Yuko Ishido

Iodine Excess as an Environmental Risk Factor for Autoimmune Thyroid Disease

The global effort to prevent iodine deficiency disorders through iodine supplementation, such as universal salt iodization, has achieved impressive progress during the last few decades. However, iodine excess, due to extensive environmental iodine exposure in addition to poor monitoring, is currently a more frequent occurrence than iodine deficiency. Iodine excess is a precipitating environmental factor in the development of autoimmune thyroid disease. Excessive amounts of iodide have been linked to the development of autoimmune thyroiditis in humans and animals, while intrathyroidal depletion of iodine prevents disease in animal strains susceptible to severe thyroiditis. Although the mechanisms by which iodide induces thyroiditis are still unclear, several mechanisms have been proposed: (1) excess iodine induces the production of cytokines and chemokines that can recruit immunocompetent cells to the thyroid; (2) processing excess iodine in thyroid epithelial cells may result in elevated levels of oxidative stress, leading to harmful lipid oxidation and thyroid tissue injuries; and (3) iodine incorporation in the protein chain of thyroglobulin may augment the antigenicity of this molecule. This review will summarize the current knowledge regarding excess iodide as an environmental toxicant and relate it to the development of autoimmune thyroid disease.

Fragments of Genomic DNA Released by Injured Cells Activate Innate Immunity and Suppress Endocrine Function in the Thyroid

Activation of innate and acquired immune responses, which can be induced by infection, inflammation, or tissue injury, may impact the development of autoimmunity. Although stimulation of cells by double-stranded DNA (dsDNA) has been shown to activate immune responses, the role of self-genomic DNA fragments released in the context of sterile cellular injury is not well understood. Using cultured thyroid cells, we show that cell injury prompts the release of genomic DNA into the cytosol, which is associated with the production of type I interferons, inflammatory cytokines, and chemokines. Molecules necessary for antigen processing and presentation to lymphocytes are also induced in thyroid cells by injury. dsDNA strongly suppressed the expression of sodium/iodide symporter and radioiodine uptake. To identify molecules responsible for sensing cytosolic dsDNA, we directly identified the cellular proteins that bound a dsDNA Sepharose column by mass spectrometry. Our analysis identified histone H2B, which was previously demonstrated to be an essential factor that mediates the activation of innate immunity induced by dsDNA. Knockdown of histone H2B using specific small interfering RNA abolished cell injury-induced innate immune activation and increased sodium/iodide symporter expression. These results indicate that genomic DNA fragments released by cell injury are recognized by extrachromosomal histone H2B, which results in the activation of genes involved in both innate and acquired immune responses in thyroid cells and suppression of thyroid function. These results suggest that sterile thyroid injury, in the absence of infection, may be sufficient to trigger autoimmune reaction and to induce thyroid dysfunction.

Clofazimine modulates the expression of lipid metabolism proteins in Mycobacterium leprae-infected macrophages.

Mycobacterium leprae (M. leprae) lives and replicates within macrophages in a foamy, lipid-laden phagosome. The lipids provide essential nutrition for the mycobacteria, and M. leprae infection modulates expression of important host proteins related to lipid metabolism. Thus, M. leprae infection increases the expression of adipophilin/adipose differentiation-related protein (ADRP) and decreases hormone-sensitive lipase (HSL), facilitating the accumulation and maintenance of lipid-rich environments suitable for the intracellular survival of M. leprae. HSL levels are not detectable in skin smear specimens taken from leprosy patients, but re-appear shortly after multidrug therapy (MDT). This study examined the effect of MDT components on host lipid metabolism in vitro, and the outcome of rifampicin, dapsone and clofazimine treatment on ADRP and HSL expression in THP-1 cells. Clofazimine attenuated the mRNA and protein levels of ADRP in M. leprae-infected cells, while those of HSL were increased. Rifampicin and dapsone did not show any significant effects on ADRP and HSL expression levels. A transient increase of interferon (IFN)-β and IFN-γ mRNA was also observed in cells infected with M. leprae and treated with clofazimine. Lipid droplets accumulated by M. leprae-infection were significantly decreased 48 h after clofazimine treatment. Such effects were not evident in cells without M. leprae infection. In clinical samples, ADRP expression was decreased and HSL expression was increased after treatment. These results suggest that clofazimine modulates lipid metabolism in M. leprae-infected macrophages by modulating the expression of ADRP and HSL. It also induces IFN production in M. leprae-infected cells. The resultant decrease in lipid accumulation, increase in lipolysis, and activation of innate immunity may be some of the key actions of clofazimine.

Rapid preparation of high-purity nuclear proteins from a small number of cultured cells for use in electrophoretic mobility shift assays.

BackgroundHighly purified nuclear protein is required when using an electrophoretic mobility shift assay (EMSA) to study transcription factors, e.g. nuclear factor-κB (NF-κB), a major transcription factor that regulates both innate and adaptive immune responses following infection. Although many protocols have been developed for nuclear protein extraction, they are not necessarily optimized for use in EMSA, often require a large number of cells and long processing times, and do not always result in complete separation of the nuclear and cytoplasmic fractions.ResultsWe have developed a simple, rapid and cost-effective method to prepare highly purified nuclear proteins from a small number of both suspended and adherent cultured cells that yields nuclear proteins comparable to those prepared by a standard large-scale method. The efficiency of the method was demonstrated by using EMSA to show the successful detection, in multilple concurrent samples, of NF-κB activation upon tetradecanoyl phorbol acetate (TPA) stimulation.ConclusionsThis method requires only a small number of cells and no specialized equipment. The steps have been simplified, resulting in a short processing time, which allows researchers to process multiple samples simultaneously and quickly. This method is especially optimized for use in EMSA, and may be useful for other applications that include proteomic analysis.

Paleopathological Evidence and Detection of Mycobacterium leprae DNA from Archaeological Skeletal Remains of Nabe-kaburi (Head-Covered with Iron Pots) Burials in Japan

The Nabe-kaburi is a unique burial method, the purpose of which is shrouded in mystery. The burials were performed during the 15th to 18th centuries in eastern Japan, and involved covering the heads of the deceased with iron pots or mortars. The identification of leprosy-specific osteological lesions among some of the excavated remains has led to the suggestion that Nabe-kaburi burials were a reflection of the social stigma against certain infectious diseases, such as leprosy, tuberculosis or syphilis. However, molecular evidence for the presence of disease has been lacking. The goal of this study was to detect Mycobacterium leprae (M. leprae) DNA in archaeological human skeletal remains from Nabe-kaburi burials. The paleopathological data from three Nabe-kaburi burials were re-evaluated before small samples were taken from affected and control areas. DNA was extracted and used as a template to target the M. leprae-specific DNA using a combination of whole genome amplification, PCR analysis and DNA sequencing. M. leprae DNA fragments were detected in the two sets of skeletal remains that had also shown paleopathological evidence of leprosy. These findings provide definitive evidence that some of the Nabe-kaburi burials were performed for people affected by leprosy. Demonstration of the presence of M. leprae DNA, combined with archeological and anthropological examinations, will aid in solving the mystery of why Nabe-kaburi burials were performed in medieval Japan.

Follicular thyroglobulin enhances gene expression necessary for thyroid hormone secretion

We have previously shown that follicular thyroglobulin (Tg) has an unexpected function as an autocrine negative-feedback regulator of thyroid hormone (TH) biosynthesis. Tg significantly suppressed the expression of genes necessary for iodide transport and TH synthesis by counteracting stimulation by TSH. However, whether follicular Tg also regulates intracellular TH transport and its secretion from thyrocytes is not known. In the present study, we examined the potential effect of follicular Tg on TH transport and secretion by quantifying the expression of two TH transporters: monocarboxylate transporter 8 (MCT8) and μ-crystallin (CRYM). Our results showed that follicular Tg at physiologic concentrations enhanced both the mRNA and protein expression levels of MCT8 and CRYM in a time- and dose-dependent manner in rat thyroid FRTL-5 cells. Although both the sodium/iodide symporter (NIS), an essential transporter of iodide from blood into the thyroid, and MCT8, a transporter of synthesized TH from the gland, were co-localized on the basolateral membrane of rat thyrocytes in vivo, Tg decreased NIS expression and increased the expression of MCT8 by counteracting TSH action. Thus, the effect of Tg on TH secretion opposed its previously described negative-feedback suppression of TH synthesis. Our results indicate that Tg mediates a complex intrinsic regulation of gene expression that is necessary to balance two opposing vectorial transport systems: the inflow of newly synthesized TH and the outflow of TH by external secretion.

Detection of Mycobacterium ulcerans subsp. shinshuense DNA from a water channel in familial Buruli ulcer cases in Japan.

AIM To determine if Mycobacterium ulcerans subsp. shinshuense is present in the common living environment in a case of familial concurrent occurrence of Buruli ulcer in Japan. MATERIALS & METHODS Environmental samples were collected primarily from a water channel in the patients residence. A combination of whole-genome amplification, touchdown PCR and DNA sequencing was used to detect M. ulcerans subsp. shinshuense DNA. RESULTS M. ulcerans subsp. shinshuense DNA was detected in a crayfish sampled in July, but not in other samples including a crayfish sampled in October. CONCLUSION These findings support an association between contaminated aquatic environments and Buruli ulcer in Japan. The data also suggest dynamic seasonal appearance of the pathogen in the environment may contribute to the seasonal variation of Buruli ulcer occurrence in Japan.

The Emerging Roles of Thyroglobulin

Thyroglobulin (Tg), the most important and abundant protein in thyroid follicles, is well known for its essential role in thyroid hormone synthesis. In addition to its conventional role as the precursor of thyroid hormones, we have uncovered a novel function of Tg as an endogenous regulator of follicular function over the past decade. The newly discovered negative feedback effect of Tg on follicular function observed in the rat and human thyroid provides an alternative explanation for the observation of follicle heterogeneity. Given the essential role of the regulatory effects of Tg, we consider that dysregulation of normal Tg function is associated with multiple human thyroid diseases including autoimmune thyroid disease and thyroid cancer. Additionally, extrathyroid Tg may serve a regulatory function in other organs. Further exploration of Tg action, especially at the molecular level, is needed to obtain a better understanding of both the physiological and pathological roles of Tg.

Comprehensive analysis of prokaryotes in environmental water using DNA microarray analysis and whole genome amplification.

The microflora in environmental water consists of a high density and diversity of bacterial species that form the foundation of the water ecosystem. Because the majority of these species cannot be cultured in vitro, a different approach is needed to identify prokaryotes in environmental water. A novel DNA microarray was developed as a simplified detection protocol. Multiple DNA probes were designed against each of the 97,927 sequences in the DNA Data Bank of Japan and mounted on a glass chip in duplicate. Evaluation of the microarray was performed using the DNA extracted from one liter of environmental water samples collected from seven sites in Japan. The extracted DNA was uniformly amplified using whole genome amplification (WGA), labeled with Cy3-conjugated 16S rRNA specific primers and hybridized to the microarray. The microarray successfully identified soil bacteria and environment-specific bacteria clusters. The DNA microarray described herein can be a useful tool in evaluating the diversity of prokaryotes and assessing environmental changes such as global warming.

Excessive Cytosolic DNA Fragments as a Potential Trigger of Graves’ Disease: An Encrypted Message Sent by Animal Models

Graves’ hyperthyroidism is caused by autoantibodies directed against the thyroid-stimulating hormone receptor (TSHR) that mimic the action of TSH. The establishment of Graves’ hyperthyroidism in experimental animals has proven to be an important approach to dissect the mechanisms of self-tolerance breakdown that lead to the production of thyroid-stimulating TSHR autoantibodies (TSAbs). “Shimojo’s model” was the first successful Graves’ animal model, wherein immunization with fibroblasts cells expressing TSHR and a major histocompatibility complex (MHC) class II molecule, but not either alone, induced TSAb production in AKR/N (H-2k) mice. This model highlights the importance of coincident MHC class II expression on TSHR-expressing cells in the development of Graves’ hyperthyroidism. These data are also in agreement with the observation that Graves’ thyrocytes often aberrantly express MHC class II antigens via mechanisms that remain unclear. Our group demonstrated that cytosolic self-genomic DNA fragments derived from sterile injured cells can induce aberrant MHC class II expression and production of multiple inflammatory cytokines and chemokines in thyrocytes in vitro, suggesting that severe cell injury may initiate immune responses in a way that is relevant to thyroid autoimmunity mediated by cytosolic DNA signaling. Furthermore, more recent successful Graves’ animal models were primarily established by immunizing mice with TSHR-expressing plasmids or adenovirus. In these models, double-stranded DNA vaccine contents presumably exert similar immune-activating effect in cells at inoculation sites and thus might pave the way toward successful Graves’ animal models. This review focuses on evidence suggesting that cell injury-derived self-DNA fragments could act as Graves’ disease triggers.