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. Author manuscript; available in PMC: 2022 Jan 30.
Published in final edited form as: Toxicology. 2020 Nov 28;448:152646. doi: 10.1016/j.tox.2020.152646

TCDD Attenuates EAE Through Induction of FasL on B Cells and Inhibition of IgG Production

Evangel Kummari 1, Erin Rushing 1, Ashleigh Nicaise 1, Amye McDonald 1, Barbara L F Kaplan 1,*
PMCID: PMC7785643  NIHMSID: NIHMS1650179  PMID: 33253778

Abstract

Previously we demonstrated that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) suppressed experimental autoimmune encephalomyelitis (EAE), a model to study multiple sclerosis (MS), through induction of regulatory T cells (Tregs) and suppression of effector T cell function in the spleen. Since B cells and specifically regulatory B cells (Bregs) have been shown to be so critical in the pathology associated with EAE and MS, we wanted to determine whether TCDD could also induce Bregs. We specifically hypothesized that a Fas ligand (FasL)+ Breg population would be induced by TCDD in EAE thereby triggering apoptosis in Fas-expressing effector T cells as one mechanism to account for inhibition of T cell function by TCDD. TCDD (0.1–2.5 μg/kg/day administered orally for 12 days) modestly increased the percentage of FasL+ B cells in the spleen and spinal cord in TCDD-treated EAE mice. However, we did not detect significant increases in percentages of FasL+ B cells using TCDD in vitro in mouse splenocytes or human peripheral blood mononuclear cells (PBMCs). Part of the modest effect by TCDD was likely related to the localized expression of FasL; for instance, in the spleen, FasL was more highly expressed by IgMhiIgDlo marginal zone (MZ) B cells, but IgMloIgDhi follicular (FO) B cells were more responsive to TCDD. Consistent with our observation of modest upregulation of FasL, we also observed modest changes in mitochondrial membrane potential in T cells co-cultured with isolated total B cells or IgM-depleted (i.e., FO-enriched) B cells from TCDD-treated EAE mice. These data suggest that while small microenvironments of apoptosis might be occurring in T cells in response to TCDD-treated B cells, it is not a major mechanism by which T cell function is compromised by TCDD in EAE. TCDD did robustly suppress IgG production systemically and in spleen and spinal cord B cells at end stage disease. Thus these studies show that TCDD’s primary effect on B cells in EAE is compromised IgG production but not FasL+ Breg induction.

Keywords: autoimmunity, AhR ligands

1. Introduction

The environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), an aryl hydrocarbon receptor (AhR) ligand, has been shown to suppress both B cell and T cell function in autoimmune diseases. TCDD completely attenuated autoimmune type 1 diabetes, which correlated with an increase in CD4+CD25+Foxp3+ T cells in pancreatic lymph nodes (Kerkvliet et al. 2009). TCDD weakened the progression of experimental autoimmune uveoretinitis by expanding the CD25+Foxp3+ Treg cell population and suppressing the activation of TH1 and TH17 cells (Zhang et al. 2010). In murine systemic lupus erythematosus, TCDD had a significant immunosuppressive effect with a decrease in CD4+ T cell population (Li and McMurray 2009). It is also well-characterized that TCDD significantly attenuated CD4+ T cells and expanded Treg cell populations in experimental autoimmune encephalomyelitis (EAE) in response to 1 μg TCDD/mouse i.p. (Duarte et al. 2013; Quintana et al. 2008) or 0.1–1 μg/kg/day over 12 days by oral gavage (0.024 – 0.24 μg/mouse total dosage) (Yang et al. 2016).

TCDD is also known to directly suppress the humoral immune response (Li et al. 2017). TCDD has been shown to impair B cell activation, B cell differentiation, and has been associated with B cell malignancies (Dooley and Holsapple 1988; Lu et al. 2011; Sherr and Monti 2013). It suppresses terminal differentiation of B cells to plasma cells, thereby reducing IgM and IgG antibody production (Sulentic et al. 1998; Sulentic and Kaminski 2011; Zhang et al. 2013; Zhou et al. 2018). Bacarelli et. al., have also shown that an increase in plasma TCDD levels is associated with a significant decrease in plasma IgG levels (Baccarelli et al. 2002). Chronic exposure to low dose TCDD causes an increase in CD19+ B cells and delayed B cell maturation (Feng et al. 2016).

In addition to direct effects on T and B cell activation and differentiation, TCDD has also been shown to induce apoptosis, which might contribute to immune suppression (Camacho et al. 2005; Dearstyne and Kerkvliet 2002; Kamath et al. 1999). TCDD-induced apoptosis was in some studies mediated by the Fas-FasL interaction (Camacho et al. 2005; Dearstyne and Kerkvliet 2002), although this has not been investigated in EAE.

Bregs can play an important role both in multiple sclerosis (MS) and EAE. For instance, Bregs control TH1 and TH17 cells in the central nervous system and lymph nodes in EAE (Matsushita et al. 2008). Bregs include glucocorticoid-induced tumor necrosis factor receptor family related gene ligand (GITRL)-positive B cells that contribute to T regulatory (Treg) cell maintenance (Ray et al. 2012), CD24+CD38+ B cells that contribute to the presence of Tregs and reduce disease (Flores-Borja et al. 2013; Song et al. 2016) and those that express FasL, which can trigger apoptosis in Fas+ target effector T cells (Cencioni et al. 2015; Fang et al. 2010; Shi et al. 2009).

Previously we showed that sub-chronic oral TCDD inhibited EAE, which was associated with induction of Tregs and suppression of effector T cells (Yang et al. 2016). Thus, we hypothesized that a FasL+ B cell population would be induced by TCDD in EAE thereby triggering apoptosis in effector T cells as one mechanism to account for inhibition of T cell function by TCDD. We utilized the EAE model and compared a single dose of 30 μg/kg TCDD given once in an i.p. injection versus oral administration over 12 days at 2.5 μg/kg/day (cumulative dose of 30 μg/kg). In addition to characterizing TCDD’s effects on FasL+ B cells, we also examined the effect of TCDD on IgG production in EAE. Together the results demonstrate differential localization of FasL expression in the spleen and modest responsiveness of precentages of FasL+ B cells to TCDD, but robust suppression of EAE-specific IgG antibody production. Although TCDD would not be developed as a therapeutic for MS, the mechanisms examined in these studies might inform the development of novel, non-toxic AhR ligands as treatments for immune-mediated diseases.

2. Materials and Methods

2.1. Animals

Three- to four-month-old female C57BL/6 mice from Envigo Laboratories were used for this study. All procedures performed on these mice were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and study protocols were approved by the Institutional Animal Care and Use Committee at Mississippi State University (17–427 and 20–219). The animals used in this project were housed in the AAALAC-accredited facilities of the College of Veterinary Medicine, Mississippi State University. They were maintained at 18–22°C with access to food and water ad libitum. Mice were monitored and access to food and water was ensured as disease progressed.

2.2. EAE induction

Mice received myelin oligodendrocyte glycoprotein (MOG) peptide (amino acids 35–55, MEVGWYRSPFSRVVHLYRNGK; MOG35–55) in Complete Freund’s Adjuvant (MOG in CFA) as described (Yang et al. 2016). On days 1–12 post immunization with MOG in CFA, mice were administered corn oil (CO) or TCDD (0.1–2.5 μg/kg/day) by oral gavage. In a separate study, mice were injected intraperitoneally (i.p.) with either 3.6% dimethylsufloxide (DMSO) in phosphate buffered saline (PBS) as the vehicle (VH) or 30 μg/kg/day of TCDD on day 1 only (Thurmond and Gasiewicz 2000). This dose was used to match the cumulative total of TCCD given orally at a dose of 2.5ug/kg/day over 12 days. Other studies have previously given a single bolus dose of 30 ug/kg of TCDD orally (Fling et al. 2020) or a 30ug/kg single i.p dose to study the effect of TCDD (Thurmond and Gasiewicz 2000) The mice were checked for clinical signs every other day for the first 10 days and then daily until day 18. Mice were scored clinically as follows: 0.5, flaccid tail; 1, awkward gait; 2, susceptible to flipping over without ability to right self; 3, single hind limb paralysis; 4, dual hind limb paralysis. Mice were not allowed to progress beyond a clinical score of 4. Spleen and spinal cord were collected at day 18 (Kummari et al. 2019).

2.3. Isolation of splenic leukocytes and B cells

The spleen was mechanically disrupted and passed through a 70-μm cell strainer to prepare a single cell suspension. Cells were centrifuged at 500 × g for 5 min at room temperature (RT) and the resulting pellet was suspended in culture media and enumerated on a Nexcelom cell counter using acridine orange/propidium iodide stain. For some experiments, B cells were isolated using the EasySep Mouse B cell isolation kit using immunomagnetic negative selection (Stemcell Technologies, Vancouver, BC). Briefly, single cell suspension of splenocytes were suspended in HBSS with 2% bovine calf serum (BCS) and 1 mM EDTA (isolation medium). For each spleen (≤1 × 108 cells) cells were suspended in 2 ml of isolation media in a polystyrene round bottom tube to which rat serum was added. Subsequently cells were incubated with antibody cocktail for 10 min at RT and RapidSpheres were added for 2.5 min at RT. The cells were then placed in the magnet, which binds all non-B cells, allowing the enriched B cells to be poured out of the tube and used for staining or assays. For JC-1 assays (below) part of the enriched B cell population was used to perform IgM depeletion to enrich the IgMloIgDhi FO B cell population. To perform this additional enrichment 20 μg biotinylated anti-mouse IgM antibody (Biolegend) was incubated per 107 B cells for 1 hr at RT while rotating the cell-antibody mix. Magnetized biotin binder beads (50 μl beader per 107 B cells) were then added to the mixture before it was placed in the magnet, allowing the IgM-depleted (IgMdepl) B cells to be poured out of the tube.

2.4. Isolation of spinal cord lymphocytes

The intact spinal cord was flushed out of the spinal column using sterile PBS and collected in RPMI. The spinal cord was then digested with collagenase D and DNase I and subjected to differential centrifugation with a 70%/30% Percoll gradient to isolate lymphocytes as described (Kummari et al. 2019).

2.5. Mouse splenic and human PBMC cultures

Healthy human PBMCs were purchased from Astarte Biologics (Bothell, WA). Human PBMCs or mouse splenocytes were seeded in culture plates in complete medium (RPMI containing 5% bovine calf serum, 1% penn-strep with 50 μM 2-mercaptoethanol or 1% glutamax for mouse or human, respectively). Cells were treated with vehicle (VH; 0.01% DMSO) or TCDD (30 nM) for 24, 48 or 72 hr.

2.6. Extracellular and Intracellular staining

Staining was performed on cells isolated from EAE mice or following in vitro treatments. Cells were stained with a viability dye (Biolegend, SanDiego, CA) in 1X PBS for 20 min and then washed in PBS. They were then incubated with Fc block for 15 min (and cells for intracellular IgG staining were also incubated with anti-IgG to block extracellular IgG), followed by fluorescentlyconjugated antibody staining for 30 min. Extracellular antibodies were all obtained from Biolegend: CD19 (PE/Cy7), FasL (PE), PD-L1 (APC), IgM (FITC) and IgD (PacBlue). The cells were washed in flow cytometry buffer (FCM; 0.1% bovine serum albumin in Hank’s buffered saline solution) and then fixed with Cytofix (BD Biosciences, San Jose, CA) for 15 min. For intracellular IgG or Cyp1a1, the cells were permeabilized and incubated with IgG (APC) or Cyp1a1 (purified; Abcam, Cambridge, MA) for 1 hr. Detection of Cyp1a1 also required incubation with a conjugated secondary prior to detection (donkey anti-rabbit AlexaFluor 647; Biolegend) for 30 min. After staining, the cells were washed and resuspended in FCM and analyzed on an ACEA Novocyte (San Diego, CA). Fluorescence minus one (FMO) controls provided guidance on gate setting. Data analysis was done with NovoExpress software.

2.7. Quantitative PCR

Splenocytes were treated with VH (0.1% DMSO) or TCDD (30 nM ) and incubated overnight. The cells were washed in PBS and pelleted at 500 × g for 5 min. RNA was isolated from cell pellets as per the manufacturer’s protocol using the RNA Easy Kit (Qiagen). The isolated RNA was quantified by Nanodrop, and all the samples were adjusted to the same concentration using nuclease-free water. Complementary DNA (cDNA) was synthesized using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, California) and used for quantitative real time-polymerase chain reaction (qRT-PCR). Data were analyzed using the delta delta Ct method with 18s rRNA as the endogenous reference. mRNA expression levels were expressed as fold change.

2.8. Prime flow

Splenocytes were treated with VH (0.1% DMSO) or TCDD (30 nM) on day 1 and incubated overnight then the PrimeFlow was initated according to the manufacturer’s protocol (Thermo Fischer Scientific) on day 2. Briefly, the cells were stained with CD19-PE/Cy7 and FasL-PE in FCM buffer containing 0.01% sodium azide and Fc block for 20 min at RT in the dark. Cells were then fixed overnight using the fixation buffers provided in the kit. On day 3, the cells were incubated with target probes Type 1-Bactin (APC) and Type 4-Cyp1a1 (FITC) at 40°C. Finally on day 4, the cells were incubated with amplification and hybridization buffers and signal was detected using fluorescent label probes. Cells were analyzed using an ACEA Novocyte and guidance for gate settings were made with FMO controls. Data analysis was done with NovoExpress software.

2.9. Immunohistochemistry

Slides with 10-micron frozen sections of spleen were air dried for 5 min and then fixed in an ice cold solution of 1:1 acetone and methanol for 5 min. The slide was then washed in PBS three times. The sections were incubated with anti-mouse CD19-AlexaFlour 488 (Southern Biotech, Birmingham, AL) and FasL-PE (Biolegend) at a dilution of 1:50 in 10% Triton-X PBS solution for 2 hr. The sections were again washed in PBS three times and incubated with DAPI for 10 min to stain nuclei, washed in PBS three more times, and coverslipped with CC mount.

2.10. ELISA

For quantification of MOG-specific IgG, a 96-well plate was coated with 100 μl per well of MOG35–55 peptide in PBS (50 μg/ml) and incubated overnight at 4°C. Washing steps were performed between each step using 0.05% Tween-20 in PBS and deionized water three times each. Plates were blocked with 3% BSA-PBS for 1 hr at RT after which serum samples were added to the wells for 1 hr at RT. MOG-specific IgG was detected using streptavidin peroxidase-conjugated IgG (Sigma, St. Louis, MO; 1:1000 dilution in 3% BSA-PBS) for 1 hr followed by addition of ABTS substrate. Color development was detected over a 1 hr-period in which absorbance values were determined every min at 405 nm on kinetic mode (SpectraMax M5, Molecular Devices, Sunnyvale, California). Data are presented as absorbance detected at 405 nm.

2.11. JC-1 Assay

Splenocytes, purified B cells and IgM-depleted B cells (i.e., FO-enriched) were isolated on day 18 from the spleens of EAE mice treated with CO or TCDD. Spleens from the SAL/CO mice were used to purify CD4+ T cells. CD4+ T cells were stained with BV650 antibodies and mixed in a 1:1 ratio with splenocytes, B cells or IgM-depleted B cells from EAE/CO or EAE/TCDD mice and allowed to incubate at 37°C overnight. The next day co-cultures were treated with JC-1 dye and CD4+ T cells were gated and analyzed for the ratio of aggregates to monomers (PE and FITC, respectively) on the ACEA Novocyte. Mitochondrial membrane changes consistent with apoptosis are detected by an increase in monomers (green or FITC signal) thereby reducing the ratio of aggregates to monomers. An example of staining detected by flow cytometry can be seen in Supplemental Figure 1 in which the mitochondrial membrane uncoupler FCCP was used as a positive control. B cell purity was assessed by staining separate samples with CD19-PE/Cy7 and IgM depletion (in experiment 2 only) was assessed by staining with IgM-FITC.

2.12. Statistical analysis

Flow cytometry data are presented as the mean percent gated ± SD of populations of interest. In vitro analyses were performed in triplicate and in vivo analyses were from at least 3 separate mice. Human PBMC data were performed on two separate days using 3 donors per experiment. Data were analyzed using a one-way or two-way ANOVA depending on the experimental design and post hoc tests were performed to assess differences between groups at p < 0.05. Percent and fold change data were transformed prior to ANOVA. Non-parametric data (clinical scores) were analyzed using a Kruskall-Wallis test. Since data for JC-1 staining were obtained from two separate experiments statistical analysis is not provided. For PrimeFlow® data, data from each column was converted to percent change using the formula (TCDD-VH/VH)*100 to generate the average change by TCDD from VH over the 4 separate experiments and statistical significance was determined using a Mann-Whitney test.

3. Results

3.1. TCDD modestly induced FasL on B cells.

Similar to our previous study using EAE without pertussis toxin (Yang et al. 2016), TCDD attenuated EAE disease following either oral or i.p. administration of TCDD (Fig. 1). We also showed previously that part of the mechanism by which TCDD attenuated EAE was inhibition of effector T cell function (Yang et al. 2016). The purpose of these studies was to determine whether TCDD could induce a population of Bregs. We focused on expression of FasL and PD-L1 as two possible Breg markers using splenocytes from our previously published study (Yang et al. 2016). We noted in this initial study that TCDD modestly increased the percentage of FasL CD19+ B cells at end-stage disease but produced no effect on programmed death-ligand 1 (PD-L1) expression (Fig 2A–B). We therefore further characterized FasL expression on B cells as they could be one possible subpopulation of regulatory B cells induced by TCDD to control other cells, such as T cells, by triggering apoptosis. Indeed, we confirmed in several studies that oral TCDD modestly increased the percentage of FasL in splenic B cells at doses up to 2.5 μg/kg/day (Fig. 3A). Interestingly, the percentage of FasL B cells was not consistently upregulated in the studies in which TCDD was administered i.p. once at the beginning of disease (Fig. 3B). However, the percent of cells expressing FasL was increased on spinal cord B cells following oral administration of TCDD, and the magnitude of expression was enhanced if the cells were also stained intracellularly with Cyp1a1 (Figs. 3C–D).

Fig. 1.

Fig. 1.

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TCDD attenuates clinical scores in EAE. EAE was induced in female C57BL/6 mice on day 0. Mice received 1 or 2.5 μg/kg/day on days 1–12 by oral gavage (n ≥ 15, A) or 30 μg/kg once on day 1 by i.p. injection (n = 19, B). Mice were scored every other day for the first 10 days then daily thereafter. Examples of clinical scores are: 0.5, flaccid tail; 1, awkward gait; 2, susceptible to flipping over without ability to right self; 3, single hind limb paralysis; 4, dual hind limb paralysis. Data are mean ± SD. Statistical differences were detected at p < 0.05 in panel A between SAL/CO and EAE/CO, a; EAE/CO and EAE/TCDD 1, b; EAE/CO and EAE/TCDD 2.5, c and in panel B between SAL/VH and EAE/VH, a; EAE/VH and EAE/TCDD 30, b.

Fig. 2.

Fig. 2.

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TCDD induced FasL on CD19+ B cells. EAE was induced in female C57BL/6 mice on day 0. Mice received 0.1 or 1 μg/kg/day on days 1–12 by oral gavage. Splenocytes were harvested on day 18 and stained for CD19 plus FasL (n ≥ 3, A) or PD-L1 (n ≥ 3, B). Cells are gated on live lymphocytes. *p < 0.05 as compared to respective corn oil (CO) vehicle. a, p < 0.05 as compared to respective SAL; b, p < 0.05 as compared to SAL/CO.

Fig. 3.

Fig. 3.

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Oral administration of TCDD induced FasL on CD19+ B cells in spleen and spinal cord. EAE was induced in female C57BL/6 mice on day 0. Mice received 1 or 2.5 μg/kg/day on days 1–12 by oral gavage (n ≥4, A; n = 3, C; n = 3, D) or 30 μg/kg once on day 1 by i.p. injection (n = 5, B). On day 18, spleens or spinal cord lymphocytes were isolated and stained for CD19 plus FasL (and intracellular Cyp1a1 in spinal cord in D). * p < 0.05 as compared to EAE/CO. SPLC, splenocytes; SC, spinal cord.

3.2. TCDD-mediated upregulation of FasL on B cells in vitro.

We next evaluated whether direct TCDD treatment of lymphocytes would increase FasL expression on B cells. We conducted two separate experiments in which mouse splenocytes or 3 separate healthy human donor PBMCs were treated with TCDD. Over the course of 72 hr, there was little upregulation of the percent cells expressing FasL on B cells by TCDD with the exception of modest, albeit not statistically significant, upregulation at 48 hr in several human donors (Figs. 4A–B). We also evaluated whether TCDD would increase Fasl mRNA expression in mouse splenocytes and found similarly modest upregulation of Fasl mRNA following in vitro TCDD treatment (Fig. 4C). Since the upregulation of FasL was so modest by TCDD we questioned whether we could increase the sensitivity of the analysis by focusing in on cells that were AhR-activated. To do this we employed PrimeFlow® so we could pre-gate on cells that were expressing Cyp1a1 mRNA then evaluated FasL protein expression on CD19+ B cells using flow cytometry (Fig. 4D). However, FasL expression on B cells was not increased, even when focusing on cells in which AhR was activated over several replicates of the experiment in which we increased sensitivity by increasing cell number (Table 1).

Fig. 4.

Fig. 4

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Effect of TCDD on FasL expression on B cells in vitro. Cells were treated with vehicle (VH, 0.01% DMSO) or TCDD (30 nM) for 24, 48 or 72 hrs. Mouse splenocytes (triplicate cultures, A) or human PBMCs (triplicate cultures from 3 donors per experiment, B) were analyzed for CD19 and FasL by flow cytometry. Mouse splenocytes were treated with VH or TCDD for 24 hr and FasL mRNA expression was assessed by real time QPCR (n = 4, C). Mouse splenocytes were treated with VH or TCDD for 24 hr and PrimeFlow was used to assess FasL protein expression in Bactin+CD19+Cyp1a1+ cells (D). Cells were gated sequentially on Bactin mRNA, CD19 protein and Cyp1a1 mRNA to examine FasL protein expression. Date from three experimental replicates are presented in Table 1. There were no statistically significant differences in these analyses.

Table 1. Effect of TCDD on FasL in AhR-activated cells.

SPLC were treated with VH (0.01% DMSO) or TCDD (30 nM) overnight followed by staining for Bactin and Cyp1a1 mRNA and CD19 and FasL protein expression by PrimeFlow. Cells were gated sequentially on Bactin, CD19 and Cyp1a1 to obtain the Cyp1a1 with FasL percent and counts. Four separate experiments were performed during which we increased cell numbers to increase sensitivity.

Sample Bactin %a Cyp1a1 %a FasL % Cyp1a1 with FasL % Cyp1a1 with FasL Count
VH 1 9.66 2.61 0.57 0.16 7
TCDD 1 7.45 5.04 0.12 0.10 4
VH 2 37.56 1.45 0.36 0.28 67
TCDD 2 22.37 0.59 0.26 0.21 24
VH 3 24.85 0.93 0.43 0.35 56
TCDD 3 13.71 1.67 1.11 1.05 58
VH 4 11.11 6.57 1.61 1.95 461
TCDD 4 8.37 8.41 1.39 1.83 302
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a

Using the percent change formula (TCDD-VH/VH)*100 to generate the average change by TCDD from VH over the 4 separate experiments, Bactin% and Cyp1a1% were significantly different at p < 0.05.

3.3. FasL is differentially expressed in the spleen.

In our various evaluations of FasL expression in mouse splenocytes, we also determined that FasL was differentially expressed in the spleen with predominant staining in marginal zone (MZ) B cells (Fig. 5A). We therefore conducted some analyses of the effect of TCDD in EAE focusing on IgMhiIgDlo (MZ) B cells versus IgMloIgDhi follicular (FO) B cells. We confirmed by flow cytometry that FasL was more highly expressed on MZ B cells (Fig. 5B). We also showed that the modest upregulation of percentage of FasL B cells by TCDD in EAE was more readily detected on the FO B cells (Figs. 5C–D). The differential expression of FasL in the spleen might account for our consistently modest upregulation of the FasL+ B cell population by TCDD.

Fig. 5.

Fig. 5.

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Differential expression of FasL in the spleen. Frozen sections of the spleen were stained with AlexaFluor 488 CD19 antibody (green) and PE FasL (red) and DAPI nuclear stain (blue) (A). Images were taken at 20X objective. Splenocytes were stained for IgM, IgD and FasL. FasL expression was assessed on MZ B cells, IgMhiIgDlo (red), versus FO B cells, IgMloIgDhi (gray) (B). EAE was induced in female C57BL/6 mice on day 0. Mice received 2.5 μg/kg/day on days 1–12 by oral gavage. Splenocytes were harvested on day 18 and stained for IgM, IgD and FasL (n > 4, C; n ≥ 4, D). Cells are gated on live lymphocytes that are MZ or FO. * p < 0.05 as compared to EAE/CO.

3.4. TCDD-treated splenocytes modestly decreased JC-1 ratio in target T cells.

Even the modest localized expression of FasL by TCDD suggests there could be TCDD-mediated apoptosis in Fas+ target cells. We confirmed that Fas was expressed on CD4+ and CD8+ T cells in mouse splenocytes (Fig. 6), suggesting they could be targets of Fas-mediated apoptosis by FasL-expressing cells. γδ T cells did not express high levels of Fas, so are not likely sensitive to FasL-mediated apoptosis (Fig 6). Thus, we evaluated changes in JC-1 dye ratio in CD4+ T cells as an indication of loss of mitochondrial membrane potential consistent with apoptosis. For these studies, we first isolated CD4+ T cells from control (SAL/CO) mouse splenocytes and stained them with CD4-BV650. We mixed the stained CD4+ T cells with cells isolated from day 18 EAE/CO or EAE/TCDD mice. We used total splenocytes, isolated B cells and IgMdepl B cells in order to enrich the IgD population (i.e., FO B cells) that had more pronounced upregulation of FasL in response to TCDD (Fig. 5D). After an overnight incubation of pre-stained CD4+ T cells with cells from EAE/CO or EAE/TCDD mice, cells were stained with JC-1 dye and CD4+ T cells were evaluated by flow cytometry based on the CD4-BV650 gate. As shown in Table 2, two separate experiments were perfomed. Splenocytes (SPLC) did not show a consistent reduction in the CD4+ T cell ratio by EAE/TCDD cells as compared to EAE/CO cells across the two experiments. However there was a small but consistent reduction in the CD4+ T cell ratio when EAE/TCDD-treated B cells or IgMdepl B cells were compared with EAE/CO-treated B cells or IgMdepl B cells. These results suggest that TCDD-treated B cells or TCDD-treated IgMdepl B cells reduced the JC-1 dye ratio in CD4+ cells consistent with apoptosis, although it was still quite modest.

Fig. 6.

Fig. 6.

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T cells express Fas. Untreated splenocytes were stained for CD4, CD8 or γδ T cells for Fas expression.

Table 2. Effect of TCDD-treated cells on CD4+ T cell mitochondrial membrane potential.

SPLC from EAE/CO or EAE/TCDD mice were harvested on day 18. SPLC were used directly or used for B cell isolation and subsequent depletion of IgM+ cells (IgMdepl). Cells (SPLC, purified B or IgMdepl B) were mixed in a 1:1 ratio with CD4+ T cells purified from SPLC obtained from the SAL/CO mice and allowed to incubate at 37°C overnight. The next day co-cultures were treated with JC-1 dye and analyzed immediately by flow cytometry. Co-culture cells were gated on CD4+ lymphocytes to determine the ratio of aggregates to monomers in T cells. Mitochondrial membrane changes consistent with apoptosis are detected by an increase in monomers (green signal) thereby reducing the ratio of aggregates to monomers. Purity of B cells (% CD19) and magnitude of IgM depletion (% IgM on CD19) were determined in separate samples. N/A, not available. Since data were obtained from two separate experiments statistical analysis is not provided.

Cells used in coculture Experiment 1 Experiment 2
CD4+ T cell Ratio (Aggregates/Monomers) % CD19 % IgM on CD19 CD4+ T cell Ratio (Aggregates/Monomers) % CD19 % IgM on CD19
SPLC
EAE/CO		 0.374 46.71% N/A 0.297 34.74% 11.98%
SPLC
EAE/TCDD		 0.305 54.39% N/A 0.683 42.69% 13.45%
B
EAE/CO		 0.406 87.34% N/A 0.425 84.81% 18.27%
B
EAE/TCDD		 0.400 93.42% N/A 0.406 87.05% 17.97%
IgMdepl
EAE/CO		 0.402 88.96% N/A 0.555 77.35% 2.27%
IgMdepl
EAE/TCDD		 0.376 93.19% N/A 0.428 86.72% 2.83%
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3.5. TCDD inhibited IgG production in EAE.

In addition to examining the effect of TCDD on regulatory B cell subsets, we determined the effect of TCDD on antibody production in EAE. Consistent with disease attenuation, TCDD suppressed circulating MOG-specific IgG (Fig. 7A–B). TCDD also suppressed intracellular IgG production in both splenocytes and spinal cord (Fig. 7C–E). Interestingly, the population of IgG that was upregulated in EAE and sensitive to suppression by TCDD was the CD19lo population (Fig. 7F).

Fig. 7.

Fig. 7.

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TCDD suppressed IgG production in EAE. Mice were euthanized at day 18 and IgG production was assessed in blood, spleen and spinal cord. A,B.) MOG-specific IgG was detected by ELISA using serum from EAE mice treated with TCDD via oral gavage over 12 days (n ≥ 7, A.) or via ip injection once (n = 5, B). ELISA plates were pre-coated with MOG35–55 peptide and detected following IgG peroxidase secondary antibody. C-E.) Intracellular IgG was detected in splenocytes (n ≥3, C; n =5, D.) or spinal cord (n = 3, E.) by flow cytometry. TCDD was administered by oral gavage (n ≥ 3; C, E.) or i.p. (n = 5; D.). Panel F shows the robust upregulation of intracellular IgG in CD19- cells, which was sensitive to inhibition with TCDD. Cells were stained with viability dye followed by Fc receptor blockade. Extracellular IgG was blocked using an unstained IgG antibody prior to permeabilization. Cells were stained with extracellular CD19-PE/Cy7 and intracellular IgG-APC. Cells are gated on live lymphocytes. * p < 0.05 as compared to EAE/CO.

4. Discussion

EAE is a well established animal model to study multiple sclerosis and the role of Bregs in MS (Dendrou et al. 2015; Lassmann 2010; Ray et al. 2012). Previous data from our lab and others have shown that TCDD suppressed EAE disease through suppression of effector T cells and induction of Tregs (Duarte et al. 2013; Quintana et al. 2008; Yang et al. 2016). TCDD is also a well-known modulator of B cell function, in particular through suppressing IgM antibody production (Sulentic and Kaminski 2011). Thus, the goal of this study was to further characterize TCDD effects on B cells and specifically determine if TCDD could induce Bregs as part of the mechanism to inhibit effector T cells, induce Tregs, or both. Several subsets of Bregs exist but the focus of this study was to examine TCDD’s effect on FasL+ B cells in EAE.

Across several replicates of the experiment in which TCDD was administered to EAE mice either via oral gavage or i.p. administration, we demonstrated that TCDD robustly attenuated disease and that a greater percentage of splenic and spinal cord B cells expressed FasL. The percentages were modest across experiments in vivo, and we did not detect an increase in FasL percentage in mouse or human B cells treated with TCDD in vitro, even in AhR-activated cells as assessed by PrimeFlow®. Together these data suggest that TCDD upregulated FasL only in the context of EAE. Interestingly in our previous study, we observed that TCDD induced Tregs in EAE mice but not in healthy mice (Yang et al. 2016), although other studies have demonstrated that TCDD treatment alone induces Tregs in vivo (Funatake et al. 2005; Zhang et al. 2010). Perhaps our inability to detect Bregs (or Tregs) in response to TCDD only in vivo is due to the timing between last administration of TCDD and evaluation of Bregs as we evaluated CD19+ FasL+ B cells 17 days after the single i.p. administration and 6 days after 12 days of oral dosing. Indeed the detection of Tregs in response to TCDD peaked at day 2 after TCDD dosing in a graft versus host disease model (Funatake et al. 2008).

Another part of the reason for the modest upregulation of percentage of FasL B cells might be attributed to the splenic localization of FasL expression as we found that FasL was expressed higher on IgMhiIgDlo MZ B cells, but it was the IgMloIgDhi FO B cells that were more responsive to TCDD. Since FasL expression changes might not be the only determinant as to whether B cells could trigger T cell apoptosis, we performed a functional assay to ascertain whether apoptosis was occurring in target cells in response to TCDD-treated cells from EAE mice. We used flow cytometry for this experiment since it required identification of T cells undergoing apoptotic changes in response to the presence of TCDD-treated splenocytes, B cells and IgM-depleted B cells (i.e., FO B cells denoted as IgMdepl). Of these populations the purified B cells and IgMdepl B cells produced mitochondrial membrane changes consistent with apoptosis in CD4+ T cells, which was consistent with our observation that TCDD upregulated the percentage of FasL FO B cells in EAE. Similar to the modest increase in CD19+FasL+ B cells, the mitochondrial membrane changes detected in target T cells was also modest, suggesting that while TCDD induced FasL+ Bregs in EAE, it is not likely a major mechanism by which TCDD attenuates EAE. We cannot exclude the possibility that the JC-1 staining merely showed changes in mitochondrial membrane potential, and not apoptosis, but since the changes were so modest, we did not pursue other apoptosis or mitochondrial membrane potential assays.

We are not the first to identify FasL upregulation as a possible mechanism for immune suppression by TCDD. Upregulation of FasL by TCDD was shown to be an important pathway for thymic involution produced by TCDD (Camacho et al. 2005; Kamath et al. 1999), although there are a couple of studies showing that FasL upregulation by TCDD was not a major contributor (Beamer et al. 2019; Nagai et al. 2006). The differences in the roles for TCDD-mediated increases in FasL in thymic involution might be related to the cell type on which FasL is expressed; for instance, FasL expression in thymic CD45- stromal cells was found to be important for thymic involution (Camacho et al. 2005). In another study of TCDD enhancement of liver injury, TCDD-mediated FasL induction on natural killer cells was found to be important for increased sensitivity to liver injury (Fullerton et al. 2013). Together these data demonstrate that cell-specific expression of FasL likely dictates the contribution that TCDD-mediated apoptosis makes in immune modulation, which is similar to what we showed here with FasL being more sensitive to TCDD in the IgMloIgDhi FO B cells as compared to the IgMhiIgDlo MZ B cells.

A role for FasL+ B cells to control effector T cells has been shown in other disease states, such as arthritis (Lundy and Fox 2009) and Schistosoma mansoni infection in mice (Lundy et al. 2001). It was interesting that a follow up study conducted in the S. mansoni model showed FasL was upregulated on B1a+ and CD5- B cells (Lundy and Boros 2002). These results are consistent with our findings that subsets of splenic B cells differentially express FasL, although we did not examine whether percentage of B1a or CD5 FasL cells was increased by TCDD in EAE.

While TCDD induction of CD19+FasL+ cells did not appear to be a major mechanism by which TCDD attenuated EAE, we did note that TCDD robustly inhibited MOG-specific IgG production in serum and intracellular IgG in splenocytes (at least in response to oral TCDD) and spinal cord. Suppressed IgG by TCDD has been reported in previous animal models (Feng et al. 2016; Harper et al. 1994; House et al. 1990; Inouye et al. 2003; Lawrence and Vorderstrasse 2004; Li and McMurray 2009; Sharma et al. 1984) and in humans in exposure incidents (Baccarelli et al. 2002; Baccarelli et al. 2004; Neubert et al. 2000; t Mannetje et al. 2018). Thus our results do not reveal TCDD-mediated IgG suppression as a novel mechanism of suppression, but they do show that intracellular IgG was suppressed by TCDD in EAE in splenic and spinal cord B cells. Moreover, the intracellular IgG was more readily detected on CD19lo cells, which confirmed our previous observation in spinal cord B cells (Kummari et al. 2019) and might be due to CD19 downregulation in terminally-differentiated plasma cells (Forsthuber et al. 2018). Targeting B cells in MS is one treatment option as the anti-CD20 monoclonal antibodies rituximab and ocrelizumab, which have been used successfully for rheumatoid arthritis, are being studied for MS and at least ocrelizumab was shown to reduce IgG in MS patients (Evertsson et al. 2020). Another recent study in which IgG-producing B cell subsets were characterized suggested that therapies that reducing IgG, in particular IgG3, might provide protection from autoimmune attacks on the central nervous system (Marsh-Wakefield et al. 2020). Although TCDD would not be developed as a therapy, these results do suggest that AhR ligands might possess efficacy in autoimmune diseases by suppressing IgG.

Overall these data show a modest TCDD-mediated increase in the percentage of FasL+ IgMloIgDhi FO B cells and suggest that while small microenvironments of apoptosis might be occurring in response to TCDD, it is not a major mechanism by which T cell function is compromised by TCDD in EAE. On the other hand, we showed that TCDD suppressed IgG production in B cells in EAE in both spleen and spinal cord, and this likely contributes to suppression of pathogenic antibodies involved in myelin destruction in EAE. Thus, these studies provide additional insights on the mechanisms by which TCDD suppressed immune function using the EAE model and might be important in development of novel, non-toxic AhR ligands as treatments for immune-mediated diseases. Moreover, while we did not detect TCDD upregulation of FasL in Cyp1a1 mRNA-expressing cells in vitro using PrimeFlow®, this methodology can be used to identify AhR ligand-dependent changes in protein expression in AhR-activated cells by first gating on cells expressing Cyp1a1 mRNA. Future studies can also focus on whether other functions of IgMloIgDhi FO B cells are specifically targeted by TCDD or other AhR ligands since this could have implications for initiation of humoral immune responses, including IgG production.

Supplementary Material

1

Highlights.

  • FasL is more highly expressed in MZ B cells (IgMhiIgDlo) in the spleen

  • TCDD modestly increased FasL in EAE, which was more readily detected on FO B cells (IgMloIgDhi)

  • TCDD suppressed IgG production in EAE

Acknowledgments

This work was supported by the National Institutes of Health grant R15027650

Abbreviations

TCDD

2,3,7,8-tetrachlorodibenzo-p-dioxin

AhR

aryl hydrocarbon receptor

EAE

experimental autoimmune encephalomyelitis

FasL

fas ligand

FO

follicular B cells

MZ

marginal zone B cells

MOG

myelin oligodendrocyte glycoprotein

Footnotes

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