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

Kawashima, Akira; Tanigawa, Kazunari; Akama, Takeshi; Wu, Huhehasi; Sue, Mariko; Yoshihara, Aya; Ishido, Yuko; Kobiyama, Kouji; Takeshita, Fumihiko; Ishii, Ken J.; Hirano, Hisashi; Kimura, Hiroaki; Sakai, Takafumi; Ishii, Norihisa; Suzuki, Koichi

doi:10.1210/jcem.96.3.zeg871a

Cited by 12 publications

(48 citation statements)

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“…The cells were grown either in Coon's modified Ham's F-12 medium supplemented with 5% bovine serum (Invitrogen) and a six-hormone mixture (1 mU TSH/mL, 10 lg insulin/mL, 10 ng somatostatin/mL, 0.36 ng hydrocortisone/mL, 5 lg transferrin/mL, and 2 ng glycyl-L-histidyl-L-lysine acetate/mL) or with 0.2% bovine serum without TSH and insulin mixture as previously described (44,45). Bovine Tg (Sigma) was used at a final concentration of 1-30 mg/mL, which was previously confirmed as having essentially the same effect as highly purified 19S Tg (30,34,35).…”

Section: Cell Culture and Treatmentmentioning

confidence: 99%

“…Total RNA isolation and real-time PCR Total RNA was purified using RNeasy Plus Mini Kit (Qiagen), and cDNA was synthesized using the HighCapacity cDNA Reverse Transcription Kits (Applied Biosystems) as described (44,45). Quantitative real-time PCR was performed using the ABI Prism 7700 Sequence Detection System (Applied Biosystems).…”

Section: Dna Microarray Analysismentioning

confidence: 99%

“…A total of 20 ng of cDNA mixed with 20 lL of FastStart Universal SYBR Green Master (Roche Diagnostics) was amplified by incubation for 10 minutes at 95°C, followed by 40 cycles consisting of 15 seconds at 95°C and 1 minute at 60°C. The mRNA levels were normalized against b-actin using the DDCt method as described (44,45). The sequences of PCR primers were as follows: Duox1 forward, 5¢-CCGTTTCTTGGGAGGTCCAGCGAT-3¢; Duox1 reverse, 5¢-CGTGGTAGCCGAAGAAGACACCCAG-3¢; Duoxa1 forward, 5¢-GCTGTTCTGGCTGCTACGGGTG-3¢; Duoxa1 reverse, 5¢-ACTGGGGTCCCTGTGAGGGTGA-3¢; Duox2 forward, 5¢-CAGCGCTACGACGGCTGGTTTA-3¢; Duox2 reverse, 5¢-CCCAAGCACTGTGCGGTTGT-3¢; Duoxa2 forward, 5¢-TCA GCGTACCGCTGCTCATCGT-3¢; and Duoxa2 reverse, 5¢-ACCAACCAGAACCAGCGCGAGT-3¢.…”

Section: Dna Microarray Analysismentioning

confidence: 99%

“…The DUOX2 promoter assay was performed with the Bright-Glo Luciferase assay systems (Promega) 48 hours after Tg stimulation. Luciferase activity was measured by FLUOstar galaxy (BMG Labtechnologies), and the data were normalized to corresponding protein concentrations, which were determined using a Bio-Rad Protein Assay Kit (Bio-Rad) (44,45).…”

Section: Plasmid Construction Transient Transfection and Reporter Gmentioning

confidence: 99%

“…Cellular protein extracts were mixed with a 4· lithium dodecyl sulfate sample buffer and a 10 · reducing agent (Invitrogen) and incubated for 10 minutes at 70°C before electrophoresis. The proteins were separated on NuPage 4%-12% Bis Tris by gel electrophoresis and transferred to i-Blot Gel transfer stacks of nitrocellulose (Invitrogen) (44,45). The membranes were washed with PBS with 0.1% Tween 20, and then incubated with either goat polyclonal anti-b-actin (Santa Cruz Biotechnology; dilution 1:1000) or goat polyclonal anti-DUOX2 antibody (sc-49939) (Santa Cruz; dilution 1:4000) for 12 hours at 4°C.…”

Section: Western Blotting Analysismentioning

confidence: 99%

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Regulation of Dual Oxidase Expression and H₂O₂Production by Thyroglobulin

Yoshihara

Hara

Kawashima

et al. 2012

Thyroid

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Background: Thyroglobulin (Tg) is a macromolecular precursor in thyroid hormone synthesis to which iodine is stably bound. Tg, which is stored in the follicular space, is also a potent negative feedback regulator of follicular function, and this is achieved by suppressing mRNA levels of thyroid-specific genes such as the sodium/iodide symporter (Slc5a5), Tg, and thyroid peroxidase. Dual oxidase 1 (DUOX1) and DUOX2, originally identified in the thyroid, are nicotinamide adenine dinucleotide phosphate (NADPH) oxidases that are necessary to produce the H 2 O 2 required for thyroid hormone biosynthesis. Since follicular Tg regulates the expression of genes that are essential for thyroid hormone synthesis, we hypothesized that Tg might also regulate DUOX expression and H 2 O 2 production. Methods: Rat thyroid FRTL-5 cells were treated with Tg, and the mRNA expression of Duox1 and Duox2 and their corresponding maturation factors Duoxa1 and Duoxa2 were evaluated by DNA microarray and real-time PCR. Duox2 promoter activity was examined by luciferase reporter gene assay. Protein levels of DUOX2 were also examined by Western blot analysis. Intracellular H 2 O 2 generation was quantified by a fluorescent dye, 5-(and-6)-chloromethyl-2¢,7¢-dichlorodihydrofluorescein diacetate, and acetyl ester (CM-H 2 DCFDA). Results: mRNA levels of Duox2 and its activation factor Duoxa2 (but not Duox1 or Duoxa1) were significantly suppressed by Tg in a dose-dependent manner and a time-dependent fashion in rat thyroid FRTL-5 cells. DUOX2 promoter activity was significantly suppressed by Tg in a dose-dependent manner. Protein levels of DUOX2 and H 2 O 2 generation in cells were also reduced by Tg treatment. Conclusions: We show that physiological concentrations of Tg suppressed the expression and function of DUOX2 in thyroid cells. These results suggest that Tg is a strong suppressor of the expression and the activity of DUOX2/DUOXA2, thereby regulating iodide organification and hormone synthesis in the thyroid. The evidence supports a reported model in which accumulated Tg in thyroid follicles plays important roles in autoregulating the function of individual follicles, which produces the basis of follicular heterogeneity.

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Section: Cell Culture and Treatmentmentioning

confidence: 99%

Section: Dna Microarray Analysismentioning

confidence: 99%

Section: Dna Microarray Analysismentioning

confidence: 99%

Section: Plasmid Construction Transient Transfection and Reporter Gmentioning

confidence: 99%

Section: Western Blotting Analysismentioning

confidence: 99%

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Regulation of Dual Oxidase Expression and H₂O₂Production by Thyroglobulin

Yoshihara

Hara

Kawashima

et al. 2012

Thyroid

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Hepatic DNA deposition drives drug‐induced liver injury and inflammation in mice

Marques¹,

Oliveira²,

Pereira³

et al. 2014

Hepatology

143

161

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Drug-induced liver injury (DILI) is an important cause of acute liver failure, with limited therapeutic options. During DILI, oncotic necrosis with concomitant release and recognition of intracellular content amplifies liver inflammation and injury. Among these molecules, self-DNA has been widely shown to trigger inflammatory and autoimmune diseases; however, whether DNA released from damaged hepatocytes accumulates into necrotic liver and the impact of its recognition by the immune system remains elusive. Here we show that treatment with two different hepatotoxic compounds (acetaminophen and thioacetamide) caused DNA release into the hepatocyte cytoplasm, which occurred in parallel with cell death in vitro. Administration of these compounds in vivo caused massive DNA deposition within liver necrotic areas, together with an intravascular DNA coating. Using confocal intravital microscopy, we revealed that liver injury due to acetaminophen overdose led to a directional migration of neutrophils to DNA-rich areas, where they exhibit an active patrolling behavior. DNA removal by intravenous DNASE1 injection or ablation of Toll-like receptor 9 (TLR9)-mediated sensing significantly reduced systemic inflammation, liver neutrophil recruitment, and hepatotoxicity. Analysis of liver leukocytes by flow cytometry revealed that emigrated neutrophils up-regulated TLR9 expression during acetaminophen-mediated necrosis, and these cells sensed and reacted to extracellular DNA by activating the TLR9/NF-jB pathway. Likewise, adoptive transfer of wild-type neutrophils to TLR9 2/2 mice reversed the hepatoprotective phenotype otherwise observed in TLR9 absence. Conclusion: Hepatic DNA accumulation is a novel feature of DILI pathogenesis. Blockage of DNA recognition by the innate immune system may constitute a promising therapeutic venue. (HEPATOLOGY 2015;61:348-360) See Editorial on Page 35 D rug-induced liver injury (DILI) is a serious condition, with a high mortality rate and limited therapeutic alternatives.1 Among these

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The Pathogenesis of Hashimoto’s Thyroiditis: Further Developments in our Understanding

2015

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indicates that HT is likely to be an extreme in this distribution and does not constitute a separate disease entity, although there are different clinical presentations across the distribution curve [3]. And while immunological techniques described below are defining discrete types of disease, such as the IgG4-related variant, for the purposes of this review we will not distinguish HT from other forms of autommune hypothyroidism except where indicated; this pragmatic approach is also necessary because of the rather loose definition that investigators apply to patients. There have been several recent reviews of this subject, in large part occasioned by the centenary of Hashimoto's discovery [4-7]. We therefore have mainly concentrated on papers since 2010, to bring readers an update on recent developments in the field, focussing firstly on predisposing factors and then on the pathogenic mechanisms responsible for tissue damage.

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

Cited by 12 publications

References 0 publications

Regulation of Dual Oxidase Expression and H₂O₂Production by Thyroglobulin

Regulation of Dual Oxidase Expression and H₂O₂Production by Thyroglobulin

Hepatic DNA deposition drives drug‐induced liver injury and inflammation in mice

The Pathogenesis of Hashimoto’s Thyroiditis: Further Developments in our Understanding

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

Cited by 12 publications

References 0 publications

Regulation of Dual Oxidase Expression and H2O2Production by Thyroglobulin

Regulation of Dual Oxidase Expression and H2O2Production by Thyroglobulin

Hepatic DNA deposition drives drug‐induced liver injury and inflammation in mice

The Pathogenesis of Hashimoto’s Thyroiditis: Further Developments in our Understanding

Contact Info

Product

Resources

About

Regulation of Dual Oxidase Expression and H₂O₂Production by Thyroglobulin

Regulation of Dual Oxidase Expression and H₂O₂Production by Thyroglobulin