Maternal regulation of biliary disease in neonates via gut microbial metabolites

Maternal seeding of the microbiome in neonates promotes a long-lasting biological footprint, but how it impacts disease susceptibility in early life remains unknown. We hypothesized that feeding butyrate to pregnant mice influences the newborn’s susceptibility to biliary atresia, a severe cholangiopathy of neonates. Here, we show that butyrate administration to mothers renders newborn mice resistant to inflammation and injury of bile ducts and improves survival. The prevention of hepatic immune cell activation and survival trait is linked to fecal signatures of Bacteroidetes and Clostridia and increases glutamate/glutamine and hypoxanthine in stool metabolites of newborn mice. In human neonates with biliary atresia, the fecal microbiome signature of these bacteria is under-represented, with suppression of glutamate/glutamine and increased hypoxanthine pathways. The direct administration of butyrate or glutamine to newborn mice attenuates the disease phenotype, but only glutamine renders bile duct epithelial cells resistant to cytotoxicity by natural killer cells. Thus, maternal intake of butyrate influences the fecal microbial population and metabolites in newborn mice and the phenotypic expression of experimental biliary atresia, with glutamine promoting survival of bile duct epithelial cells.

Ontogeny of RORγt+ cells in the intestine of newborns and its role in the development of experimental necrotizing enterocolitis

Background Neonates possess an immature and plastic immune system, which is a major cause of some diseases in newborns. Necrotizing enterocolitis (NEC) is a severe and devastating intestinal disease that typically affects premature infants. However, the development of intestinal immune cells in neonates and their roles in the pathological process of NEC have not been elucidated. Results We examined the ontogeny of intestinal lamina propria lymphocytes in the early life of mice and found a high percentage of RORγt+ cells (containing inflammatory Th17 and ILC3 populations) during the first week of life. Importantly, the proportion of RORγt+ cells of intestinal lamina propria further increased in both NEC mice and patients tissue than the control. Furthermore, the application of GSK805, a specific antagonist of RORγt, inhibited IL-17A release and ameliorated NEC severity. Conclusions Our data reveal the high proportion of RORγt+ cells in newborn mice may directly contribute to the development of NEC.

ADAR1 RNA editing regulates endothelial cell functions via the MDA-5 RNA sensing signaling pathway

The RNA-sensing signaling pathway has been well studied as an essential antiviral mechanism of innate immunity. However, its role in non-infected cells is yet to be thoroughly characterized. Here, we demonstrated that the RNA sensing signaling pathway also reacts to the endogenous cellular RNAs in endothelial cells (ECs), and this reaction is regulated by the RNA-editing enzyme ADAR1. Cellular RNA sequencing analysis showed that EC RNAs endure extensive RNA editing, especially in the RNA transcripts of short interspersed nuclear elements. The EC-specific deletion of ADAR1 dramatically reduced the editing level on short interspersed nuclear element RNAs, resulting in newborn death in mice with damage evident in multiple organs. Genome-wide gene expression analysis revealed a prominent innate immune activation with a dramatically elevated expression of interferon-stimulated genes. However, blocking the RNA sensing signaling pathway by deletion of the cellular RNA receptor MDA-5 prevented interferon-stimulated gene expression and rescued the newborn mice from death. This evidence demonstrated that the RNA-editing/RNA-sensing signaling pathway dramatically modulates EC function, representing a novel molecular mechanism for the regulation of EC functions.

CCRL2 Modulates Physiological and Pathological Angiogenesis During Retinal Development

Chemerin is a multifunctional protein involved in the regulation of inflammation, metabolism, and tumorigenesis. It binds to three receptors, CMKLR1, GPR1 and CCRL2. CMKLR1 is a fully functional receptor mediating most of the known activities of chemerin. CCRL2 does not seem to couple to any intracellular signaling pathway and is presently considered as an atypical receptor able to present the protein to cells expressing CMKLR1. CCRL2 is expressed by many cell types including leukocyte subsets and endothelial cells, and its expression is strongly upregulated by inflammatory stimuli. We recently reported that chemerin can negatively regulate the angiogenesis process, including during the development of the vascular network in mouse retina. The role of CCRL2 in angiogenesis was unexplored so far. In the present work, we demonstrate that mice lacking CCRL2 exhibit a lower density of vessels in the developing retina and this phenotype persists in adulthood, in a CMKLR1-dependent manner. Vascular sprouting was not affected, while vessel pruning, and endothelial cell apoptosis were increased. Pathological angiogenesis was also reduced in CCRL2-/- mice in a model of oxygen-induced retinopathy. The phenotype closely mimics that of mice overexpressing chemerin, and the concentration of chemerin was found elevated in the blood of newborn mice, when the retinal vasculature develops. CCRL2 appears therefore to regulate the distribution and concentration of chemerin in organs, regulating thereby its bioactivity.

Developmentally Regulated Modulation of Lumbar Motoneurons by Metabotropic Glutamate Receptors: A Cellular and Behavioral Analysis in Newborn Mice

The present study explores the impact of metabotropic glutamate receptor (mGluR) activation on activity-dependent synaptic plasticity (ADSP) and the intrinsic membrane properties of lumbar motoneurons (MNs) using a combination of biochemical, pharmacological, electrophysiological and behavioral techniques. Using spinal cord slices from C57BL/6JRJ mice at two developmental stages, 1-3 and 8-12 postnatal days (P1-P3; P8-P12, respectively), we found that ADSP expressed at glutamatergic synapses between axons conveyed in the ventrolateral funiculus (VLF) and MNs, involved mGluR activation. Using specific agonists of the three groups of mGluRs, we observed that mGluR stimulation causes subtype-specific and developmentally regulated modulation of the ADSP and synaptic transmission at VLF-MN synapses as well as the intrinsic membrane properties of MNs. RT-qPCR analysis revealed a downregulation of mGluR gene expression with age in the ventral part of the lumbar spinal cord. Interestingly, the selective harvest by laser microdissection of MNs innervating the Gastrocnemius and Tibialis anterior muscles unraveled that the level of Grm2 expression is higher in Tibialis MNs compared to Gastrocnemius MNs suggesting a specific mGluR gene expression profile in these two MN pools. Finally, we assessed the functional impact of mGluR modulation on electrically induced bouts of fictive locomotion in the isolated spinal cord preparation of P1-P3 mice, and in vivo during spontaneous episodes of swimming activity in both P1-P3 and P8-P12 mouse pups. We observed that the mGluR agonists induced distinct and specific effects on the motor burst amplitudes and period of the locomotor rhythms tested and that their actions are function of the developmental stage of the animals. Altogether our data show that the metabotropic glutamatergic system exerts a complex neuromodulation in the developing spinal lumbar motor networks and provide new insights into the expression and modulation of ADSP in MNs.

Coumarin Derivative N6 as a Novel anti-hantavirus Infection Agent Targeting AKT

Hantaviruses are globally emerging zoonotic viruses that can cause hemorrhagic fever with renal syndrome (HFRS) in Asia and Europe, which is primarily caused by Hantaan virus (HTNV) infection, results in profound morbidity and mortality. However, no specific treatment is available for this disease. Coumarin derivatives have been reported as antiviral molecules, while studies about the bioactivity of coumarin derivatives against HTNV infection are limited. To study the potential antiviral activity of coumarin derivatives, 126 coumarin derivatives are synthesized, and their inhibitory activity against HTNV is analyzed in vitro. Among these compounds, N6 inhibits HTNV with relatively high selectivity index at 10.9, and the viral titer of HTNV is reduced significantly after 5, 10, and 20 μM N6 treatments. Furthermore, the administration of N6 at the early stage of HTNV infection can inhibit the replication and production of infectious HTNV in host cell, this therapeutic efficacy is confirmed in HTNV-infected newborn mice at the early stage of infection. The molecular docking results show that N6 forms interactions with the key amino acid residues at its active site, and reveals several molecular interactions responsible for the observed affinity, and the treatment of N6 can inhibit the expression of p (Ser473)Akt and HTNV nucleocapsid protein significantly. As such, these observations demonstrate that coumarin derivative N6 might be used as a potential agent against HTNV infection.

Thyroid hormone-dependent regulation of metabolism and heart regeneration

While adult zebrafish and newborn mice possess a robust capacity to regenerate their hearts, this ability is generally lost in adult mammals. The logic behind the diversity of cardiac regenerative capacity across the animal kingdom is not well understood. We have recently reported that animal metabolism is inversely correlated to the abundance of mononucleated diploid cardiomyocytes in the heart, which retain proliferative and regenerative potential. Thyroid hormones are classical regulators of animal metabolism, mitochondrial function, and thermogenesis and a growing body of scientific evidence demonstrates that these hormonal regulators also have direct effects on cardiomyocyte proliferation and maturation. We propose that thyroid hormones dually control animal metabolism and cardiac regenerative potential through distinct mechanisms, which may represent an evolutionary tradeoff for the acquisition of endothermy and loss of heart regenerative capacity. In this review, we describe the effects of thyroid hormones on animal metabolism and cardiomyocyte regeneration, and highlight recent reports linking the loss of mammalian cardiac regenerative capacity to metabolic shifts occurring after birth.

Isolation and phenotyping of cardiac-derived progenitor cells from neonatal mice

Dysfunctions of resident progenitor cells play a significant role in the pathogenesis of decreased myocardial contractility in heart failure, so the most promising approaches for the treatment of heart disease are cardiac-derived stem/progenitor cells (CSCs). Materials and methods. Protocols for progenitor cell cultures from different parts of the heart of newborn FVB/N mice have been developed and their proliferative potential has been characterized. Comparative analysis of the expression of CD31, CD34, CD44, CD45, CD73, CD90, CD105, CD117, CD309 and troponin I by cells from native myocardial biopsies and in the obtained cultures was performed by flow cytometric immunophenotyping. Results. The expression of mesenchymal markers CD44 and CD90 in the absence of the hematopoietic marker CD45 was demonstrated in early passages in mouse myocardial progenitor cell cultures. Relatively high expression of CD34 and CD31 was found. The presence of a minor population of CD44+117+ cells which correspond to the phenotype of cardiac progenitor cells, was detected. Expression of troponin I as one of the key markers of cardiomyocytes as well as the vascular endothelial growth factor receptor has been confirmed in terminally differentiated cultures of cells with contractile activity. Conclusions. It was found that newborn mice in the myocardial tissue contain more cells with the expression of markers of cardiac progenitors than in adult animals. The relative content of such cells is higher in the atria than in the ventricles. Cardiac progenitor cells in neonatal mice derived from the atrial appendages have better proliferative potential than cell cultures isolated from the ventricles.

Hypothermia modulates myeloid cell polarization in neonatal hypoxic–ischemic brain injury

Background Neonatal encephalopathy due to hypoxia–ischemia (HI) is a leading cause of death and disability in term newborns. Therapeutic hypothermia (HT) is the only recommended therapy. However, 30% still suffer from neurological deficits. Inflammation is a major hallmark of HI pathophysiology with myeloid cells being key players, participating either in progression or in resolution of injury-induced inflammation. In the present study, we investigated the impact of HT on the temporal and spatial dynamics of microglia/macrophage polarization after neonatal HI in newborn mice. Methods Nine-day-old C57BL/6 mice were exposed to HI through occlusion of the right common carotid artery followed by 1 h hypoxia. Immediately after HI, animals were cooled for 4 h or kept at physiological body core temperature. Analyses were performed at 1, 3 and 7 days post HI. Brain injury, neuronal cell loss, apoptosis and microglia activation were assessed by immunohistochemistry. A broad set of typical genes associated with classical (M1) and alternative (M2) myeloid cell activation was analyzed by real time PCR in ex vivo isolated CD11b+ microglia/macrophages. Purity and composition of isolated cells was determined by flow cytometry. Results Immediate HT significantly reduced HI-induced brain injury and neuronal loss 7 days post HI, whereas only mild non-significant protection from HI-induced apoptosis and neuronal loss were observed 1 and 3 days after HI. Microglia activation, i.e., Iba-1 immunoreactivity peaked 3 days after HI and was not modulated by HT. However, ex vivo isolated CD11b+ cells revealed a strong upregulation of the majority of M1 but also M2 marker genes at day 1, which was significantly reduced by HT and rapidly declined at day 3. HI induced a significant increase in the frequency of peripheral macrophages in sorted CD11b+ cells at day 1, which deteriorated until day 7 and was significantly decreased by HT. Conclusion Our data demonstrate that HT-induced neuroprotection is preceded by acute suppression of HI-induced upregulation of inflammatory genes in myeloid cells and decreased infiltration of peripheral macrophages, both representing potential important effector mechanisms of HT.

Soluble Brain Homogenates from Diverse Human and Mouse Sources Preferentially Seed Diffuse Aβ Plaque Pathology When Injected into Newborn Mouse Hosts

Background Seeding of pathology related to Alzheimer’s disease (AD) and Lewy body disease (LBD) by the injection of tissue homogenates, purified proteins, or recombinant proteins into model systems has revealed prion-like seeding of the protein aggregates that define these disorders. Most commonly these homogenates are injected into adult mice stereotaxically. Injection of brain lysates into newborn mice represents an alternative approach of delivering seeds that could be used to direct the evolution of amyloid-β (Aβ) pathology co-mixed with either tau or α-synuclein (αSyn) pathology in vulnerable mouse models. Methods Homogenates of human pre-frontal cortex were prepared and injected into the lateral ventricles of newborn (P0) mice expressing a mutant humanized amyloid precursor protein (APP), human P301L tau, human wild type αSyn, or combinations thereof. The injected brain homogenates were prepared from AD and AD/LBD cases displaying variable degrees of Aβ pathology and co-existing tau and αSyn deposits. Behavioral assessments of APP transgenic mice injected with AD brain lysates were conducted. Results We observed that the lysates from the brains of individuals with AD (Aβ+, tau+), AD/LBD (Aβ+, tau+, αSyn+), or Pathological Aging (Aβ+, tau-, αSyn-) efficiently seeded diffuse Aβ deposits, composed primarily of Aβ42 peptides, in our transgenic host animals. Moderate seeding of cerebral amyloid angiopathy (CAA) was also observed. No animal of any genotype developed discernable tau or αSyn pathology. Fear conditioning, cognitive, outcome was not significantly altered in APP transgenic animals injected with AD brain lysates compared to nontransgenic controls. Conclusions These findings demonstrate that diffuse Aβ pathology, which is a common feature of AD, AD/LBD, and PA brains, can be easily induced by injecting newborn APP mice with crude brain homogenates. Seeding of tau or αSyn comorbidities was disappointingly inefficient in the models we used, indicating additional methodological refinement will be needed to efficiently seed AD or AD/LBD mixed pathologies by injecting newborn mice.