oBJective: In NOD.H2 h4 mice, high dietary iodine intake has been known to cause Iodineaccelerated spontaneous Autoimmune thyroiditis (IsAt) via an unknown mechanism. the aim of the study was to examine whether the NOD.H2 h4 genetic background predisposes to enhanced iodine organification in thyroglobulin (tg), a target autoantigen in IsAt. DEsIGN: to avoid issues associated with an ongoing anti-tg antibody response, we assessed tg iodination levels in iodine-fed, b-cell deficient NOD.H2 h4 mice. Additionally, we tested whether humoral or cellular immune responses of iodine-fed NOD.H2 h4 mice are preferentially directed to tg with increased iodine content (I-tg) or known pathogenic tg peptides that contained iodine. rEsULts: the iodine content of tg was not significantly different between control (9.0±2.7 I atoms per monomer) and iodine-fed mice (10.9±0.3 I atoms per monomer). Furthermore, in iodine-fed NOD.H2 h4 mice developing IsAt, strong but equivalent serum IgG responses were detected to both tg or I-tg, whereas their lymphoid cells were stimulated weakly but equally well by tg or I-tg in vitro and did not show reactivity against a panel of five pathogenic tg peptides that contained iodine. cONcLUsIONs: the results suggest that development of IsAt in NOD.H2 h4 mice is not associated with enhanced iodine organification or differential b-or t-cell responses to iodinated determinants in tg.

In the field of autoimmune thyroiditis, NOD.H2(h4) mice have attracted significant and increasing attention since they not only develop spontaneous disease but they present thyroiditis with accelerated incidence and severity if they ingest iodide through their drinking water. This animal model highlights the interplay between genetic and dietary factors in the triggering of autoimmune disease and offers new opportunities to study immunoregulatory parameters influenced by both genes and environment. Here, we review experimental findings with this mouse model of thyroiditis.

Background: Enhanced iodide intake in NOD.H2 h4 mice accelerates the incidence and severity of spontaneous autoimmune thyroiditis (SAT) via an unknown mechanism. A plausible hypothesis is that iodide-induced apoptosis of thyrocytes can create imbalances in antigenic load and/or disruption of immunoregulatory mechanisms that facilitate activation of autoreactive T cells in cervical lymph nodes draining the thyroid. Methods: We examined whether NOD.H2 h4 thyrocytes, exposed to low NaI concentrations in vitro, are more susceptible to apoptosis compared to thyrocytes from CBA/J mice, which are resistant to iodide-accelerated SAT (ISAT). We also looked, at the transcriptional level, for differential activation of genes involved in apoptosis or oxidative stress pathways that may account for potential differences in iodide-mediated apoptosis between NOD.H2 h4 and CBA/J thyrocytes. Results: We report that NOD.H2 h4 thyrocytes, cultured for 24 h at very low (4-8 lM) concentrations of NaI, exhibit high levels (40-55%) of apoptosis, as assessed microscopically following staining with fluorescent caspase inhibitors. Similar treatment of thyrocytes from CBA/J mice, which are resistant to ISAT, yielded significantly lower (10-20%) apoptotic rates. Expression analysis by real-time polymerase chain reaction using arrays of apoptosis-and oxidative stress-related genes showed that NaI intake upregulates the expression of 22 genes involved in ROS metabolism and/or antioxidant function in CBA/J thyrocytes, whereas only two of these genes were upregulated in NOD.H2 h4 thyrocytes. Among the set of overexpressed genes were those encoding thyroid peroxidase (Tpo; 5.77-fold), glutathione peroxidases (Gpx2, Gpx4, Gpx7; 2.03-3.14-fold), peroxiredoxins (Prdx1, Prdx2, Prdx5; 2.27-2.97-fold), superoxide dismutase 1 (Sod1; 3.57-fold), thioredoxin 1 (Txn1; 2.13-fold), and the uncoupling proteins 2 and 3 (Ucp2, Ucp3; 2.01-2.15-fold).Conclusions: The results demonstrate that an impaired control of oxidative stress mechanisms is associated with the observed high susceptibility of NOD.H2 h4 thyrocytes to NaI-mediated apoptosis, and suggest a contributing factor for the development of ISAT in this strain.

Summary Splenic Ly6C high monocytes are innate immune cells involved in the regulation of central nervous system-related diseases. Recent studies have reported the shaping of peripheral immune responses by the gut microbiome via mostly unexplored pathways. In this study, we report that a 4-day antibiotic treatment eliminates certain families of the Bacteroidetes, Firmicutes, Tenericutes, and Actinobacteria phyla in the gut and reduces the levels of multiple pattern recognition receptor (PRR) ligands in the serum. Reduction of PRR ligands was associated with reduced numbers and perturbed function of splenic Ly6C high monocytes, which acquired an immature phenotype producing decreased levels of inflammatory cytokines and exhibiting increased phagocytic and anti-microbial abilities. Addition of PRR ligands in antibiotic-treated mice restored the number and functions of splenic Ly6C high monocytes. Our data identify circulating PRR ligands as critical regulators of the splenic Ly6C high monocyte behavior and suggest possible intervention pathways to manipulate this crucial immune cell subset.

Enhanced iodide ingestion is known to accelerate the incidence and severity of spontaneous autoimmune thyroiditis [iodide-accelerated spontaneous autoimmune thyroiditis (ISAT)] in NOD.H2h4 mice. CD4+ cells are required for the development and maintenance of ISAT, but their target epitopes remain unknown. In this study, we show that the previously identified thyroglobulin (Tg) T cell epitope p2549–2560 containing thyroxine at position 2553 (T4p2553) induces thyroiditis as well as strong specific T and B cell responses in NOD.H2h4 mice. In ISAT, activated CD4+ T cells specific for T4p2553 are detected before the disease onset in thyroid-draining cervical lymph nodes only in mice placed on an iodide-rich diet and not in age-matched controls. In addition, selective enrichment of CD4+ IFN-γ+ T4p2553-specific cells is observed among cervical lymph node cells and intrathyroidal lymphocytes. T4p2553 was equally detectable on dendritic cells obtained ex vivo from cervical lymph node cells of NaI-fed or control mice, suggesting that the iodide-rich diet contributes to the activation of autoreactive cells rather than the generation of the autoantigenic epitope. Furthermore, spontaneous T4p2553-specific IgG are not detectable within the strong Tg-specific autoantibody response. To our knowledge, these data identify for the first time a Tg T cell epitope as a spontaneous target in ISAT.

Experimental autoimmune thyroiditis, induced in mice after challenge with thyroglobulin (Tg), is known to be under the genetic control of the H2Ak locus. Because cathepsins are known to influence proteolytic processing of Tg in vivo, we examined in this study whether putative H2Ak-binding Tg epitopes, located near cathepsin cleavage sites within mouse Tg, have immunopathogenic properties. Cathepsin L, B, and D cleavage sites in mouse Tg were predicted based on homology with known cathepsin cleavage sites in rabbit Tg. We used an algorithm-based approach to identify H2Ak-binding motifs within 20-aa residue segments adjacent to cathepsin cleavage sites, and five 12mer peptides encompassing these sequences were synthesized. Two of them, p2369 (aa 2369–2380) and p2439 (aa 2439–2450) were immunogenic, eliciting significant proliferative T cell responses using lymph node cells from peptide-primed mice and production of IL-2 and IFN-γ in recall assays in vitro. Both peptides induced experimental autoimmune thyroiditis upon direct challenge of CBA/J mice with peptide in CFA and by adoptive transfer of peptide-primed lymph node cells into naive recipient hosts, but neither peptide was characterized as dominant.

SummaryRecent studies have shown that high salt (HS) intake exacerbates experimental autoimmune encephalomyelitis and have raised the possibility that a HS diet may comprise a risk factor for autoimmune diseases in general. In this report, we have examined whether a HS diet regimen could exacerbate murine autoimmune thyroiditis, including spontaneous autoimmune thyroiditis (SAT) in non-obese diabetic (NOD.H2 h4 ) mice, experimental autoimmune thyroiditis (EAT) in C57BL/6J mice challenged with thyroglobulin (Tg) and EAT in CBA/J mice challenged with the Tg peptide (2549-2560). The physiological impact of HS intake was confirmed by enhanced water consumption and suppressed aldosterone levels in all strains. However, the HS treatment failed to significantly affect the incidence and severity of SAT or EAT or Tg-specific immunoglobulin (Ig)G levels, relative to control mice maintained on a normal salt diet. In three experimental models, these data demonstrate that HS intake does not exacerbate autoimmune thyroiditis, indicating that a HS diet is not a risk factor for all autoimmune diseases.

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