Aled H. Bryant

Bioenergetic analysis of human peripheral blood mononuclear cells

Leucocytes respond rapidly to pathogenic and other insults, with responses ranging from cytokine production to migration and phagocytosis. These are bioenergetically expensive, and increased glycolytic flux provides adenosine triphosphate (ATP) rapidly to support these essential functions. However, much of this work is from animal studies. To understand more clearly the relative role of glycolysis and oxidative phosphorylation in human leucocytes, especially their utility in a translational research setting, we undertook a study of human peripheral blood mononuclear cells (MNCs) bioenergetics. Glycolysis was essential during lipopolysaccharide (LPS)‐mediated interleukin (IL)−1β, IL‐6 and tumour necrosis factor (TNF)‐α production, as 2‐deoxy‐D‐glucose decreased significantly the output of all three cytokines. After optimizing cell numbers and the concentrations of all activators and inhibitors, oxidative phosphorylation and glycolysis profiles of fresh and cryopreserved/resuscitated MNCs were determined to explore the utility of MNCs for determining the bioenergetics health profile in multiple clinical settings. While the LPS‐induced cytokine response did not differ significantly between fresh and resuscitated cells from the same donors, cryopreservation/resuscitation significantly affected mainly some measures of oxidative phosphorylation, but also glycolysis. Bioenergetics analysis of human MNCs provides a quick, effective means to measure the bioenergetics health index of many individuals, but cryopreserved cells are not suitable for such an analysis. The translational utility of this approach was tested by comparing MNCs of pregnant and non‐pregnant women to reveal increased bioenergetics health index with pregnancy but significantly reduced basal glycolysis and glycolytic capacity. More detailed analysis of discrete leucocyte populations would be required to understand the relative roles of glycolysis and oxidative phosphorylation during inflammation and other immune responses.

Production and regulation of interleukin-1 family cytokines at the materno-fetal interface

HighlightsLPS modulates multiple IL‐1 family cytokines from placenta, choriodecidua and amnion.IL‐1&bgr;/IL‐18 production is caspase‐dependent; IL‐1&agr; production is calpain‐dependent.Amnion has broadest responsiveness to IL‐1 family members.Complex IL‐1 family network at materno‐fetal interface. Abstract IL‐1 family members regulate innate immune responses, are produced by gestation‐associated tissues, and have a role in healthy and adverse pregnancy outcomes. To better understand their role at the materno‐fetal interface we used a human tissue explant model to map lipopolysaccharide (LPS)‐stimulated production of IL‐1&agr;, IL‐1&bgr;, IL‐18, IL‐33, IL‐1Ra, IL‐18BPa, ST2 and IL‐1RAcP by placenta, choriodecidua and amnion. Caspase‐dependent processing of IL‐1&agr;, IL‐1&bgr;, IL‐18, and IL‐33 and the ability of IL‐1&agr;, IL‐1&bgr;, IL‐18, and IL‐33 to regulate the production of IL‐1RA, IL‐18BPa, ST2 and IL‐1RAcP was also determined. LPS acted as a potent inducer of IL‐1 family member expression especially in the placenta and choriodecidua with the response by the amnion restricted to IL‐1&bgr;. Caspases‐1, 4 and 8 contributed to LPS‐stimulated production of IL‐1&bgr; and IL‐18, whereas calpain was required for IL‐1&agr; production. Exogenous administration of IL‐1&agr;, IL‐1&bgr;, IL‐18, and IL‐33 lead to differential expression of IL‐1Ra, IL‐18BPa, ST2 and IL‐1RAcP across all tissues examined. Most notable were the counter‐regulatory effect of LPS on IL‐1&bgr; and IL‐1Ra in the amnion and the broad responsiveness of the amnion to IL‐1 family cytokines for increased production of immunomodulatory peptides and soluble receptors. The placenta and membranes vary not only in their output of various IL‐1 family members but also in their counter‐regulatory mechanisms through endogenous inhibitory peptides, processing enzymes and soluble decoy receptors. This interactive network of inflammatory mediators likely contributes to innate defence mechanisms at the materno‐fetal interface to limit, in particular, the detrimental effects of microbial invasion.

Interleukin 4 and interleukin 13 downregulate the lipopolysaccharide-mediated inflammatory response by human gestation-associated tissues†

Abstract Inflammation is a key feature of preterm and term labor. Proinflammatory mediators are produced by gestation-associated tissues in response to pathogen-associated molecular patterns and damage-associated molecular patterns. Interleukin (IL)4, IL10, and IL13 are anti-inflammatory cytokines with potential as anti-inflammatory therapies to prevent preterm birth. The objective of this study was to determine if IL4 and IL13 exert anti-inflammatory effects on lipopolysaccharide (LPS)-stimulated production of proinflammatory cytokines produced by human term gestation-associated tissues (placenta, choriodecidua, and amnion). Both IL4 and IL13 reduced LPS-stimulated IL1B and macrophage inflammatory protein1A; this effect diminished with delay to exposure to either cytokine. There was no effect on LPS-stimulated prostaglandin production. Interleukin 4 receptor alpha (IL4RA) was expressed throughout the placenta, choriodecidua, and amnion, and the inhibitory effects of IL4 and IL13 were IL4RA dependent. Combined IL4 and IL13 did not enhance the anti-inflammatory potential of either cytokine; however, a combination of IL4 and IL10 had a greater anti-inflammatory effect than either cytokine alone. These findings demonstrate that human term gestation-associated tissues are responsive to the anti-inflammatory cytokines IL4 and IL13, which could downregulate LPS-induced cytokine production in these tissues. Antiinflammatory cytokines might offer an adjunct to existing therapeutics to prevent adverse obstetric outcome. Summary Sentence The anti-inflammatory cytokines interleukin (IL)4 and IL13 can downregulate the lipopolysaccharide-induced inflammatory response by human gestation-associated tissues in an IL4 receptor alpha-dependent manner.

Cytokines and the Innate Immune Response at the Materno-Fetal Interface

Labour is the climax of pregnancy resulting in the expulsion of the fetus from the uterus. It is a complex process and the mechanisms involved in the initiation of labour are poorly understood despite decades of investigation. Generally, labour is not a sudden occurrence but one for which the body prepares: numerous physiological, biochemical and immunological events take place at the materno-fetal interface, and in both mother and fetus in the lead up to parturition (Hendricks, Brenner et al. 1970). These same processes might be accelerated or other pathways brought into play when labour occurs prematurely. The bulk of perinatal morbidity and mortality is associated with premature labour and delivery of a preterm infant (Tucker and McGuire 2004; Steer 2005). Understanding the mechanisms of labour in health and in adverse obstetric outcomes should provide insight into the pathogenesis of preterm birth.

Inflammation, Obesity, and Neuromodulation in Pregnancy and Fetal Development

For over 50 years investigators in reproductive immunology have sought to identify the mechanisms that explain how the immunocompetent mother tolerates the semi-allogeneic fetus. The current immunological paradigm of pregnancy is of active immunological tolerance of fetal cells by the pregnant woman. A perturbed tolerance response has been hypothesised to underlie adverse pregnancy outcomes. Also there is much interest in how antenatal determinants related to the maternal environment impact on these immunological mechanisms and thereby the development and long term health of the offspring. Environmental insults acting during fetal development can program the structure and function of tissues, organs and body systems. Maternal obesity may be one such insult. While the impact of maternal obesity on immune function of mother and child is relatively unknown, animal models reveal that obesity and/or high-fat feeding during pregnancy have detrimental effects on the development of the pancreas, liver, cardiovascular system, and the central and peripheral nervous systems. Many of these changes relate to regulation of energy balance and metabolism but there is a growing appreciation of the impact of maternal obesity on inflammatory responses in many tissues of both mother and child.

Expression and function of NOD-like receptors by human term gestation-associated tissues

INTRODUCTION Nucleotide-binding oligomerization domain (NOD)-like receptors or NOD-like receptors (NLRs) have been implicated in several disease pathologies associated with inflammation. Since local and systemic inflammation is a hallmark of both term and preterm labour, a role for NLRs at the materno-fetal interface has been postulated. METHODS Gene expression and immunolocalisation of NLR family members in human placenta, choriodecidua, and amnion were examined. Tissue explants were used to examine the response to activators of NOD1 (Tri-DAP), NOD2 (MDP) and NLRP3 (nigericin). Cell/tissue-free supernatants were examined for the production of interleukin (IL)-1β, IL-6, IL-8 and IL-10 using specific ELISAs. RESULTS Expression of transcripts for NOD1, NOD2, NLRP3, NLRC4, NLRX1, NLRP1 and NAIP and protein expression of NOD1, NOD2 and NLRP3 were a broad feature of all term gestation-associated tissues. Production of cytokines was increased significantly in response to all ligands in placenta and choriodecidua, except for MDP-induced IL-10. Similarly, there was a significant in the amnion except for MDP induced IL-1β and IL-10 response to either agonist. IL-1β production was dependent on caspase-1 regardless of agonist used or tissue examined. DISCUSSION Term human gestation-associated tissues express functional NLRs which likely play a role in both sterile and pathogen-driven inflammatory responses at the materno-fetal interface.

Human gestation-associated tissues express functional cytosolic nucleic acid sensing pattern recognition receptors

The role of viral infections in adverse pregnancy outcomes has gained interest in recent years. Innate immune pattern recognition receptors (PRRs) and their signalling pathways, that yield a cytokine output in response to pathogenic stimuli, have been postulated to link infection at the maternal–fetal interface and adverse pregnancy outcomes. The objective of this study was to investigate the expression and functional response of nucleic acid ligand responsive Toll‐like receptors (TLR‐3, −7, −8 and −9), and retinoic acid‐inducible gene 1 (RIG‐I)‐like receptors [RIG‐I, melanoma differentiation‐associated protein 5 (MDA5) and Laboratory of Genetics and Physiology 2(LGP2)] in human term gestation‐associated tissues (placenta, choriodecidua and amnion) using an explant model. Immunohistochemistry revealed that these PRRs were expressed by the term placenta, choriodecidua and amnion. A statistically significant increase in interleukin (IL)‐6 and/or IL‐8 production in response to specific agonists for TLR‐3 (Poly(I:C); low and high molecular weight), TLR‐7 (imiquimod), TLR‐8 (ssRNA40) and RIG‐I/MDA5 (Poly(I:C)LyoVec) was observed; there was no response to a TLR‐9 (ODN21798) agonist. A hierarchical clustering approach was used to compare the response of each tissue type to the ligands studied and revealed that the placenta and choriodecidua generate a more similar IL‐8 response, while the choriodecidua and amnion generate a more similar IL‐6 response to nucleic acid ligands. These findings demonstrate that responsiveness via TLR‐3, TLR‐7, TLR‐8 and RIG‐1/MDA5 is a broad feature of human term gestation‐associated tissues with differential responses by tissue that might underpin adverse obstetric outcomes.