igh regulatory region (3’IghRR). TCDD inhibits mouse 3’IghRR activation and induces aryl hydrocarbon receptor (AhR) binding to dioxin response elements (DREs) within the 3’IghRR enhancers: hs1,2 and hs4. The human hs1,2 enhancer (hu-hs1,2) is polymorphic due to the presence of one to four invariant sequences (IS), which have been correlated with several autoimmune diseases. The IS also contains a DRE-core motif. Therefore, the objective was to determine if hu-hs1,2 activity is sensitive to TCDD. Utilizing a mouse B-cell line (CH12.LX), we compared the effects of TCDD on mouse (mo-hs1,2) versus hu-hs1,2 activity. TCDD inhibited mo-hs1,2 similar to the mouse 3’IghRR. In contrast, hu-hs1,2 was activated by TCDD and antagonist studies supported an AhR-dependent activation, which was replicated in a human B-cell line (IM-9). Absence of Pax5 binding sites is a major difference between the human and mouse hs1,2 sequence. Insertion of the high affinity Pax5 site in hu-hs1,2 markedly blunted reporter activity but did not alter TCDD’s effect (i.e. no shift from activation to inhibition). Additionally, deletional analysis demonstrated a significant IS contribution to hu-hs1,2 basal activity but TCDD-induced activity was not strictly IS number-dependent. Taken together our results suggest that hu-hs1,2 is a significant target of TCDD and support species differences in hs1,2 regulation. Therefore, sensitivity of hu-hs1,2 to chemical-induced modulation may influence the occurrence and/or severity of human diseases associated with hu-hs1,2.

Allelic frequencies of the hs1.2 enhancer within the immunoglobulin heavy chain region in Dayton, Ohio patients screened for celiac disease with duodenal biopsy

“…Furthermore, there is a binding site (i.e. dioxin response element, DRE) for the aryl hydrocarbon receptor (AhR), which can be activated by a variety of structurally diverse ligands, including dietary and microbiota-derived ligands [23,30]. Indeed, it is becoming increasingly acknowledged that the AhR is an important environmental sensor and regulator of mucosal immunity and AhR knockout mouse models have demonstrated an essential role of the AhR in the structural integrity of the gut (reviewed by [31]).…”

“…DNA was extracted from buccal cells using the GenElute Mammalian Genomic DNA Kit following the manufacturer's instructions (Sigma-Aldrich, St. Louis, MO). Nested PCR was completed as previously outlined [17,23]. Briefly, for the first round of PCR, 1 μg of genomic DNA was used as a template to selectively amplify a 5.4 kb fragment of the 3′ IGH RR-1 (Fig.…”

“…Primers and PCR conditions were as described previously [23]. The nested PCR products were stained for 40 minutes in SYBR Green I Nucleic Acid and visualized using an 8% polyacrylamide gel and the LAS3000 Fuji imager (Fujifilm Medical Systems USA, Stamford, CT) [23]. Alleles 1, 2, 3, and 4 were associated with the following sizes: 297, 350, 403, and 456 bp, respectively.…”

“…Six members of the research team independently analyzed all genotyping gels for allele assignments. Additionally, allele assignments were confirmed by sequencing (Retrogen, San Diego, CA) a random sampling of nested PCR products as described previously [23]. …”

Impact of environmental factors on alloimmunity and transplant fate

“…Tolerance induction was associated with expansion of CD4 + C-D25 + Foxp3 + Tregs. While TCDD has been shown to inhibit B cell differentiation and antibody production in vitro (91)(92)(93)(94), no studies have yet demonstrated a potential benefit in transplantation. In humans, there is some observational evidence that traffic air pollution may shorten the life of lung transplants through the development of bronchiolitis obliterans syndrome (95).…”