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. 2022 Jan 9;11(1):4.
doi: 10.3390/antib11010004.

TCDD Inhibition of IgG1 Production in Experimental Autoimmune Encephalomyelitis (EAE) and In Vitro

Ashleigh J Nicaise  1 , Amye McDonald  1 , Erin Rushing Sears  1 , Trell Sturgis  1 , Barbara L F Kaplan  1
Affiliations

TCDD Inhibition of IgG1 Production in Experimental Autoimmune Encephalomyelitis (EAE) and In Vitro

Ashleigh J Nicaise et al. Antibodies (Basel). .

Abstract

The environmental contaminant 2,3,7,8-tetrachlorodibenzo-para-dioxin (TCDD) is a ligand for the aryl hydrocarbon receptor (AhR). TCDD is well-characterized to produce immunotoxicity, including suppression of antibody production. Previously we showed that TCDD inhibited myelin oligodendrocyte glycoprotein (MOG) peptide-specific IgG and attenuated disease in experimental autoimmune encephalomyelitis (EAE) model in mice. Thus, the purpose of this study was to characterize the effects of TCDD on IgG subclasses in EAE and in vitro and assess effects in B cells derived from various tissues. TCDD modestly suppressed intracellular IgG expression in splenocytes (SPLC), but not bone marrow (BM) or lymph node (LN) cells. To further understand TCDD's effects on IgG, we utilized LPS and LPS + IL-4 in vitro to stimulate IgG3 and IgG1 production, respectively. TCDD preferentially suppressed IgG1+ cell surface expression, especially in SPLC. However, TCDD was able to suppress IgG1 and IgG3 secretion from SPLC and B cells, but not BM cells. Lastly, we revisited the EAE model and determined that TCDD suppressed MOG-specific IgG1 production. Together these data show that the IgG1 subclass of IgG is a sensitive target of suppression by TCDD. Part of the pathophysiology of EAE involves production of pathogenic antibodies that can recruit cytolytic cells to destroy MOG-expressing cells that comprise myelin, so inhibition of IgG1 likely contributes to TCDD's EAE disease attenuation.

Keywords: TCDD; aryl hydrocarbon receptor; experimental autoimmune encephalomyelitis.

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Conflict of interest statement

B.L.F.K. has funding from NanoMed Systems, Inc. for a distinct project but these funds were not used for the current studies.

Figures

Figure 1
Figure 1
TCDD modestly suppressed i.c. IgG in SPLC in EAE. EAE was induced in mice using active immunization with MOG peptide on day 0. TCDD (2.5 µg/kg/day) in corn oil (CO) was administered via oral gavage for 12 days for a cumulative dosage of 30 µg/kg. At day 18, mice were sacrificed and SPLC were stained for extracellular B cell markers and i.c. IgG. Positive or negative B cell markers plus i.c. IgG double positive percentages were normalized in each of two experiments to the average of the EAE/CO groups (n = 3 per experiment) to which all other groups were compared in that experiment (total n = 6). Graphs represent average ± SD of normalized values from the two separate experiments. * p < 0.05 as compared to EAE/CO using Friedman’s ANOVA and Dunn’s post hoc test. i.c., intracellular; SPLC, splenocytes; SAL, saline.
Figure 2
Figure 2
TCDD effect on i.c. IgG in LN in EAE. Samples were obtained as in Figure 1, but from LN. * p < 0.05 as compared to EAE/CO using Friedman’s ANOVA and Dunn’s post hoc test. i.c., intracellular; LN, lymph nodes.
Figure 3
Figure 3
TCDD modestly reversed EAE-induced downregulation of i.c. IgG in BM in EAE. Samples were obtained as in Figure 1, but from BM. * p < 0.05 as compared to EAE/CO using Friedman’s ANOVA and Dunn’s post hoc test. i.c., intracellular; BM, bone marrow.
Figure 4
Figure 4
Overlap between CD19-i.c. IgG+, B220-i.c. IgG+ and CD5-i.c. IgG+ populations. Using one of the SAL/CO samples, CD19-i.c. IgG+ cells were identified in the dot plot as red so that overlapping populations could be detected across gates and tissues. Data from one of the SAL/CO samples of one of the two experiments are representative.
Figure 5
Figure 5
TCDD inhibited the percentage of CD19+IgG1+ cells. SPLC or B cells were treated with VH (0.01% DMSO) or TCDD (30 nM) for 30 min then were stimulated with LPS (5 µg/mL) alone or with IL-4 (10 ng/mL). Cells were incubated for 4 days then stained for CD19, B220, IgG1 and IgG3. Cells were gated on live single lymphocytes. (A) CD19+IgG1+; (B) B220+IgG1+; (C) CD19+IgG3+; (D) B220+IgG3+. Bars represent mean ± SD from triplicate samples. S, SPLC; B, B cells; Stim, LPS or LPS + IL-4; VH, vehicle; Untx, untreated. * p < 0.05 as compared to respective Stim + VH control within cell type. Experiments were repeated at least twice.
Figure 6
Figure 6
Percent of CD19+IgG1+ and B220+IgG1+ cells were correlated. Percent CD19+IgG1+ and B220+IgG1+, regardless of treatment, were plotted. Data were obtained from one of the representative in vitro experiments in which IgG1 was evaluated on CD19+ and B220+ cells. The positive correlation regression line is significant at p < 0.0001.
Figure 7
Figure 7
CD19 and B220 were highly co-expressed. Using LPS + IL-4-stimulated SPLC from day 4, cells were pre-gated for total IgG1 or IgG3 from the live single lymphocyte population then assessed for CD19, B220 and CD5. Top, IgG1; bottom, IgG3. Data from one of the LPS + IL-4 samples of one of at least two experiments are representative.
Figure 8
Figure 8
TCDD inhibited the percentage of CD19+IgG1+ cells in B cells at day 4. SPLC, B cells or BM cells were treated with VH (0.01% DMSO) or TCDD (30 nM) for 30 min then were stimulated with LPS + IL-4 (5 µg/mL + 10 ng/mL). Cells were incubated for 1, 2, 3 or 4 days then stained for CD19, B220, CD5 IgG1 and IgG3 (top, IgG1; bottom, IgG3; CD19 shown only). Cells were gated on live single lymphocytes. Bars represent mean ± SD from triplicate samples. Untx, untreated; VH, vehicle. * p < 0.05 as compared to respective LPS + IL-4 + VH control within day. Experiments were repeated at least twice.
Figure 9
Figure 9
TCDD inhibited the percentage of B220+IgG1+ cells in SPLC and B cells at day 4. SPLC, B cells or BM cells were treated with VH (0.01% DMSO) or TCDD (30 nM) for 30 min then were stimulated with LPS + IL-4 (5 µg/mL + 10 ng/mL). Cells were incubated for 1, 2, 3 or 4 days then stained for CD19, B220, CD5, IgG1 and IgG3 (top, IgG1; bottom, IgG3; B220 shown only). Cells were gated on live single lymphocytes. Bars represent mean ± SD from triplicate samples. Untx, untreated; VH, vehicle. * p < 0.05 as compared to respective LPS + IL-4 + VH control within day. Experiments were repeated at least twice.
Figure 10
Figure 10
TCDD did not inhibit the percentage of CD5+IgG1+ or CD5+IgG3+ cells. SPLC, B cells or BM cells were treated with VH (0.01% DMSO) or TCDD (30 nM) for 30 min then were stimulated with LPS + IL-4 (5 µg/mL + 10 ng/mL). Cells were incubated for 1, 2, 3 or 4 days then stained for CD19, B220, CD5 IgG1 and IgG3 (top, IgG1; bottom, IgG3; CD5 shown only). Cells were gated on live single lymphocytes. Bars represent mean ± SD from triplicate samples. Untx, untreated; VH, vehicle. * p < 0.05 as compared to respective LPS + IL-4 + VH control within day. Experiments were repeated at least twice.
Figure 11
Figure 11
TCDD inhibited percentage of cells co-expressing IgG1 and IgG3. SPLC, B cells or BM cells were treated with VH (0.01% DMSO) or TCDD (30 nM) for 30 min then were stimulated with LPS + IL-4 (5 µg/mL + 10 ng/mL). Cells were incubated for 1, 2, 3 or 4 days then stained for CD19, B220, CD5 IgG1 and IgG3. Cells were gated on live single lymphocytes then the double positive population for IgG1 and IgG3 was calculated on day 4 only. Bars represent mean ± SD from triplicate samples. Untx, untreated; VH, vehicle. * p < 0.05 as compared to respective LPS + IL-4 + VH control within cell type. Data were obtained from one of the representative in vitro experiments in which IgG1 and IgG3 were evaluated on CD19+ and B220+ cells.
Figure 12
Figure 12
TCDD inhibited the MFI of IgG1 in SPLC. SPLC, B cells or BM cells were treated with VH (0.01% DMSO) or TCDD (30 nM) for 30 min then were stimulated with LPS + IL-4 (5 µg/mL + 10 ng/mL). Cells were incubated for 1, 2, 3 or 4 days then stained for CD19, B220, CD5 IgG1 and IgG3 (top, IgG1; bottom, IgG3; day 4 shown only). Cells were gated on live single lymphocytes. Bars represent mean ± SD from triplicate samples. Untx, untreated; VH, vehicle. * p < 0.05 as compared to respective LPS + IL-4 + VH control within B cell marker. Data were obtained from one of the representative in vitro experiments in which IgG1 and IgG3 were evaluated on CD19+ and B220+ cells.
Figure 13
Figure 13
TCDD inhibited IgG1 and IgG3 antibody secretion. SPLC, B cells or BM cells were treated with VH (0.01% DMSO) or TCDD (30 nM) for 30 min then were stimulated with LPS + IL-4 (5 µg/mL + 10 ng/mL). Cells were incubated for 1, 2, 3 or 4 days then supernatants were collected and assayed by ELISA (top, IgG1; bottom, IgG3). Antibody levels were quantified as a concentration compared to a standard curve then normalized based on cell counts obtained from the Novocyte flow cytometer on each day. Bars represent mean ± SD from triplicate samples. Untx, untreated; VH, vehicle. * p < 0.05 as compared to respective LPS + IL-4 + VH control within day.
Figure 14
Figure 14
Effect of TCDD on gene expression. SPLC were treated with VH (0.01% DMSO) or TCDD (30 nM) for 30 min then were stimulated with LPS + IL-4 (5 µg/mL + 10 ng/mL). Cells were incubated for 2 or 4 days then total RNA was isolated. RT-qPCR was performed for Cyp1a1 and Aicda. Bars represent mean ± SD from triplicate samples. Untx, untreated; VH, vehicle. * p < 0.05 as compared to respective LPS + IL-4 + VH control within day.
Figure 15
Figure 15
TCDD inhibited MOG-specific IgG1 in EAE. Mice were immunized with MOG peptide in CFA on day 0. Mice received CO or 2.5 µg TCDD/kg/day via oral gavage for 12 days. On day 18, blood was collected, and serum was isolated from individual mice. Serum was assayed in an ELISA. Bars represent mean ± SD from separate mice (n = 3 or 6). Results are representative of 3 separate experiments. SAL, saline; CO, corn oil. * p < 0.05 as compared to EAE/CO.
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