Unstable Foxp3 Regulatory T Cells and Altered Dendritic Cells Are
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BIOLOGY OF REPRODUCTION (2015) 93(4):95, 1–14 Published online before print 29 July 2015. DOI 10.1095/biolreprod.115.128694 Unstable Foxp3þ Regulatory T Cells and Altered Dendritic Cells Are Associated with Lipopolysaccharide-Induced Fetal Loss in Pregnant Interleukin 10-Deficient Mice1 Jelmer R. Prins,3,4,5 Bihong Zhang,3,4 John E. Schjenken,4 Leigh R. Guerin,4 Simon C. Barry,4 and Sarah A. Robertson2,4 4The Robinson Research Institute, School of Medicine, University of Adelaide, Adelaide, South Australia, Australia 5Department of Obstetrics and Gynaecology, University Medical Center Groningen, University of Groningen, Downloaded from https://academic.oup.com/biolreprod/article/93/4/95, 1-14/2434263 by guest on 30 September 2021 Groningen, The Netherlands ABSTRACT INTRODUCTION Maternal interleukin (IL) 10 deficiency elevates susceptibility Inheritance by the conceptus of maternal and paternal to fetal loss induced by the model Toll-like receptor agonist transplantation antigens results in a semiallogeneic challenge to lipopolysaccharide, but the mechanisms are not well elucidated. the mother. Substantial adaptations in the maternal immune Here, we show that Il10 null mutant (Il10À/À) mice exhibit system are required to tolerate the fetus and suppress altered local T cell responses in pregnancy, exhibiting pro- deleterious inflammatory responses that cause gestational nounced hyperplasia in para-aortic lymph nodes draining the disorders and fetal demise. Secretion of immune-regulatory + + uterus with .6-fold increased CD4 and CD8 T cells compared Downloaded from www.biolreprod.org. + + cytokines and hormones by placental trophoblasts and uterine with wild-type controls. Among these CD4 cells, Foxp3 T cells [1–3] constrain inflammation and limit type 1 immunity, regulatory (Treg) cells were substantially enriched, with 11-fold particularly in the gestational tissues and local lymph nodes higher numbers at Day 9.5 postcoitum. Lymph node hypertro- À/À (LNs). Prominent among these cytokines is interleukin (IL) 10 phy in Il10 mice was associated with more activated [1, 4], which stimulates and reinforces modifications to the phenotypes in dendritic cells and macrophages, with elevated innate and adaptive immune system, including induction of expression of MHCII, scavenger receptor, and CD80. Affymetrix regulatory phenotypes in the dendritic cell (DC), macrophage, microarray revealed an altered transcriptional profile in Treg À/À and T lymphocyte compartments [5–7]. Experiments in Il10 cells from pregnant Il10 mice, with elevated expression of À/À Ctse (cathepsin E), Il1r1, Il12rb2, and Ifng. In vitro, Il10À/À Treg null mutant (Il10 ) mice demonstrate that in the absence of maternal IL10, neither allogeneic nor syngeneic pregnancies cells showed reduced steady-state Foxp3 expression, and À/À polyclonal stimulation caused greater loss of Foxp3 and reduced are compromised [8–10]. However, pregnant Il10 mice are capacity to suppress IL17 in CD4+Foxp3À T cells. We conclude highly vulnerable to inflammatory challenge, with administra- that despite a substantially expanded Treg cell pool, the tion of low-dose lipopolysaccharide (LPS) or other Toll-like diminished stability of Treg cells, increased numbers of effector receptor (TLR) ligands causing elevated rates of fetal T cells, and altered phenotypes in dendritic cells and macro- resorption (miscarriage) [11, 12] or preterm delivery [13, 14], phages in pregnancy all potentially confer vulnerability to depending on gestational timing of the insult. inflammation-induced fetal loss in Il10À/À mice. These findings The mechanisms through which IL10 controls inflammatory suggest that IL10 has a pivotal role in facilitating robust immune mediators have not been defined, although uterine natural killer protection of the fetus from inflammatory challenge and that (uNK) cells and tumor necrosis factor (TNF) appear to be IL10 deficiency could contribute to human gestational disorders involved in promoting fetal loss when pregnant Il10À/À mice in which altered T cell responses are implicated. are challenged [11]. We postulate that in the absence of IL10, cytokines, dendritic cells, fetal loss, immunology, inflammation, anti-inflammatory mechanisms protecting the fetus are com- interleukin 10, preeclampsia, pregnancy, regulatory T cells promised. One of the key leukocyte populations likely to be affected by absence of IL10 are T regulatory (Treg) cells, a subset of anti-inflammatory and immune suppressive CD4þ T lymphocytes defined by their expression of the transcription factor fork-head box P3 (Foxp3) [15, 16]. The critical role of Treg cells in limiting inflammation and mediating immune tolerance is demonstrated in Foxp3 null mutant mice (Scurfy 1This study was supported by project and fellowship grants from the mice) that develop a lethal multiorgan lymphoproliferative NHMRC (Australia). The microarray data discussed in this publication disorder [17]. IL10 is implicated in the generation of Treg cells have been deposited in the National Center for Biotechnology Information’s Gene Expression Omnibus and are accessible through and is prominent in aspects of their suppressive function [7, GEO Series accession no. GSE71494 (www.ncbi.nlm.nih.gov/geo/ 18], including in pregnancy, where neutralizing IL10 abrogates query/acc.cgi?acc¼GSE71494). the protective effects of Treg cells in a murine abortion model 2Correspondence: Sarah A. Robertson, The Robinson Research Insti- [19]. IL10 also influences macrophages and DCs, which have tute, School of Medicine, University of Adelaide, Adelaide, SA 5005, important roles in sustaining pregnancy independently of T Australia. E-mail: [email protected] 3 cells [20] as well as through specific antigen-presenting and These authors contributed equally to this work. immune-regulatory functions, including control of the gener- ation of induced Treg cells from naı¨ve T lymphocyte Received: 28 January 2015. precursors [7]. First decision: 28 February 2015. During murine pregnancy, Treg cells are elevated in the Accepted: 24 July 2015. Ó 2015 by the Society for the Study of Reproduction, Inc. gestational tissues and systemic circulation by approximately eISSN: 1529-7268 http://www.biolreprod.org 50% in midgestation [10, 21], and similar changes are evident ISSN: 0006-3363 in pregnant women [22, 23]. Experiments in Treg cell-depleted 1 Article 95 PRINS ET AL. mice [24, 25] and abortion-prone mice [26] show these cells Flow Cytometry are essential for establishing allogeneic pregnancy. Their immune regulatory actions are most crucial around the time Para-aortic and inguinal LNs were excised from female mice between 0900 and 1100 h at estrus or at various time points in pregnancy. Single-cell of embryo implantation, when their abundance is a limiting suspensions were prepared by mechanical dispersion between glass microscope factor in implantation and placental development [27]. slides. Lymphocytes were suspended in 0.1% BSA/PBS with 0.05% sodium Experiments using tetramers demonstrate the majority of the azide (pH 7.4; fluorescence-activated cell sorting [FACS] buffer). Aliquots of maternal Treg cells that expand in pregnancy are fetal antigen- 106 cells were incubated with anti-Fc-cIIR (FcBlock; BD Biosciences) for 5 specific [10], and dependence on the CNS1 regulatory region min at 48C, then incubated with combinations of phycoerythrin (PE) anti-CD4 in the Foxp3 gene confirms this is at least partly the result of (L3T4; BD Biosciences), fluorescein isothiocyanate (FITC) anti-CD8 (Ly-2; extrathymic generation of inducible (peripheral) Treg cells BD Biosciences), and PE-Cy7 anti-CD25 (PC61; eBioscience) for 30 min at 48C to identify T cell subsets. Following T cell surface staining, cells were responding to fetal alloantigen [28] after initial priming in fixed and permeabilized using Foxp3 Staining Buffer Set (eBioscience) response to paternal seminal fluid at conception [29]. Treg cells according to the manufacturer’s instructions. Permeabilized cells were have recently been shown to protect the fetus from preterm incubated with anti-Fc-cIIR for 5 min at 48C and then with APC anti-Foxp3 Downloaded from https://academic.oup.com/biolreprod/article/93/4/95, 1-14/2434263 by guest on 30 September 2021 delivery induced by the TLR4 ligand bacterial LPS [30] and (FJK-16s; eBioscience) for 30 min at 48C. from fetal loss induced by the TLR9 ligand DNA motif CpG To analyze other leukocyte subsets and to phenotype DCs and [31]. Although Treg cells can develop and execute at least macrophages, aliquots were incubated with anti-Fc-cIIR antibody plus combinations of APC anti-CD11b (M1/70; eBioscience), AF488 anti-CD11c some protolerance functions independently of IL10 [10, 32, (N418; eBioscience), PE-Cy7 anti-CD19b (eBio 1D3; eBioscience), PE or 33], to our knowledge the effect of IL10 deficiency on Treg biotin anti-F4/80 (BM8; eBioscience), PE anti-CD4 (L3T4; BD Biosciences), cell populations in pregnancy has not been investigated. biotin anti-major histocompatability class II (MHCII; M5/114.15.2; eBio- In the present study, we demonstrate significant alterations science), biotin anti-MHCII, scavenger receptor class A (macrophage scavenger in the maternal Treg cell response to allogeneic pregnancy in receptor [MSR]; 2F8; AbD Serotec), biotin anti-CD80 (16-10A1; BD female mice with genetic IL10 deficiency. We report Biosciences), and APC anti-CD86 (GL1; BD Biosciences) for 30 min at 48C, followed by PerCp Streptavidin (BD Biosciences). After washing, cells were substantial