Genes expression profile of innate immune response mediators in the uNK cell of normal pregnancy and after embryo lesion

Author(s)

Karina Yumi Degaki

• Document type: Master's Dissertation
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia
• Defense date: 2008-04-08

Examining board members

Aureo Tatsumi Yamada; Silvia Daher; Sebastian San Martin Henriquez

• Advisor: Aureo Tatsumi Yamada

Abstract

The uterine natural killer cells (uNK) actively modulate the maternal-fetal interface and homeostasis of pregnant uterus, without triggering the typical cytolytic activity of innate immune response. However, experimentally induced embryo lesion in the pregnant mouse uterus, changes the uterine homeostasis with commitment of uNK cell and possible induction of its cytolytic activity. The present work evaluated the gene expression of mediators related either to the activation and effectors molecules of uNK cells cytotoxic activity. It was evaluated the genes of perforin, granzyme-A, IFN-y, IL-15, IL-18, TNF-a, IL-6, IL-2, DAP-12, Fas, Fas-L by RT-PCR (Reverse Transcriptase Polymerase Chain Reaction), using RNA isolated from normal pregnant mice uterus on gestational day (gd) 9 and after 0.5, 1, 2, 6, 12 and 24h of abnormal pregnancy provoked by surgical lesion of the embryo (SLE) and also from RNA obtained from uNK cells isolated from normal pregnant mice. Differences in the DAP12, IFN-??and TNF-a ?genes expressions were also investigated with RNA isolated from immature and mature subtypes of uNK cells dissected from cryosections of mesometrial regions in the laser capture microscope (LCM) and evaluated by quantitative Real Time-PCR (qRT-PCR). The results of RT-PCR confirmed the expression of all genes evaluated in the uterine tissue homogenates and in the uNK cells were not detected the only expression of IL-15 and IL-2 genes. By semi-quantitative analysis of RT-PCR amplicons, the genes of cytolytic proteins, granzymes-A and perforin all together with pro-inflammatory cytokines IFN-y ?and TNF-a ?showed significant increasing at 1h after SLE and decreasing to the level near of normal pregnancy after 6h of SLE. The increasing expression of these genes related to innate immune response of NK cells suggest the quick changes of uNK cells behavior in response to the SLE, which activation could involve the expression of DAP12 gene that also showed increasing at the same period after SLE. On the other hand, the expression of IL-15, IL-2, IL-18 and IL-6 genes related to the migration, proliferation and differentiation of uNK cells did not showed significant differences among the SLE samples and normal pregnancy. The significant decreasing of FasL after SLE could down regulate the triggering of apoptotic cell death pathway in the uNK cells and therefore, increasing the viability of these cells in the uterine microenvironment affected by SLE. These results prove that SLE induces fast up-regulation genes expression related to the cytotoxic pathway of uNK cells innate immune response ability. Based on analysis of qRT-PCR of DAP12, TNF-a ?and IFN-y ?genes performed with RNA obtained from uNK cells dissected in the LCM, it was identified differences in the expression of these genes between now a days classified as immature and mature forms of uNK cells. These expressions differences attest the occurrence of functionally heterogeneous subpopulations of uNK cells not restricted to the differentiation stages and possible specific distribution domains in the uterine environment (AU)