The decidualized endometrium harbors abundant leukocyte populations that are proposed to regulate critical processes at the maternal fetal interface including transformation of decidual spiral arteries. The work in this thesis investigated the leukocyte subtypes in the decidua throughout the course of this vascular transformation. A particular focus was the role of the uterine Natural Killer (uNK) cells and macrophages in an in vitro model of vascular remodeling. A significant infiltration of uNK cells and macrophages, matrix metalloproteinase-2/9 activity, and evidence of apoptosis and phagocytosis were observed in remodeling arterioles. From first to second trimester, FACS analysis demonstrated dramatic changes in the decidual leukocyte subpopulations, including the decline of uNK cells and macrophages and substantial increase in T lymphocytes and neutrophils. These data demonstrate an integral role of uNK cells and macrophages in early vascular remodeling and provide evidence of unique and complex immune interactions in the decidual microenvironment during human pregnancy.

The decidualized endometrium harbors abundant leukocyte populations that are proposed to regulate critical processes at the maternal fetal interface including transformation of decidual spiral arteries. The work in this thesis investigated the leukocyte subtypes in the decidua throughout the course of this vascular transformation. A particular focus was the role of the uterine Natural Killer (uNK) cells and macrophages in an in vitro model of vascular remodeling. A significant infiltration of uNK cells and macrophages, matrix metalloproteinase-2/9 activity, and evidence of apoptosis and phagocytosis were observed in remodeling arterioles. From first to second trimester, FACS analysis demonstrated dramatic changes in the decidual leukocyte subpopulations, including the decline of uNK cells and macrophages and substantial increase in T lymphocytes and neutrophils. These data demonstrate an integral role of uNK cells and macrophages in early vascular remodeling and provide evidence of unique and complex immune interactions in the decidual microenvironment during human pregnancy.

Orientador: Áureo Tatsumi Yamada / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-16T23:37:33Z (GMT). No. of bitstreams: 1 Bianco_JuaresEdnaldoRomero_D.pdf: 17105644 bytes, checksum: 64a116dab9ebcd72e51d45d800243157 (MD5) Previous issue date: 2018-08-16T20:37:19Z / Resumo: As células Natural Killer uterinas (uNK) participam da interação materno fetal atuando ativamente como elemento imuno-modulador do micro-ambiente uterino na gestação bem sucedida. Apesar das uNK possuírem um potencial lítico, relacionado com a sua resposta imune inata, este não se manifesta em uma gestação normal. Por outro lado, diversos relatos sugerem o envolvimento destas células em abortos e interrupções da gestação. No presente trabalho, foi avaliado o possível envolvimento das células uNK em atividade citotóxica sob a influência "in loco" do líquido amniótico no útero pseudogestante de camundongos. Para tanto, camundongos fêmeas foram acasaladas com machos vasectomizados, e os cornos uterinos injetados com óleo mineral para indução do útero pseudogestante. O líquido aminótico coletado em condições assépticas dos sítios de desenvolvimento embrionário normal no 10° dia de gestação (dg), foi inoculado no volume constante de 3uL através de micro-injeção na parede uterina da região antímesometrial nosnódulos de decidualização dos animais no 9o dia de pseudogestação (dpg). Amostras dos sítios inoculados com líquido amniótico foram coletadas nos intervalos de 30 minutos, 1, 2 e 6 horas para análises morfológicas, citoquímicas, imunocitoquímicas, imunoquímica e expressões gênicas. Como controles, foram utilizados úteros gestantes normais no 8o dg e úteros pseudogestantes submetidos à introdução de agulha hipodérmica na parede do corno uterino e/ou inoculados com 3uL de solução fisiológica estéril.Pelas análises morfológicas realizadas em cortes histológicos avaliados pela coloração de hematoxilina-eosina, o endométrio decidualizado da região mesometrial e antímesometrial do útero pseudogestante inoculado com salina ou simples introdução da agulha hipodérmica não apresentaram variações. Igualmente, a morfologia das células uNK através da citoquímica com lectina DBA, não apresentou diferenças entre os grupos controles. Ainoculação de LA no útero pseudogestante promoveuhiperemia local após 30 minutos que, em cortes histológicos, foi constatada como estagnação do sangue e dilatação dos vasos venosos no endométrio. Nestes locais foram constatadas alterações degenerativas nas células uNK com perda de reatividade da superfície celular e no conteúdo dos grânulos através da lectina DBA. A análise immunocitoquímica, RT-PCR e ELISA para as citocinas IFN-y e TNF-a e, das proteínas perforina e granzima-A revelaram que, o líquido amniótico acentuava a expressão destas moléculas ou dos seus genes relacionados à resposta pró-inflamatória, enquanto não alteravam a expressão de moléculas anti-inflamatórias como a IL-10. A geração do óxido nítrico (NO), através da quantificação de nitrato e nitrito, demonstrou um aumento nas concentrações, principalmente do nitrito após a inoculação do líquido amniótico. O teste de TÚNEL e a imunocitoquímica para anti-caspase-3 revelaram aumento de marcações positivas nas células uNK e deciduais sob o efeito do líquido amniótico em relação aos grupo controles, enquanto se verificava uma relação inversa para a imunocitoquímica com o anti-PCNA aos 2 a 6 horas após líquido amniótico. O conjunto dos resultados obtidos comprovam que as células uNK são sensíveis à ação local do líquido amniótico, com padrão de resposta compatível à ativação da sua capacidade citotóxica e pró-inflamatória relacionada com a resposta imune inata. / Abstract: Uterine Natural Killer (uNK) cells participate on the maternal fetus interaction actively playing as a immunomodulator element of the uterine micro-enviroment during a well successful gestation. Despite uNK having a lytic potencial that is related to its innate immune responsive, this does not manifest in a normal gestation. Moreover, several reports suggest the involvement of these cells in abortions and interruptions of gestation. In this study, we investigated the possible involvement of uNK cells in cytotoxic activity under the influence "in loco" of amniotic fluid in the uterus of mice pseudopregnant. For this, female mice were mated with vasectomized males and the uterine horns injected with mineral oil for induction of the pseudopregnant uterus. The amniotic fluid collected aseptically from sites of normal embryonic development in the 10 th day of gestation (dg) was inoculated on the constant volume of 3ul_ through microinjection on the uterine wall in the antimesometrial region into uterine nodules from animals on the 9th day of pseudopregnancy (dpg). Samples of inoculated sites with amniotic fluid were collected at intervals of 30 minutes, 1, 2 and 6 hours for morphological, cytochemical, immunocytochemical, immunochemical and relative gene expressions analyses. As controls, normal pregnant uteri on the 8th dg, and uterus on 9th dpg were submitted to the introduction of a hypodermic needle into the wall of the uterus and/or inoculated with 3uL of sterile saline.For the morphological analysis performed on histological sections evaluated by hematoxylin-eosin, the decidualized endometrium of the mesometrial and antimesometrial regions of the pseudopregnant uterus were inoculated with saline or simple introduction of the hypodermic needle showed no variations. Also, the morphology of uNK cells by DBA lectin cytochemistry, evidenced no differences among control groups.The amniotic fluid inoculation on the pseudopregnant uterus promote Hyperaemia after 30 min, in histological slides that was observed as blood interruption flow and dilatation of the vessels on the endometrium. On these areas it was observed degenerative modifications on uNK cells with loss in the positive reaction to DBA lectin in the cellular membranes and in the granules.The immunocytochemical analysis, RT-PCR and ELISA for the cytokines IFN-y and TNF-a, and the perforin and granzyme-A protein revealed that the amniotic fluid enhanced the expression of these molecules or their genes related to pro-inflammatory response, while not altering the expression of anti-inflammatory molecules such as IL-10.The generation of nitric oxide (NO), was observed through the quantification of nitrate and nitrite that demonstrated an increase in concentrations, primarily of nitrite after inoculation of amniotic fluid.The TUNEL assay and immunocytochemistry for anti-caspase-3 revealed an increase on positive uNK and decidual cells under the effect of amniotic fluid in relation to the control group, while there was an inverse relationship to immunocytochemistry with anti-PCNA at 2 and 6 hours after the amniotic fluid. The overall results show that uNK cells are sensitive to local action of amniotic fluid, compatible with standard response to the activation of their cytotoxic and proinflammatory that are related to the innate immune response. / Doutorado / Biologia Celular / Doutor em Biologia Celular e Estrutural

Orientadores: Áureo Tatsumi Yamada, Michael Joseph Soares / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-18T04:12:34Z (GMT). No. of bitstreams: 1 Lippe_ElianaMaraOliveira_D.pdf: 1508385 bytes, checksum: f80492236b91aa11f6167dbfd5369199 (MD5) Previous issue date: 2011 / Resumo: Um processo de remodelação progressiva da vascularização uterina precede a placentação em humanos e roedores para prover o suprimento sanguíneo adequado na interface materno-fetal, tendo as células NK um papel modulador através da produção de citocinas como o IFN'gama', fatores de crescimento como o VEGF e radicais livres como o óxido nítrico (NO). A regulação das células uNK nesta atividade tem sido atribuída à influência de fatores exógenos como a hipóxia e parácrinos como a prolactin-like protein-A (PLP-A). Contudo, como estes mecanismos são integrados no controle da angiogênese e vasculogênese da interface materno-fetal envolvendo as células uNK não são plenamente compreendidos. No presente trabalho, foram investigadas experimentalmente a resposta das células uNK relacionadas com os mecanismos de regulação dos fatores angiogênicos sob influência da hipóxia no período pré-placentário. Para tanto, foram avaliados camundongos CD1 e geneticamente modificados IL15-/-, PLPA-/- e IL15-/-/PLPA-/- gestantes no 8° dia de gestação (dg), mantidos em hipóxia (42 0Torr - 11%O2) durante 48 horas. Em comparação com os animais mantidos em normóxia (760Torr - 21%O2) a quantidade de sítios anormais apresentou incremento estatisticamente significativo sob hipóxia nos animais CD1, porém este índice era substancialmente maior nos animais depletados dos genes IL15 e PLPA. As amostras dos sítios uterinos de desenvolvimento embrionários coletados foram processados para obtenção de criocortes destinados às reações citoquímicas, imunocitoquímicas e hibridização in situ, e homogeneizados teciduais para extração do RNAm ou proteínas. Conforme esperado, os resultados da citoquímica com lectina DBA e imunocitoquímica de perforina comprovam a ausência de células uNK nos animais IL15-/- e IL15-/-/PLPA-/-, enquanto nos animais PLPA-/-, a incidência das células uNK perforina positivas não difere dos CD1 em normóxia ou hipóxia. A concentração protéica de VEGF e dos genes das isoformas VEGFA, VEGFB e VEGFC, assim como do IFN? e das isoformas iNOS e eNOS, o do TNF? e seus receptores TNFR1 e TNFR2 não apresentaram variações em suas concentrações ou níveis de expressões com padrões definidas de regulação negativa ou positiva entre os animais avaliados. Contudo a imunocitoquímica demonstrou redução de marcação de células endoteliais endoglinpositivas no endométrio e aumento no potencial invasivo de células trofoblásticas TROMA-I positivas dos animais IL15-/-. O conjunto destes resultados confirma que a ausência das células uNK afeta a vascularização normal do endométrio, a qual pode resultar em perdas gestacionais e induz a hipertrofia placentária, sem a participação direta da atividade citotóxica destas células. Comprovam também que os mecanismos de controle da expressão de fatores angiogênicos no útero gestante são multifatoriais, não sendo dependente de uma via única como o da PLPA/VEGF, ou exclusivamente das células uNK como fontes de fatores que modulam a angiogênese na interface maternofetal do útero gestante / Abstract: A gradual process of remodeling of uterine vasculature precedes placentation in humans and rodents to provide adequate blood supply in maternal-fetal interface, where NK cells producing cytokines like growth factor (VEGF) and IFN'gama' and free radical as nitric oxide (NO). Regulation of this uNK cells activity seems to be under influence of exogenous and endogenous factors such as hypoxia as paracrine effects of prolactin-like protein-A (PLPA). Nevertheless, how these mechanisms are integrated in the control of maternal-fetal interface angiogenesis and vasculogenesis involving uNK cells are not fully understood. In this study, we investigated experimentally the uNK cells response related to the mechanisms of regulation of angiogenic factor under hypoxia influence in pre-placental period. Thus, we evaluated genetically modified mice and CD1-IL15-/-, PLPA-/- and IL15-/- /PLPA-/-pregnant on the 8th day of gestation (dg), maintained in hypoxia (420Torr-11%O2) for 48h. When compared to the control animals in normoxia (760Torr-21%O2) the amount of abnormal embryo developing sites showed statistically significant increase under hypoxia in CD1 animals, but this rate was substantially higher in the PLPA and IL15 gene depleted animals. Uterine samples were processed to obtain cryosection for cytochemical, immunocytochemical and in situ hybridization reactions and extraction of mRNA or protein for PCR or ELISA reactions, respectively. As expected, the results of cytochemistry and immunocitochemistry with DBA lectin and perforin prove the absence of uNK cells in IL15-/- and IL15-/-/PLPA-/-animals, while in PLPA-/-animals, the incidence of uNK cells perforinpositive did not differ from CD1 animlas in normoxia or hypoxia. The concentration of protein VEGF and the gene isoforms expression of VEGF (A, B and C), as well as, IFN?, iNOS and eNOS, TNF? and their receptors TNFR1 and TNFR2 did not show constancy in the pattern of variations. However, the immunocytochemistry showed reduced staining of endoglin-positive endothelial cells in the endometrium and increase in invasive potential of trophoblast cells by TROMA-I positive in IL15-/- animals. This set of results confirms that the absence of uNK cells affects the normal vasculature of the endometrium which may increased intrauterine growth restriction (IUGR) or pregnancy failure rates and induces placental hypertrophy. This abnormality at the maternal-fetal interface does not seems to be involves direct participation of cytotoxic activity of uNK cells. Furthemore, the control mechanisms of angiogenic factors expression in the pregnant uterus are multifactorial rather than dependent of a single pathway like the PLP-A/VEGF, or limited to uNK cells as a source of factors that modulate angiogenesis in the maternal-fetal interface of pregnant uterus / Doutorado / Histologia / Doutor em Biologia Celular e Estrutural

Orientador: Áureo Tatsumi Yamada / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-20T04:02:31Z (GMT). No. of bitstreams: 1 Lima_PatriciaDanieleAzevedo_D.pdf: 2840118 bytes, checksum: 470a2cf0b7d63e81fd8f48aba5af44fc (MD5) Previous issue date: 2012 / Resumo: Células Natural Killer uterina (uNK) produzem moléculas angiogênicas e citocinas críticas ao sucesso da gestação , assim como proteínas citolíticas relacionadas à resposta imune inata. Contudo, se as capacidades angiogênicas e citolíticas são provenientes de diferentes subpopulações de células uNK não é conhecido; da mesma forma, estes fenótipos ainda não são estabelecidos. Assim, a proposta inicial deste trabalho foi avaliar...Observação: O resumo, na íntegra, poderá ser visualizado no texto completo da tese digital / Abstract: Angiogenic and cytokine molecules produced by uterine natural killer (uNK) cells are critical for successful pregnancy. Cytolytic proteins are also express by uNK cells. However, it is unknown whether the angiogenic and cytolytic capacities are from different uNK subsets, or the same cells. Thus, we initially proposed to evaluate...Note: The complete abstract is available with the full electronic document / Doutorado / Histologia / Doutor em Biologia Celular e Estrutural

Uterine NK (uNK) cells are a distinct subset of NK cells in the decidua of humans and rodents during pregnancy, which are essential for remodelling of the spiral arteries supplying the feto-placental unit. Similarly to peripheral NK cells, uNK cells express Natural Killer receptors (NKRs) that engage MHC class I molecules. Evidence from human genetic association studies suggests that, in the presence of allogeneic cognate paternal MHC class I ligands, inhibitory uterine NKRs are associated with disorders of pregnancy arising from impaired decidual vascular remodelling. Conversely, enhancement of human uNK cell activity through activating NKRs is associated with high birth weight. Evidence from mouse models corroborates that uNK cell activity is modulated by interactions between NKRs and MHC class I, but has largely focussed on the effect of paternal MHC. In this study, the contribution of maternal immunogenetic regulation of NK cell function to reproductive outcome was assessed independently of parental MHC disparity in mice. To evaluate the role of NKR genes in isolation, I used congenic B6.BALB-TC1 (TC1) mice that differ from C57BL/6 (B6) mice only within the region of chromosome six encoding NKRs that recognise MHC class I. Absence of a major inhibitory NKR for self-MHC, Ly49I, in TC1 mice causes a compensatory shift in the NKR repertoire expressed and preserves a majority subpopulation of educated NK cells. B6 and TC1 splenic and uterine NK cells are similarly functionally reactive and mature, and no significant differences could be detected in spiral arterial remodelling or fetal growth between these strains in MHC-syngeneic matings. This supports data from human immunogenetic studies showing that maternal uterine NKRs are not associated with differences in pregnancy outcome in the absence of novel paternal MHC class I ligands, and highlights the importance of maternal and paternal co-regulation of uNK cell activity during pregnancy. No mouse models of uNK cell activation are currently available with which to corroborate human immunogenetic associations between activating uterine NKRs and high birth weight. Male m157-transgenic (m157-Tg) mice, which ubiquitously express viral m157 glycoprotein ligands for the activating NKR Ly49H, were mated with B6 females. Exclusive expression of m157 glycoprotein by trophoblast improved placental efficiency, but did not enhance fetal growth. Some fertility clinics surmise that uNK cell activation initiates the pathogenesis of spontaneous abortion. It has been suggested that this may occur due to reduced expression by human uNK cells of miR-483-3p, which stimulates endogenous insulin-like growth factor (IGF)-1 production and uNK cell cytotoxicity in vitro. It is demonstrated here that neither miR-483-3p nor IGF-1 regulate murine NK cell development, maturation or function. No discernible reproductive phenotype is evident in miR-483 deficient females. It can be inferred that post-transcriptional control by miR-483 is not biologically relevant to murine NK cell function. Although m157-Tg mice may provide an interesting model to further study uNK cell-mediated placental adaptations, it remains important to identify a murine model of enhanced uNK cell function to corroborate human immunogenetic associations with high birth weight and to challenge the supposition that uNK cell activation is harmful to pregnancy.