Severe Autoimmune Thyroiditis in Transgenic NOD.H2h4 Mice Expressing Interleukin-4 in the Thyroid.

Background: Hashimoto's thyroiditis is a common autoimmune thyroid disorder characterized by thyroid lymphocytic infiltrates and autoreactive antibodies against thyroglobulin (TgAbs) and thyroperoxidase. Final evolution of the disease can lead to hypothyroidism with destruction of the thyroid architecture. Interleukin-4 (IL-4) is involved in the humoral immune response and B cell activation required in autoimmune thyroiditis (AT) progression. We used our mouse model overexpressing IL-4 by thyrocytes (Thyr-IL4) to study the impact of a local IL-4 expression in AT using transgenic nonobese diabetic (NOD.H2h4) derived animals treated with iodide-supplemented water to increase the incidence of spontaneous AT (SAT). Methods: Thyr-IL4 NOD.H2h4 and nonpathogenic C57BL/6 animals aged 8 weeks were exposed to 0.05% sodium iodide (NaI) in their drinking water for 8 and 16 weeks. Circulating TgAbs and expression of intrathyroidal cytokines were quantified. Thyroid inflammation was assessed by classical histological analyses, including identification of some immune cell populations. The most sensitive parameter to evaluate the thyroid function, serum thyrotropin (TSH), was also measured at the end of the treatment. Results: Relative to wild-type (WT) animals, Thyr-IL4 NOD.H2h4 mice developed severe accelerated SAT with elevated serum TgAbs and numerous thyroid infiltrates mainly composed of CD4+/CD8+ T cells, B lymphocytes, and monocytes/macrophages. Thyroid expression of T helper (Th) Th1/Th2 cytokines was also enhanced, as well as IL-17. In contrast, excessive iodide supply did not induce TgAbs in WT and Thyr-IL4 SAT-resistant C57BL/6 animals. However, moderate leukocyte infiltrations in transgenic thyroids were evident compared to WT, but associated with a limited number of T and B cells and a different cytokine profile from Thyr-IL4 NOD.H2h4 mice. Finally, and despite their diverse immune responses, both transgenic strains presented marked thyroid enlargement and elevated serum TSH at the end of the treatment in contrast to their WT littermates. Conclusions: These findings demonstrated that ectopic expression of IL-4 from thyrocytes enhanced the severity of accelerated SAT in disease-prone Thyr-IL4 NOD.H2h4 animals and promoted thyroid leukocyte infiltration in SAT-resistant transgenic C57BL/6 mice. Moreover, impaired thyroid function emerged in both transgenic strains during the progression of the disease.

Murine Thyroid IL-4 Expression Worsens Hypothyroidism on Iodine Restriction and Mitigates Graves Disease Development.

Cytokines are known to perturb thyroid function and the role of interleukin-4 (IL-4) in the pathogenesis of Graves disease (GD) remains controversial. In our mouse model overexpressing IL-4 in thyrocytes (Thyr-IL4), we have reported that adult mice preserved normal serum thyroxine despite an iodide uptake defect. In the present work, we evaluated if iodine restriction could uncover the thyroid deficiency in Thyr-IL4 animals as well as the role of pendrin overexpression as a compensatory mechanism. Moreover, using an experimental model of GD we investigated the effect of a local expression of IL-4 on the incidence of hyperthyroidism. Thyr-IL4 mice developed more rapidly elevated serum thyrotropin under low-iodine supply with thyroid enlargement and classical histological modifications. These hallmarks of hypothyroidism were all enhanced in Thyr-IL4 mice with complete pendrin invalidation. Following immunization, a lower proportion of Thyr-IL4 animals developed hyperthyroidism. Surprisingly, immunized Thyr-IL4 animals presented numerous leukocyte infiltrates, associated with increased intrathyroidal expression of IFN-γ. We have demonstrated that thyroid deficiency in Thyr-IL4 mice is partially compensated for by the excessive iodide content of the standard chow and the overexpression of pendrin in these animals. Furthermore, we have shown that the local expression of IL-4 in the thyroid attenuates GD progression, which was associated with enhanced thyroid infiltration by immune cells that could negatively affect thyroid function.

DUOX1-mediated hydrogen peroxide release regulates sodium transport in H441 bronchiolar epithelial cells.

AIM: Dexamethasone has been shown to induce the formation of epithelial domes by bronchiolar H441 cells. It stimulates the expression of both amiloride inhibitable epithelial sodium channels (ENaC) and dual oxidase-1 (DUOX1). We therefore ask the question whether DUOX1 expression and production of submillimolar amounts of H2 O2 is instrumental for the sodium channel upregulation observed in H441 cells. METHODS: In vitro cell culture, nystatin-perforated whole-cell patch-clamp technique, immunocytochemistry and RT-PCR methods have been used. RESULTS: Cells forming epithelial domes induced by dexamethasone (0.1 µmol L-1 , 24 hours) and by 5-aza-2'-deoxytidine (1 µmol L-1 , 48 hours) expressed more DUOX1 protein compared with other cells in the monolayer. Dome formation could be inhibited by exogenous catalase in a concentration-dependent manner and by the NADPH oxidase inhibitor diphenyliodonium, which suggested the involvement of H2 O2 . While single application of 0.2 mmol L-1 H2 O2 induced transient dome formation, lower doses were ineffective and higher doses disrupted the cell monolayer. Hydrogen peroxide (0.1 mmol L-1 ) activated acutely amiloride-sensitive whole-cell currents from 3.91 ± 0.79 pA pF-1 to 4.76 ± 0.98 pA pF-1 in dome-forming cells and had no effect in cells outside of domes. ENaC but not DUOX1 transcription was potentiated by catalase in the presence of dexamethasone, which suggested negative feedback of H2 O2 on ENaC gene expression. CONCLUSION: Our observations suggest that tonic production of H2 O2 by DUOX1 participates in maintaining the level of vectorial sodium transport by lung epithelial cells. Moreover, the system appears to be well tuned as it would allow H2 O2 -dependent innate immunity without inducing airway/alveolar sodium and fluid hyperabsorption.

Expression, Localization, and Regulation of the Sodium Bicarbonate Cotransporter NBCe1 in the Thyroid.

BACKGROUND: The intrafollicular space of thyroid follicles is the storage compartment for thyroid hormones. Its pH has been established at around 7.6 at least after thyrotropin (TSH) stimulation. This alkaline intrafollicular pH is thought to be critical for iodide coupling to thyroglobulin and internalization of iodinated thyroglobulin. At least in mice, this alkalinization requires the expression of pendrin (Slc26a4) within the apical membrane, and a lack of pendrin results in acidic follicular lumen pH. Yet, the mechanism importing HCO3- into the cytoplasm is unknown. This study investigated whether the rather ubiquitous sodium bicarbonate cotransporter NBCe1 (SLC4A4) might play this role. It also examined which variant was expressed and where it was localized in both rat and human thyroid tissue. Lastly, the dependence of its expression on TSH was studied. METHODS: Reverse transcription polymerase chain reaction, immunofluorescence, and Western blotting were used to test whether TSH stimulated NBCe1 protein expression in vivo. Subcellular localization of NBCe1 was performed using immunofluorescence in both rat and human thyroid. Cultured thyroid cells were also used to attempt to define how TSH affects NBCe1 expression. RESULTS: Only transcripts of the NBCe1-B variant were detected in both rat and human thyroid. Of interest, NBCe1-C was not detected in human tissues, not even in the brain. On immunofluorescence microscopy, the immunostaining of NBCe1 mainly appeared in the basolateral membrane upon stimulation with TSH. This TSH induction of basolateral membrane expression of NBCe1 protein was confirmed in vivo in rat thyroid and in vitro on human thyroid slices. CONCLUSIONS: This study demonstrates the expression of the sodium bicarbonate cotransporter NBCe1-B in rat and human thyroid. Additionally, the data suggest that TSH blocks the degradation of NBCe1 protein by trafficking it to the basolateral membrane. Hence, TSH increases NBCe1 half-life without increasing its synthesis.