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Chorioamnionitis is a common precipitant of preterm birth and is associated with many of the morbidities of prematurity, including necrotizing enterocolitis (NEC). However, a mechanistic link between these two conditions remains yet to be discovered. We have adopted a murine model of chorioamnionitis involving lipopolysaccharide (LPS)-induced fetal exposure to maternal inflammation (FEMI). This model of FEMI induces a sterile maternal, placental, and fetal inflammatory cascade, which is also present in many cases of clinical chorioamnionitis. Although models exist that utilize live bacteria and more accurately mimic the pathophysiology of an ascending infection resulting in chorioamnionitis, these methods may cause indirect effects on development of the immature intestinal tract and the associated developing microbiome. Using this protocol, we have demonstrated that LPS-induced FEMI results in a dose-dependent increase in pregnancy loss and preterm birth, as well as disruption of normal intestinal development in offspring. Further, we have demonstrated that FEMI significantly increases intestinal injury and serum cytokines in offspring, while simultaneously decreasing goblet and Paneth cells, both of which provide a first line of innate immunity against intestinal inflammation. Although a similar model of LPS-induced FEMI has been used to model the association between chorioamnionitis and subsequent abnormalities of the central nervous system, to our knowledge, this protocol is the first to attempt to elucidate a mechanistic link between chorioamnionitis and later perturbations in intestinal development as a potential link between chorioamnionitis and NEC.
Fetal exposure to chorioamnionitis can impact the outcomes of the developing fetus both at the time of birth and in the subsequent neonatal period. Infants exposed to chorioamnionitis have a higher incidence of gastrointestinal (GI) pathology, including necrotizing enterocolitis (NEC); however, the mechanism remains undefined. To simulate the fetal exposure to maternal inflammation (FEMI) induced by chorioamnionitis, pregnant mice (C57BL/6J, , or ) were injected intraperitoneally on embryonic day (E)15.5 with lipopolysaccharide (LPS; 100 µg/kg body weight). Pups were delivered at term, and reared to postnatal day (P)0, P7, P14, P28 or P56. Serum and intestinal tissue samples were collected to quantify growth, inflammatory markers, histological intestinal injury, and goblet and Paneth cells. To determine whether FEMI increased subsequent susceptibility to intestinal injury, a secondary dose of LPS (100 µg/kg body weight) was given on P5, prior to tissue harvesting on P7. FEMI had no effect on growth of the offspring or their small intestine. FEMI significantly decreased both goblet and Paneth cell numbers while simultaneously increasing serum levels of IL-1β, IL-10, KC/GRO (CXCL1 and CXCL2), TNF and IL-6. These alterations were IL-6 dependent and, importantly, increased susceptibility to LPS-induced intestinal injury later in life. Our data show that FEMI impairs normal intestinal development by decreasing components of innate immunity and simultaneously increasing markers of inflammation. These changes increase susceptibility to intestinal injury later in life and provide novel mechanistic data to potentially explain why preterm infants exposed to chorioamnionitis prior to birth have a higher incidence of NEC and other GI disorders.
© 2019. Published by The Company of Biologists Ltd.
INTRODUCTION - Metabolic stress (e.g., gestational diabetes mellitus (GDM) and obesity) and infections are common during pregnancy, impacting fetal development and the health of offspring. Such antenatal stresses can differentially impact male and female offspring. We sought to determine how metabolic stress and maternal immune activation (MIA), either alone or in combination, alters inflammatory gene expression within the placenta and whether the effects exhibited sexual dimorphism.
METHODS - Female C57BL/6 J mice were fed a normal diet or a high fat diet for 6 weeks prior to mating, with the latter diet inducing a GDM phenotype during pregnancy. Dams within each diet group at gestational day (GD) 12.5 received either an intraperitoneal injection of the viral mimic, polyinosinic:polycytidylic acid (poly(I:C)) or saline. Three hours post injection; placentae were collected and analyzed for changes in the expression of 248 unique immune genes.
RESULTS - Placental immune gene expression was significantly altered by GDM, MIA and the combination of the two (GDM+MIA). mRNA expression was generally lower in placentae of mice exposed to GDM alone compared with the other experimental groups, while mice exposed to MIA exhibited the highest transcript levels. Notably, fetal/placental sex influenced the responses of many immune genes to both metabolic and inflammatory stress.
DISCUSSION - GDM and MIA provoke inflammatory responses within the placenta and such effects exhibit sexual dimorphism. The combination of these stressors impacts the placenta differently than either condition alone. These findings may help explain sexual dimorphism observed in adverse pregnancy outcomes in human offspring exposed to similar stressors.
Copyright © 2019. Published by Elsevier Ltd.
BACKGROUND/PURPOSE - Wilms tumor (WT) is the most common childhood kidney cancer globally. Our prior unbiased proteomic screen of WT disparities revealed increased expression of Fragile X-Related 1 (FXR1) in Kenyan specimens where survival is dismal. FXR1 is an RNA-binding protein that associates with poor outcomes in multiple adult cancers. The aim of this study therefore was to validate and characterize the FXR1 expression domain in WT.
METHODS - Quantitative FXR1 gene expression was compared between WT, adjacent, adult, and fetal kidney specimens. The cellular and subcellular expression domain of FXR1 was characterized across these tissues using immunoperoxidase staining. RNA-sequencing of FXR1 was performed from WT and other pediatric malignancies to examine its broader target potential.
RESULTS - FXR1 was detected in all clinical WT specimens evaluated (n = 82), and as a result appeared independent of demographic, histology, or adverse event. Specific cytosolic staining was strongest in blastema, intermediate and variable in epithelia, and weakest in stroma. When present, areas of skeletal muscle differentiation stained strongly for FXR1. qPCR revealed increased FXR1 expression in WT compared to adult and adjacent kidney (p < 0.0002) but was similar to fetal kidney (p = 0.648). RNA-sequencing revealed expression of FXR1 in multiple pediatric tumors, greatest in rhabdomyosarcoma and WT.
CONCLUSIONS - FXR1 was expressed consistently across this broad sampling of WT and most robustly in the primitive blastema. Notably, FXR1 labeled a specific self-renewing progenitor population of the fetal kidney.
Copyright © 2019 Elsevier Inc. All rights reserved.
During the dermal wound healing process, the mechanical rigidity of the newly deposited extracellular matrix and transforming growth factor-β1 promote the transition of fibroblasts into myofibroblasts. Myofibroblasts generate large cellular forces that contract and remodel the extracellular matrix leading to scar formation. In contrast, myofibroblasts are not detected in fetal dermal wounds which are more compliant and contain less transforming growth factor-β1 than adult wounds. Instead, fetal fibroblasts orchestrate scarless healing of dermal wounds resulting in healed tissues that resemble uninjured dermis. While these biomechanical differences suggest that the fetal wound environment promotes smaller cellular forces which enable regeneration, previous studies indicate that fetal fibroblasts have unique contractile properties that may facilitate scarless dermal repair. Therefore, we tested whether physiologic wound rigidities and transforming growth factor-β1 induce contractile forces and myofibroblast differentiation of fetal dermal fibroblasts. In comparison to their adult dermal counterparts, we found that fetal fibroblasts exhibit a deficient contractile response to rigid extracellular matrix and transforming growth factor-β1. Our data suggest that the contractile phenotype of fetal dermal fibroblasts limits their cellular force production and prevents their ability to differentiate into myofibroblasts.
© 2018 The Authors. Wound Repair and Regeneration published by Wiley Periodicals, Inc. on behalf of by the Wound Healing Society.
Wnt/β-catenin signaling is necessary for normal lung development, and abnormal Wnt signaling contributes to the pathogenesis of both bronchopulmonary dysplasia (BPD) and idiopathic pulmonary fibrosis (IPF), fibrotic lung diseases that occur during infancy and aging, respectively. Using a library of human normal and diseased human lung samples, we identified a distinct signature of nuclear accumulation of β-catenin phosphorylated at tyrosine 489 and epithelial cell cytosolic localization of β-catenin phosphorylated at tyrosine 654 in early normal lung development and fibrotic lung diseases BPD and IPF. Furthermore, this signature was recapitulated in murine models of BPD and IPF. Image analysis of immunofluorescence colocalization demonstrated a consistent pattern of elevated nuclear phosphorylated β-catenin in the lung epithelium and surrounding mesenchyme in BPD and IPF, closely resembling the pattern observed in 18-week fetal lung. Nuclear β-catenin phosphorylated at tyrosine 489 associated with an increased expression of Wnt target gene AXIN2, suggesting that the observed β-catenin signature is of functional significance during normal development and injury repair. The association of specific modifications of β-catenin during normal lung development and again in response to lung injury supports the widely held concept that repair of lung injury involves the recapitulation of developmental programs. Furthermore, these observations suggest that β-catenin phosphorylation has potential as a therapeutic target for the treatment and prevention of both BPD and IPF.
Copyright © 2018 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.
In preterm infants, soluble inflammatory mediators target lung mesenchymal cells, disrupting airway and alveolar morphogenesis. However, how mesenchymal cells respond directly to microbial stimuli remains poorly characterized. Our objective was to measure the genome-wide innate immune response in fetal lung mesenchymal cells exposed to the bacterial endotoxin lipopolysaccharide (LPS). With the use of Affymetrix MoGene 1.0st arrays, we showed that LPS induced expression of unique innate immune transcripts heavily weighted toward CC and CXC family chemokines. The transcriptional response was different between cells from E11, E15, and E18 mouse lungs. In all cells tested, LPS inhibited expression of a small core group of genes including the VEGF receptor Although best characterized in vascular endothelial populations, we demonstrated here that fetal mouse lung mesenchymal cells express and respond to VEGF-A stimulation. In mesenchymal cells, VEGF-A increased cell migration, activated the ERK/AKT pathway, and promoted FOXO3A nuclear exclusion. With the use of an experimental coculture model of epithelial-mesenchymal interactions, we also showed that VEGFR2 inhibition prevented formation of three-dimensional structures. Both LPS and tyrosine kinase inhibition reduced three-dimensional structure formation. Our data suggest a novel mechanism for inflammation-mediated defects in lung development involving reduced VEGF signaling in lung mesenchyme.
Copyright © 2017 the American Physiological Society.
Zika virus (ZIKV) is an emerging mosquito-transmitted flavivirus that can cause severe disease, including congenital birth defects during pregnancy. To develop candidate therapeutic agents against ZIKV, we isolated a panel of human monoclonal antibodies from subjects that were previously infected with ZIKV. We show that a subset of antibodies recognize diverse epitopes on the envelope (E) protein and exhibit potent neutralizing activity. One of the most inhibitory antibodies, ZIKV-117, broadly neutralized infection of ZIKV strains corresponding to African and Asian-American lineages. Epitope mapping studies revealed that ZIKV-117 recognized a unique quaternary epitope on the E protein dimer-dimer interface. We evaluated the therapeutic efficacy of ZIKV-117 in pregnant and non-pregnant mice. Monoclonal antibody treatment markedly reduced tissue pathology, placental and fetal infection, and mortality in mice. Thus, neutralizing human antibodies can protect against maternal-fetal transmission, infection and disease, and reveal important determinants for structure-based rational vaccine design efforts.
To discover mechanisms that mediate plasticity in mammary cells, we characterized signaling networks that are present in the mammary stem cells responsible for fetal and adult mammary development. These analyses identified a signaling axis between FGF signaling and the transcription factor Sox10. Here, we show that Sox10 is specifically expressed in mammary cells exhibiting the highest levels of stem/progenitor activity. This includes fetal and adult mammary cells in vivo and mammary organoids in vitro. Sox10 is functionally relevant, as its deletion reduces stem/progenitor competence whereas its overexpression increases stem/progenitor activity. Intriguingly, we also show that Sox10 overexpression causes mammary cells to undergo a mesenchymal transition. Consistent with these findings, Sox10 is preferentially expressed in stem- and mesenchymal-like breast cancers. These results demonstrate a signaling mechanism through which stem and mesenchymal states are acquired in mammary cells and suggest therapeutic avenues in breast cancers for which targeted therapies are currently unavailable.
Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
Stem cells of the gastrointestinal tract, pancreas, liver and other columnar epithelia collectively resist cloning in their elemental states. Here we demonstrate the cloning and propagation of highly clonogenic, 'ground state' stem cells of the human intestine and colon. We show that derived stem-cell pedigrees sustain limited copy number and sequence variation despite extensive serial passaging and display exquisitely precise, cell-autonomous commitment to epithelial differentiation consistent with their origins along the intestinal tract. This developmentally patterned and epigenetically maintained commitment of stem cells is likely to enforce the functional specificity of the adult intestinal tract. Using clonally derived colonic epithelia, we show that toxins A or B of the enteric pathogen Clostridium difficile recapitulate the salient features of pseudomembranous colitis. The stability of the epigenetic commitment programs of these stem cells, coupled with their unlimited replicative expansion and maintained clonogenicity, suggests certain advantages for their use in disease modelling and regenerative medicine.