Transport of the Proinflammatory Chemokines C-C Motif Chemokine Ligand 2 (MCP-1) and C-C Motif Chemokine Ligand 5 (RANTES) across the Intact Mouse Blood-Brain Barrier Is Inhibited by Heparin and Eprodisate and Increased with Systemic Inflammation.

One important function of the vascular blood-brain barrier (BBB) is to facilitate neuroimmune communication. The BBB fulfills this function, in part, through its ability to transport cytokines and chemokines. C-C motif chemokine receptor 2 (CCL2) (MCP-1) and C-C motif chemokine receptor 5 (CCL5) (RANTES) are proinflammatory chemokines that mediate neuroimmune responses to acute insults and aspects of brain injury and neurodegenerative diseases; however, a blood-to-brain transport system has not been evaluated for either chemokine in vivo. Therefore, we determined whether CCL2 and CCL5 in blood can cross the intact BBB and enter the brain. Using CD-1 mice, we found that 125I-labeled CCL2 and CCL5 crossed the BBB and entered the brain parenchyma. We next aimed to identify the mechanisms of 125I-CCL2 and 125I-CCL5 transport in an in situ brain perfusion model. We found that both heparin and eprodisate inhibited brain uptake of 125I-CCL2 and 125I-CCL5 in situ, whereas antagonists of their receptors, CCR2 or CCR5, respectively, did not, suggesting that heparan sulfates at the endothelial surface mediate BBB transport. Finally, we showed that CCL2 and CCL5 transport across the BBB increased following a single injection of 0.3 mg/kg lipopolysaccharide. These data demonstrate that CCL2 and CCL5 in the brain can derive, in part, from the circulation, especially during systemic inflammation. Further, binding to the BBB-associated heparan sulfate is a mechanism by which both chemokines can cross the intact BBB, highlighting a novel therapeutic target for treating neuroinflammation. SIGNIFICANCE STATEMENT: Our work demonstrates that C-C motif chemokine ligand 2 (CCL2) and C-C motif chemokine ligand 5 (CCL5) can cross the intact blood-brain barrier and that transport is robustly increased during inflammation. These data suggest that circulating CCL2 and CCL5 can contribute to brain levels of each chemokine. We further show that the transport of both chemokines is inhibited by heparin and eprodisate, suggesting that CCL2/CCL5-heparan sulfate interactions could be therapeutically targeted to limit accumulation of these chemokines in the brain.

Harnessing and delivering microbial metabolites as therapeutics via advanced pharmaceutical approaches.

Microbial metabolites have emerged as key players in the interplay between diet, the gut microbiome, and host health. Two major classes, short-chain fatty acids (SCFAs) and tryptophan (Trp) metabolites, are recognized to regulate inflammatory, immune, and metabolic responses within the host. Given that many human diseases are associated with dysbiosis of the gut microbiome and consequent reductions in microbial metabolite production, the administration of these metabolites represents a direct, multi-targeted treatment. While a multitude of preclinical studies showcase the therapeutic potential of both SCFAs and Trp metabolites, they often rely on high doses and frequent dosing regimens to achieve systemic effects, thereby constraining their clinical applicability. To address these limitations, a variety of pharmaceutical formulations approaches that enable targeted, delayed, and/or sustained microbial metabolite delivery have been developed. These approaches, including enteric encapsulations, esterification to dietary fiber, prodrugs, and nanoformulations, pave the way for the next generation of microbial metabolite-based therapeutics. In this review, we first provide an overview of the roles of microbial metabolites in maintaining host homeostasis and outline how compromised metabolite production contributes to the pathogenesis of inflammatory, metabolic, autoimmune, allergic, infectious, and cancerous diseases. Additionally, we explore the therapeutic potential of metabolites in these disease contexts. Then, we provide a comprehensive and up-to-date review of the pharmaceutical strategies that have been employed to enhance the therapeutic efficacy of microbial metabolites, with a focus on SCFAs and Trp metabolites.

Prolonged culturing of iPSC-derived brain endothelial-like cells is associated with quiescence, downregulation of glycolysis, and resistance to disruption by an Alzheimer's brain milieu.

BACKGROUND: Human induced pluripotent stem cell (hiPSC)-derived brain endothelial-like cells (iBECs) are a robust, scalable, and translatable model of the human blood-brain barrier (BBB). Prior works have shown that high transendothelial electrical resistance (TEER) persists in iBECs for at least 2 weeks, emphasizing the utility of the model for longer term studies. However, most studies evaluate iBECs within the first few days of subculture, and little is known about their proliferative state, which could influence their functions. In this study, we characterized iBEC proliferative state in relation to key BBB properties at early (2 days) and late (9 days) post-subculture time points. METHODS: hiPSCs were differentiated into iBECs using fully defined, serum-free medium. The proportion of proliferating cells was determined by BrdU assays. We evaluated TEER, expression of glycolysis enzymes and tight and adherens junction proteins (TJP and AJP), and glucose transporter-1 (GLUT1) function by immunoblotting, immunofluorescence, and quantifying radiolabeled tracer permeabilities. We also compared barrier disruption in response to TNF-α and conditioned medium (CM) from hiPSC-derived neurons harboring the Alzheimer's disease (AD)-causing Swedish mutation (APPSwe/+). RESULTS: A significant decline in iBEC proliferation over time in culture was accompanied by adoption of a more quiescent endothelial metabolic state, indicated by downregulation of glycolysis-related proteins and upregulation GLUT1. Interestingly, upregulation of GLUT1 was associated with reduced glucose transport rates in more quiescent iBECs. We also found significant decreases in claudin-5 (CLDN5) and vascular endothelial-cadherin (VE-Cad) and a trend toward a decrease in platelet endothelial cell adhesion molecule-1 (PECAM-1), whereas zona occludens-1 (ZO-1) increased and occludin (OCLN) remained unchanged. Despite differences in TJP and AJP expression, there was no difference in mean TEER on day 2 vs. day 9. TNF-α induced disruption irrespective of iBEC proliferative state. Conversely, APPSwe/+ CM disrupted only proliferating iBEC monolayers. CONCLUSION: iBECs can be used to study responses to disease-relevant stimuli in proliferating vs. more quiescent endothelial cell states, which may provide insight into BBB vulnerabilities in contexts of development, brain injury, and neurodegenerative disease.

The S1 protein of SARS-CoV-2 crosses the blood-brain barrier in mice.

It is unclear whether severe acute respiratory syndrome coronavirus 2, which causes coronavirus disease 2019, can enter the brain. Severe acute respiratory syndrome coronavirus 2 binds to cells via the S1 subunit of its spike protein. We show that intravenously injected radioiodinated S1 (I-S1) readily crossed the blood-brain barrier in male mice, was taken up by brain regions and entered the parenchymal brain space. I-S1 was also taken up by the lung, spleen, kidney and liver. Intranasally administered I-S1 also entered the brain, although at levels roughly ten times lower than after intravenous administration. APOE genotype and sex did not affect whole-brain I-S1 uptake but had variable effects on uptake by the olfactory bulb, liver, spleen and kidney. I-S1 uptake in the hippocampus and olfactory bulb was reduced by lipopolysaccharide-induced inflammation. Mechanistic studies indicated that I-S1 crosses the blood-brain barrier by adsorptive transcytosis and that murine angiotensin-converting enzyme 2 is involved in brain and lung uptake, but not in kidney, liver or spleen uptake.