Effect of Age on Xenobiotic-Induced Autoimmunity

Abstract

Aging is associated with increased spontaneous autoantibody production and chronic inflammation, yet its impact on xenobiotic-induced autoimmunity remains unexplored. This study investigates the effect of age on mercury-induced autoimmunity (HgIA) in B10.S mice, a model of xenobiotic-induced autoimmunity characterized by anti-nucleolar autoantibodies (ANoA). Mature (3 months), adult (6 months), middle-aged (12 months), and old-age (24 months) mice were exposed to mercury (HgCl₂) or phosphate-buffered saline (PBS) for 4-5 weeks. While spontaneous anti-nuclear antibodies (ANA) increased with age in PBS-treated mice (34% in middle-aged, 57% in old age mice), HgIA incidence declined in old age mice, with only 59% (26/44) developing significant ANoA titers compared to 91-100% in younger cohorts. Notably, 56% (10/18) of initially ANoA-negative old mice had detectable ANoA at a lower dilution, indicating a reduced but not absent response. ANoA negativity in old age mice was associated with lower immunoglobulin levels, reduced anti-chromatin antibodies, and diminished germinal center formation, suggestive of immunosenescence. Flow cytometry revealed age-related declines in CD4⁺ T cells, with mercury exposure augmenting T-cell differentiation in younger but not old mice. These findings demonstrate that aging enhances spontaneous autoimmunity but impairs xenobiotic-induced autoimmunity, with a subset of old age mice retaining partial responsiveness at lower dilutions, highlighting the complex interplay between immunosenescence and environmental triggers.

Figure 1.. Age and HgCl₂-induced changes in autoantibodies and hypergammaglobulinemia in B10.S mice.

Sera assessment of B10.S mice at 3 (n=17/18), 6 (n=19/20), 12 (n=56/57) or 24 (n=48/44) months of age treated with PBS (gray) or HgCl₂ (blue). Mice treatment and assays performed as described in Materials & Methods. Statistical comparisons between age groups were performed with Kruskal-Wallis tests. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Statistical comparisons of PBS and HgCl₂ treatment groups were performed with Mann-Whitney U tests. a, p<0.05; b, p<0.01; c, p<0.001; d, p<0.0001.

Figure 2.. Comparison of humoral immune responses in ANA negative and positive middle aged and old age B10.S mice.

Sera assessment of ANA negative (gray) and positive (green) 12 (n=32/19) and 24 (n=20/28) month old mice treated with PBS. Mice treatment and assays performed as described in Materials & Methods. Statistical comparisons between age groups were performed with Kruskal-Wallis tests. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Statistical comparisons of ANA negative and positive treatment groups were performed with Mann-Whitney U tests. a, p<0.05; b, p<0.01; c, p<0.001; d, p<0.0001.

Figure 3.. The ANoA response requires HgCl₂-exposure but does not occur in all animals with age.

Assessment of ANoA in B10.S mice at 3 (n=17/18), 6 (n=19/20), 12 (n=56/57) or 24 (n=48/44) months of age treated with PBS (gray) or HgCl₂ (blue). Mice treatment and assays performed as described in Materials & Methods. Statistical comparisons between age groups were performed with Kruskal-Wallis tests. *P<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Statistical comparisons of PBS and HgCl₂ treatment groups were performed with Mann-Whitney U tests. a, p<0.05; b, p<0.01; c, p<0.001; d, p<0.0001.

Figure 4.. Comparison of humoral immune responses in ANoA negative and positive HgCl₂ exposed old age mice.

Sera assessment of ANoA negative (n=18) and ANoA positive (n=26) 24 month old mice treated with HgCl₂. Mice treatment and assays performed as described in Materials & Methods. Statistical comparisons were performed with Mann-Whitney U tests. *P<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Figure 5.. Changes in T cell subsets with age and HgIA.

Multicolor flow cytometry on the spleens of B10.S mice at 3 (n=18/18), 6 (n=12/12), 12 (13/13) or 24 months (n=19/23) of age treated with PBS (gray) or HgCl₂ (blue). Mice treatment and assays performed as described in Materials & Methods. Statistical comparisons between age groups were performed with Kruskal-Wallis tests. *P<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Statistical comparisons of PBS and HgCl₂ treatment groups were performed with Mann-Whitney U tests. a, p<0.05; b, p<0.01; c, p<0.001; d, p<0.0001.

Figure 6.. Activation of CD4⁺ T cell subsets with age and HgIA.

Multicolor flow cytometry on the spleens of B10.S mice at 3 (n=18/18), 6 (n=12/12), 12 (13/13) or 24 (n=19/23) months of age treated with PBS (gray) or HgCl₂ (blue). Mice treatment and assays performed as described in Materials & Methods. Statistical comparisons between age groups were performed with Kruskal-Wallis tests. *P<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Statistical comparisons of PBS and HgCl₂ treatment groups were performed with Mann-Whitney U tests. a, p<0.05; b, p<0.01; c, p<0.001; d, p<0.0001.

Figure 7.. Germinal center T follicular helper cells with HgIA.

Multicolor flow cytometry on the spleens of B10.S mice at 12 months of age treated with PBS (n=3) (gray) or HgCl₂ (n=4) (blue). Mice treatment and assays performed as described in Materials & Methods. Statistical comparisons between PBS and HgCl₂ treatment groups were performed with unpaired t-tests (black *). *P<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Figure 8.. B cell subset changes with age and HgIA.

Multicolor flow cytometry on the spleens of B10.S mice at 3 (n=18/18), 6 (n=12/12), 12 (13/13) or 24 (n=19/23) months of age treated with PBS (gray) or HgCl₂ (blue). Mice treatment and assays performed as described in Materials & Methods. Statistical comparisons between age groups were performed with Kruskal-Wallis tests. *P<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Statistical comparisons of PBS and HgCl₂ treatment groups were performed with Mann-Whitney U tests. a, p<0.05; b, p<0.01; c, p<0.001; d, p<0.0001.

Figure 9.. Age-associated B cells with age and HgIA.

Multicolor flow cytometry on the spleens of B10.S mice at 3 (n=3/4), 6 (n=7/7) or 12 (11/8) months of age treated with PBS (gray) or HgCl₂ (blue). Mice treatment and assays performed as described in Materials & Methods. Statistical comparisons between age groups were performed with one-way ANOVA. *P<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Statistical comparisons between PBS and HgCl₂ treatment groups were performed with unpaired t-tests. a, p<0.05; b, p<0.01; c, p<0.001; d, p<0.0001.

Figure 10.. non T or B cell type changes with age and mHgIA.

Multicolor flow cytometry on the spleens of B10.S mice at 3 (n=18/18), 6 (n=12/12), 12 (13/13) or 24 (n=19/23) months of age treated with PBS (gray) or HgCl₂ (blue). Mice treatment and assays performed as described in Materials & Methods. Statistical comparisons between age groups were performed with Kruskal-Wallis tests. *P<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Statistical comparisons of PBS and HgCl₂ treatment groups were performed with Mann-Whitney U tests. a, p<0.05; b, p<0.01; c, p<0.001; d, p<0.0001.

Figure 11.. Spontaneous ANA is reflected in spleen cell populations in middle aged mice.

Multicolor flow cytometry on the spleens of ANA− (gray) and ANA+ (green) 12 (10/3) and 24 (11/10) month old B10.S mice treated with PBS. Mice treatment and assays performed as described in Materials & Methods. Statistical comparisons between age groups were performed with ANOVA tests. *P<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Statistical comparisons of PBS and HgCl₂ treatment groups were performed with unpaired t-tests. a, p<0.05; b, p<0.01; c, p<0.001; d, p<0.0001.

Figure 12.. ANoA− and ANoA+ responses to HgCl₂ are reflected in spleen cell populations in old mice.

Multicolor flow cytometry on the spleens of ANoA− (n=7) and ANoA+ (n=16) 24 month old B10.S mice. Mice treatment and assays performed as described in Materials & Methods. Statistical comparisons between ANoA negative and positive mice were performed with unpaired t-tests. *P<0.05, **p<0.01, ***p<0.001, ****p<0.0001.