Gentaro Izumi

Toll-like Receptors at the Maternal-Fetal Interface in Normal Pregnancy and Pregnancy Complications

Toll‐like receptors (TLRs) form the major family of pattern recognition receptors (PRRs) that are involved in innate immunity. Innate immune responses against microorganisms at the maternal‐fetal interface may have a significant impact on the success of pregnancy, as intrauterine infections have been shown to be strongly associated with certain complications of pregnancy. At the maternal‐fetal interface, TLRs are expressed not only in the immune cells but also in non‐immune cells such as trophoblasts and decidual cells; moreover, their expression patterns vary according to the stage of pregnancy. Here, we will update potential functions of TLRs in these cells, their recognition and response to microorganisms, and their involvement in the innate immunity. The impact of TLR‐mediated innate immune response will be discussed via animal model studies, as well as clinical observations.

Dienogest reduces proliferation, aromatase expression and angiogenesis, and increases apoptosis in human endometriosis

Abstract Dienogest is a novel progestin that is highly selective for progesterone receptors and inhibits endometriosis. However, it remains unknown how the administration of dienogest to patients with endometriosis impacts on their lesion tissues. The aim of this study was to evaluate the in vivo effect of dienogest on endometriosis tissue. We collected endometrioma tissues from patients treated with dienogest (N = 7) or not treated (N = 11, controls). Cell proliferation, aromatase expression and blood vessel density were evaluated by staining for Ki67, aromatase and the von Willebrand factor, respectively. Apoptosis was detected using the TUNEL assay. The proportion of Ki67 and aromatase positive epithelial cells was significantly lower in the dienogest group than in controls (p < 0.05, respectively). The number of TUNEL positive cells was significantly higher in the dienogest group (p < 0.05). The density of blood vessels in endometrioma was marginally lower in the dienogest group compared with controls (p = 0.20). Our study demonstrates that endometrioma taken from patients treated with dienogest show remarkable histological features such as reduction of proliferation, aromatase expression and angiogenesis, and increase of apoptosis. This study clarified the impact of dienogest on local histological events that explain its therapeutic effect on endometriosis. Chinese abstract 地诺孕素是一种新型的孕激素，具有孕激素受体高度选择性，可抑制子宫内膜异位症。但是，地诺孕素作用于内异症患者受损组织的机理现在仍未明确。本研究的目的在于评估地诺孕素在体内对子宫内膜异位组织的影响。我们采集经地诺孕素治疗（N = 7）与未经治疗（N = 11，对照组）的患者的子宫内膜异位组织。通过对Ki67染色、芳香化酶及von Willebrand因子的测定，评估细胞增殖、芳香化酶表达及血管密度。细胞凋亡情况通过TUNEL检验测定。结果显示，地诺孕素组的Ki67与芳香化酶阳性上皮细胞比例显著低于对照组（均为p < 0.05）。地诺孕素组的TUNEL阳性细胞数目显著高于对照组（p < 0.05）。地诺孕素组子宫内膜异位组织中血管密度略低于对照组（p = 0.20）。本研究结果表明，经地诺孕素治疗的子宫内膜异位症患者可出现显著的组织学特征改变，如降低细胞增殖、芳香化酶表达及血管生成减少及细胞凋亡增加。研究明确了地诺孕素对局部组织的影响，证实了其治疗子宫内膜异位症的效果。

Activin-A is induced by interleukin-1β and tumor necrosis factor-α and enhances the mRNA expression of interleukin-6 and protease-activated receptor-2 and proliferation of stromal cells from endometrioma

OBJECTIVE To examine the regulation and the function of activin-A in stromal cells derived from endometrioma. DESIGN Molecular studies. SETTING University research laboratory. PATIENT(S) Endometrioma stromal cells (EoSC) were obtained from 28 patients with ovarian endometrioma undergoing laparoscopy. INTERVENTION(S) EoSC were cultured with inflammatory stimuli or recombinant activin-A, followed by RNA extraction. MAIN OUTCOME MEASURE(S) Activin mRNA expression was evaluated by real-time reverse transcription-polymerase chain reaction (RT-PCR), and activin-A concentration of supernatant of cultured EoSC was evaluated by ELISA. Also, the effect of activin-A on EoSC was evaluated with real-time RT-PCR and cell proliferation assay. RESULT(S) Inflammatory stimuli, interleukin (IL) -1β, and tumor necrosis factor (TNF) -α induced inhibin/activin-βA subunit mRNA and activin-A protein expression in EoSC. Additionally, activin-A enhanced EoSC proliferation and increased the expression of IL-6 and protease-activated receptor (PAR)-2 mRNA. CONCLUSION(S) An in vitro study revealed that activin-A, which is induced by IL-1β or TNF-α, might promote endometriosis by stimulating IL-6 and PAR-2 mRNA expression and increasing the proliferation of EoSC.

Dienogest, a new conservative strategy for extragenital endometriosis: a pilot study

Extragenital endometriosis severely impairs the quality of life for affected women but its standard management has not yet been well established because of its relatively low incidence. As extragenital organs, intestine, followed by urinary tract, is the most common place affected by endometriosis, for which surgical treatment is sometimes difficult and accompanied by severe complications. Recently, dienogest, a novel progestin, has emerged as a new alternative for endometriosis, especially for endometriosis-associated pain. In this report, we presented four cases with rectosigmoidal and one with bladder endometriosis, treated with oral 2 mg/day dienogest for over 6 months. For all cases, the measurable extragenital lesions exhibited the reduction in their size after 10 to 11 months of use, accompanied with immediate relief of subjective symptoms related with extragenital lesions. This report suggests that dienogest can be a novel conservative alternative for extragenital endometriosis.

Toll-Like Receptor-3 Ligation-Induced Indoleamine 2, 3-Dioxygenase Expression in Human Trophoblasts

Indoleamine 2,3-dioxygenase (IDO) is an enzyme that degrades an essential amino acid, tryptophan, and plays a role in inhibiting the proliferation of T cells and intracellular pathogens. Inhibiting IDO in mice leads to fetal rejection, suggesting its significance in establishing pregnancy. Toll-like receptor 3 (TLR-3) is a key component of the innate immune system that recognizes viral double-stranded RNA and triggers immune reactions by producing type I interferon. Using a human trophoblast cell culture system, we studied the effect of TLR-3 ligation on IDO expression and function by treating trophoblasts with polyinosinic-polycytidylic acid [poly(I:C)] (a synthetic double stranded RNA, which mimics viral RNA). Real-time PCR and Western blot analysis revealed that IDO mRNA and protein expression was significantly induced by poly(I:C). The activity of IDO was also increased by poly(I:C) given that the L-kynurenine concentrations were elevated in conditioned media. Conditioned media from poly(I:C)-treated trophoblasts were found to inhibit the proliferation of human T cells significantly. Poly(I:C) was also shown to induce interferon (IFN)-β mRNA expression in trophoblasts. Recombinant human IFN-β increased IDO mRNA expression in trophoblasts more rapidly than poly(I:C). Pretreating with neutralizing antibody against IFN-β significantly suppressed IDO induction by poly(I:C). Collectively we have demonstrated that ligation of TLR-3 by poly(I:C) induces IDO expression in human first-trimester trophoblasts via an IFN-β-dependent pathway. These findings suggest that upon viral infection, trophoblasts induce IDO and in turn contribute to antimicrobial activity and maintenance of fetomaternal tolerance.

Effects of 1,25-Dihydroxy Vitamin D3 on Endometriosis

CONTEXT Endometriosis is an estrogen-dependent, chronic inflammatory disease. Recent studies have shown that vitamin D (VD) is an effective modulator of the immune system and plays an important role in controlling many inflammatory diseases. OBJECTIVE The objective of the study was to clarify the in vitro effects of 1,25-dihydroxy vitamin D3 (1,25[OH]2D3) on human endometriotic stromal cells (ESCs) and to determine the serum levels of VD in endometriosis patients. DESIGN, PATIENTS, AND MAIN OUTCOME MEASURES ESCs were isolated from ovarian endometrioma and cultured with 1,25(OH)2D3. Gene expression of IL-8, cyclooxygenase-2, microsomal prostaglandin E synthase-1, microsomal prostaglandin E synthase-2, cytosolic prostaglandin E synthase, 15-hydroxyprostaglandin dehydrogenase, matrix metalloproteinase (MMP)-2, and MMP-9 was examined using quantitative RT-PCR. The production of IL-8 and prostaglandin E2 was measured using an ELISA and an enzyme immunoassay. Viable cell number was assessed using a cell-counting assay, and DNA synthesis was assessed using the bromodeoxyuridine incorporation assay. Apoptosis was assessed using flow cytometry. The expression of inhibitory-κBα protein was detected using Western blotting. The serum levels of 25-hydroxyvitamin D3 and 1,25(OH)2D3 were measured by a RIA. RESULTS In vitro studies showed that 1,25(OH)2D3 significantly reduced IL-1β- or TNF-α-induced inflammatory responses, such as IL-8 expression and prostaglandin activity. 1,25(OH)2D3 also reduced viable ESC numbers and DNA synthesis but did not affect apoptosis. MMP-2 and MMP-9 expressions were reduced by 1,25(OH)2D3. 1,25(OH)2D3 inhibited nuclear factor-κB activation. The serum 25-hydroxyvitamin D3 levels were significantly lower in women with severe endometriosis than in the controls and women with mild endometriosis. Serum 1,25(OH)2D3 levels were not different between groups. CONCLUSIONS VD modulates inflammation and proliferation in endometriotic cells, and a lower VD status is associated with endometriosis. Taken together, VD supplementation could be a novel therapeutic strategy for managing endometriosis.

Interleukin-1β stimulates the secretion of thymic stromal lymphopoietin (TSLP) from endometrioma stromal cells: possible involvement of TSLP in endometriosis

STUDY QUESTION Is thymic stromal lymphopoietin (TSLP) involved in the pathophysiology of endometriosis? SUMMARY ANSWER TSLP is up-regulated by interleukin (IL)-1β and may be involved in the development of endometriosis. WHAT IS KNOWN ALREADY Endometriosis is a chronic inflammatory disease in which the Th2 immune response is activated and has been suggested to promote the disease. TSLP is a master cytokine that drive Th2 immune response. STUDY DESIGN, SIZE, DURATION A laboratory study. PARTICIPANTS/MATERIALS, SETTING, METHODS Primary cultures of endometrioma stromal cells (ESCs) were treated with IL-1β, a typical inflammatory cytokine associated with endometriosis. Gene expression of TSLP in ESCs and secretion of TSLP protein from ESCs were studied using quantitative PCR and a specific ELISA. Interferon γ (IFNγ), a typical Th1 cytokine, and IL-4, a typical Th2 cytokine, were added to the culture to evaluate their effect on the IL-1β-induced secretion of TSLP. Inhibitors of p38 mitogen-activated protein kinase (MAPK), p42/44 MAPK and stress-activated protein kinase/Jun amino-terminal kinase (SAPK/JNK) were added to the culture to examine intracellular signals involved in IL-1β-induced TSLP secretion. The expression of TSLP in endometrioma tissue was examined by immunohistochemistry. The concentration of TSLP in the serum and peritoneal fluid (PF) of women with or without endometriosis was measured with a specific ELISA. MAIN RESULTS AND THE ROLE OF CHANCE IL-1β stimulated the expression of TSLP mRNA and secretion of TSLP protein from ESCs. IL-4 enhanced the IL-1β-induced TSLP secretion from ESCs, while IFNγ reduced it. Inhibitors of p42/44 MAPK, p38 MAPK and SAPK/JNK suppressed the IL-1β-induced secretion of TSLP from ESCs. Positive immunostaining of TSLP was observed in the stroma of endometrioma tissue. TSLP concentrations in the serum and PF were both higher in women with endometriosis compared with those without endometriosis. LIMITATIONS, REASONS FOR CAUTION The present study was only in vitro. The samples used for culture were endometrioma tissues, not including other types of endometriosis. Therefore, the present findings should be interpreted with caution. WIDER IMPLICATIONS OF THE FINDINGS This study provided new insights in the Th2 immune response-related mechanism in endometriosis. STUDY FUNDING This study is partly supported by grants from the Ministry of Health, Labour and Welfare, and the Ministry of Education, Culture, Sports, Science and Technology. The authors have no conflicts of interest to declare.

Cyclic Stretch Augments Production of Neutrophil Chemokines and Matrix Metalloproteinase-1 in Human Uterine Smooth Muscle Cells

The aim of this study was to investigate the impact of uterine contraction on the immune environment within the uterus during parturition.

Interleukin-4 and Prostaglandin E2 Synergistically Up-Regulate 3β-Hydroxysteroid Dehydrogenase Type 2 in Endometrioma Stromal Cells

CONTEXT Endometriosis is a chronic inflammatory disease in which immune response and production of estrogen in endometriotic tissues are involved in the development of the disease. Prostaglandin E2 (PGE2) stimulates aromatase (P450arom) expression in endometrioma stromal cells (ESCs) and increases the production of estrogens. On the other hand, an accumulating amount of evidence suggests that IL-4, a typical Th2 cytokine, plays important roles in the disease. OBJECTIVE The objective of the investigation was to study the effect of IL-4 on the expression of 3β-hydroxysteroid dehydrogenase (HSD3B2), a pivotal enzyme for estrogen production, in ESCs. DESIGN, PATIENTS, AND MAIN OUTCOME MEASURES ESCs were isolated from ovarian endometrioma tissues and cultured with IL-4 and PGE2. CP-690550, a Janus protein tyrosine kinase 3 inhibitor, and HSD3B2 small interfering RNA were added to the culture. Gene expression of HSD3B2 and P450arom was examined by quantitative RT-PCR. Dehydroepiandrosterone (DHEA) was added to the culture, and then the combined enzyme activity of HSD3B2, which converts DHEA to androstenedione, and P450arom, which converts androstenedione to estrone, was examined by measuring estrone concentration in the supernatants with a specific enzyme immunoassay. RESULTS IL-4 increased the expression of HSD3B2 mRNA in a dose-dependent manner. CP-650550 inhibited the IL-4-induced increase in HSD3B2 mRNA expression. PGE2 also increased the expression of HSD3B2 mRNA, and the combination of IL-4 and PGE2 synergistically increased the expression of HSD3B2 mRNA. IL-4 had no effect on the expression of P450arom mRNA, whereas PGE2 increased the expression of P450arom mRNA. Although PGE2 alone increased the production of estrone from DHEA, the combination of IL-4 and PGE2 significantly augmented the production of estrone from DHEA. The enhanced production of estrone by the combination of IL-4 and PGE2 was inhibited by CP-690550 and HSD3B2 small interfering RNA. CONCLUSIONS IL-4 in combination with PGE2 may enhance estrogen production in endometriotic tissues, implying an elaborate mechanism that Th2 immune response augments inflammation-dependent progression of the disease.

Simultaneous Detection and Evaluation of Four Subsets of CD4+ T Lymphocyte in Lesions and Peripheral Blood in Endometriosis.

The proportion of CD4+ T lymphocytes, Th1, Th2, Th17, and regulatory T cells in endometriosis lesions and peripheral blood are not known.