The role of inflammasome in the innate and adaptive immunity in pregnant women with preeclampsia

Abstract

Preeclampsia (PE) is an obstetric pathology, which appears between 3% and 8% of pregnancies and is considered as a major cause of morbidity and mortality, both maternal and fetal. The literature suggests that PE is characterized by a state of maladaptive immune tolerance, identified by oxidative stress and abnormal activation of the innate and adaptive immune system. Plasma of pregnant women with PE has elevated levels of molecular structures associated with stress and cell death, termed damage associated molecular patterns (DAMPs) as heat shock protein (Hsp70), HMGB1 (High mobility group box 1), hyaluronan (HA) and Uric Acid, seem to contribute directly to the pathogenesis of this disease. These DAMPs bind to receptors as Toll-like (TLR) and Nod-like (NLR) present in cells of innate immunity and can activate an intracellular complex called inflammasome, a multi-protein complex, important for processing and release of interleukin-1beta (IL-1beta) and IL-18. These cytokines are potent inflammatory mediators and important in the activation of the adaptive immune response, and are responsible for T cell differentiation into Th1 and Th17, respectively. In PE, the occurrence of systemic inflammatory response seems to result from deficiency in the control of effector T cells by regulatory T cells (Treg). Therefore, the balance between Treg and Th17 cells may be critical for tolerance to the fetus and for the prevention of disease. This project, which aims at better understand the pathophysiology of PE, has the following objectives: a) To determine the concentration of DAMPs (Hsp70, HMGB1, hyaluronan and Uric Acid) in the plasma of pregnant women with PE; b) to assess the state of activation both endogenous and induced by DAMPS (Hsp70, HA and monosodium urate) in monocytes of these patients, by identifying the presence of inflammasome NLRP3 and its association with the production of IL-1beta, tumor necrosis factor-alpha (TNF-alpha) and IL-18; c) to assess the involvement of T cell subpopulations (Th1, Th2, Th17 and Treg) by analyzing the profile of cytokines and transcription factors produced by these cell subpopulations; d) to correlate inflammasome activation with the profile of T cell subpopulations. Forty pregnant women including 20 normotensive and 20 with preeclampsia, matched for gestational age as well as 20 healthy non-pregnant women will be studied. Blood samples of these patients will be centrifuge and plasma separate and store at -80°C for determination of DAMPs (Hsp70, HMGB1, hyaluronan and uric acid). Peripheral blood monocytes will be incubated at 37ºC in of 5% CO2 constant atmosphere in the presence or absence of DAMPs (Hsp70, hyaluronan and monosodium urate). The supernatant obtained after 18h of culture will be harvested and employed for IL-1beta, IL-18 and TNF-alpha determination by enzyme immunoassay (ELISA). The presence of inflammasome in these cells will be evaluated by gene expression of NLRP3, caspase-1, IL-1beta, IL-18 and TNF-alpha after blood collection by quantitative real-time PCR (RT-qPCR) (endogenous expression) or after in vitro activation with monosodium urate (stimulated expression). To study the subpopulations of T cells, peripheral blood mononuclear cells (PBMC) will be evaluated for production of pro and anti-inflammatory cytokines and expression of transcription factors involved in activation of these cells. For determination of cytokines, the cells will be incubating in the absence or presence of DAMPs (Hsp70, hyaluronan and monosodium urate). The supernatant obtained after 24 h and 48 h of culture will be used to determine the cytokine profile of Th1 (IFN-³ and TNF-±), Th2 (IL-4), Treg (IL-10 and TGF-²1) and Th17 (IL -17 and IL-22) by ELISA. The expression of intracytoplasmic transcription factors T-bet (Th1), GATA3 (Th2), RORc(Th17) and Foxp3(Treg) will be evaluate by flow cytometry, using specific monoclonal antibodies labeled with fluorochromes after blood collection(endogenous expression). (AU)