Exosomes are 50‐ to 150‐nm‐diameter extracellular vesicles secreted by all mammalian cells except mature red blood cells and contribute to diverse physiological and pathological functions within the body. Many methods have been used to isolate and analyze exosomes, resulting in inconsistencies across experiments and raising questions about how to compare results obtained using different approaches. Questions have also been raised regarding the purity of the various preparations with regard to the sizes and types of vesicles and to the presence of lipoproteins. Thus, investigators often find it challenging to identify the optimal exosome isolation protocol for their experimental needs. Our laboratories have compared ultracentrifugation and commercial precipitation‐ and column‐based exosome isolation kits for exosome preparation. Here, we present protocols for exosome isolation using two of the most commonly used methods, ultracentrifugation and precipitation, followed by downstream analyses. We use NanoSight nanoparticle tracking analysis and flow cytometry (Cytek^®) to determine exosome concentrations and sizes. Imaging flow cytometry can be utilized to both size exosomes and immunophenotype surface markers on exosomes (ImageStream^®). High‐performance liquid chromatography followed by nano‐flow liquid chromatography–mass spectrometry (LCMS) of the exosome fractions can be used to determine the presence of lipoproteins, with LCMS able to provide a proteomic profile of the exosome preparations. We found that the precipitation method was six times faster and resulted in a ∼2.5‐fold higher concentration of exosomes per milliliter compared to ultracentrifugation. Both methods yielded extracellular vesicles in the size range of exosomes, and both preparations included apoproteins. © 2020 Wiley Periodicals LLC.Basic Protocol 1: Pre‐analytic fluid collection and processingBasic Protocol 2: Exosome isolation by ultracentrifugationAlternate Protocol 1: Exosome isolation by precipitationBasic Protocol 3: Analysis of exosomes by NanoSight nanoparticle tracking analysisAlternate Protocol 2: Analysis of exosomes by flow cytometry and imaging flow cytometryBasic Protocol 4: Downstream analysis of exosomes using high‐performance liquid chromatographyBasic Protocol 5: Downstream analysis of the exosome proteome using nano‐flow liquid chromatography–mass spectrometry

Functional chemokine receptors and chemokines are expressed by glial cells within the CNS, though relatively little is known about the patterns of neuronal chemokine receptor expression and function. We developed monoclonal antibodies to the CCR1, CCR2, CCR3, CCR6, CXCR2, CXCR3 and CXCR4 chemokine receptors to study their expression in human fetal neurons cultured from brain tissue as well as the clonally derived NT2.N human neuronal cell line (NTera 2/cl.D1). Specific monoclonal antibody labeling demonstrated expression of CCR2, CXCR2, CXCR3 and CXCR4 on neurons from both sources. Co-labeling studies revealed strong expression of CXCR3 and CXCR4 on both dendritic and axonal processes, with a weaker expression of CXCR2 and CCR2. Reverse transcriptase-polymerase chain reaction analysis of pure NT2.N neurons confirmed RNA expression for CCR2, CXCR2, CXCR3 and CXCR4. No changes in the neuronal labeling pattern of chemokine receptor expression were noted when NT2.N neurons were grown on a supporting layer of astrocytes, again consistent with similar patterns seen in primary human fetal brain cultures. Analysis of single-cell calcium transients revealed a robust response to stromal derived factor-1alpha (CXCR4) and melanocyte growth-stimulating activity (CXCR2), and variable response to monocyte chemoattractant protein-1 (CCR2) or interferon-gamma inducible protein-10 (CXCR3). Finally, we detected the release of monocyte chemoattractant protein-1 from pure cultures of NT2.N neurons, but not undifferentiated NT2 cells. These data indicate that individual neurons may not only co-express multiple functional chemokine receptors, but also that neurons themselves produce chemokines which may influence cellular function within the central nervous system.

Rationale: Idiopathic pulmonary fibrosis (IPF) is a lethal lung disease characterized by lung epithelial cell injury, increased (myo)fibroblast activation, and extracellular matrix deposition. Extracellular vesicles (EVs) regulate intercellular communication by carrying a variety of signaling mediators, including WNT (wingless/integrated) proteins. The relevance of EVs in pulmonary fibrosis and their potential contribution to disease pathogenesis, however, remain unexplored. Objectives: To characterize EVs and study the role of EV-bound WNT signaling in IPF. Methods: We isolated EVs from BAL fluid (BALF) from experimental lung fibrosis as well as samples from IPF, non-IPF interstitial lung disease (ILD), non-ILD, and healthy volunteers from two independent cohorts. EVs were characterized by transmission electron microscopy, nanoparticle tracking analysis, and Western blotting. Primary human lung fibroblasts (phLFs) were used for EV isolation and analyzed by metabolic activity assays, cell counting, quantitative PCR, and Western blotting upon WNT gain- and loss-of-function studies. Measurements and Main Results: We found increased EVs, particularly exosomes, in BALF from experimental lung fibrosis as well as from patients with IPF. WNT5A was secreted on EVs in lung fibrosis and induced by transforming growth factor-β in primary human lung fibroblasts. The phLF-derived EVs induced phLF proliferation, which was attenuated by WNT5A silencing and antibody-mediated inhibition and required intact EV structure. Similarly, EVs from IPF BALF induced phLF proliferation, which was mediated by WNT5A. Conclusions: Increased EVs function as carriers for signaling mediators, such as WNT5A, in IPF and thus contribute to disease pathogenesis. Characterization of EV secretion and composition may lead to novel approaches to diagnose and develop treatments for pulmonary fibrosis.

Introduction: Inflammatory markers have long been observed in the brain, cerebrospinal fluid (CSF), and plasma of Alzheimer's disease (AD) patients, suggesting that inflammation contributes to AD and might be a therapeutic target. However, non‐steroidal anti‐inflammatory drug trials in AD and mild cognitive impairment (MCI) failed to show benefit. Our previous work seeking to understand why people with the inflammatory disease rheumatoid arthritis are protected from AD found that short‐term treatment of transgenic AD mice with the pro‐inflammatory cytokine granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) led to an increase in activated microglia, a 50% reduction in amyloid load, an increase in synaptic area, and improvement in spatial memory to normal. These results called into question the consensus view that inflammation is solely detrimental in AD. Here, we tested our hypothesis that modulation of the innate immune system might similarly be used to treat AD in humans by investigating the ability of GM‐CSF/sargramostim to safely ameliorate AD symptoms/pathology. Methods: A randomized, double‐blind, placebo‐controlled trial was conducted in mild‐to‐moderate AD participants (NCT01409915). Treatments (20 participants/group) occurred 5 days/week for 3 weeks plus two follow‐up (FU) visits (FU1 at 45 days and FU2 at 90 days) with neurological, neuropsychological, blood biomarker, and imaging assessments. Results: Sargramostim treatment expectedly changed innate immune system markers, with no drug‐related serious adverse events or amyloid‐related imaging abnormalities. At end of treatment (EOT), the Mini‐Mental State Examination score of the sargramostim group increased compared to baseline ( P = .0074) and compared to placebo ( P = .0370); the treatment effect persisted at FU1 ( P = .0272). Plasma markers of amyloid beta (Aβ40 [decreased in AD]) increased 10% ( P = .0105); plasma markers of neurodegeneration (total tau and UCH‐L1) decreased 24% ( P = .0174) and 42% ( P = .0019), respectively, after sargramostim treatment compared to placebo. Discussion: The innate immune system is a viable target for therapeutic intervention in AD. An extended treatment trial testing the long‐term safety and efficacy of GM‐CSF/sargramostim in AD is warranted.

Age‐related macular degeneration (AMD) is a leading cause of blindness worldwide. Drusen are key contributors to the etiology of AMD and the ability to modulate drusen biogenesis could lead to therapeutic strategies to slow or halt AMD progression. The mechanisms underlying drusen biogenesis, however, remain mostly unknown. Here we demonstrate that under homeostatic conditions extracellular vesicles (EVs) secreted by retinal pigment epithelium (RPE) cells are enriched in proteins associated with mechanisms involved in AMD pathophysiology, including oxidative stress, immune response, inflammation, complement system and drusen composition. Furthermore, we provide first evidence that drusen‐associated proteins are released as cargo of extracellular vesicles secreted by RPE cells in a polarised apical:basal mode. Notably, drusen‐associated proteins exhibited distinctive directional secretion modes in homeostatic conditions and, differential modulation of this directional secretion in response to AMD stressors. These observations underpin the existence of a finely‐tuned mechanism regulating directional apical:basal sorting and secretion of drusen‐associated proteins via EVs, and its modulation in response to mechanisms involved in AMD pathophysiology. Collectively, our results strongly support an active role of RPE‐derived EVs as a key source of drusen proteins and important contributors to drusen development and growth.