Sten F.W.M. Libregts

Extracellular vesicles (EVs) are small, heterogeneous and difficult to measure. Flow cytometry (FC) is a key technology for the measurement of individual particles, but its application to the analysis of EVs and other submicron particles has presented many challenges and has produced a number of controversial results, in part due to limitations of instrument detection, lack of robust methods and ambiguities in how data should be interpreted. These complications are exacerbated by the field's lack of a robust reporting framework, and many EV-FC manuscripts include incomplete descriptions of methods and results, contain artefacts stemming from an insufficient instrument sensitivity and inappropriate experimental design and lack appropriate calibration and standardization. To address these issues, a working group (WG) of EV-FC researchers from ISEV, ISAC and ISTH, worked together as an EV-FC WG and developed a consensus framework for the minimum information that should be provided regarding EV-FC. This framework incorporates the existing Minimum Information for Studies of EVs (MISEV) guidelines and Minimum Information about a FC experiment (MIFlowCyt) standard in an EV-FC-specific reporting framework (MIFlowCyt-EV) that supports reporting of critical information related to sample staining, EV detection and measurement and experimental design in manuscripts that report EV-FC data. MIFlowCyt-EV provides a structure for sharing EV-FC results, but it does not prescribe specific protocols, as there will continue to be rapid evolution of instruments and methods for the foreseeable future. MIFlowCyt-EV accommodates this evolution, while providing information needed to evaluate and compare different approaches. Because MIFlowCyt-EV will ensure consistency in the manner of reporting of EV-FC studies, over time we expect that adoption of MIFlowCyt-EV as a standard for reporting EV- FC studies will improve the ability to quantitatively compare results from different laboratories and to support the development of new instruments and assays for improved measurement of EVs.

OBJECTIVE: To explore the involvement of synovial macrophages in early cartilage damage in osteoarthritis (OA), and to identify the role of matrix metalloproteinase 3 (MMP-3) in the pathology of early and late OA. METHODS: The role of synovial macrophages in MMP-mediated damage in OA was studied by depleting synovial macrophages prior to elicitation of a collagenase-induced instability model of OA. The expression of MMP in synovium and cartilage was monitored using TaqMan analysis. In spontaneous and induced OA, cartilage pathology was scored in MMP-3-knockout mice and control mice, by histologic assessment and VDIPEN staining. RESULTS: On day 14 following induction of OA, MMP-mediated neoepitopes were detected in cartilage from mice with mild experimental OA (mean +/- SD positively stained surface area 20 +/- 3.2%). Remarkably, by depleting synovial macrophages prior to induction of OA, the generation of MMP-induced neoepitopes was largely prevented (mean +/- SD positively stained surface area 5 +/- 1%; P< 0.001), indicating an important role for synovial macrophages in the occurrence of MMP-mediated cartilage damage. We observed a strong decrease in MMP-3 and MMP-9 expression in synovial but not cartilage tissue in macrophage-depleted joints. Among 2-year-old mice, spontaneous OA-like changes in the lining layer were significantly decreased in MMP-3-knockout mice compared with control mice. Even more striking was the 67% reduction in the occurrence of severe cartilage damage in MMP-3-knockout mice. In addition, MMP-mediated VDIPEN expression was significantly decreased, indicating reduced MMP-mediated cartilage breakdown. CONCLUSION: The results of this study prove that MMP-3 is involved in the generation of severe cartilage damage in murine OA. Synovial macrophages are crucial in early MMP activity and appear to mediate MMP production in synovium rather than cartilage.

Anemia of chronic disease is a complication accompanying many inflammatory diseases. The proinflammatory cytokine IFN-γ has been implicated in this form of anemia, but the underlying mechanism remains unclear. Here we describe a novel mouse model for anemia of chronic disease, in which enhanced CD27-mediated costimulation strongly increases the formation of IFN-γ-producing effector T cells, leading to a progressive anemia. We demonstrate that the anemia in these mice is fully dependent on IFN-γ and that this cytokine reduces both the life span and the formation of red blood cells. Molecular analysis revealed that IFN-γ induces expression of the transcription factors of interferon regulatory factor-1 (IRF-1) and PU.1 in both murine and human erythroid precursors. We found that, on IFN-γ stimulation, IRF-1 binds to the promoter of SPI.1 (PU.1) and induces PU.1 expression, leading to inhibition of erythropoiesis. Notably, down-regulation of either IRF-1 or PU.1 expression is sufficient to overcome IFN-γ-induced inhibition of erythropoiesis. These findings reveal a molecular mechanism by which chronic exposure to IFN-γ induces anemia.

BACKGROUND. Cell-free circulating nucleic acids, including 22-nt microRNAs (miRNAs), represent noninvasive biomarkers for treatment response monitoring of cancer patients. While the majority of plasma miRNA is bound to proteins, a smaller, less well-characterized pool is associated with extracellular vesicles (EVs). Here, we addressed whether EV-associated miRNAs reflect metabolic disease in classical Hodgkin lymphoma (cHL) patients. METHODS. With standardized size-exclusion chromatography (SEC), we isolated EV-associated extracellular RNA (exRNA) fractions and protein-bound miRNA from plasma of cHL patients and healthy subjects. We performed a comprehensive small RNA sequencing analysis and validation by TaqMan qRT-PCR for candidate discovery. Fluorodeoxyglucose-PET (FDG-PET) status before treatment, directly after treatment, and during long-term follow-up was compared directly with EV miRNA levels. RESULTS. The plasma EV miRNA repertoire was more extensive compared with protein-bound miRNA that was heavily dominated by a few abundant miRNA species and was less informative of disease status. Purified EV fractions of untreated cHL patients and tumor EVs had enriched levels of miR24-3p, miR127-3p, miR21-5p, miR155-5p, and let7a-5p compared with EV fractions from healthy subjects and disease controls. Serial monitoring of EV miRNA levels in patients before treatment, directly after treatment, and during long-term follow-up revealed robust, stable decreases in miRNA levels matching a complete metabolic response, as observed with FDG-PET. Importantly, EV miRNA levels rose again in relapse patients. CONCLUSION. We conclude that cHL-related miRNA levels in circulating EVs reflect the presence of vital tumor tissue and are suitable for therapy response and relapse monitoring in individual cHL patients. FUNDING. Cancer Center Amsterdam Foundation (CCA-2013), Dutch Cancer Society (KWF-5510), Technology Foundation STW (STW Perspectief CANCER-ID).

Steady-state hematopoiesis is altered on infection, but the cellular and molecular mechanisms driving these changes are largely unknown. Modulation of hematopoiesis is essential to increase the output of the appropriate type of effector cell required to combat the invading pathogen. In the present study, we demonstrate that the pro-inflammatory cytokine IFNγ is involved in orchestrating inflammation-induced myelopoiesis. Using both mouse models and in vitro assays, we show that IFNγ induces the differentiation of monocytes over neutrophils at the level of myeloid progenitors. Infection with lymphocytic choriomeningitis virus induces monopoiesis in wild-type mice, but causes increased neutrophil production in IFNγ(-/-) mice. We demonstrate that IFNγ enhances the expression of the monopoiesis-inducing transcription factors IRF8 and PU.1 in myeloid progenitor cells, whereas it reduces G-CSF-driven neutrophil differentiation via a SOCS3-dependent inhibition of STAT3 phosphorylation. These results establish a critical role for IFNγ in directing monocyte versus neutrophil development during immune activation.