Deep proteomics and phosphoproteomics reveal novel biological pathways perturbed by morphine, morphine-3-glucuronide and morphine-6-glucuronide in human astrocytes.

Tolerance and hyperalgesia associated with chronic exposure to morphine are major limitations in the clinical management of chronic pain. At a cellular level, neuronal signaling can in part account for these undesired side effects, but unknown mechanisms mediated by central nervous system glial cells are likely also involved. Here we applied data-independent acquisition mass spectrometry to perform a deep proteome and phosphoproteome analysis of how human astrocytes responds to opioid stimulation. We unveil time- and dose-dependent effects induced by morphine and its major active metabolites morphine-3-glucuronide (M3G) and morphine-6-glucuronide that converging on activation of mitogen-activated protein kinase and mammalian target of rapamycin signaling pathways. We also find that especially longer exposure to M3G leads to significant dysregulation of biological pathways linked to extracellular matrix organization, antigen presentation, cell adhesion, and glutamate homeostasis, which are crucial for neuron- and leukocyte-astrocyte interactions.

Profiling the proteomic inflammatory state of human astrocytes using DIA mass spectrometry.

BACKGROUND: Astrocytes are the most abundant cells in the central nervous system and are responsible for a wide range of functions critical to normal neuronal development, synapse formation, blood-brain barrier regulation, and brain homeostasis. They are also actively involved in initiating and perpetuating neuroinflammatory responses. However, information about their proteomic phenotypes under inflammation is currently limited. METHOD: Data-independent acquisition mass spectrometry was applied to extensively characterize the profile of more than 4000 proteins in immortalized human fetal astrocytes under distinct inflammatory conditions induced by TNF, IL-1ß, and LPS, while multiplex immunoassay-based screening was used to quantify a wide range of cytokines released under these inflammatory conditions. Then, immunocytochemistry and western blotting were used to verify the activation of canonical and non-canonical NF-κB upon exposure to the different stimuli. Finally, an in vitro model of the blood-brain barrier consisting of a co-culture of primary human brain microvascular endothelial cells and primary human astrocytes was used to verify the inflammatory response of astrocytes upon LPS exposure in a more complex in vitro system. RESULTS: We reported on a set of 186 proteins whose levels were significantly modulated by TNF, IL-1ß, and LPS. These three stimuli induced proteome perturbations, which led to an increased abundance of key inflammatory proteins involved in antigen presentation and non-canonical NF-κB pathways. TNF and IL-1ß, but not LPS, also activated the canonical NF-κB pathway, which in turn led to an extensive inflammatory response and dysregulation of cytoskeletal and adhesion proteins. In addition, TNF and LPS, but not IL-1ß, increased the abundance of several interferon-stimulated gene products. Finally, TNF and IL-1ß similarly upregulated the secretion of several cytokines and chemokines, whereas LPS only induced a moderate increase in IL-8, IFN-γ, and IL-1ß secretion. Upregulation of proteins associated with type I IFN and non-canonical NF-κB signaling was also observed in primary astrocytes co-cultured with primary brain microvascular endothelial cells exposed to LPS. CONCLUSIONS: The present study provides comprehensive information about the proteomic phenotypes of human astrocytes upon exposure to inflammatory stimuli both in monoculture and in co-culture with human brain microvascular endothelial cells.

Regression of Breast Cancer Metastases Following Treatment with Irradiated SV-BR-1-GM, a GM-CSF Overexpressing Breast Cancer Cell Line: Intellectual Property and Immune Markers of Response.

BACKGROUND: SV-BR-1-GM, derived from a patient with grade 2 (moderately differentiated) breast cancer, is a GM-CSF-secreting breast cancer cell line with properties of antigen-presenting cells. SV-BR-1-GM and next-generation versions are covered by several pending and granted patents. METHODS: We report findings from an open-label phase I, single-arm pilot study with irradiated SV-BR-1-GM cells in 3 breast and 1 ovarian cancer subjects. Inoculations were preceded by lowdose intravenous cyclophosphamide and followed by interferon-alpha2b injections into the SVBR- 1-GM inoculation sites. We assessed both cellular and humoral immune responses, and measured expression levels of SV-BR-1-GM HLA alleles. RESULTS: Treatment was generally safe and well tolerated. Immune responses were elicited universally. Overall survival was more than 33 months for three of the four patients. As previously reported, one patient had prompt regression of metastases in lung, breast, and soft tissue. Following cessation of treatment, the patient relapsed widely, including in the brain. Upon retreatment, rapid tumor response was again seen, including complete regression of brain metastases. Consistent with a role of Class II HLA in contributing to SV-BR-1-GM's mechanism of action, this patient allele-matched SV-BR-1-GM at the HLA-DRB1 and HLA-DRB3 loci. We are in the process of developing next-generation SV-BR-1-GM, expressing patient-specific HLAs. Patent applications were filed in various jurisdictions. Thus far, one is granted, in Japan. CONCLUSION: A whole-cell immunotherapy regimen with SV-BR-1-GM cells induced regression of metastatic breast cancer. We develop intellectual property based on SV-BR-1-GM's predicted mechanism of action to develop additional whole-cell immunotherapies for cancer patients.

Cerebrospinal Fluid-Derived Microvesicles From Sleeping Sickness Patients Alter Protein Expression in Human Astrocytes.

Human African trypanosomiasis (HAT) caused by the extracellular protozoon Trypanosoma brucei, is a neglected tropical disease affecting the poorest communities in sub-Saharan Africa. HAT progresses from a hemolymphatic first stage (S1) to a meningo-encephalitic late stage (S2) when parasites reach the central nervous system (CNS), although the existence of an intermediate stage (Int.) has also been proposed. The pathophysiological mechanisms associated with the development of S2 encephalopathy are yet to be fully elucidated. Here we hypothesized that HAT progression toward S2 might be accompanied by an increased release of microvesicles (MVs), sub-micron elements (0.1-1 µm) involved in inflammatory processes and in the determination of the outcome of infections. We studied the morphology of MVs isolated from HAT cerebrospinal fluid (CSF) by transmission electron microscopy (TEM) and used flow cytometry to show that total-MVs and leukocyte derived-CD45+ MVs are significantly increased in concentration in S2 patients' CSF compared to S1 and Int. samples (n = 12 per group). To assess potential biological properties of these MVs, immortalized human astrocytes were exposed, in vitro, to MVs enriched from S1, Int. or S2 CSF. Data-independent acquisition mass spectrometry analyses showed that S2 MVs induced, compared to Int. or S1 MVs, a strong proteome modulation in astrocytes that resembled the one produced by IFN-γ, a key molecule in HAT pathogenesis. Our results indicate that HAT S2 CSF harbors MVs potentially involved in the mechanisms of pathology associated with HAT late stage. Such vesicles might thus represent a new player to consider in future functional studies.

Characterisation of extracellular vesicle-subsets derived from brain endothelial cells and analysis of their protein cargo modulation after TNF exposure.

Little is known about the composition and functional differences between extracellular vesicle (EV) subsets, such as microvesicles (MVs) and exosomes (EXOs), nor to what extent their cargo reflects the phenotypic state of the cell of origin. Brain endothelial cells are the constitutive part of the blood-brain barrier (BBB), a selective barrier that maintains brain homeostasis. BBB impairment is associated with several neuroinflammatory diseases with the pro-inflammatory cytokine tumour necrosis factor (TNF) often playing a key role. In the present study, shotgun proteomics and parallel reaction monitoring (PRM)-based targeted mass spectrometry were used to characterise brain endothelial cell-released EVs, and to study how TNF exposure modulated EV protein cargoes. MVs were found to be enriched in mitochondrial and cytoskeletal proteins, whereas EXOs were enriched in adhesion, histone and ribosomal proteins. After stimulation with TNF, several proteins involved in TNF and NF-κB signalling pathways, that were found to be differentially expressed in cells, were also differentially expressed in both MVs and EXOs. Thus, our results revealed some novel proteins as potentially useful candidates for discriminating between MVs and EXOs, together with additional evidence that cells "package" proteins in EVs systematically and according to their phenotypic state.