Monocarboxylate transporter 8 modulates the viability and invasive capacity of human placental cells and fetoplacental growth in mice

Monocarboxylate transporter 8 (MCT8) is a well-established thyroid hormone (TH) transporter. In humans, MCT8 mutations result in changes in circulating TH concentrations and X-linked severe global neurodevelopmental delay. MCT8 is expressed in the human placenta throughout gestation, with increased expression in trophoblast cells from growth-restricted pregnancies. We postulate that MCT8 plays an important role in placental development and transplacental TH transport. We investigated the effect of altering MCT8 expression in human trophoblast in vitro and in a Mct8 knockout mouse model. Silencing of endogenous MCT8 reduced T3 uptake into human extravillous trophoblast-like cells (SGHPL-4; 40%, P<0.05) and primary cytotrophoblast (15%, P<0.05). MCT8 over-expression transiently increased T3 uptake (SGHPL-4∶30%, P<0.05; cytotrophoblast: 15%, P<0.05). Silencing MCT8 did not significantly affect SGHPL-4 invasion, but with MCT8 over-expression T3 treatment promoted invasion compared with no T3 (3.3-fold; P<0.05). Furthermore, MCT8 silencing increased cytotrophoblast viability (∼20%, P<0.05) and MCT8 over-expression reduced cytotrophoblast viability independently of T3 (∼20%, P<0.05). In vivo, Mct8 knockout reduced fetal:placental weight ratios compared with wild-type controls at gestational day 18 (25%, P<0.05) but absolute fetal and placental weights were not significantly different. The volume fraction of the labyrinthine zone of the placenta, which facilitates maternal-fetal exchange, was reduced in Mct8 knockout placentae (10%, P<0.05). However, there was no effect on mouse placental cell proliferation in vivo. We conclude that MCT8 makes a significant contribution to T3 uptake into human trophoblast cells and has a role in modulating human trophoblast cell invasion and viability. In mice, Mct8 knockout has subtle effects upon fetoplacental growth and does not significantly affect placental cell viability probably due to compensatory mechanisms in vivo

The maternal microbiome during pregnancy and allergic disease in the offspring

There is substantial epidemiological and mechanistic evidence that the increase in allergic disease and asthma in many parts of the world in part relates to changes in microbial exposures and diet acting via the composition and metabolic products of the intestinal microbiome. The majority of research in this field has focused on the gut microbiome during infancy, but it is increasingly clear that the maternal microbiome during pregnancy also has a key role in preventing an allergy-prone immune phenotype in the offspring. The mechanisms by which the maternal microbiome influences the developing fetal immune system include alignment between the maternal and infant regulatory immune status and transplacental passage of microbial metabolites and IgG. Interplay between microbial stimulatory factors such as lipopolysaccharides and regulatory factors such as short-chain fatty acids may also influence on fetal immune development. However, our understanding of these pathways is at an early stage and further mechanistic studies are needed. There are also no data from human studies relating the composition and metabolic activity of the maternal microbiome during pregnancy to the offspring's immune status at birth and risk of allergic disease. Improved knowledge of these pathways may inform novel strategies for tackling the increase in allergic disorders in the modern world

MINocyclinE to Reduce inflammation and blood brain barrier leakage in small Vessel diseAse (MINERVA) trial study protocol.

BACKGROUND: Cerebral small vessel disease (SVD) is a common cause of stroke and cognitive impairment. Recent data has implicated neuroinflammation and increased blood-brain barrier (BBB) permeability in its pathogenesis, but whether such processes are causal and can be therapeutically modified is uncertain. In a rodent model of SVD, minocycline was associated with reduced white matter lesions, inflammation and BBB permeability. AIMS: To determine whether blood-brain barrier permeability (measured using dynamic contrast-enhanced MRI) and microglial activation (measured by positron emission tomography using the radioligand 11C-PK11195) can be modified in SVD. DESIGN: Phase II randomised double blind, placebo-controlled trial of minocycline 100 mg twice daily for 3 months in 44 participants with moderate to severe SVD defined as a clinical lacunar stroke and confluent white matter hyperintensities. OUTCOMES: Primary outcome measures are volume and intensity of focal increases of blood-brain barrier permeability and microglial activation determined using PET-MRI imaging. Secondary outcome measures include inflammatory biomarkers in serum, and change in conventional MRI markers and cognitive performance over 1 year follow up. DISCUSSION: The MINERVA trial aims to test whether minocycline can influence novel pathological processes thought to be involved in SVD progression, and will provide insights into whether central nervous system inflammation in SVD can be therapeutically modulated

MINocyclinE to Reduce inflammation and blood-brain barrier leakage in small Vessel diseAse (MINERVA): A phase II, randomized, double-blind, placebo-controlled experimental medicine trial

Publication status: PublishedFunder: Association of British Neurologists / Guarantors of BrainBackground: Cerebral small vessel disease (SVD) is a common cause of stroke and a major contributor to vascular dementia, but there are few treatments to delay disease progression. Neuroinflammation and increased blood-brain barrier (BBB) permeability have been suggested to play a role in pathogenesis, and both can be visualised in man using advanced imaging. However, whether they drive disease progression and can be targeted therapeutically is uncertain. In a rodent model of SVD, minocycline treatment reduced white matter damage and markers of inflammation and BBB permeability, and improved behavioural outcomes. We determined whether minocycline had a similar effect in patients with moderate to severe SVD. Methods: MINERVA (ISRCTN15483452) was a phase II randomised, double blind, placebo-controlled trial performed in a single UK centre. The study included 44 participants with moderate to severe SVD defined as a clinical lacunar stroke and confluent MRI white matter hyperintensities, who were randomly assigned 1:1 to either minocycline 100mg twice daily or placebo for three months. The co-primary outcome measures were volume of focal areas of increased microglial signal (determined using 11C-PK11195 positron emission tomography) and increased blood-brain barrier permeability (measured using dynamic contrast-enhanced [DCE] MRI). Secondary outcome measures included change in inflammatory biomarkers in serum. Results: 44 participants were recruited between September 2019 and June 2022. All 44 were included in a modified intention-to-treat analysis: 23 in the minocycline group and 21 in the placebo group. Mean age was 73.0 ± 8.9 years in the treatment group and 66.5 ± 11.9 in the placebo group (p = 0.04), but other baseline demographics were balanced. Participants were recruited at a mean of 23.1 ± 24.7 months post stroke. Hotspots of increased 11C-PK11195 binding and increased BBB permeability were present in the participants. There was no treatment effect of minocycline on the change in percentage of 11C-PK11195 binding hotspots (RR 1.01, 95% CI 0.98 – 1.04), or on BBB permeability hotspots (RR 0.97, 95% CI 0.91 – 1.03). All secondary analyses, as well as the per-protocol analysis, showed no treatment effect. There was also no significant treatment effect on C-reactive protein levels or on any of the individual biomarkers in a proteomics panel targeting vascular inflammation and endothelial activation. Interpretation: Although increased 11C-PK11195 binding consistent with microglial activation, and increased BBB permeability on DCE-MRI, are both present in SVD, treatment with minocycline did not reduce measurements of either process. Our findings do not support a phase 3 trial of minocycline therapy to delay SVD progression. Whether these pathophysiological mechanisms are causal, or secondary to tissue damage, remains to be determined.This work was funded by a Medical Research Council experimental medicine grant. Infrastructural support was provided by the Cambridge British Heart Foundation Centre of Research Excellence (RE/18/1/34212) and Cambridge University Hospitals NIHR Biomedical Research Centre (BRC-1215-20014). The views expressed in this publication are those of the authors and not necessarily those of the NIHR, NHS, or UK Department of Health and Social Care

Stereohistological analysis of male wild-type (+/y) and male Mct8 knockout (−/y) E18.5 placentae.

<p><b>A</b>: Representative picture of a placental section (wild-type), stained with hematoxylin and eosin, and photographed using a 2× objective lens. The different regions of the mouse placenta are annotated: db = decidua basalis, Lz = labyrinthine zone, Jz = junctional zone and c = chorionic plate. <b>B–C</b>: Volume fractions (B) of the placental regions relative to the whole placenta and absolute volumes (C) of the placental regions in wild-type and litter-matched Mct8 knockout samples. Six placentae were assessed per genotype. The bars represent the mean ±SEM for each group. Significant differences between groups are indicated by *P<0.05.</p

Absolute fetal (A and C), absolute placental (B and D) weights and fetal to placental weight ratios (E and F) of male wild-type (+/y) and male Mct8 knockout (−/y) fetuses at gestational day 14 (E14.5; n = 17 litters) and 18 (E18.5; n = 9 litters).

<p>Each dot represents the weight of individual samples and the lines represent the mean ±SEM for each group. Significant differences between groups are indicated by *P<0.05.</p

T3 uptake in primary cytotrophoblast cells following MCT8 silencing (A,C) or MCT8 over-expression (B,D).

<p><b>A and B</b>: T3 uptake time course from one representative experiment. Cytotrophoblast cells were incubated for 0 to 30 minutes with [¹²⁵I]-T3. The amount of intracellular radioactivity was quantified and expressed as a percentage of the total radioactivity added. <b>C and D</b>: Relative T3 uptake following incubation for 10 minutes with [¹²⁵I]-T3. Bars represent average of four experiments +SEM. Within each experiment, each condition was performed in duplicate. The mean uptake for the control within each experiment has been assigned the value of 100%. Statisticallysignificant differences are indicated by *P<0.05 and **P<0.01.</p

Effect of MCT8 silencing (A,C,E) and MCT8 over-expression (B,D,F) on the viability of the EVT-like SGHPL-4 cells.

<p>assessed by MTT assay (A–B), and apoptosis by quantification of Caspase 3/7 activity (C–D) and counting the number of apoptotic cells by time lapse microscopy (E–F). The assays were performed 72 hours following silencing of endogenous MCT8 or transfection with human wild-type MCT8, and 48 hours post-treatment with 0 or 10 nM T3. <b>A and B</b>: The absorbance for each sample was normalized to the average absorbance measured in cells transfected with control (control siRNA or VO) and treated with 0 nM T3. The bars represent average of five experiments +SEM. Four replicates were performed per condition within each experiment. <b>C and D</b>: Results were normalized to the MTT results in order to correct for cell numbers. Bars represent average of three experiments +SEM. Within each experiment, each condition was performed in triplicate. <b>E and F:</b> Bars represent average of three experiments +SEM. Within each experiment 20 cells were counted per condition.</p