An in vitro model of human hematopoiesis identifies a regulatory role for the aryl hydrocarbon receptor.

In vitro models to study simultaneous development of different human immune cells and hematopoietic lineages are lacking. We identified and characterized, using single-cell methods, an in vitro stromal cell-free culture system of human hematopoietic stem and progenitor cell (HSPC) differentiation that allows concurrent development of multiple immune cell lineages. The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor influencing many biological processes in diverse cell types. Using this in vitro model, we found that AHR activation by the highly specific AHR ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin, drives differentiation of human umbilical cord blood-derived CD34+ HSPCs toward monocytes and granulocytes with a significant decrease in lymphoid and megakaryocyte lineage specification that may lead to reduced immune competence. To our knowledge, we also discovered for the first time, using single-cell modalities, that AHR activation decreased the expression of BCL11A and IRF8 in progenitor cells, which are critical genes involved in hematopoietic lineage specification processes at both transcriptomic and protein levels. Our in vitro model of hematopoiesis, coupled with single-cell tools, therefore allows for a better understanding of the role played by AHR in modulating hematopoietic differentiation.

Role of Programmed Cell Death Protein-1 and Lymphocyte Specific Protein Tyrosine Kinase in the Aryl Hydrocarbon Receptor- Mediated Impairment of the IgM Response in Human CD5+ Innate-Like B Cells.

Innate-like B cells (ILBs) are a heterogeneous population B cells which participate in innate and adaptive immune responses. This diverse subset of B cells is characterized by the expression of CD5 and has been shown to secrete high levels of immunoglobulin M (IgM) in the absence of infection or vaccination. Further, CD5+ ILBs have been shown to express high basal levels of lymphocyte specific protein tyrosine kinase (LCK) and programmed cell death protein-1 (PD-1), which are particularly sensitive to stimulation by interferon gamma (IFNγ). Previous studies have demonstrated that activation of the aryl hydrocarbon receptor (AHR), a cytosolic ligand-activated transcription factor, results in suppressed IgM responses and is dependent on LCK. A recent study showed that CD5+ ILBs are particularly sensitive to AHR activation as evidenced by a significant suppression of the IgM response compared to CD5- B cells, which were refractory. Therefore, the objective of this study was to further investigate the role of LCK and PD-1 signaling in AHR-mediated suppression of CD5+ ILBs. In addition, studies were conducted to establish whether IFNγ alters the levels of LCK and PD-1 in CD5+ ILBs. We found that AHR activation led to a significant upregulation of total LCK and PD-1 proteins in CD5+ ILBs, which correlated with suppression of IgM. Interestingly, treatment with recombinant IFNγ reduced LCK protein levels and reversed AHR-mediated IgM suppression in CD5+ ILBs in a similar manner as LCK inhibitors. Collectively, these results support a critical role for LCK and PD-1 in AHR-mediated suppression of the IgM response in human CD5+ ILBs.

Cannabidiol selectively modulates interleukin (IL)-1ß and IL-6 production in toll-like receptor activated human peripheral blood monocytes.

Cannabidiol (CBD) is a major non-euphoric cannabis-derived compound that has become popular in its over-the-counter use. CBD possesses low affinity for cannabinoid receptors, while the primary molecular target(s) by which it mediates biological activity remain poorly defined. Individuals commonly self-medicate using CBD products with little knowledge of its specific immunopharmacological effects on the human immune system; however, research has established primarily in rodent models that CBD possesses immune modulating properties. The objective of this study was to evaluate whether CBD modulates the innate immune response by human primary monocytes activated through toll-like receptors (TLR) 1-9. Monocytes were activated through each TLR and treated with CBD (0.5-10 µM) for 22 h. Monocyte secretion profiles for 13 immune mediators were quantified including: IL-4, IL-2, IP-10, IL-1ß, TNFα, MCP-1, IL-17a, IL-6, IL-10, IFNγ, IL-12p70, IL-8, and TGF-ß1. CBD treatment significantly suppressed secretion of proinflammatory cytokine IL-1ß by monocytes activated through most TLRs, apart from TLRs 3 and 8. Additionally, CBD treatment induced significant modulation of IL-6 production by monocytes activated through most TLRs, except for TLRs 1 and 3. Most other monocyte-derived factors assayed were refractory to CBD modulation. Overall, CBD selectively altered monocyte-derived IL-1ß and IL-6 when activated through most TLRs. This study is of particular importance as it provides a direct and comprehensive assessment of the effects of CBD on TLR-activated primary human monocytes at a time when CBD containing products are being widely used by the public.

Identification of a Sensitive Human Immunological Target of Aryl Hydrocarbon Receptor Activation: CD5+ Innate-Like B Cells.

Xenobiotic-mediated activation of the aryl hydrocarbon receptor (AHR) is immunotoxic in a number of immune cell types, with the B cell being a well-established sensitive target. Recent advances have provided evidence that the B cell repertoire is a heterogeneous population, with subpopulations exhibiting vastly different cellular and functional phenotypes. Recent work from our laboratory identified the T cell specific kinase lck as being differentially regulated by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), which is a potent activator of AHR. While LCK is primarily expressed in T cells, a subset of CD5+ B cells also express LCK. CD5 positivity describes a broad class of B lymphocytes termed innate-like B cells (ILBs) that are critical mediators of innate immunity through constitutive secretion of polyvalent natural immunoglobulin M (IgM). We hypothesized that CD5+ ILBs may be sensitive to AHR-mediated immunotoxicity. Indeed, when CD5+ B cells were isolated from the CD19+ pool and treated with TCDD, they showed increased suppression of the CD40 ligand-induced IgM response compared to CD5- B cells. Further, characterization of the CD5+ population indicated increased basal expression of AHR, AHR repressor (AHRR), and cytochrome p450 family 1 member a1 (CYP1A1). Indeed the levels of AHR-mediated suppression of the IgM response from individual donors strongly correlated with the percentage of the B cell pool that was CD5+, suggesting that CD5+ B cells are more sensitive to AHR-mediated impairment. Together these data highlight the sensitive nature of CD5+ ILBs to AHR activation and provide insight into mechanisms associated with AHR activation in human B cells.

Δ9-Tetrahydrocannabinol (THC) Impairs CD8+ T Cell-Mediated Activation of Astrocytes.

CD8+ T cells can contribute to neuroinflammation by secretion of inflammatory cytokines like interferon γ (IFNγ) and tumor necrosis factor α (TNFα). Astrocytes, a glial cell in the brain, can be stimulated by IFNγ and TNFα to secrete the inflammatory cytokines, monocyte chemotactic protein 1 (MCP-1), interleukin 6 (IL-6), and interferon-γ inducible protein 10 (IP-10). Δ9-Tetrahydrocannabinol (THC), the primary psychoactive cannabinoid in Cannabis sativa, possesses potent anti-inflammatory activity. The objective of this investigation was to assess the effects of THC treatment on CD8+ T cell-mediated activation of astrocytes. CD3/CD28/IFNα- stimulated CD8+ T cells were treated with vehicle (0.03% EtOH) or THC and cocultured with U251 astrocytes. IP-10+, MCP-1+, and IL-6+ astrocytes were quantified by flow cytometry. LegendPlex™ was used to measure cytokine secretion by CD8+ T cells and flow cytometry was employed to quantify IFNγ, TNFα, and lysosomal-associated membrane protein 1 (LAMP-1) expression. Recombinant TNFα and IFNγ were used to stimulate MCP-1, IP-10, IL-6 responses in U251 astrocytes, which were measured by flow cytometry. Treatment with THC reduced CD8+ T cell-mediated induction of IP-10 and IL-6 responses in U251 astrocytes but had no effect on MCP-1. THC treatment differentially affected T cell effector functions such that IFNγ and degranulation responses were sensitive to THC-mediated ablation while TNFα was not. Lastly, THC treatment reduced the IFNγ-induced IP-10 response but had no effect on TNFα-induced MCP-1 response in U251 astrocytes. The results suggest that cannabinoid treatment can selectively reduce certain CD8+ T cell responses that contribute to stimulation of astrocytes. Graphical Abstract Treatment with THC can abate CD8+ T cell-dependent neuroinflammatory processes by inhibiting CD8+ cell differentiation into effector cells, suppressing CD8+ effector cell function, and reducing activation of astrocytes by CD8+ T cell-derived inflammatory cytokines.

Evaluation of immunologic and intestinal effects in rats administered an E 171-containing diet, a food grade titanium dioxide (TiO2).

The toxicity of dietary E 171, a food grade titanium dioxide was evaluated. A recent study reported rats receiving E 171 in water developed inflammation and aberrant crypt foci (ACF) in the gastrointestinal tract. Here, rats received food containing E 171 (7 or 100 days). The 100-day study included feeding E 171 after dimethylhydrazine (DMH) or vehicle only pretreatment. Food consumption was similar between treatment groups with maximum total cumulative E 171 exposure being 2617 mg/kg in 7 days and 29,400 mg/kg in 100 days. No differences were observed due to E 171 in the percentage of dendritic, CD4+ T or Treg cells within Peyer's patches or the periphery, or in cytokine production in plasma, sections of jejunum, and colon in 7- or 100-day E 171 alone fed rats. Differences were observed for IL-17A in colon (400 ppm E 171 + DMH) and IL-12p70 in plasma (40 ppm E 171 + DMH). E 171 had no effect on histopathologic evaluations of small and large intestines, liver, spleen, lungs, or testes, and no effects on ACF, goblet cell numbers, or colonic gland length. Dietary E 171 administration (7- or 100-day), even at high doses, produced no effect on the immune parameters or tissue morphology.

Characterization of Adult Canine Kidney Epithelial Stem Cells That Give Rise to Dome-Forming Tubular Cells.

Dome formation can occur in cultured tubular epithelial cells originating from various tissues, including the mammary gland and the kidney. The isolation and characterization of normal kidney epithelial stem cells that give rise to dome-forming tubular cells have never been reported. We attempted to isolate and characterize canine kidney epithelial stem cells using a simple cell culture method that we have previously used to isolate other adult human stem cells. Dome-forming kidney epithelial cells were derived from dissociated adult canine kidney tissues that were cultured in a modified keratinocyte serum-free medium supplemented with N-acetyl-l-cysteine, l-ascorbic acid 2-phosphate, nicotinamide, and fetal bovine serum. These cells exhibited high self-renewal capacity in long-term culture (growth for >13 months and 30 cumulative population doublings) and exhibited characteristics of stem cells, including (1) deficiency in gap junctional intercellular communication, (2) anchorage-independent growth, (3) expression of stem cell markers octamer-binding transcription factor 4 and SRY (sex determining region Y)-box 2, (4) expression of cell surface markers CD24 and CD133, and (5) multipotent differentiation into osteoblasts, adipocytes, chondrocytes, and dome-forming tubular cells. Most of these characteristics are shared by the well-known canine renal tubule-derived immortalized Madin-Darby Canine Kidney cell line. Furthermore, the putative canine kidney stem cells developed in this study formed budding tubule-like organoids on Matrigel and required high cell density (>4,000 cells/cm2) for sustained growth and confluency for dome formation. The signal transducer and activator of transcription-3 (STAT3) phosphorylation inhibitor, AG490, inhibited colony-forming efficiency and dome formation, whereas lipopolysaccharide, an activator of STAT3, increased colony-forming efficiency in a dose-dependent manner. These results are consistent with the hypothesis that high cell density induces STAT3 expression, which promotes both stem cell self-renewal and differentiation into tubular cells. Our novel cell culture method should be useful for the future development of normal human kidney stem cells for clinical applications and for studying mechanisms of nephrotoxicity.

Δ9-Tetrahydrocannabinol Suppresses Monocyte-Mediated Astrocyte Production of Monocyte Chemoattractant Protein 1 and Interleukin-6 in a Toll-Like Receptor 7-Stimulated Human Coculture.

Cannabis is widely used in the United States, with an estimated prevalence of 9.5%. Certain cannabinoids in Cannabis sativa, Δ9-tetrahydrocannabinol (THC) in particular, possess immune-modulating and anti-inflammatory activity. Depending on the context, the anti-inflammatory activity of cannabinoids may be beneficial (e.g., in treating inflammatory diseases) or detrimental to normal immune defense against pathogens. The potential beneficial effect of cannabinoids on chronic neuroinflammation has gained recent attention. Monocyte migration to the brain has been implicated as a key event in chronic neuroinflammation and in the etiology of central nervous system diseases including viral infection (e.g., human immunodeficiency virus-associated neurocognitive disorder). In the brain, monocytes can contribute to neuroinflammation through interactions with astrocytes, including inducing astrocyte secretion of cytokines and chemokines. In a human coculture system, monocyte-derived interleukin (IL)-1ß due to Toll-like receptor 7 (TLR7) activation has been identified to promote astrocyte production of monocyte chemoattractant protein (MCP)-1 and IL-6. THC treatment of the TLR7-stimulated coculture suppressed monocyte secretion of IL-1ß, resulting in decreased astrocyte production of MCP-1 and IL-6. Furthermore, THC displayed direct inhibition of monocytes, as TLR7-stimulated monocyte monocultures treated with THC also showed suppressed IL-1ß production. The cannabinoid receptor 2 (CB2) agonist, JWH-015, impaired monocyte IL-1ß production similar to that of THC, suggesting that THC acts, in part, through CB2. THC also suppressed key elements of the IL-1ß production pathway, including IL1B mRNA levels and caspase-1 activity. Collectively, this study demonstrates that the anti-inflammatory properties of THC suppress TLR7-induced monocyte secretion of IL-1ß through CB2, which results in decreased astrocyte secretion of MCP-1 and IL-6. SIGNIFICANCE STATEMENT: Because cannabis use is highly prevalent in the United States and has putative anti-inflammatory properties, it is important to investigate the effect of cannabinoids on immune cell function. Furthermore, cannabinoids have garnered particular interest due to their potential beneficial effects on attenuating viral-induced chronic neuroinflammation. This study utilized a primary human coculture system to demonstrate that the major psychotropic cannabinoid in cannabis, Δ9-tetrahydrocannabinol, and a cannabinoid receptor-2 selective agonist suppress specific monocyte-mediated astrocyte inflammatory responses.

Imiquimod and interferon-alpha augment monocyte-mediated astrocyte secretion of MCP-1, IL-6 and IP-10 in a human co-culture system.

Toll-like receptor 7 (TLR7)-activation has been implicated as a significant mechanism of neuroinflammation triggered by ssRNA viruses. Infiltration of monocytes into the brain and astrocyte activation occurs during in vivo TLR7-mediated neuroinflammation. The objective here was to determine whether the TLR7 agonist, imiquimod, and interferon-alpha (IFN-α), promote monocyte-mediated astrocyte secretion of pro-inflammatory factors. Using a human primary co-culture system, we demonstrate that monocytes, together with imiquimod and IFN-α, promote astrocyte secretion of MCP-1, IL-6 and IP-10. Furthermore, TLR7-induced monocyte-derived IL-1ß is critical for promoting the astrocyte response. Overall, this study provides a potential mechanism for TLR7-mediated neuroinflammation.

Suppression of CpG-ODN-mediated IFNα and TNFα response in human plasmacytoid dendritic cells (pDC) by cannabinoid receptor 2 (CB2)-specific agonists.

Plasmacytoid dendritic cells (pDC) compose 0.2-0.5% of circulating leukocytes but play a significant role in mounting host immune responses. Elevated and chronic activation of pDC are implicated in autoimmune disease like systemic lupus erythematosus and rheumatoid arthritis. Δ9-tetrahydrocannabinol (THC) is a well characterized cannabinoid with potent anti-inflammatory activity, but acceptance of THC as a treatment for autoimmune disorders has been hindered due to psychotropic activity. The psychotropic effects of THC are mediated through cannabinoid receptor 1 (CB1) expressed in the central nervous system while the immunomodulatory effects of THC result from THC binding to CB1 and CB2 on immune cells. Synthetic CB2-selective agonists have been developed to explore immune modulation by cannabinoids in the absence of psychotropic effects. The goal of these studies was to determine if the CB2-selective agonists, JWH-015 and JWH-133, have comparable efficacy to THC in modulating IFNα and TNFα responses by primary human pDC. Treatment with JWH-133 and JWH-015 inhibited CpG-induced IFNα and TNFα responses by pDC. Further, the phosphorylation of IRF7, TBK1, NFκB, and IKKγ, key events in pDC activation, were suppressed by THC, JWH-133, and JWH-015. Likewise, the phosphorylation of AKT at the S473 and T308 residues were differentially modulated by treatment with THC and both JWH compounds. Collectively, these results demonstrate the potential for CB2 targeted therapeutics for treatment of inflammatory conditions involving aberrant pDC activity.

Lymphocyte-Specific Protein Tyrosine Kinase (LCK) is Involved in the Aryl Hydrocarbon Receptor-Mediated Impairment of Immunoglobulin Secretion in Human Primary B Cells.

The aryl hydrocarbon receptor (AHR) is a cytosolic ligand-activated transcription factor involved in xenobiotic sensing, cell cycle regulation, and cell development. In humans, the activation of AHR by 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), a high affinity AHR-ligand, impairs the secretion of immunoglobulin M (IgM) to suppress humoral immunity. However, the mechanisms bridging the activation of AHR and the impairment of IgM secretion by human primary B cells remain poorly understood. Recent transcriptomic analysis revealed upregulation of lymphocyte-specific protein tyrosine kinase (LCK) in AHR-activated human primary B cells. LCK is a well-characterized tyrosine kinase that phosphorylates critical signaling proteins involved in activation and cytokine production in T cells. Conversely, the role of LCK in human primary B cells is not well understood. In the current studies, we have verified the transcriptomic finding by detecting AHR-mediated upregulation of LCK protein in human primary B cells. We also confirmed the role of AHR in the upregulation of LCK by using a specific AHR antagonist, which abolished the AHR-mediated increase of LCK. Furthermore, we have confirmed the role of LCK in the AHR-mediated suppression of IgM by using LCK specific inhibitors, which restored the IgM secretion by human B cells in the presence of TCDD. Collectively, the current studies demonstrate a novel role of LCK in IgM response and provide new insights into the mechanism for AHR-mediated impairment of immunoglobulin secretion by human primary B cells.

CLARITY-BPA: Effects of chronic bisphenol A exposure on the immune system: Part 2 - Characterization of lymphoproliferative and immune effector responses by splenic leukocytes.

Bisphenol A (BPA) is commonly used in the manufacturing of a wide range of consumer products, including polycarbonate plastics, epoxy resin that lines beverage and food cans, and some dental sealants. Consumption of food and beverages containing BPA represents the primary route of human BPA exposure, which is virtually ubiquitous. An increasing number of studies have evaluated the effects of BPA on immune responses in laboratory animals that have reported a variety of effects some of which have been contradictory. To address the divergent findings surrounding BPA exposure, a comprehensive chronic treatment study of BPA was conducted in Sprague-Dawley rats, termed the Consortium Linking Academic and Regulatory Insights on Toxicity of BPA (CLARITY-BPA). As a participant in the CLARITY-BPA project, our studies evaluated the effects of BPA on a broad range of immune function endpoints using spleen cells isolated from BPA or vehicle treated rats. This comprehensive assessment included measurements of lymphoproliferation in response to mitogenic stimuli, immunoglobulin production by B cells, and cellular activation of T cells, NK cells, monocytes, granulocytes, macrophages and dendritic cells. In total, 630 different measurements in BPA treated rats were performed of which 35 measurements were statistically different from vehicle controls. The most substantive alteration associated with BPA treatment was the augmentation of lymphoproliferation in response to pokeweed mitogen stimulations in 1 year old male rats, which was also observed in the reference estrogen ethinyl estradiol treated groups. With the exception of the aforementioned, the statistically significant changes associated with BPA treatment were mostly sporadic and not dose-dependent with only one out of five BPA dose groups showing a statistical difference. In addition, the observed BPA-associated alterations were mostly moderate in magnitude and showed no persistent trend over the one-year time period. Based on these findings, we conclude that the observed BPA-mediated changes observed in this study are unlikely to alter immune competence in adult rats.

Aryl Hydrocarbon Receptor Activation Suppresses EBF1 and PAX5 and Impairs Human B Lymphopoiesis.

Aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that mediates biological responses to endogenous and environmental chemical cues. Increasing evidence shows that the AHR plays physiological roles in regulating development, homeostasis, and function of a variety of cell lineages in the immune system. However, the role of AHR in human B cell development has not been investigated. Toward this end, an in vitro feeder-free human B cell developmental model system was employed using human cord blood CD34+ hematopoietic stem/progenitor cells. Using this model, we found that AHR activation by the high-affinity ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin significantly suppressed the generation of early B cells and pro-B cells from hematopoietic stem/progenitor cells, indicating the impairment of B cell lineage specification and commitment. Addition of an AHR antagonist reversed 2,3,7,8-tetrachlorodibenzo-p-dioxin-elicited suppression of early B and pro-B cells, suggesting a role of AHR in regulating B lymphopoiesis. Gene expression analysis revealed a significant decrease in the messenger RNA level of early B cell factor 1 (EBF1) and paired box 5, two critical transcription factors directing B cell lineage specification and commitment. Additionally, binding of the ligand-activated AHR to the putative dioxin response elements in the EBF1 promoter was demonstrated by EMSAs and chromatin immunoprecipitation analysis, suggesting transcriptional regulation of EBF1 by AHR. Taken together, this study demonstrates a role for the AHR in regulating human B cell development, and it suggests that transcriptional alterations of EBF1 by the AHR are involved in the underlying mechanism.

Aryl hydrocarbon receptor activation by 2,3,7,8-tetrachlorodibenzo-p-dioxin impairs human B lymphopoiesis.

The homeostasis of peripheral B cell compartment requires lifelong B lymphopoiesis from hematopoietic stem cells (HSC). As a result, the B cell repertoire is susceptible to disruptions of hematopoiesis. Increasing evidence, primarily from rodent models, shows that the aryl hydrocarbon receptor (AHR) regulates hematopoiesis. To study the effects of persistent AHR activation on human B cell development, a potent AHR agonist and known environmental contaminant, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was utilized. An in vitro B cell development model system was established by co-culturing human cord blood-derived HSCs with irradiated human primary bone marrow stromal cells. Using this in vitro model, we found that TCDD significantly suppressed the total number of hematopoietic stem and progenitor cells (HSPC) in a concentration-dependent manner. Cell death analysis demonstrated that the decrease in cell number was not due to cytotoxicity by TCDD. In addition, TCDD markedly decreased CD34 expression on HSPCs. Structure-activity relationship studies using dioxin congeners demonstrated a correlation between the relative AHR binding affinity and the magnitude of decrease in the number of HSPCs and CD34 expression, suggesting that AHR mediates the observed TCDD-elicited changes in HSPCs. Moreover, a significant reduction in lineage committed B cell-derived from HSCs was observed in the presence of TCDD, indicating impairment of human B cell development. Similar effects of TCDD were observed regardless of the use of stromal cells in cultures indicating a direct effect of TCDD on HSCs. Collectively, we demonstrate that AHR activation by TCDD on human HSCs impairs early stages of human B lymphopoiesis.

SHP-1 is directly activated by the aryl hydrocarbon receptor and regulates BCL-6 in the presence of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).

The environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), which is a strong AHR agonist, causes significant suppression of human B cell activation and differentiation. The current studies describe the identification of Src homology phosphatase 1 (SHP-1) encoded by the gene PTPN6 as a putative regulator of TCDD-mediated suppression of B cell activation. Shp-1 was initially identified through a genome-wide analysis of AHR binding in mouse B cells in the presence of TCDD. The binding of AHR to the PTPN6 promoter was further confirmed using electrophoretic mobility shift assays in which, specific binding of AHR was detected at four putative DRE sites within PTPN6 promoter. Time-course measurements performed in human B cells highlighted a significant increase in SHP-1 mRNA and protein levels in the presence of TCDD. The changes in the protein levels of SHP-1 were also observed in a TCDD concentration-dependent manner. The increase in SHP-1 levels was also seen to occur due to a change in early signaling events in the presence of TCDD. We have shown that BCL-6 regulates B cell activation by repressing activation marker CD80 in the presence of TCDD. TCDD-treatment led to a significant increase in the double positive (SHP-1hi BCL-6hi) population. Interestingly, treatment of naïve human B cells with SHP-1 inhibitor decreased BCL-6 protein levels suggesting possible regulation of BCL-6 by SHP-1 for the first time. Collectively, these results suggest that SHP-1 is regulated by AHR in the presence of TCDD and may, in part through BCL-6, regulate TCDD-mediated suppression of human B cell activation.

The Influence of Human Interindividual Variability on the Low-Dose Region of Dose-Response Curve Induced by 2,3,7,8-Tetrachlorodibenzo-p-Dioxin in Primary B Cells.

The influence of interindividual variability is not typically assessed in traditional toxicological studies. Given that chemical exposures occur in heterogeneous populations, this knowledge gap has the potential to cause undue harm within the realms of public health and industrial and municipal finances. A recent report from the National Research Council (NRC) suggests that when accounting for interindividual variation in responses, traditionally assumed nonlinear dose-response relationships (DRRs) for noncancer-causing endpoints would better be explained with a linear relationship within the low-dose region. To address this knowledge gap and directly test the NRC's assumption, this study focused on assessing the DRR between 2,3,7,8-tetracholorodibenzo-p-dioxin (TCDD) exposure and immune suppression in a cohort of unique human donors. Human B cells were isolated from 51 individual donors and treated with logarithmically increasing concentrations of TCDD (0-30 nM TCDD). Two endpoints sensitive to TCDD were assessed: (1) number of IgM-secreting B cells and (2) quantity of IgM secreted. The results show that TCDD significantly suppressed both the number of IgM-secreting B cells and the quantity of IgM secreted (P < .05). Statistical model comparisons indicate that the low-dose region of the two DRRs is best explained with a nonlinear relationship. Rather than assuming low-dose linearity for all noncancer-causing DRRs, our study suggests the need to consider the specific mode of action of toxicants and pharmaceuticals during risk-management decision making.

Suppression of human B cell activation by 2,3,7,8-tetrachlorodibenzo-p-dioxin involves altered regulation of B cell lymphoma-6.

The environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) produces marked suppression of the primary humoral immune response in virtually every animal species evaluated thus far. In addition, epidemiological studies performed in areas of dioxin contamination have identified an association between TCDD exposure and an increased incidence of non-Hodgkin's lymphoma (NHL). Recent studies using an in vitro CD40 ligand model of human B cell differentiation have shown that TCDD impairs both B cell activation and differentiation. The present study extends these findings by identifying B cell lymphoma-6 [BCL-6] as a putative cellular target for deregulation by TCDD, which may contribute to suppression of B cell function as well as NHL. BCL-6 is a multifunctional transcriptional repressor frequently mutated in NHLs and known to regulate critical events of B cell activation and differentiation. In the presence of TCDD, BCL-6 protein levels were elevated and concurrently the same population of cells with high BCL-6 levels showed decreased CD80 and CD69 expression indicative of impaired cellular activation. The elevated BCL-6 levels resulted in a concomitant increase in BCL-6 DNA binding activity at its cognate binding site within an enhancer region for CD80. Furthermore, a small molecule inhibitor of BCL-6 activity reversed TCDD-mediated suppression of CD80 expression in human B cells. In the presence of a low-affinity ligand of the aryl hydrocarbon receptor (AHR), suppression of B cell activation and altered BCL-6 regulation were not observed. These results provide new mechanistic insights into the role of BCL-6 in the suppression of human B cell activation by TCDD.

Differential modulation by delta9-tetrahydrocannabinol (∆9)-THC) of CD40 ligand (CD40L) expression in activated mouse splenic CD4+ T cells.

The anti-inflammatory activity of cannabinoids has been widely demonstrated in experimental animal models and in humans. CD40-CD40-ligand (L) interactions are among the most crucial initiators of inflammation. This study investigated the effects of ∆(9)-THC on CD40L expression in mouse splenic T cells after activation with various stimuli. Time course studies demonstrated that peak surface expression of CD40L by CD4(+) T cells after anti-CD3/CD28 or phorbol ester plus calcium ionophore (PMA/Io) occurred 8 h post activation. Peak CD40L mRNA levels were observed at 2 h post PMA/Io treatment and at 4 h post anti-CD3/CD28 treatment. Pretreatment with ∆(9)-THC significantly impaired the upregulation of CD40L induced by anti-CD3/CD28 at both the protein and mRNA level. By contrast, ∆(9)-THC did not affect PMA/Io-induced surface CD40L expression on CD4(+) T cells. Additionally, ∆(9)-THC also attenuated anti-CD3/CD28-induced CD40L expression on CD4(+) T cells derived from CB1(-/-)/CB2(-/-) mice. We investigated whether the mechanism by which ∆(9)-THC suppressed CD40L expression involved putative cannabinoid activation of the glucocorticoid receptor (GR). Although activation of GR resulted in suppression of CD40L induction by anti-CD3/CD28, no interaction between ∆(9)-THC and GR was observed by a glucocorticoid response element (GRE) luciferase reporter assay in HEK293T cells. Collectively, these results suggest that ∆(9)-THC targets proximal T cell receptor-associated signaling in a cannabinoid receptor- and glucocorticoid receptor-independent manner. These findings identify suppression of CD40L expression as a novel part of the mechanism by which ∆(9)-THC exerts anti-inflammatory activity.

2,3,7,8-Tetrachlorodibenzo-p-dioxin-mediated disruption of the CD40 ligand-induced activation of primary human B cells.

Suppression of the primary antibody response is particularly sensitive to suppression by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in mice; however, surprisingly little is known concerning the effects of TCDD on humoral immunity or B cell function in humans. Results from a limited number of previous studies, primarily employing in vitro activation models, suggested that human B cell effector function is suppressed by TCDD. The present study sought to extend these findings by investigating, in primary human B cells, the effects of TCDD on several critical stages leading to antibody secretion including activation and plasmacytic differentiation using an in vitro CD40 ligand activation model. These studies revealed important differences in the response of human and mouse B cells to TCDD, the most striking being altered expression of plasmacytic differentiation regulators, B lymphocyte-induced maturation protein 1 and paired box protein 5, in mouse but not human B cells. The activation of human B cells was profoundly impaired by TCDD, as evidenced by decreased expression of activation markers CD80, CD86, and CD69. The impaired activation correlated with decreased cell viability, which prevented the progression of human B cells toward plasmacytic differentiation. TCDD treatment also attenuated the early activation of mitogen-activated protein kinases (MAPK) and Akt signaling in human B cells. Collectively, the present study provided experimental evidence for novel mechanisms by which TCDD impairs the effector function of primary human B cells.

The aryl hydrocarbon receptor interacts with ATP5α1, a subunit of the ATP synthase complex, and modulates mitochondrial function.

Dioxins, including 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD), produce a wide range of toxic effects in mammals. Most, if not all, of these toxic effects are regulated by the aryl hydrocarbon receptor (AHR). The AHR is a ligand activated transcription factor that has been shown to interact with numerous proteins capable of influencing the receptor's function. The ability of secondary proteins to alter AHR-mediated transcriptional events, a necessary step for toxicity, led us to determine whether additional interacting proteins could be identified. To this end, we have employed tandem affinity purification (TAP) of the AHR in Hepa1c1c7 cells. TAP of the AHR, followed by mass spectrometry (MS) identified ATP5α1, a subunit of the ATP synthase complex, as a strong AHR interactor in the absence of ligand. The interaction was lost upon exposure to TCDD. The association was confirmed by co-immunoprecipitation in multiple cell lines. In addition, cell fractionation experiments showed that a fraction of the AHR is found in the mitochondria. To ascribe a potential functional role to the AHR:ATP5α1 interaction, TCDD was shown to induce a hyperpolarization of the mitochondrial membrane in an AHR-dependent and transcription-independent manner. These results suggest that a fraction of the total cellular AHR pool is localized to the mitochondria and contributes to the organelle's homeostasis.