Antenatal Ureaplasma Infection Causes Colonic Mucus Barrier Defects: Implications for Intestinal Pathologies.

Chorioamnionitis is a risk factor for necrotizing enterocolitis (NEC). Ureaplasma parvum (UP) is clinically the most isolated microorganism in chorioamnionitis, but its pathogenicity remains debated. Chorioamnionitis is associated with ileal barrier changes, but colonic barrier alterations, including those of the mucus barrier, remain under-investigated, despite their importance in NEC pathophysiology. Therefore, in this study, the hypothesis that antenatal UP exposure disturbs colonic mucus barrier integrity, thereby potentially contributing to NEC pathogenesis, was investigated. In an established ovine chorioamnionitis model, lambs were intra-amniotically exposed to UP or saline for 7 d from 122 to 129 d gestational age. Thereafter, colonic mucus layer thickness and functional integrity, underlying mechanisms, including endoplasmic reticulum (ER) stress and redox status, and cellular morphology by transmission electron microscopy were studied. The clinical significance of the experimental findings was verified by examining colon samples from NEC patients and controls. UP-exposed lambs have a thicker but dysfunctional colonic mucus layer in which bacteria-sized beads reach the intestinal epithelium, indicating undesired bacterial contact with the epithelium. This is paralleled by disturbed goblet cell MUC2 folding, pro-apoptotic ER stress and signs of mitochondrial dysfunction in the colonic epithelium. Importantly, the colonic epithelium from human NEC patients showed comparable mitochondrial aberrations, indicating that NEC-associated intestinal barrier injury already occurs during chorioamnionitis. This study underlines the pathogenic potential of UP during pregnancy; it demonstrates that antenatal UP infection leads to severe colonic mucus barrier deficits, providing a mechanistic link between antenatal infections and postnatal NEC development.

Hypoxia-Driven Changes in a Human Intestinal Organoid Model and the Protective Effects of Hydrolyzed Whey.

Many whey proteins, peptides and protein-derived amino acids have been suggested to improve gut health through their anti-oxidant, anti-microbial, barrier-protective and immune-modulating effects. Interestingly, although the degree of hydrolysis influences peptide composition and, thereby, biological function, this important aspect is often overlooked. In the current study, we aimed to investigate the effects of whey protein fractions with different degrees of enzymatic hydrolysis on the intestinal epithelium in health and disease with a novel 2D human intestinal organoid (HIO) monolayer model. In addition, we aimed to assess the anti-microbial activity and immune effects of the whey protein fractions. Human intestinal organoids were cultured from adult small intestines, and a model enabling apical administration of nutritional components during hypoxia-induced intestinal inflammation and normoxia (control) in crypt-like and villus-like HIO was established. Subsequently, the potential beneficial effects of whey protein isolate (WPI) and two whey protein hydrolysates with a 27.7% degree of hydrolysis (DH28) and a 50.9% degree of hydrolysis (DH51) were assessed. In addition, possible immune modulatory effects on human peripheral immune cells and anti-microbial activity on four microbial strains of the whey protein fractions were investigated. Exposure to DH28 prevented paracellular barrier loss of crypt-like HIO following hypoxia-induced intestinal inflammation with a concomitant decrease in hypoxia inducible factor 1 alpha (HIF1α) mRNA expression. WPI increased Treg numbers and Treg expression of cluster of differentiation 25 (CD25) and CD69 and reduced CD4+ T cell proliferation, whereas no anti-microbial effects were observed. The observed biological effects were differentially mediated by diverse whey protein fractions, indicating that (degree of) hydrolysis influences their biological effects. Moreover, these new insights may provide opportunities to improve immune tolerance and promote intestinal health.

Antenatal Ureaplasma infection induces ovine small intestinal goblet cell defects: a strong link with NEC pathology.

Disruption of the intestinal mucus barrier and intestinal epithelial endoplasmic reticulum (ER) stress contribute to necrotizing enterocolitis (NEC). Previously, we observed intestinal goblet cell loss and increased intestinal epithelial ER stress following chorioamnionitis. Here, we investigated how chorioamnionitis affects goblet cells by assessing their cellular characteristics. Importantly, goblet cell features are compared with those in clinical NEC biopsies. Mucus thickness was assessed as read-out of goblet cell function. Fetal lambs were intra-amniotically (IA) infected for 7d at 122 gestational age with Ureaplasma parvum serovar-3, the main microorganism clinically associated with chorioamnionitis. After preterm delivery, mucus thickness, goblet cell numbers, gut inflammation, epithelial proliferation and apoptosis and intestinal epithelial ER stress were investigated in the terminal ileum. Next, goblet cell morphological alterations (TEM) were studied and compared to human NEC samples. Ileal mucus thickness and goblet cell numbers were elevated following IA UP exposure. Increased pro-apoptotic ER stress, detected by elevated CHOP-positive cell counts and disrupted organelle morphology of secretory cells in the intestinal epithelium, was observed in IA UP exposed animals. Importantly, comparable cellular morphological alterations were observed in the ileum from NEC patients. In conclusion, UP-driven chorioamnionitis leads to a thickened ileal mucus layer and mucus hypersecretion from goblet cells. Since this was associated with pro-apoptotic ER stress and organelle disruption, mucus barrier alterations seem to occur at the expense of goblet cell resilience and may therefore predispose to detrimental intestinal outcomes. The remarkable overlap of these in utero findings with observations in NEC patients underscores their clinical relevance.

Thiol-ene conjugation of VEGF peptide to electrospun scaffolds as potential application for angiogenesis.

Vascular endothelial growth factor (VEGF) plays a vital role in promoting attachment and proliferation of endothelial cells, and induces angiogenesis. In recent years, much research has been conducted on functionalization of tissue engineering scaffolds with VEGF or VEGF-mimetic peptide to promote angiogenesis. However, most chemical reactions are nonspecific and require organic solvents, which can compromise control over functionalization and alter peptide/protein activity. An attractive alternative is the fabrication of functionalizable electrospun fibers, which can overcome these hurdles. In this study, we used thiol-ene chemistry for the conjugation of a VEGF-mimetic peptide to the surface of poly (ε-caprolactone) (PCL) fibrous scaffolds with varying amounts of a functional PCL-diacrylate (PCL-DA) polymer. 30% PCL-DA was selected due to homogeneous fiber morphology. A VEGF-mimetic peptide was then immobilized on PCL-DA fibrous scaffolds by a light-initiated thiol-ene reaction. 7-Mercapto-4-methylcoumarin, RGD-FITC peptide and VEGF-TAMRA mimetic peptide were used to validate the thiol-ene reaction with fibrous scaffolds. Tensile strength and elastic modulus of 30% PCL-DA fibrous scaffolds were significantly increased after the reaction. Conjugation of 30% PCL-DA fibrous scaffolds with VEGF peptide increased the surface water wettability of the scaffolds. Patterned structures could be obtained after using a photomask on the fibrous film. Moreover, in vitro studies indicated that scaffolds functionalized with the VEGF-mimetic peptide were able to induce phosphorylation of VEGF receptor and enhanced HUVECs survival, proliferation and adhesion. A chick chorioallantoic membrane (CAM) assay further indicated that the VEGF peptide functionalized scaffolds are able to promote angiogenesis in vivo. These results show that scaffold functionalization can be controlled via a simple polymer mixing approach, and that the functionalized VEGF peptide-scaffolds have potential for vascular tissue regeneration.

Electrospun Scaffolds Functionalized with a Hydrogen Sulfide Donor Stimulate Angiogenesis.

Tissue-engineered constructs are currently limited by the lack of vascularization necessary for the survival and integration of implanted tissues. Hydrogen sulfide (H2S), an endogenous signaling gas (gasotransmitter), has been recently reported as a promising alternative to growth factors to mediate and promote angiogenesis in low concentrations. Yet, sustained delivery of H2S remains a challenge. Herein, we have developed angiogenic scaffolds by covalent attachment of an H2S donor to a polycaprolactone (PCL) electrospun scaffold. These scaffolds were engineered to include azide functional groups (on 1, 5, or 10% of the PCL end groups) and were modified using a straightforward click reaction with an alkyne-functionalized N-thiocarboxyanhydride (alkynyl-NTA). This created H2S-releasing scaffolds that rely on NTA ring-opening in water followed by conversion of released carbonyl sulfide into H2S. These functionalized scaffolds showed dose-dependent release of H2S based on the amount of NTA functionality within the scaffold. The NTA-functionalized fibrous scaffolds supported human umbilical vein endothelial cell (HUVEC) proliferation, formed more confluent endothelial monolayers, and facilitated the formation of tight cell-cell junctions to a greater extent than unfunctionalized scaffolds. Covalent conjugation of H2S donors to scaffolds not only promotes HUVEC proliferation in vitro, but also increases neovascularization in ovo, as observed in the chick chorioallantoic membrane assay. NTA-functionalized scaffolds provide localized control over vascularization through the sustained delivery of a powerful endogenous angiogenic agent, which should be further explored to promote angiogenesis in tissue engineering.

Intestinal Goblet Cell Loss during Chorioamnionitis in Fetal Lambs: Mechanistic Insights and Postnatal Implications.

Chorioamnionitis, an important cause of preterm birth, is linked to necrotizing enterocolitis (NEC). NEC is characterized by a disrupted mucus barrier, goblet cell loss, and endoplasmic reticulum (ER) stress of the intestinal epithelium. These findings prompted us to investigate the mechanisms underlying goblet cell alterations over time in an ovine chorioamnionitis model. Fetal lambs were intra-amniotically (IA) exposed to lipopolysaccharides (LPS) for 5, 12, or 24 h, or 2, 4, 8, or 15 d before premature delivery at 125 d gestational age (GA). Gut inflammation, the number, distribution, and differentiation of goblet cells, ER stress, and apoptosis were measured. We found a biphasic reduction in goblet cell numbers 24 h-2 d after, and 15 d after IA LPS exposure. The second decrease of goblet cell numbers was preceded by intestinal inflammation, apoptosis, and crypt ER stress, and increased SAM-pointed domain-containing ETS transcription factor (SPDEF)-positive cell counts. Our combined findings indicated that ER stress drives apoptosis of maturating goblet cells during chorioamnionitis, ultimately reducing goblet cell numbers. As similar changes have been described in patients suffering from NEC, these findings are considered to be clinically important for understanding the predecessors of NEC, and targeting ER stress in this context is interesting for future therapeutics.

Corrigendum: Chronic Intra-Uterine Ureaplasma parvum Infection Induces Injury of the Enteric Nervous System in Ovine Fetuses.

[This corrects the article DOI: 10.3389/fimmu.2020.00189.].

Chronic Intra-Uterine Ureaplasma parvum Infection Induces Injury of the Enteric Nervous System in Ovine Fetuses.

Background: Chorioamnionitis, inflammation of the fetal membranes during pregnancy, is often caused by intra-amniotic (IA) infection with single or multiple microbes. Chorioamnionitis can be either acute or chronic and is associated with adverse postnatal outcomes of the intestine, including necrotizing enterocolitis (NEC). Neonates with NEC have structural and functional damage to the intestinal mucosa and the enteric nervous system (ENS), with loss of enteric neurons and glial cells. Yet, the impact of acute, chronic, or repetitive antenatal inflammatory stimuli on the development of the intestinal mucosa and ENS has not been studied. The aim of this study was therefore to investigate the effect of acute, chronic, and repetitive microbial exposure on the intestinal mucosa, submucosa and ENS in premature lambs. Materials and Methods: A sheep model of pregnancy was used in which the ileal mucosa, submucosa, and ENS were assessed following IA exposure to lipopolysaccharide (LPS) for 2 or 7 days (acute), Ureaplasma parvum (UP) for 42 days (chronic), or repetitive microbial exposure (42 days UP with 2 or 7 days LPS). Results: IA LPS exposure for 7 days or IA UP exposure for 42 days caused intestinal injury and inflammation in the mucosal and submucosal layers of the gut. Repetitive microbial exposure did not further aggravate injury of the terminal ileum. Chronic IA UP exposure caused significant structural ENS alterations characterized by loss of PGP9.5 and S100ß immunoreactivity, whereas these changes were not found after re-exposure of chronic UP-exposed fetuses to LPS for 2 or 7 days. Conclusion: The in utero loss of PGP9.5 and S100ß immunoreactivity following chronic UP exposure corresponds with intestinal changes in neonates with NEC and may therefore form a novel mechanistic explanation for the association of chorioamnionitis and NEC.

Electrical stimulation promotes the angiogenic potential of adipose-derived stem cells.

Autologous fat transfer (AFT) is limited by post-operative volume loss due to ischemia-induced cell death in the fat graft. Previous studies have demonstrated that electrical stimulation (ES) promotes angiogenesis in a variety of tissues and cell types. In this study we investigated the effects of ES on the angiogenic potential of adipose-derived stem cells (ASC), important progenitor cells in fat grafts with proven angiogenic potential. Cultured human ASC were electrically stimulated for 72 hours after which the medium of stimulated (ES) and non-stimulated (control) ASC was analysed for angiogenesis-related proteins by protein array and ELISA. The functional effect of ES on angiogenesis was then assessed in vitro and in vivo. Nine angiogenesis-related proteins were detected in the medium of electrically (non-)stimulated ASC and were quantified by ELISA. The pro-angiogenic proteins VEGF and MCP-1 were significantly increased following ES compared to controls, while the anti-angiogenic factor Serpin E1/PAI-1 was significantly decreased. Despite increased levels of anti-angiogenic TSP-1 and TIMP-1, medium of ES-treated ASC significantly increased vessel density, total vessel network length and branching points in chorio-allantoic membrane assays. In conclusion, our proof-of-concept study showed that ES increased the angiogenic potential of ASC both in vitro and in vivo.

Protection of the Ovine Fetal Gut against Ureaplasma-Induced Chorioamnionitis: A Potential Role for Plant Sterols.

Chorioamnionitis, clinically most frequently associated with Ureaplasma, is linked to intestinal inflammation and subsequent gut injury. No treatment is available to prevent chorioamnionitis-driven adverse intestinal outcomes. Evidence is increasing that plant sterols possess immune-modulatory properties. Therefore, we investigated the potential therapeutic effects of plant sterols in lambs intra-amniotically (IA) exposed to Ureaplasma. Fetal lambs were IA exposed to Ureaplasma parvum (U. parvum, UP) for six days from 127 d-133 d of gestational age (GA). The plant sterols ß-sitosterol and campesterol, dissolved with ß-cyclodextrin (carrier), were given IA every two days from 122 d-131 d GA. Fetal circulatory cytokine levels, gut inflammation, intestinal injury, enterocyte maturation, and mucosal phospholipid and bile acid profiles were measured at 133 d GA (term 150 d). IA plant sterol administration blocked a fetal inflammatory response syndrome. Plant sterols reduced intestinal accumulation of proinflammatory phospholipids and tended to prevent mucosal myeloperoxidase-positive (MPO) cell influx, indicating an inhibition of gut inflammation. IA administration of plant sterols and carrier diminished intestinal mucosal damage, stimulated maturation of the immature epithelium, and partially prevented U. parvum-driven reduction of mucosal bile acids. In conclusion, we show that ß-sitosterol and campesterol administration protected the fetus against adverse gut outcomes following UP-driven chorioamnionitis by preventing intestinal and systemic inflammation.

The Paradoxical Effects of Chronic Intra-Amniotic Ureaplasma parvum Exposure on Ovine Fetal Brain Development.

Chorioamnionitis is associated with adverse neurodevelopmental outcomes in preterm infants. Ureaplasma spp. are the microorganisms most frequently isolated from the amniotic fluid of women diagnosed with chorioamnionitis. However, controversy remains concerning the role of Ureaplasma spp. in the pathogenesis of neonatal brain injury. We hypothesize that reexposure to an inflammatory trigger during the perinatal period might be responsible for the variation in brain outcomes of preterms following Ureaplasma-driven chorioamnionitis. To investigate these clinical scenarios, we performed a detailed multimodal study in which ovine neurodevelopmental outcomes were assessed following chronic intra-amniotic Ureaplasma parvum (UP) infection either alone or combined with subsequent lipopolysaccharide (LPS) exposure. We show that chronic intra-amniotic UP exposure during the second trimester provoked a decrease in astrocytes, increased oligodendrocyte numbers, and elevated 5-methylcytosine levels. In contrast, short-term LPS exposure before preterm birth induced increased microglial activation, myelin loss, elevation of 5-hydroxymethylcytosine levels, and lipid profile changes. These LPS-induced changes were prevented by chronic preexposure to UP (preconditioning). These data indicate that chronic UP exposure has dual effects on preterm brain development in utero. On the one hand, prolonged UP exposure causes detrimental cerebral changes that may predispose to adverse postnatal clinical outcomes. On the other, chronic intra-amniotic UP exposure preconditions the brain against a second inflammatory hit. This study demonstrates that microbial interactions and the timing and duration of the inflammatory insults determine the effects on the fetal brain. Therefore, this study helps to understand the complex and diverse postnatal neurological outcomes following UP driven chorioamnionitis.

Hypoxia-induced contraction of chicken embryo mesenteric arteries: mechanisms and developmental changes.

The fetal cardiovascular responses to acute hypoxia include a redistribution of the cardiac output toward the heart and the brain at the expense of other organs, such as the intestine. We hypothesized that hypoxia exerts a direct effect on the mesenteric artery (MA) that may contribute to this response. Using wire myography, we investigated the response to hypoxia (Po2 ~2.5 kPa for 20 min) of isolated MAs from 15- to 21-day chicken embryos (E15, E19, E21), and 1- to 45-day-old chickens (P1, P3, P14, P45). Agonist-induced pretone or an intact endothelium were not required to obtain a consistent and reproducible response to hypoxia, which showed a pattern of initial rapid phasic contraction followed by a sustained tonic contraction. Phasic contraction was reduced by elimination of extracellular Ca2+ or by presence of the neurotoxin tetrodotoxin, the α1-adrenoceptor antagonist prazosin, or inhibitors of L-type voltage-gated Ca2+ channels (nifedipine), mitochondrial electron transport chain (rotenone and antimycin A), and NADPH oxidase (VAS2870). The Rho-kinase inhibitor Y27632 impaired both phasic and tonic contraction and, when combined with elimination of extracellular Ca2+, hypoxia-induced contraction was virtually abolished. Hypoxic MA contraction was absent at E15 but present from E19 and increased toward the first days posthatching. It then decreased during the first weeks of life and P45 MAs were unable to sustain hypoxia-induced contraction over time. In conclusion, the results of the present study demonstrate that hypoxic vasoconstriction is an intrinsic feature of chicken MA vascular smooth muscle cells during late embryogenesis and the perinatal period.

Morphological and functional alterations of the ductus arteriosus in a chicken model of hypoxia-induced fetal growth retardation.

The hypoxic conditions in which children with intrauterine growth retardation (IUGR) develop are hypothesized to alter the development of the ductus arteriosus (DA). We aimed to evaluate the effects of in ovo hypoxia on chicken DA morphometry and reactivity. Hypoxia (15% O2 from day 6 to 19 of the 21-d incubation period) produced a reduction in the body mass of the 19-d fetuses and a shortening of right and left DAs. However, ductal lumen and media cross-sectional areas were not affected by hypoxia. The ductal contractions induced by oxygen, KCl, H2O2, 4-aminopyridine, and endothelin-1 were similar in control and hypoxic fetuses. In contrast, the DAs from the hypoxic fetuses showed increased contractile responses to norepinephrine and phenylephrine and impaired relaxations to acetylcholine, sodium nitroprusside, and isoproterenol. The relaxations induced by 8-Br-cGMP, forskolin, Y-27632, and hydroxyfasudil were not altered by chronic hypoxia. In conclusion, chronic in ovo hypoxia-induced growth retardation in fetal chickens and altered the response of the DA to adrenergic agonists and to endothelium-dependent and -independent relaxing agents. Our observations support the concept that prolonged patency of the DA in infants with IUGR may be partially related with hypoxia-induced changes in local vascular mechanisms.

Chronic moderate hypoxia and protein malnutrition both induce growth retardation, but have distinct effects on arterial endothelium-dependent reactivity in the chicken embryo.

Deviations in the rate of intrauterine growth may change organ system development, resulting in cardiovascular disease in adult life. Arterial endothelial dysfunction often plays an important role in these diseases. The effects of two interventions that reduce fetal growth, chronic hypoxia and protein malnutrition, on arterial endothelial function were investigated. Eggs of White Leghorn chickens were incubated either in room air or in 15% O2 from d 6 until d 19 of the 21-d incubation. Protein malnutrition was induced by removal of 10% of the total albumen content at d 0. In vitro reactivity of the femoral artery in response to vasodilators was measured at d 19. Both chronic hypoxia and protein malnutrition reduced embryonic body weight at d 19 by 14% without affecting relative brain weight. Chronic hypoxia or protein malnutrition did not change sensitivity to the exogenous nitric oxide donor, sodium nitroprusside (5.74 +/- 0.15 versus 5.85 +/- 0.23 and 6.05 +/- 0.18 versus 6.01 +/- 0.34, respectively). Whereas protein malnutrition did not modify arterial sensitivity to acetylcholine (7.00 +/- 0.10 versus 7.12 +/- 0.05), chronic hypoxia reduced sensitivity to this endothelium-dependent vasodilator (6.57 +/- 0.07 versus 7.02 +/- 0.06). In the presence of Nomega-nitro-l-arginine methyl ester, this difference in sensitivity to acetylcholine was no longer apparent (6.31 +/- 0.13 versus 6.27 +/- 0.06), indicating that chronic exposure to hypoxia reduced sensitivity to acetylcholine by lowering nitric oxide release. In additional experiments, a decrease in basal nitric oxide release in arteries of 3- to 4-wk-old chickens that had been exposed to in ovo chronic hypoxia was observed (increase in K+ contraction: -0.16 +/- 0.33 N/m versus 0.68 +/- 020 N/m). Protein malnutrition and chronic hypoxia both induce disproportionate growth retardation, but only the latter impairs arterial endothelial function. Intrauterine exposure to chronic hypoxia induces changes in arterial endothelial properties that may play a role in the development of cardiovascular disease in adult life.