Regulatory T Cells and IFNγ in Mercury-Induced Autoimmunity: Insights from Adoptive Transfer in B10.S Mice

Simple Summary

Autoimmune diseases occur when the immune system mistakenly attacks the body’s own tissues. This can happen due to a mix of genetic and environmental factors. Special immune cells called regulatory T cells (Tregs) normally keep the immune system in balance, while a molecule called interferon-gamma (IFNγ) can both promote and regulate inflammation depending on the situation. In the present study, we studied mercury-induced autoimmunity (HgIA) in mice, a condition triggered by mercury exposure that causes harmful antibodies and kidney damage. We tested whether Tregs could reduce this disease and whether IFN-γ was important for their function. When Tregs from mercury-exposed mice were transferred to other mice, they greatly reduced harmful antibodies and kidney injury. However, Tregs from mice lacking IFNγ were much less effective, and those mice still developed signs of autoimmunity. The study shows that Tregs can protect against mercury-related autoimmune damage, but they need IFNγ to work properly. This highlights IFNγ as a key partner for Tregs in controlling harmful immune responses during chronic exposure to environmental toxins like mercury.

Abstract

Autoimmune diseases result from a breakdown of immune tolerance influenced by genetic and environmental factors. Regulatory T cells (Tregs) maintain immune homeostasis, while interferon-γ (IFNγ) has context-dependent proinflammatory and regulatory roles. In B10.S mice, mercury-induced autoimmunity (HgIA) emerges within approximately 4 weeks of Hg exposure and is marked by antinucleolar antibody (ANoA) production, polyclonal B-cell activation, and deposition of immune complexes in the kidney. We investigated whether Tregs attenuate HgIA and evaluated IFNγ’s role in this regulation. Female WT and IFNγ^−/− B10.S mice received HgCl₂ or water for 4 weeks until all mice developed ANoA. CD4⁺CD25⁺Foxp3⁺ Tregs or CD4⁺CD25⁻Foxp3⁻ cells were transferred into HgCl₂-exposed WT recipients and monitored for 13 weeks. Compared with Hg-primed non-Tregs, Hg-primed WT Tregs were statistically associated with significantly reduced autoantibody levels, lower IgG1/IgG2a, and significantly decreased glomerular IgG/C3c deposition, suggesting that Hg exposure may modulate Treg function. Conversely, both water- and Hg-primed Tregs and non-Tregs from IFNγ^−/− donors elicited profoundly diminished autoantibody production and renal pathology in recipients. IFNγ^−/− mice lacked fibrillarin-specific responses, highlighting its requirement for HgIA initiation. While non-Treg transfer failed to suppress HgIA, Treg transfer reduced HgIA and highlighted relevance for immune-regulatory therapies, especially where environmental toxicants may drive autoimmune disease.

1. Introduction

Autoimmune diseases result from a breakdown in immune tolerance, where the immune system erroneously targets self-antigens, leading to chronic inflammation and tissue damage [1]. This loss of tolerance is driven by a complex interplay between genetic predisposition and environmental factors [2]. Among the key regulators of immune homeostasis are regulatory T cells (Tregs), a specialized subset of CD4⁺ T cells characterized by the expression of the transcription factor Foxp3. Tregs are essential for maintaining peripheral tolerance and preventing autoimmune pathology [3]. Tregs include thymic Tregs enforcing self-tolerance, peripherally induced Tregs generated from CD4⁺ T cells by tolerogenic cues, and Th1-like Tregs adopting inflammatory traits to regulate specialized immune environments [4]. Tregs exert their suppressive functions through a variety of mechanisms, including the secretion of anti-inflammatory cytokines such as IL-10, TGF-β, and IL-35; metabolic disruption through adenosine production; cytolysis mediated by perforin and granzymes; and modulation of antigen-presenting cells via interactions with CTLA-4 and LAG-3 [5,6]. Their ability to suppress excessive immune responses is crucial for preventing tissue damage, as underscored by Sakaguchi et al. [7]. Environmental agents such as inorganic mercury (Hg²⁺) have been shown to trigger autoimmune responses in genetically susceptible individuals [8,9,10]. The mercury-induced autoimmunity (HgIA) model in mice recapitulates several features of human systemic autoimmune diseases, including the production of antinuclear antibodies (ANA), polyclonal B cell activation (PBA), and immune-complex (IC) depositions [2]. Loss of effective Treg control enables sustained autoreactive T- and B-cell activity, increasing autoantibody production and renal immune-complex (IC) deposition that drives lupus-like glomerular inflammation [11,12]. IFN-γ further amplifies IC-mediated injury by enhancing antigen presentation, promoting class-switched autoantibodies, and increasing inflammatory cell influx into glomeruli. Recent work shows IFN-γ–linked pathways modulate T- and NK-cell behavior in inflamed kidneys, worsening IC-driven glomerulonephritis [13].

A hallmark of HgIA is the MHC class II-restricted autoantibody response to fibrillarin, a nucleolar protein involved in ribosomal RNA processing. This response mirrors human autoimmunity and targets a conserved epitope [14]. Mercury exposure can modify self-antigens such as fibrillarin, but the immunogenicity appears to be driven by proteolytic fragments generated during mercury-induced cell death rather than neoantigen formation [15]. Importantly, the development of anti-fibrillarin autoantibodies (AFA) is critically dependent on interferon-gamma (IFNγ). Mice lacking the IFNγ gene fail to produce autoantibodies against fibrillarin following mercury exposure, demonstrating that a Th1-driven immune response, mediated by IFNγ, is essential for autoimmune activation in HgIA [16,17]. Anti-fibrillarin autoantibodies (AFA) occur in ~10% of systemic sclerosis (SSc) patients and are linked to severe disease, with environmental factors implicated in their development [18,19,20,21]. Renal immune-complex deposition likewise characterizes human lupus nephritis, where glomerular IC accumulation drives inflammation and tissue injury. Recent studies show that IC deposition promotes inflammatory cell recruitment and glomerular damage in SLE [13], and urinary IC-based biomarkers closely mirror renal pathology [22]. Murine Hg-induced autoimmunity (HgIA) is a chronic-exposure model in which repeated low-dose mercury triggers sustained autoantibody production and immune-complex pathology [2]. Its prolonged course provides a controlled setting to examine how IFN-γ shapes Treg adaptation or dysfunction, allowing for mechanistic insight into IFN-γ-dependent regulatory failure during persistent environmental immune activation. IFNγ is a central cytokine in both innate and adaptive immunity, primarily produced by CD4⁺/CD8⁺ T cells and NK cells, with contributions from macrophages, dendritic cells, and B cells.

The relationship between Tregs and IFNγ is complex. While IFNγ is traditionally viewed as pro-inflammatory, it can also support Treg function under certain conditions [23,24,25]. Th1-like Tregs, which produce IFNγ, retain suppressive capacity in Th1-dominated environments. However, prolonged IFNγ exposure may destabilize Tregs by downregulating Foxp3, impairing their suppressive function [26].

A growing body of recent work demonstrates that adoptive transfer of ex vivo–expanded or antigen-specific Tregs effectively suppresses pathogenic effector responses and limits autoimmune pathology. Layland and colleagues [27] showed that xenobiotic-primed CD4⁺CD25⁺ T cells prevented drug-induced autoantibody formation, whereas CD4⁺CD25⁻ T cells induced de novo ANA production, providing an early adoptive-transfer framework relevant to HgIA. Building on this foundation, Treg-based adoptive cell transfer has become a promising strategy for restoring self-tolerance, with early clinical studies [28] of polyclonal Tregs demonstrating safety and feasibility [29]. More recent work shows that antigen-specific Tregs provide greater precision and therapeutic efficacy, driving current efforts toward engineered or targeted Treg therapies [30].

The aim of this study was to investigate the role of regulatory T cells (Tregs) and the contribution of IFNγ to Treg-mediated immune regulation during HgIA, with particular emphasis on the development of antinucleolar antibodies (ANoA). To address this, Tregs were isolated from water- or mercury-treated wild-type (WT) B10.S mice or IFNγ-deficient B10.S (IFNγ^−/−) mice and adoptively transferred into WT recipient mice. The recipients were subsequently exposed to mercury and monitored for up to 13 weeks.

In this study, we present that adoptive transfer of Tregs from mercury- or water-treated WT donors reduced autoantibody levels, B cell activation, and kidney immune deposits. In contrast, Tregs from IFNγ-deficient mice showed impaired suppression, with minimal differences between treated groups. Early after mercury exposure, Tregs from IFNγ^−/− mice had limited effect, failing to significantly alter autoimmunity. These findings highlight IFNγ’s critical role in Treg-mediated suppression of mercury-induced autoimmunity.

2. Materials and Methods

2.1. Animals

Female wildtype B10.S (B10.S WT) mice (H-2^s) purchased from Taconic M&B (Ry, Ejby, Denmark), were 8–12 weeks of age at the onset of the study. The B10.S with targeted mutation (knockout, KO) for IFNγ (B10.S IFNγ^−/−) were obtained from Scripps Research Institute [31], La Jolla, CA, USA and maintained by brother–sister mating in the animal facilities of the Faculty of Health Sciences, Linköping. All mice were housed in steel-wire cages in pathogen-free animal facilities at Linköping University. All mice were maintained at 22 ± 2 °C with a 50 ± 10% relative humidity and under a 12 h dark/light cycle and had ad libitum access to standard mouse pellets (CRME rodent, Special Diets, Witham, UK). All animal procedures were approved by the Laboratory Animal Ethics Committee, Linkoping, Sweden.

2.2. Treatment and Experimental Design

2.2.1. Donors

Twenty female WT and twenty female IFNγ^−/− B10.S-mice were given, ad libitum, drinking water (controls) or water containing 4 mg HgCl₂/L equivalent to an internal dose of 74 μg Hg/kg body weight per day during a period of 4 weeks. This can be compared with WHO data, for a LOAEL (lowest-observed-adverse-effect-level) ranging from 0.23 to 0.63 mg/kg body weight per day [32]. Administration of this dose results in the induction of ANoA and a broad spectrum of autoimmune parameters in H-2^s mice [33]. At the time of sacrifice, all mice were euthanized using isoflurane^® (Abbott, Solna, Sweden). During the necropsy procedure, blood samples were collected from retro-orbital plexus behind the eye, and sera were immediately isolated and stored at −80 °C until further analysis. Retro-orbital blood collection was used to obtain sufficient volumes of high-quality serum while minimizing handling time. The procedure was selected to reduce animal discomfort and was performed by trained personnel under anesthesia in accordance with institutional and national animal welfare guidelines to minimize pain and distress. Spleens were removed and processed for single-cell isolation and immunological analyses (Figure 1).

Splenic single-cell suspensions from Hg-treated mice were prepared as previously described [34]. Dead cells (5–10%) were gated using 7-Amino-actinomycin D (ViaProbe, BD). Purity assessment was performed according to the manufacturer’s instructions. The positively selected CD4⁺CD25⁺FoxP3⁺ fraction underwent two rounds of magnetic separation using MS Columns, after which magnetically retained cells were eluted to obtain an enriched CD4⁺CD25⁺ T-cell population. Surface staining for CD4 (FITC) and CD25 (APC), followed by fixation, permeabilization, and intracellular FoxP3 (PE) staining, confirmed the isolation of a highly purified regulatory T-cell population (Supplementary Figure S1).

Purified CD4⁺CD25⁺Foxp3⁺ regulatory T-cells (Tregs) and CD4⁺CD25⁻ Foxp3⁻ T-cells (non-Tregs) from Hg-treated mice were identified using a detection kit (Miltenyi Biotec GmbH, Lund, Sweden). Cells obtained from 20 WT B10.S and 20 IFN-γ⁻/⁻ B10.S mice were pooled into four separate tubes for transfer to new groups of recipient mice. Pooling the cells ensured that sufficient cell numbers were available for each transfer, as the yield varied between individual mice within each group.

2.2.2. Recipients

Isolated Treg and non-Tregs then diluted in phosphate-buffered saline (PBS) to achieve the desired concentration. Subsequently, 100 μL of the cell suspension, containing approximately 1 × 10⁶ cells, was administered to recipient mice via intravenous (i.v.) injection (Figure 1). Recipient mouse groups received regular drinking water or water containing 4 mg HgCl₂/L. Depending on treatment, four groups of recipient mice were formed (Figure 1). Recipients that received non-Treg cells from water-treated or HgCl₂-treated WT B10.S or IFN-γ^−/− mice were assigned to groups A, C, E, and G, respectively. Recipients that received Tregs from water-treated or HgCl₂-treated WT B10.S or IFN-γ⁻/⁻ mice were assigned to groups B, D, F, and H.

2.3. Blood and Tissue Sampling

Blood was collected from the retro-orbital plexus under isoflurane^® anesthesia (Abbott, Sweden), with approximately 0.5 mL obtained per bleeding, prior to mercury exposure and again at weeks 5, 7, 9, and 13. All samples were anonymized and coded numerically. At the end of the study (week 13), all mice were euthanized under anesthesia. At necropsy, blood was sampled, and spleen and kidney were collected. In each case, sera were isolated immediately upon blood collection and stored at −80 °C until used for analysis.

2.4. Serum Antinuclear Antibodies (ANA) Assessed by Indirect Immunofluorescence

Serum antinuclear antibodies (ANA) were assessed by indirect immunofluorescence using HEp-2 cells (Binding Site Ltd., Birmingham, UK) as a substrate [33]. In brief, serum samples were serially diluted 1:80–1:5120 with phosphate-buffered saline (PBS, pH 7.4) and placed on slides containing a monolayer of HEp-2 cells for 30 min at 21 °C. Thereafter, the slides were incubated with fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse antibody against immunoglobulin total IgG₁, IgG_2a, or IgG_2b, (1:50 dilution; Southern Biotechnology, Birmingham, AL, USA). The titer was defined as the highest serum dilution that gave a distinct nuclear staining. No staining at a serum dilution of 1:80 was considered a negative result (0). A pool of sera from young, individually ANA-negative mice was used as a negative control. Titers and patterns were assessed using a Nikon incident-light fluorescence microscope (Nikon Instech Co. Ltd., Kanagawa, Japan). All observations were conducted with coded samples. ANoA patterns were classified according to the International Consensus on ANoA Patterns (ICAP) [35].

To compare the presence and titers of ANoA in the IgG₁ and IgG_2a isotypes, an IgG_2a–IgG₁ ANoA index was calculated based on titer steps. No specific staining of HEp-2 cells at a serum dilution of 1:40 with anti-mouse IgG₁ or IgG_2a antibodies was recorded as “0”. Specific staining at 1:40 was assigned one titer step (+1), with two steps at 1:80 (+2), and so forth. The index for each serum sample was obtained by subtracting the IgG₁ titer steps from the IgG_2a titer steps. A negative index value indicated a predominance of IgG₁ ANoA, whereas a positive value indicated a predominance of IgG_2a ANoA.

2.5. Serum Anti-Chromatin Antibodies Assessed by Enzyme-Linked Immunosorbent Assay (ELISA)

Serum anti-chromatin antibodies were measured as described previously [36]. Calf thymus chromatin was added to microtiter plates (Nunc, Copenhagen, Denmark). After overnight incubation at 4 °C, the plates were blocked with 0.1% gelatin overnight at 4 °C. Next, serum samples diluted 1:400 in PBS were added for 90 min at room temperature in duplicate. Using ALP-conjugated goat anti-mouse IgG antibodies (Caltag Laboratories, Burlingame, CA, USA) and pNPP (p-Nitrophenyl Phosphate) substrate, the optical density (OD) was measured at 405 nm. A pool of sera from strong ANA-positive NZB/W F1 mice (homogeneous pattern) and a pooled serum from weakly ANA-positive NZB/W F1 sera were used as positive controls, and a pooled serum from young ANA-negative mice was used as negative control.

2.6. Investigation of Serum Antinucleolar Antibodies (ANoA) Specificity by Western Blotting

To assess the specificity of serum autoantibodies, Western blotting was performed on sera as previously described [35]. Briefly, nuclear extract isolated from mouse liver [37] was separated by SDS-PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) using 12.5% and 18% Tris-HCl gels (Bio-Rad, Hercules, CA, USA). The separated protein bands were then blotted onto 0.45 µm nitrocellulose membranes (Bio-Rad) at 0.8 mA/cm² under water cooling (Criterion Blotter; Bio-Rad) for 30 min. The membranes were blocked, overnight at 4 °C, using 5% non-fat dry milk (Bio-Rad). The lanes on the membranes were then cut, using a blade, into separate strips and each incubated with diluted mice sera for 60 min. Following repeated washing steps, HRP-conjugated goat anti-mouse IgG (Southern Biotech.) was added, followed by chemiluminescent HRP substrate (Immobilon Western, Millipore Corporation; Billerica, MA, USA). High-performance chemiluminescence films (Amersham Hyperfilm ECL; Amersham, GE Healthcare, Buckinghamshire, UK) were then exposed to the membrane strips and developed with Kodak D-19 developer (Eastman Kodak; Rochester, NY, USA). Reference sera human fibrillarin (Binding Site) was used.

2.7. Detection of Anti-DNP Antibodies with ELISA

The method used has been described previously [35]. Microtiter plates (Nunc) were coated with DNP-albumin (Albumin Human Dinitrophenyl, Sigma, St. Louis, MO, USA) overnight. Sera diluted 1:100 were added to the wells. Pooled sera from NZB-NZW mice (Harlan Scandinavia, Allerød, Denmark) was used as positive control. Goat anti-mouse-IgG-ALP (polyvalent IgG, IgA, IgM) (Sigma-Aldrich Chemie, Steinheim, Germany) was added. Plates were washed before the substrate was added and the optical density (OD) at 405 nm was measured after 20 min; the reaction was stopped with 3 M NaOH after 55 min. Background values in wells coated with PBS were subtracted.

2.8. Detection of Anti-ssDNA Antibodies with ELISA

The method used has been described previously [37]. Microtiter plates (Nunc) were coated with single-stranded DNA (ssDNA) overnight followed by repeated washes. Sera, diluted 1:150, were added to the wells. Pooled sera from NZB-NZW mice (Harlan Scandinavia, Denmark) were used as positive control. Following a 1 h incubation, plates were washed and goat anti-mouse-IgG-ALP (polyvalent IgG, IgA, IgM) (Sigma-Aldrich Chemie, Steinheim, Germany), diluted 1:1000, was added for a 2 h incubation. Plates were washed before the substrate was added and the optical density at 405 nm was measured after 20 min; the reaction was stopped with 3 M NaOH after 45 min and measured once more. Background values in wells coated with PBS were subtracted.

2.9. Serum IgG₁ and IgG_2a Antibodies Assessed by Enzyme-Linked Immunosorbent Assay (ELISA)

Serum IgG₁ levels were determined as described in Puente et al. [33]. In brief, wells of microtiter plates (Nunc, Copenhagen, Denmark) were coated overnight at 4 °C with rat anti-mouse IgG₁ monoclonal antibody (mAb; LO-IMEX, Brussels, Belgium) followed by washing and blocking. Then, mice sera (1:1000 dilution) were added to wells and incubated at 37 °C for 2 h. After rinsing with PBST, any bound IgG₁ was detected using a horseradish peroxidase (HRP)-conjugated rat anti-mouse IgG₁ mAb (1:2000 dilution; LO-IMEX). The optical density (OD) in the wells was measured at 450 nm in a Multiskan Ascent 96/384 plate reader (Thermo Labsystems, Helsinki, Finland). Background values were subtracted using PBS instead of serum.

Serum IgG_2a levels were determined as described in Puente et al. [33]. Using the same approaches as above, wells were coated with purified anti-mouse Ig κ Light Chain (BD Biosciences, Piscataway, NJ, USA). After washing and blocking, diluted sera were added to wells and any bound IgG_2a ultimately detected using an alkaline phosphatase (AKP)-anti-mouse IgG_2a (1:200 dilution, BD Biosciences) and OD measured at 405 nm. The concentration of IgG_2a and IgG₁ in each sample was calculated by extrapolation from the standard curves generated in parallel using purified anti-mouse IgG_2a (BD Biosciences) and mouse myeloma proteins (IgG₁ isotype; LO-IMEX).

2.10. Tissue Immune Complex Deposition

To assess immune complex deposition, sections from kidney and spleen were stained using FITC-conjugated goat anti-mouse IgG (Southern Biotech.), as well as anti-C3c antibodies (Organon Technica, West Chester, PA, USA) as described [38]. The titer for IgG and C3c were determined by serial dilution of the antibodies.

2.11. Statistical Methods

All statistical calculations were performed using GraphPad Software Inc, Version 10.5.0. The difference between the titers of ANoA between the different groups was tested by using the Kruskal–Wallis test followed by Dunn’s post hoc test (more than 2 groups) and Mann–Whitney test (2 groups). Mice groups were compared regarding anti-ssDNA and anti-DNP, anti-chromatin, and IgG₁ and IgG_2a abs, as well as immune-complex deposition in kidneys by using the Kruskal–Wallis statistics test followed by Dunn’s post hoc test and a non-parametric Mann–Whitney test. p-value < 0.05 was considered statistically significant. A clustering heatmap was constructed using ClustVis software, version 2.0 (https://biit.cs.ut.ee/clustvis/, accessed on 1 October 2025). Columns with identical annotations were merged by calculating the median value within each group. Rows were mean-centered and scaled to unit variance. Missing values were estimated using imputation. Both rows and columns were subsequently clustered using correlation distance and average linkage.

3. Results

3.1. Serum Antinucleolar Antibodies (ANoA) Pattern, in Donor WT- or IFNγ B10.S Mice

Following 30 days of 4 mg HgCl₂/L treatment, all wild-type (WT) B10.S donor mice exhibited a consistent development of antinucleolar antibodies (ANoA) with a mean titer of 640, characterized by a distinct clumpy nucleolar staining pattern (Figure 2A) [35]. In stark contrast, B10.S donor mice with a targeted knockout (KO) of the interferon-gamma gene (IFNγ^−/−) demonstrated no detectable nucleolar staining (Figure 2B) under identical experimental conditions.

3.2. Antinucleolar Serum Antibodies of IgG Isotypes in Recipient WT B10.S Mice

To investigate the role of Tregs in mercury-induced autoimmunity (HgIA), we assessed the development of antinucleolar antibodies (ANoA) in WT B10.S recipient mice. Recipients received either non-Treg cells (CD4⁺CD25⁻Foxp3⁻; groups A, C, E, and G) or Treg cells (CD4⁺CD25⁺Foxp3⁺; groups B, D, F, and H). Donor cells were obtained from both WT B10.S and B10.S IFNγ^−/− mice that were either untreated (H₂O-treated) or exposed to 4 mg HgCl₂/L. Cells from untreated donors were transferred to groups A, B, E, and F, whereas cells from HgCl₂-exposed donors were transferred to groups C, D, G, and H (Figure 1). Following adoptive transfer, all recipient mice were exposed to 4 mg HgCl₂/L for 13 weeks to induce HgIA.

3.3. Adoptive Transfer of Treg Cells from Water-Primed WT B10.S Mice Reduce the Formation of ANoA in Syngeneic Recipient Mice

As shown in

Table 1. Reciprocal serum ANoA titers of total IgG and IgG isotypes in female B10.S mice that received either CD4⁺CD25⁻ or CD4⁺CD25⁺ T cells from H₂O-treated donor mice. Following cell transfer, recipient mice were exposed to 4 mg HgCl₂/L for 13 weeks. Serum samples were collected at baseline (week 0) and at weeks 5, 7, 9, and 13 of treatment.

Donors Treatment	Recipients * Treated with 4 mg HgCl2/L
	Type of Received T-Cell (Group)	Treatm. Time (Weeks)	ANoA
			IgG tot.er (%)	IgG1 (%)	IgG2a (%)	IgG2b (%)
H2O	CD4+CD25− (A)	5	180 ± 20 # (100)	110 ± 20 (70)	160 ± 40 (80)	40 ± 10 (60)
		7	320 ± 60 (100)	160 ± 20 (100)	230 ± 30 (100)	80 ± 10 (90)
		9	500 ± 100 (100)	210 ± 20 (100)	330 ± 60 (100)	110 ± 10 (100)
		13	780 ± 140 (100)	270 ± 50 (100)	370 ± 50 (100)	180 ± 20 (100)
H2O	CD4+CD25+ (B)	5	220 ± 30 (90)	90 ± 20 (80)	170 ± 40 (80)	50 ± 10 (60)
		7	220 ± 80 (70)	120 ± 30 (70)	150 ± 40 (70)	60 ± 10 (60)
		9	220 ± 80 a (70)	140 ± 40 (70)	170 ± 60 a (70)	50 ± 20 (50)
		13	190 ± 30 b (70)	180 ± 70 (60)	180 ± 70 a (60)	30 ± 20 (30)

As all mice tested negative for ANoA at the start of treatment (week 0), these data are not included in the table. * Number of mice = 10. # Mean of IgG titer ± SEM. ^a,b = p value ≤ 0.05, 0.001 indicates differences in the corresponding week in group A.

and Figure 3, WT B10.S mice recipients of Treg cells from H₂O-primed donors (group B) and subsequently exposed to 4 mg HgCl₂/L for 13 weeks displayed a significant reduction in ANoA titers at weeks 9 and 13 compared with group A mice, which received non-Treg cells.

At week 5, ANoA titers were already detectable both in group A and B. In group A, all mice (100%) developed total IgG ANoA, with titers increasing steadily from 180 at week 5 to 780 at week 13. All IgG subclasses (IgG₁, IgG_2a, IgG_2b) reached 100% positivity by week 9, with titers rising consistently over time (Table 1).

In contrast, group B (recipients of Tregs) exhibited a weaker response. Although 90% of mice were positive for total IgG ANoA at week 5, this proportion remained at 70% through week 13, with titers peaking at 220 at week 5 and significantly declining to 190 by week 13 (Figure 3A). IgG ANoA subclass positivity also declined over time in group B, with IgG_2a especially demonstrating a notable decrease in titer compared with group A. The IgG_2b ANoA-fraction showed the most pronounced reduction, from 60% at week 5 to 30% at week 13. Corresponding titers for IgG_2b dropped from 50 to 30.

3.4. Adoptive Transfer of Treg Cells from Hg-Primed WT B10.S Mice Partially Suppressed the Formation of ANoA in Syngeneic Recipient Mice

In group C, recipients of non-Treg cells and exposed to 4 mg HgCl₂/L, all mice (100%) developed ANoA of total IgG and all IgG subclasses by week 5 (

Table 2. Reciprocal serum ANoA titers of total IgG and IgG isotypes in female B10.S mice that received either CD4⁺CD25⁻ or CD4⁺CD25⁺ T cells from HgCl₂-treated donor mice. Following cell transfer, recipient mice were exposed to 4 mg HgCl₂/L for 13 weeks. Serum samples were collected at baseline (week 0) and at weeks 5, 7, 9, and 13 of treatment.

Donors Treatment	Recipients * Treated with 4 mg HgCl2/L
	Type of Received T-Cell (Group)	Treatm. Time (Weeks)	ANoA
			IgG tot. (%)	IgG1 (%)	IgG2a (%)	IgG2b (%)
HgCl2	CD4+CD25− (C)	5	620 ± 160 # (100)	200 ± 40 (90)	300 ± 80 (90)	100 ± 30 (60)
		7	960 ± 210 (100)	250 ± 60 (90)	660 ± 120 (100)	230 ± 30 (100)
		9	1860 ± 410 (100)	510 ± 50 (100)	900 ± 110 (100)	380 ± 60 (100)
		13	2690 ± 510 (100)	480 ± 50 (100)	1760 ± 440 (100)	340 ± 60 (100)
HgCl2	CD4+CD25+ (D)	5	500 ± 110 (100)	270 ± 70 (100)	370 ± 80 (100)	130 ± 10 (100)
		7	300 ± 80 b (80)	90 ± 30 a (60)	180 ± 60 b (80)	40 ± 10 c (50)
		9	170 ± 70 c (50)	30 ± 20 c (30)	20 ± 10 c (30)	10 ± 10 c (10)
		13	20 ± 20 c (30)	20 ± 20 c (10)	10 ± 10 c (10)	0 c

As all mice tested negative for ANoA at the start of treatment (week 0), these data are not included in the table. * Number of mice = 10. ^# Mean of IgG titer ± SEM. ^a,b,c = indicate statistically significant differences compared to the corresponding week in group C, with p values ≤ 0.05, 0.01, and 0.001, respectively.

). Titers increased progressively over time, with total IgG titers rising from 620 at week 5 to 2690 at week 13. IgG_2a showed the most pronounced increase, reaching 1760 at week 13. IgG₁ and IgG_2b titers also rose steadily with the treatment time (Figure 3A).

In contrast, mice group D, Treg recipients, displayed a significantly reduced ANoA response. Although all mice were initially positive in week 5, titers declined sharply thereafter. By week 13, total IgG titers had dropped to 20, with IgG₁ and IgG_2a titers reduced to 20 and 10, respectively, while the IgG_2b titers were undetectable (0). The fraction of ANoA-positive mice also declined significantly, with only 30% positive for total IgG and 10% or fewer for all subclasses at week 13 (Table 2 and Figure 3A).

Statistical analysis revealed significant differences between groups C and D at multiple time points. Reductions in titers in group D were significant as early as week 7 and became highly significant by weeks 9 and 13 (p ≤ 0.05, 0.01, and 0.001), particularly for IgG₁, IgG_2a, and IgG_2b.

Analysis of the IgG_2a–IgG₁ index revealed distinct differences between experimental groups. Mice recipients of non-Treg cells from Hg-primed donors exhibited higher IgG_2a–IgG₁ index values, indicating a predominance of Th1-associated IgG_2a ANoA responses. In contrast, recipients of Treg cells generally showed lower index values, suggesting a regulatory effect that favored Th2-associated IgG₁responses (Figure 3B).

3.5. Mice Recipients of Treg Cells from Hg-Primed WT B10.S Donors Showed No Corresponding Antibodies Against Antifibrillarin in the Western Blot Analysis

The adoptive transfer of Treg cells from Hg-primed WT-mice to syngeneic mice following Hg treatment appears to suppress the presence of autoantibodies to fibrillarin, as evidenced by the lack of reactivity in lanes 4–6 (Figure 4). In response to Hg treatment serum antibodies from mice, recipients of non-Treg cells, reacted with a 34 kDa protein corresponding to human antifibrillarin antibody (lane 3) as it appears in the mouse serum from lanes 9–12. The intensity of the autoimmune response correlates with the ANoA titer, with stronger responses in highly positive mice (lanes 11–12) (Figure 4).

3.6. Treg Cells from Hg-Primed Donor WT B10.S Changed the Levels of Antibodies Against Chromatin, DNP, and ssDNA in Hg-Treated Syngeneic Mice

Adoptive transfer of Treg cells from water-primed donor mice did not significantly alter anti-chromatin antibody levels in Hg-treated WT B10.S recipients compared with mice recipients of non-Treg cells. In contrast, group D, which received CD4⁺CD25⁺Foxp3⁺ Tregs from Hg-primed donors, showed a significant reduction in anti-chromatin antibodies over time. Antibody levels in these mice increased steadily from week 7 to week 13 but remained significantly lower than in group C, which received non-Treg cells (Figure 5). Serum antibodies generated by polyclonal B-cell activation (BPA), including those targeting DNP and ssDNA, were also influenced by the type of transferred T cell. Mice recipients of Tregs from Hg-primed donors exhibited markedly lower serum DNP antibody levels after 9 and 13 weeks of treatment compared with mice recipients of non-Treg cells. No notable anti-DNP antibody levels were detected in groups A or B at any time point (Figure 5). Mice in group C displayed a significant and sustained increase in anti-ssDNA antibodies from week 5 through week 13; however, the difference reached statistical significance only at week 13.

3.7. Adoptive Transfer of Treg Cells Resulted in a Significant Decrease in Serum Immunoglobulin Concentration in Recipient Hg-Treated Syngeneic Mice

Serum concentrations of IgG1 and IgG2a in Hg-exposed WT B10.S recipient mice were strongly influenced by the type of transferred T cell. Mice recipients of non-Treg cells showed a rapid and significant increase in IgG1 levels, peaking around week 7 and remaining high through week 13, in contrast to group D, which received Tregs from Hg-primed donors (Figure 6). Group B, which received Tregs from water-primed donors, also displayed lower IgG1 levels than the corresponding non-Treg recipients throughout the study, although the difference reached statistical significance only at week 13.

Overall, Treg cells, particularly those from Hg-primed donors (group D), effectively suppressed the production of both IgG1 and IgG2a antibodies in Hg-treated recipient mice.

3.8. Decline in Glomerular Immune-Complex Deposition in Hg-Treated WT B10.S Recipient Mice Depends on the Type of Transferred T Cells

Adoptive transfer of Treg cells, whether derived from water-primed or Hg-primed donors (groups B and D), resulted in markedly reduced titers of both C3c and IgG glomerular immune-complex deposits (Figure 7).

3.9. The Immune Response in B10.S Mice Recipients of T Cells from IFN-g^−/− Knockout Mice

Mice in groups E, F, G, and H, which received T cells from IFNγ-deficient (IFNγ^−/−) donors, showed greatly reduced or absent immune responses (Figure 8; Supplementary Tables S1 and S2). In the absence of IFNγ, the downstream pathways that normally drive autoantibody production and B-cell activation were not initiated, resulting in uniformly low antibody levels.

No significant differences were observed between recipients of Tregs and non-Tregs when the transferred cells lacked IFNγ, indicating that both subsets require IFNγ to exert their typical immunomodulatory effects. Consequently, transfer of IFNγ-deficient T cells produced a uniformly muted autoimmune phenotype, regardless of T-cell type. These findings highlight IFNγ as a critical driver of mercury-induced immune activation.

3.10. Summary of the Results

Hg-primed Treg cells exerted a pronounced suppressive effect on HgCl₂-induced immune responses, most clearly observed at weeks 9 and 13, as indicated by the lighter shades in the heatmap (Figure 9). In contrast, mice that received Tregs from IFNγ-deficient donors displayed minimal or no immune activation. As the study progressed, the differences between groups became increasingly evident with continued HgCl₂ exposure. Notably, mice that received non-Treg cells from Hg-primed donors showed heightened immune activity, reflected by the red and orange intensities in the heatmap (Figure 9).

4. Discussion

The present study investigates the intricate interplay between mercury exposure, regulatory T cells (Tregs), non-regulatory T cells (non-Tregs), and interferon-gamma (IFNγ) signaling in the modulation of mercury-induced autoimmunity (HgIA) using the susceptible B10.S mouse strain. Our findings elucidate critical aspects of immune regulation and tolerance breakdown in environmentally induced autoimmune diseases. Our results clarify key checkpoints of immune regulation and tolerance failure in environmentally driven autoimmunity.

Adoptive transfer of Tregs from water-primed and Hg-primed WT B10.S donors reduced antinucleolar antibodies (ANoA), polyclonal B-cell activation (PBA), and renal immune-complex (IC) deposition relative to non-Treg recipients. This aligns with the canonical Treg mechanisms, IL-10/TGF-β production, metabolic disruption, and APC modulation via CTLA-4 and LAG-3, described previously [3,39,40]. Interestingly, Tregs from Hg-primed donors appeared to show greater suppressive capacity, as indicated by a trend toward reduced ANoA titers and renal IC deposition relative to Tregs from water-primed donors. This pattern supports the possibility that Hg exposure may involve epigenetic or metabolic programs that endow Tregs with a memory-like regulatory phenotype [23,25,33,41]. Consistent with reports that xenobiotics can transiently expand but ultimately destabilize Tregs under chronic exposure, our data suggest exposure-dependent tuning of Treg potency [42,43]. Non-Tregs, in contrast to Tregs, often exhibit pro-inflammatory characteristics and can exacerbate autoimmune responses. In the context of HgIA, non-Tregs from both water-primed and Hg-primed donors failed to suppress ANoA formation, PBA, and renal IC deposition effectively. This suggests that non-Tregs may contribute to the breakdown of immune tolerance by providing costimulatory signals that promote B-cell activation and antibody production. Their inability to suppress autoimmune markers highlights the specificity and importance of Treg-mediated regulation in maintaining immune homeostasis [44,45].

A pivotal observation is the dependence of Treg-mediated protection on IFNγ. Tregs from IFNγ-deficient (IFNγ^−/−) donors showed little suppressive efficacy: ANoA, PBA markers (anti-chromatin, anti-DNP, anti-ssDNA), and renal IC deposition were not substantially reduced in recipients. This agrees with evidence that IFNγ supports Treg specialization within Th1-biased environments [24,46]. An explicit early-versus-late phase interpretation is crucial for understanding how IFNγ-dependent regulation and Treg efficacy may evolve during prolonged mercury exposure. Early in Hg exposure, IFNγ may play a dual role by promoting initial Treg activation and localization to sites of inflammation while also contributing to the pro-inflammatory milieu. Over time, chronic Hg exposure could lead to Treg exhaustion and a shift in the balance towards pro-inflammatory responses, despite the presence of IFNγ. This phase transition underscores the dynamic nature of immune regulation and the potential for therapeutic intervention at different stages of disease progression [47]. Recent work highlights that IFNγ-driven remodeling of the Treg compartment is not merely supportive of early regulatory activation but may constitute a determinant checkpoint in disease progression, wherein IFNγ shapes a Th1-like Treg subset through sustained induction of T-bet and CXCR3. Such IFNγ-imprinted Tregs preferentially migrate into CXCL9/10-rich inflammatory niches and interact intimately with dendritic cells, thereby modulating Th1 responses in a highly tissue-specific manner [46,48]. This emerging evidence suggests that the Th1–Treg transition is a double-edged process: while early acquisition of T-bet and CXCR3 can enhance the capacity of Tregs to suppress Th1 activity and limit excessive inflammation, chronic IFNγ exposure may destabilize or “fatigue” these Th1-like Tregs, ultimately reducing suppressive potency and contributing to autoimmune amplification [49]. IFNγ induces T-bet and CXCR3 in Tregs, facilitating their trafficking to inflamed tissues and contact-dependent suppression of Th1 effectors [24,46]. While IFNγ deficiency does not abolish PBA, it attenuates autoreactive IgG subclass switching and reduces ANoA, implying selective control of B-cell tolerance checkpoints, affinity maturation and isotype diversification by IFNγ [2,50]. Together, these recent insights position the IFNγ-mediated Th1-Treg transition as a critical nexus in environmentally triggered systemic autoimmunity. Rather than functioning as a linear suppressive pathway, the IFNγ→T-bet→CXCR3 axis appears to enable stage-specific regulatory “reprogramming,” with protective effects early in disease but pathological consequences during chronic exposure [48]. This dynamic spectrum, from early IFNγ-supported regulatory containment to late-phase Treg fragility, suggests that therapeutic strategies may need to discriminate between beneficial Th1-Treg imprinting and harmful chronic IFNγ signaling. Targeting chemokine-guided Treg positioning (e.g., CXCR3-dependent tissue infiltration) or modulating STAT1-driven Treg stability may offer stage-adapted interventions for Hg-induced and lupus-like autoimmunity [49].

Mercury skews toward Th2-type humoral responses and perturbs regulation [51,52]. Our data mirror this, including changes in the IgG2a/IgG1 index in Hg-exposed recipients. The coexistence of Th2-biased antibodies with an IFNγ requirement indicates that IFNγ modulates the quality rather than the polarity of B-cell help: even in Th2-skewed settings, IFNγ signals shape autoreactive antibody generation [2].

Hg may act directly on T-cell differentiation and indirectly via APC activation. By interacting with thiol-containing signaling molecules, Hg perturbs NF-κB pathways downstream of TLRs [51], enhancing co-stimulation and antigen presentation and intensifying T–B cross-talk [53]. In Tregs, Hg could potentially influence epigenetic regulatory circuits and might reduce LAG-3–MHC II–mediated restraint, a pathway thought to be important for limiting DC activation. These possibilities remain speculative and should be viewed as hypotheses requiring further experimental confirmation [53,54,55]. Our findings are compatible with progressive erosion of Treg stability and function under chronic exposure, echoing xenobiotic-driven lupus models [42,43]. Genetic susceptibility (H-2s and NF-κB-regulated loci such as Bank1 and Nfkb1) likely lowers thresholds for tolerance loss under Hg stress [56]. Together, the adoptive-transfer data position Tregs as a central integrator of these risks, with efficacy contingent on IFNγ and LAG-3–dependent control.

The relationship between serological autoantibodies and tissue pathology in HgIA is complex [57]. Elevated ANoA titers correlate with increased renal IC deposition, suggesting a direct link between autoantibody production and tissue damage [37,58]. Rising anti-chromatin, anti-DNP, and anti-ssDNA titers closely paralleled increased renal immune-complex deposition across groups, indicating a functional link between circulating autoantibodies and tissue pathology. Suppression of these serological markers in Treg recipients corresponded to reduced deposition, whereas increases in non-Treg recipients aligned with more prominent glomerular involvement.

Human data parallel these observations: asymptomatic ANA⁺ individuals often show expanded adaptive Tregs and elevated TGF-β1 (compensatory regulation), whereas SARD patients display Th2/Th17 skewing, plasmablast hyperactivity, and myeloid priming [59,60]. Longitudinal studies identify LAG-3⁺ Tregs with increased IFN-α and TGF-β1 as early indicators of immune stress before flares [61], supporting the concept that intact IFNγ-Treg collaboration and LAG-3–mediated restraint preserve homeostasis during environmental challenge.

Future work should invert the design using IFNγ-deficient B10.S recipients to test whether WT Hg-primed Tregs and non-Tregs can, respectively, restore regulation or break tolerance under Hg exposure. Integrated serologic and cellular readouts (Th1/Th2 cytokines, isotype switching) will clarify how IFNγ, Tregs, and non-Tregs collectively shape HgIA.

In summary, HgIA is governed by a regulatory triad in which Tregs and IFNγ signaling restrain pathogenic immunity. IFNγ sustains Treg competence in inflamed tissues, whereas chronic Hg exposure undermines this stability. Restoring these axes, via adoptive Treg therapy, cytokine pathway tuning, or reinforcing co-inhibitory receptors, may reverse tolerance breakdown in environmentally induced autoimmunity [62].

5. Conclusions

Collectively, our findings show that Hg-primed non-Tregs (Group C) amplify, whereas Hg-primed Tregs (Group D) attenuate, environmentally induced autoimmunity. Tregs markedly reduce HgIA severity, and IFNγ serves as a key cofactor enabling effective Treg control of autoantibody responses and renal immunopathology. Although mechanisms such as memory-like features in non-Tregs and potential epigenetic influences on Treg function remain undefined, the patterns observed in our study may point to provisional, testable hypotheses rather than established mechanistic conclusions. These interpretations should therefore be viewed as speculative frameworks that warrant further experimental validation. The IFNγ-dependent effects identified appear novel and warrant continued mechanistic investigation. Our study is limited by the lack of detailed phenotypic and stability characterization of IFNγ^−/− Tregs, and additional markers indicative of lineage stability. Addressing these gaps will be essential to determine whether the observed suppressive effects in IFNγ^−/− B10.S mice reflect defects in IFNγ-dependent signaling during the suppressive process itself, or whether they instead arise from broader alterations in Treg development and ontogeny.

6. In Memoriam

During the preparation of this manuscript, our co-author, Dr. Mehdi Amirhosseini, who played a significant role both in conducting the analyses and in the scientific writing, sadly passed away at a young age. This loss has left us in deep sorrow.

Dr. Amirhosseini was a highly valued and respected colleague. Through his meticulous work, sharp scientific insight, and strong commitment, he quickly established himself as an indispensable member of our research team. From the very design phase of the study, Mehdi contributed actively to the development and progress of the project.

We will remember Mehdi Aamirhosseini not only for his important contributions to research and scientific advancement, but above all for his personal warmth, integrity, and humanity. His passing is a profound loss to us all.

This work is dedicated to his memory and his invaluable contributions to science.