Farsky update log4/10/2023 Human neutrophils treated with estriol and progesterone display a distinct phenotype and induce Tregs from a naïve T cell population. Neutrophils cultured alone did produce cytokines, confirming their purity and the absence of contaminating monocytes ( SI Appendix, Fig. These T cells produced significant levels of IL-10, IL-17, IL-2, and IL-5, but not of IFN-γ, compared with T cells activated in the presence of vehicle-treated neutrophils or on their own ( Fig. The addition of E3P neutrophils to autologous carboxyfluorescein succinimidyl ester (CFSE)-labeled T cells (+ anti-CD3, anti-CD28 mAbs), enhanced T-cell proliferation ( Fig. 1 A), typical of an anti-inflammatory/quiescent neutrophil status ( 14, 18). Treatment of neutrophils for 30 min with E3P led to a distinct CD16 loCD62L loCD11b loAnxA1 hi phenotype ( Fig. S1 B) or the placental estrogen estriol (E3), or in combination (hereinafter E3P), at 100 ng/mL, physiological pregnancy levels. S1 A) were incubated with either progesterone (P SI Appendix, Fig. Male cells express the same levels of estrogen and progesterone receptors on leukocytes as females ( 17), and thus respond to both hormones. Neutrophils from healthy male donors were used to minimize any effects from endogeneous progesterone and estrogen. To study a potential role for neutrophils in maternal tolerance, we tested whether neutrophils exposed to pregnancy hormones could affect T-cell responses. To check for congenic markers after adoptive transfer of donor CD45.1 neutrophils, cells were stained with Alexa Fluor 488-conjugated anti-mouse CD45.1 (Biolegend clone A20), along with Alexa Fluor 450-conjugated CD45.2 (eBioscience clone 104). Mice were originally obtained from Charles River Italy. Female CD45.1 (Ly5.1) mice were kindly provided by Andrew Cope, King’s College London. Following an initial lentiviral transduction (24 h) to knock down forkhead box protein 1 (FOXO1), cells were washed and incubated in Iscove’s modified Dulbecco’s medium supplemented with 20% FCS and 100 ng/mL recombinant murine GCSF for 5 d. Progenitors were isolated via negative selection (Stem Cell Technology). Neutrophils were made from bone marrow progenitors. Neutrophils were reconstituted at indicated times during the pregnancy. Following identification of a vaginal plug, circulating maternal neutrophils were depleted at days 5 and 8 using a monoclonal neutralizing antibody (Biolegend, clone IA8 50 μg i.v.). Here 8- to 12-wk-old Balb/C male mice were mated with aged-matched C57 BL/6 females. These data define a nonredundant function of neutrophil–T cell interactions in the regulation of vascularization at the maternal–fetal interface.Īll in vivo experiments were conducted under the Home Office regulation following approval by the Queen Mary University of London Ethics Committee. Finally, neutrophil-selective FOXO1 knockdown leads to defective placentation and compromised embryo development, similar to that resulting from neutrophil depletion. Unlike in women with healthy pregnancies, neutrophils from blood and placental samples of preeclamptic women fail to induce niT cells as a direct consequence of their inability to transfer FOXO1 to T cells. Using CD45 congenic cells, we show that induction of niT cells and their regulatory function occurs via transfer of apoptotic neutrophil-derived proteins, including forkhead box protein 1 (FOXO1), to T cells. Neutrophil depletion during murine pregnancy leads to abnormal development of the fetal-maternal unit and reduced empbryo development, with placental architecture displaying poor trophoblast invasion and spiral artery development in the maternal decidua, accompanied by significantly attenuated niT cell numbers in draining lymph nodes. Neutrophil-induced T (niT) cells produce IL-10, IL-17, and VEGF and promote vessel growth in vitro. We report that human neutrophils exposed to pregnancy hormones progesterone and estriol promote the establishment of maternal tolerance through the induction of a population of CD4 + T cells displaying a GARP +CD127 loFOXP3 + phenotype following antigen activation. Although neutrophils are known to be fundamental in controlling innate immune responses, their role in regulating adaptive immunity is just starting to be appreciated.
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