RESUMO
IL-36 cytokines (the agonists IL-36α, IL-36ß, IL-36γ, and the antagonist IL-36Ra) are expressed in the mouse uterus and associated with maternal immune response during pregnancy. Here, we characterize the expression of IL-36 members in human primary trophoblast cells (PTC) and trophoblastic cell lines (HTR-8/SVneo and JEG-3) and upon treatment with bacterial and viral components. Effects of recombinant IL-36 on the migration capacity of trophoblastic cells, their ability to interact with endothelial cells and the induction of angiogenic factors and miRNAs (angiomiRNAs) were examined. Constitutive protein expression of IL-36 (α, ß, and γ) and their receptor (IL-36R) was found in all cell types. In PTC, transcripts for all IL-36 subtypes were found, whereas in trophoblastic cell lines only for IL36G and IL36RN. A synthetic analog of double-stranded RNA (poly I:C) and lipopolysaccharide (LPS) induced the expression of IL-36 members in a cell-specific and time-dependent manner. In HTR-8/SVneo cells, IL-36 cytokines increased cell migration and their capacity to interact with endothelial cells. VEGFA and PGF mRNA and protein, as well as the angiomiRNAs miR-146a-3p and miR-141-5p were upregulated as IL-36 response in PTC and HTR-8/SVneo cells. In conclusion, IL-36 cytokines are modulated by microbial components and regulate trophoblast migration and interaction with endothelial cells. Therefore, a fundamental role of these cytokines in the placentation process and in response to infections may be expected.
Assuntos
Regulação da Expressão Gênica/genética , Interleucina-1/genética , Neovascularização Fisiológica/genética , Trofoblastos/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Movimento Celular/genética , Movimento Celular/fisiologia , Células Cultivadas , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Interleucina-1/metabolismo , Interleucinas/genética , Interleucinas/metabolismo , Lipopolissacarídeos/farmacologia , MicroRNAs/genética , Neovascularização Fisiológica/fisiologia , Poli I-C/farmacologia , Prostaglandinas F/genética , Prostaglandinas F/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Trofoblastos/citologia , Trofoblastos/fisiologia , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Fluoxetine (FLX) is a pharmaceutical acting as a selective serotonin reuptake inhibitor and is used to treat depression in humans. Fluoxetine and the major active metabolite norfluoxetine (NFLX) are released to aquatic systems via sewage-treatment effluents. They have been found to bioconcentrate in wild fish, raising concerns over potential endocrine disrupting effects. The objective of this study was to determine effects of waterborne FLX, including environmental concentrations, on the reproductive axis in sexually mature male goldfish. We initially cloned the goldfish serotonin transporter to investigate tissue and temporal expression of the serotonin transporter, the FLX target, in order to determine target tissues and sensitive exposure windows. Sexually mature male goldfish, which showed the highest levels of serotonin transporter expression in the neuroendocrine brain, were exposed to FLX at 0.54µg/L and 54µg/L in a 14-d exposure before receiving vehicle or sex pheromone stimulus consisting of either 4.3nM 17,20ß-dihydroxy-4-pregnene-3-one (17,20P) or 3nM prostaglandin F2(α) (PGF2(α)). Reproductive endpoints assessed included gonadosomatic index, milt volume, and blood levels of the sex steroids testosterone and estradiol. Neuroendocrine function was investigated by measuring blood levels of luteinizing hormone, growth hormone, pituitary gene expression of luteinizing hormone, growth hormone and follicle-stimulating hormone and neuroendocrine brain expression of isotocin and vasotocin. To investigate changes at the gonadal level of the reproductive axis, testicular gene expression of the gonadotropin receptors, both the luteinizing hormone receptor and the follicle-stimulating hormone receptor, were measured as well as expression of the growth hormone receptor. To investigate potential impacts on spermatogenesis, testicular gene expression of the spermatogenesis marker vasa was measured and histological samples of testis were analyzed qualitatively. Estrogen indices were measured by expression and activity analysis of gonadal aromatase, as well as liver expression analysis of the estrogenic marker, esr1. After 14d, basal milt volume significantly decreased at 54µg/L FLX while pheromone-stimulated milt volume decreased at 0.54µg/L and 54µg/L FLX. Fluoxetine (54µg/L) inhibited both basal and pheromone-stimulated testosterone levels. Significant concentration-dependent reductions in follicle-stimulating hormone and isotocin expression were observed with FLX in the 17,20P- and PGF2(α)-stimulated groups, respectively. Estradiol levels and expression of esr1 concentration-dependently increased with FLX. This study demonstrates that FLX disrupts reproductive physiology of male fish at environmentally relevant concentrations, and potential mechanisms are discussed.