Survival and Pulmonary Injury After Neonatal Sepsis: PD1/PDL1's Contributions to Mouse and Human Immunopathology

Morbidity and mortality associated with neonatal sepsis remains a healthcare crisis. PD1−/− neonatal mice endured experimental sepsis, in the form of cecal slurry (CS), and showed improved rates of survival compared to wildtype (WT) counterparts. End-organ injury, particularly of the lung, contributes to the devastation set forth by neonatal sepsis. PDL1−/− neonatal mice, in contrast to PD1−/− neonatal mice did not have a significant improvement in survival after CS. Because of this, we focused subsequent studies on the impact of PD1 gene deficiency on lung injury. Here, we observed that at 24 h post-CS (but not at 4 or 12 h) there was a marked increase in pulmonary edema (PE), neutrophil influx, myeloperoxidase (MPO) levels, and cytokine expression sham (Sh) WT mice. Regarding pulmonary endothelial cell (EC) adhesion molecule expression, we observed that Zona occludens-1 (ZO-1) within the cell shifted from a membranous location to a peri-nuclear location after CS in WT murine cultured ECs at 24hrs, but remained membranous among PD1−/− lungs. To expand the scope of this inquiry, we investigated human neonatal lung tissue. We observed that the lungs of human newborns exposed to intrauterine infection had significantly higher numbers of PD1+ cells compared to specimens who died from non-infectious causes. Together, these data suggest that PD1/PDL1, a pathway typically thought to govern adaptive immune processes in adult animals, can modulate the largely innate neonatal pulmonary immune response to experimental septic insult. The potential future significance of this area of study includes that PD1/PDL1 checkpoint proteins may be viable therapeutic targets in the septic neonate.

Site-specific phosphorylation of PSD-95 dynamically regulates the post-synaptic density as observed by phase separation

SUMMARYPostsynaptic density protein 95 is a key scaffolding protein in the postsynaptic density of excitatory glutamatergic neurons, organizing signaling complexes primarily via its three PSD-95/Discs-large/Zona occludens domains. PSD-95 is regulated by phosphorylation, but technical challenges have limited studies of the molecular details. Here, we genetically introduced site-specific phosphorylations in single, tandem and full-length PSD-95 and generated a total of 11 phosphorylated protein variants. We examined how these phosphorylations affected binding to known interaction partners and the impact on phase separation of PSD-95 complexes, and identified two new phosphorylation sites with opposing effects. Phosphorylation of Ser78 inhibited phase separation with the glutamate receptor subunit GluN2B and the auxiliary protein stargazin, whereas phosphorylation of Ser116 induced phase separation with stargazin only. Thus, by genetically introducing phosphoserine site-specifically and exploring the impact on phase separation, we have provided new insights into the regulation of PSD-95 by phosphorylation and the dynamics of the PSD.Graphical Abstract: Phosphorylation of PSD-95.Illustration of the interaction between the PDZ domains of PSD-95 and the NMDA receptor (NMDAR) (PDB: 4PE5) at the plasma membrane. PSD-95 interacts with the AMPA receptor (AMPAR) (PDB: 3KG2) through Stargazin (PDB: 5KBU). Using the software Ranch, a model of FL PSD-95 was generated using known structural domains of PSD-95 connected via a fully-flexible linker (PDZ1-2, PDB: 3GSL; PDZ3, PDB: 5JXB; SH3-GK, PDB: 1KJW).Illustration of the effect of phosphorylation of PSD-95 on the formation of condensates at the PSD. PSD-95 can phase separate with receptors and intracellular proteins, and phosphorylation is can affect this interaction and lead to alterations, both negative and positive, in the formation of condensates.

Hyperglycemic Condition Causes Pro-Inflammatory and Permeability Alterations Associated with Monocyte Recruitment and Deregulated NFκB/PPARγ Pathways on Cerebral Endothelial Cells: Evidence for Polyphenols Uptake and Protective Effect

Hyperglycemia alters the function of cerebral endothelial cells from the blood-brain barrier, increasing the risk of cerebrovascular complications during diabetes. This study evaluated the protective effect of polyphenols on inflammatory and permeability markers on bEnd3 cerebral endothelial cells exposed to high glucose concentration. Results show that hyperglycemic condition increased nuclear factor kappa B (NFκB) activity, deregulated the expression of interleukin-1 beta (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), interleukin-10 (IL-10) and endothelial-leukocyte adhesion molecule (E-selectin) genes, raised MCP-1 secretion and elevated monocyte adhesion and transendothelial migration. High glucose decreased occludin, claudin-5, zona occludens-1 (ZO-1) and zona occludens-2 (ZO-2) tight junctions production and altered the endothelial permeability. Characterized polyphenolic extracts from the French medicinal plants Antirhea borbonica, Ayapana triplinervis, Dodonaea viscosa and Terminalia bentzoe, and their major polyphenols quercetin, caffeic, chlorogenic and gallic acids limited the pro-inflammatory and permeability alterations caused by high glucose. Peroxisome proliferator-activated receptor gamma (PPARγ) agonist also attenuated these damages while PPARγ antagonist aggravated them, suggesting PPARγ protective action. Interestingly, polyphenols improved PPARγ gene expression lowered by high glucose. Moreover, polyphenols were detected at the intracellular level or membrane-bound to cells, with evidence for breast cancer resistance protein (BCRP) efflux transporter role. Altogether, these findings emphasize the ability of polyphenols to protect cerebral endothelial cells in hyperglycemic condition and their relevance for pharmacological strategies aiming to limit cerebrovascular disorders in diabetes.

Vascular permeability disruption explored in the proteomes of mouse lungs and human microvascular cells following acute bromine exposure

Bromine (Br2) is an organohalide found in nature and is integral to many manufacturing processes. Br2 is toxic to living organisms, and high concentrations can prove fatal. To meet industrial demand, large amounts of purified Br2 are produced, transported, and stored worldwide, providing a multitude of interfaces for potential human exposure through either accidents or terrorism. To identify the key mechanisms associated with acute Br2 exposure, we have surveyed the lung proteomes of C57BL/6 male mice and human lung-derived microvascular endothelial cells (HMECs) at 24 h following exposure to Br2 in concentrations likely to be encountered in the vicinity of industrial accidents. Global discovery proteomics applications combined with systems biology analysis identified robust and highly significant changes in proteins associated with three biological processes: 1) exosome secretion, 2) inflammation, and 3) vascular permeability. We focused on the latter, conducting physiological studies on isolated perfused lungs harvested from mice 24 h after Br2 exposure. These experiments revealed significant increases in the filtration coefficient ( Kf) indicating increased permeability of the pulmonary vasculature. Similarly, confluent monolayers of Br2 and Br-lipid-treated HMECs exhibited differential levels of zona occludens-1 that were found to be dissociated from cell wall localization, an increase in phosphorylation and internalization of E-cadherin, as well as increased actin stress fiber formation, all of which are consistent with increased permeability. Taken as a whole, our discovery proteomics and systems analysis workflow, combined with physiological measurements of permeability, revealed both profound and novel biological changes that contribute to our current understanding of Br2 toxicity.

Hydroxychloroquine Mitigates the Production of 8-Isoprostane and Improves Vascular Dysfunction: Implications for Treating Preeclampsia

In preeclampsia, widespread maternal endothelial dysfunction is often secondary to excessive generation of placental-derived anti-angiogenic factors, including soluble fms-like tyrosine kinase-1 (sFlt-1) and soluble endoglin (sEng), along with proinflammatory cytokines such as tumour necrosis factor-α (TNF-α) and activin A, understanding of which offers potential opportunities for the development of novel therapies. The antimalarial hydroxychloroquine is an anti-inflammatory drug improving endothelial homeostasis in lupus. It has not been explored as to whether it can improve placental and endothelial function in preeclampsia. In this in vitro study, term placental explants were used to assess the effects of hydroxychloroquine on placental production of sFlt-1, sEng, TNF-α, activin A, and 8-isoprostane after exposure to hypoxic injury or oxidative stress. Similarly, human umbilical vein endothelial cells (HUVECs) were used to assess the effects of hydroxychloroquine on in vitro markers of endothelial dysfunction. Hydroxychloroquine had no effect on the release of sFlt-1, sEng, TNF-α, activin A, or 8-isoprostane from placental explants exposed to hypoxic injury or oxidative stress. However, hydroxychloroquine mitigated TNF-α-induced HUVEC production of 8-isoprostane and Nicotinanamide adenine dinucleotide phosphate (NADPH) oxidase expression. Hydroxychloroquine also mitigated TNF-α and preeclamptic serum-induced HUVEC monolayer permeability and rescued the loss of zona occludens protein zona occludens 1 (ZO-1). Although hydroxychloroquine had no apparent effects on trophoblast function, it may be a useful endothelial protectant in women presenting with preeclampsia.

Prolyl Hydroxylase Inhibition Mitigates Pouchitis

Background Pouchitis is the most common long-term complication after restorative proctocolectomy with ileal pouch–anal anastomosis (IPAA) for ulcerative colitis (UC) or familial adenomatous polyposis (FAP), which can eventually progress to pouch failure, necessitating permanent stoma construction. Hypoxia-inducible transcription factor prolyl hydroxylase–containing enzymes (PHD1, PHD2, and PHD3) are molecular oxygen sensors that control adaptive gene expression through hypoxia-inducible factor (HIF). Emerging evidence supports PHDs as being therapeutic targets in intestinal inflammation. However, pharmacological inhibition of PHDs has not been validated as a treatment strategy in pouchitis. Methods PHD1-3 mRNA and protein expression were analyzed in mucosal pouch and prepouch ileal patient biopsies. After establishment of a preclinical IPAA model in rats, the impact of the pan-PHD small-molecule inhibitor dimethyloxalylglycine (DMOG) on dextran sulfate sodium (DSS)–induced pouchitis was studied. Clinical and molecular parameters were investigated. Results PHD1, but not PHD2 or PHD3, was overexpressed in pouchitis in biopsies of patients with IPAA for UC but not FAP. In addition, PHD1 expression correlated with disease activity. DMOG treatment profoundly mitigated DSS-induced pouchitis in a rodent IPAA model. Mechanistically, DMOG restored intestinal epithelial barrier function by induction of tight junction proteins zona occludens-1 and claudin-1 and alleviation of intestinal epithelial cell apoptosis, thus attenuating pouch inflammation. Conclusions Together, these results establish a strong therapeutic rationale for targeting PHD1 with small-molecule inhibitors in pouchitis after IPAA for UC.

How the dual PDZ domain from Postsynaptic density protein 95 clusters ion channels and receptors

PSD-95 is a member of Membrane Associated Guanylate Kinase class of proteins which form scaffolding interactions with partner proteins including ion and receptor channels. PSD-95 is directly implicated in modulating the electrical responses of excitable cells. The first two PSD-95/Disks Large/Zona Occludens domains of PSD-95 have been shown to be the key component in the formation of channel clusters. We report crystal structures of the dual domain in both in apo and ligand-bound form; thermodynamic analysis of ligand association and Small Angle X-ray Scattering of the dual domain in the absence and presence of ligands. These experiments reveal that the ligated double domain forms a scaffold in the complete sense of the word. The concentration of the components in this study is comparable to those found in compartments of excitable cells such as the postsynaptic density and juxta-paranodes of Ranvier. The properties of the dual domain explain the basis of the scaffolding function of PSD-95, and provide a more detailed understanding of the integration of key components of neuronal specializations involved in nervous signal transmission.