SummaryLeucocytes 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)21b, IL-6 and tumour necrosis factor (TNF)-a 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.

Summary 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.

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.

Pro-inflammatory cytokine output in response to multiple TLR ligands was decreased in the preterm compared to the term placenta but gene expression for each TLR tended to be similar. Reduced cytokine production by the preterm placenta in response to stimulation of TLRs therefore must be regulated at the post-transcriptional level in a gestational age dependent manner.

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-1α, IL-1β, IL-18, IL-33, IL-1Ra, IL-18BPa, ST2 and IL-1RAcP by placenta, choriodecidua and amnion. Caspase-dependent processing of IL-1α, IL-1β, IL-18, and IL-33 and the ability of IL-1α, IL-1β, 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-1β. Caspases-1, 4 and 8 contributed to LPS-stimulated production of IL-1β and IL-18, whereas calpain was required for IL-1α production. Exogenous administration of IL-1α, IL-1β, 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-1β 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.