Myelin down-regulates myelin phagocytosis by microglia and macrophages through interactions between CD47 on myelin and SIRPα (signal regulatory protein-α) on phagocytes

<p>Abstract</p> <p>Background</p> <p>Traumatic injury to axons produces breakdown of axons and myelin at the site of the lesion and then further distal to this where Wallerian degeneration develops. The rapid removal of degenerated myelin by phagocytosis is advantageous for repair since molecules in myelin impede regeneration of severed axons. Thus, revealing mechanisms that regulate myelin phagocytosis by macrophages and microglia is important. We hypothesize that myelin regulates its own phagocytosis by simultaneous activation and down-regulation of microglial and macrophage responses. Activation follows myelin binding to receptors that mediate its phagocytosis (e.g. complement receptor-3), which has been previously studied. Down-regulation, which we test here, follows binding of myelin CD47 to the immune inhibitory receptor SIRPα (signal regulatory protein-α) on macrophages and microglia.</p> <p>Methods</p> <p>CD47 and SIRPα expression was studied by confocal immunofluorescence microscopy, and myelin phagocytosis by ELISA.</p> <p>Results</p> <p>We first document that myelin, oligodendrocytes and Schwann cells express CD47 without SIRPα and further confirm that microglia and macrophages express both CD47 and SIRPα. Thus, CD47 on myelin can bind to and subsequently activate SIRPα on phagocytes, a prerequisite for CD47/SIRPα-dependent down-regulation of CD47^+/+myelin phagocytosis by itself. We then demonstrate that phagocytosis of CD47^+/+myelin is augmented when binding between myelin CD47 and SIRPα on phagocytes is blocked by mAbs against CD47 and SIRPα, indicating that down-regulation of phagocytosis indeed depends on CD47-SIRPα binding. Further, phagocytosis in serum-free medium of CD47^+/+myelin is augmented after knocking down SIRPα levels (SIRPα-KD) in phagocytes by lentiviral infection with SIRPα-shRNA, whereas phagocytosis of myelin that lacks CD47 (CD47^-/-) is not. Thus, myelin CD47 produces SIRPα-dependent down-regulation of CD47^+/+myelin phagocytosis in phagocytes. Unexpectedly, phagocytosis of CD47^-/-myelin by SIRPα-KD phagocytes, which is not altered from normal when tested in serum-free medium, is augmented when serum is present. Therefore, both myelin CD47 and serum may each promote SIRPα-dependent down-regulation of myelin phagocytosis irrespective of the other.</p> <p>Conclusions</p> <p>Myelin down-regulates its own phagocytosis through CD47-SIRPα interactions. It may further be argued that CD47 functions normally as a marker of "self" that helps protect intact myelin and myelin-forming oligodendrocytes and Schwann cells from activated microglia and macrophages. However, the very same mechanism that impedes phagocytosis may turn disadvantageous when rapid clearance of degenerated myelin is helpful.</p

Blood-Brain Barrier Cellular Responses Toward Organophosphates: Natural Compensatory Processes and Exogenous Interventions to Rescue Barrier Properties

Organophosphorus compounds (OPs) are highly toxic chemicals widely used as pesticides (e.g., paraoxon (PX)- the active metabolite of the insecticide parathion) and as chemical warfare nerve agents. Blood-brain barrier (BBB) leakage has been shown in rodents exposed to PX, which is an organophosphate oxon. In this study, we investigated the cellular mechanisms involved in BBB reaction after acute exposure to PX in an established in vitro BBB system made of stem-cell derived, human brain-like endothelial cells (BLECs) together with brain pericytes that closely mimic the in vivo BBB. Our results show that PX directly affects the BBB in vitro both at toxic and non-toxic concentrations by attenuating tight junctional (TJ) protein expression and that only above a certain threshold the paracellular barrier integrity is compromised. Below this threshold, BLECs exhibit a morphological coping mechanism in which they enlarge their cell area thus preventing the formation of meaningful intercellular gaps and maintaining barrier integrity. Importantly, we demonstrate that reversal of the apoptotic cell death induced by PX, by a pan-caspase-inhibitor ZVAD-FMK (ZVAD) can reduce PX-induced cell death and elevate cell area but do not prevent the induced BBB permeability, implying that TJ complex functionality is hindered. This is corroborated by formation of ROS at all toxic concentrations of PX and which are even higher with ZVAD. We suggest that while lower levels of ROS can induce compensating mechanisms, higher PX-induced oxidative stress levels interfere with barrier integrity

Non-Invasive Low Pulsed Electrical Fields for Inducing BBB Disruption in Mice—Feasibility Demonstration

The blood&ndash;brain barrier (BBB) is a major hurdle for the treatment of central nervous system disorders, limiting passage of both small and large therapeutic agents from the blood stream into the brain. Thus, means for inducing BBB disruption (BBBd) are urgently needed. Here, we studied the application of low pulsed electrical fields (PEFs) for inducing BBBd in mice. Mice were treated by low PEFs using electrodes pressed against both sides of the skull (100&ndash;400 square 50 &micro;s pulses at 4 Hz with different voltages). BBBd as a function of treatment parameters was evaluated using MRI-based treatment response assessment maps (TRAMs) and Evans blue extravasation. A 3D numerical model of the mouse brain and electrodes was constructed using finite element software, simulating the electric fields distribution in the brain and ensuring no significant temperature elevation. BBBd was demonstrated immediately after treatment and significant linear regressions were found between treatment parameters and the extent of BBBd. The maximal induced electric field in the mice brains, calculated by the numerical model, ranged between 62.4 and 187.2 V/cm for the minimal and maximal applied voltages. These results demonstrate the feasibility of inducing significant BBBd using non-invasive low PEFs, well below the threshold for electroporation

Caspase-1 has a critical role in blood-brain barrier injury and its inhibition contributes to multifaceted repair

International audienceExcessive inflammation might activate and injure the blood-brain barrier (BBB), a common feature of many central nervous system (CNS) disorders. We previously developed an in vitro BBB injury model in which the organophosphate paraoxon (PX) affects the BBB endothelium by attenuating junctional protein expression leading to weakened barrier integrity. The objective of this study was to investigate the inflammatory cellular response at the BBB to elucidate critical pathways that might lead to effective treatment in CNS pathologies in which the BBB is compromised. We hypothesized that caspase-1, a core component of the inflammasome complex, might have important role in BBB function since accumulating evidence indicates its involvement in brain inflammation and pathophysiology

BBB opening by low pulsed electric fields, depicted by delayed-contrast MRI, enables efficient delivery of therapeutic doxorubicin doses into mice brains

Abstract Background Pharmacological treatment of CNS diseases is limited due to the presence of the blood-brain barrier (BBB). Recent years showed significant advancement in the field of CNS drug delivery enablers, with technologies such as MR-guided focused ultrasound reaching clinical trials. This have inspired researchers in the field to invent novel brain barriers opening (BBo) technologies that are required to be simple, fast, safe and efficient. One such technology, recently developed by us, is BDF (Barrier Disrupting Fields), based on low pulsed electric fields (L-PEFs) for opening the BBB in a controlled, safe, reversible and non-invasive manner. Here, we conducted an in vivo study to show that BDF is a feasible technology for delivering Doxorubicin (Doxo) into mice brain. Means for depicting BBBo levels were developed and applied for monitoring the treatment and predicting response. Overall, the goals of the presented study were to demonstrate the feasibility for delivering therapeutic Doxo doses into naïve and tumor-bearing mice brains and applying delayed–contrast MRI (DCM) for monitoring the levels of BBBo. Methods L-PEFs were applied using plate electrodes placed on the intact skull of naïve mice. L-PEFs/Sham mice were scanned immediately after the procedure by DCM (“MRI experiment”), or injected with Doxo and Trypan blue followed by delayed (4 h) perfusion and brain extraction (“Doxo experiment”). Doxo concentrations were measured in brain samples using confocal microscopy and compared to IC50 of Doxo in glioma cell lines in vitro. In order to map BBBo extent throughout the brain, pixel by pixel MR image analysis was performed using the DCM data. Finally, the efficacy of L-PEFs in combination with Doxo was tested in nude mice bearing intracranial human glioma tumors. Results Significant amount of Doxo was found in cortical regions of all L-PEFs-treated mice brains (0.50 ± 0.06 µg Doxo/gr brain) while in Sham brains, Doxo concentrations were below or on the verge of detection limit (0.03 ± 0.02 µg Doxo/gr brain). This concentration was x97 higher than IC50 of Doxo calculated in gl261 mouse glioma cells and x8 higher than IC50 of Doxo calculated in U87 human glioma cells. DCM analysis revealed significant BBBo levels in the cortical regions of L-PEFs-treated mice; the average volume of BBBo in the L-PEFs-treated mice was x29 higher than in the Sham group. The calculated BBBo levels dropped exponentially as a function of BBBo threshold, similarly to the electric fields distribution in the brain. Finally, combining non-invasive L-PEFs with Doxo significantly decreased brain tumors growth rates in nude mice. Conclusions Our results demonstrate significant BBBo levels induced by extra-cranial L-PEFs, enabling efficient delivery of therapeutic Doxo doses into the brain and reducing tumor growth. As BBBo was undetectable by standard contrast-enhanced MRI, DCM was applied to generate maps depicting the BBBo levels throughout the brain. These findings suggest that BDF is a promising technology for efficient drug delivery into the brain with important implications for future treatment of brain cancer and additional CNS diseases

Endothelial Iron Homeostasis Regulates Blood-Brain Barrier Integrity via the HIF2α—Ve-Cadherin Pathway

International audienceThe objective of this study was to investigate the molecular response to damage at the blood brain barrier (BBB) and to elucidate critical pathways that might lead to effective treatment in central nervous system (CNS) pathologies in which the BBB is compromised. We have used a human, stem-cell derived in-vitro BBB injury model to gain a better understanding of the mechanisms controlling BBB integrity. Chemical injury induced by exposure to an organophosphate resulted in rapid lipid peroxidation, initiating a ferroptosis-like process. Additionally, mitochondrial ROS formation (MRF) and increase in mitochondrial membrane permeability were induced, leading to apoptotic cell death. Yet, these processes did not directly result in damage to barrier functionality, since blocking them did not reverse the increased permeability. We found that the iron chelator, Desferal© significantly decreased MRF and apoptosis subsequent to barrier insult, while also rescuing barrier integrity by inhibiting the labile iron pool increase, inducing HIF2α expression and preventing the degradation of Ve-cadherin specifically on the endothelial cell surface. Moreover, the novel nitroxide JP4-039 significantly rescued both injury-induced endothelium cell toxicity and barrier functionality. Elucidating a regulatory pathway that maintains BBB integrity illuminates a potential therapeutic approach to protect the BBB degradation that is evident in many neurological diseases

Additional file 2 of BBB opening by low pulsed electric fields, depicted by delayed-contrast MRI, enables efficient delivery of therapeutic doxorubicin doses into mice brains

Supplementary Material 2: Video 1a. Growth of GL261 cells without treatment

Additional file 6 of BBB opening by low pulsed electric fields, depicted by delayed-contrast MRI, enables efficient delivery of therapeutic doxorubicin doses into mice brains

Supplementary Material 6: Video 2b. Growth of U87 cells treated with 200nM Doxo

Additional file 1 of BBB opening by low pulsed electric fields, depicted by delayed-contrast MRI, enables efficient delivery of therapeutic doxorubicin doses into mice brains

Supplementary Material 1: Figure 1. Micrographs of U87 and GL261 glioma cells. Microscopic pictures of the two cell lines treated with different concentrations of Doxo are shown at day 5. Bar = 400µm