Analysis of Protease Activity in Live Antigen-presenting Cells Shows Regulation of the Phagosomal Proteolytic Contents During Dendritic Cell Activation

Here, we describe a new approach designed to monitor the proteolytic activity of maturing phagosomes in live antigen-presenting cells. We find that an ingested particle sequentially encounters distinct protease activities during phagosomal maturation. Incorporation of active proteases into the phagosome of the macrophage cell line J774 indicates that phagosome maturation involves progressive fusion with early and late endocytic compartments. In contrast, phagosome biogenesis in bone marrow–derived dendritic cells (DCs) and macrophages preferentially involves endocytic compartments enriched in cathepsin S. Kinetics of phagosomal maturation is faster in macrophages than in DCs. Furthermore, the delivery of active proteases to the phagosome is significantly reduced after the activation of DCs with lipopolysaccharide. This observation is in agreement with the notion that DCs prevent the premature destruction of antigenic determinants to optimize T cell activation. Phagosomal maturation is therefore a tightly regulated process that varies according to the type and differentiation stage of the phagocyte

Invariant Chain Controls the Activity of Extracellular Cathepsin L

Secretion of proteases is critical for degradation of the extracellular matrix during an inflammatory response. Cathepsin (Cat) S and L are the major elastinolytic cysteine proteases in mouse macrophages. A 65 amino acid segment of the p41 splice variant (p4165aa) of major histocompatibility complex class II–associated invariant chain (Ii) binds to the active site of CatL and permits the maintenance of a pool of mature enzyme in endosomal compartments of macro-phages and dendritic cells (DCs). Here we show that interaction of p4165aa with mature CatL allows extracellular accumulation of the active enzyme. We detected mature CatL as a complex with p4165aa in culture supernatants from antigen-presenting cells (APCs). Extracellular accumulation of mature CatL is up-regulated by inflammatory stimuli as observed in interferon (IFN)-γ–treated macrophages and lipopolysaccharide (LPS)-activated DCs. Despite the neutral pH of the extracellular milieu, released CatL associated with p4165aa is catalytically active as demonstrated by active site labeling and elastin degradation assays. We propose that p4165aa stabilizes CatL in the extracellular environment and induces a local increase in the concentration of matrix-degrading enzymes during inflammation. Through its interaction with CatL, Ii may therefore control the migratory response of APCs and/or the recruitment of effectors of the inflammatory response

Endothelial cell invasion is controlled by dactylopodia

Copyright © 2021 the Author(s). Published by PNAS. This open access article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND).Sprouting angiogenesis is fundamental for development and contributes to cancer, diabetic retinopathy, and cardiovascular diseases. Sprouting angiogenesis depends on the invasive properties of endothelial tip cells. However, there is very limited knowledge on how tip cells invade into tissues. Here, we show that endothelial tip cells use dactylopodia as the main cellular protrusion for invasion into nonvascular extracellular matrix. We show that dactylopodia and filopodia protrusions are balanced by myosin IIA (NMIIA) and actin-related protein 2/3 (Arp2/3) activity. Endothelial cell-autonomous ablation of NMIIA promotes excessive dactylopodia formation in detriment of filopodia. Conversely, endothelial cell-autonomous ablation of Arp2/3 prevents dactylopodia development and leads to excessive filopodia formation. We further show that NMIIA inhibits Rac1-dependent activation of Arp2/3 by regulating the maturation state of focal adhesions. Our discoveries establish a comprehensive model of how endothelial tip cells regulate its protrusive activity and will pave the way toward strategies to block invasive tip cells during sprouting angiogenesis.C.A.F. was supported by a European Research Council starting grant (679368), the European Union H2020-TWINN-2015—Twinning (692322), the Fundação para a Ciência e a Tecnologia funding (grants IF/00412/2012, EXPL/BEX-BCM/2258/2013, PTDC/MED-PAT/31639/2017, and PTDC/BIA-CEL/32180/2017 and fellowships CEECIND/04251/2017 and CEECIND/02589/2018), and a grant from the Fondation Leducq (17CVD03).info:eu-repo/semantics/publishedVersio

The actin-based motor protein myosin II regulates MHC class II trafficking and BCR-driven antigen presentation

Antigen (Ag) capture and presentation onto major histocompatibility complex (MHC) class II molecules by B lymphocytes is mediated by their surface Ag receptor (B cell receptor [BCR]). Therefore, the transport of vesicles that carry MHC class II and BCR–Ag complexes must be coordinated for them to converge for processing. In this study, we identify the actin-associated motor protein myosin II as being essential for this process. Myosin II is activated upon BCR engagement and associates with MHC class II–invariant chain complexes. Myosin II inhibition or depletion compromises the convergence and concentration of MHC class II and BCR–Ag complexes into lysosomes devoted to Ag processing. Accordingly, the formation of MHC class II–peptides and subsequent CD4 T cell activation are impaired in cells lacking myosin II activity. Therefore, myosin II emerges as a key motor protein in BCR-driven Ag processing and presentation

Cell migration and antigen capture are antagonistic processes coupled by myosin II in dendritic cells

The immune response relies on the migration of leukocytes and on their ability to stop in precise anatomical locations to fulfil their task. How leukocyte migration and function are coordinated is unknown. Here we show that in immature dendritic cells, which patrol their environment by engulfing extracellular material, cell migration and antigen capture are antagonistic. This antagonism results from transient enrichment of myosin IIA at the cell front, which disrupts the back-to-front gradient of the motor protein, slowing down locomotion but promoting antigen capture. We further highlight that myosin IIA enrichment at the cell front requires the MHC class II-associated invariant chain (Ii). Thus, by controlling myosin IIA localization, Ii imposes on dendritic cells an intermittent antigen capture behaviour that might facilitate environment patrolling. We propose that the requirement for myosin II in both cell migration and specific cell functions may provide a general mechanism for their coordination in time and space

Myosin II Activity Is Selectively Needed for Migration in Highly Confined Microenvironments in Mature Dendritic Cells

Upon infection, mature dendritic cells (mDCs) migrate from peripheral tissue to lymph nodes (LNs) to activate T lymphocytes and initiate the adaptive immune response. This fast and tightly regulated process is tuned by different microenvironmental factors, such as the physical properties of the tissue. Mechanistically, mDCs migration mostly relies on acto-myosin flow and contractility that depend on non-muscular Myosin IIA (MyoII) activity. However, the specific contribution of this molecular motor for mDCs navigation in complex microenvironments has yet to be fully established. Here, we identified a specific role of MyoII activity in the regulation of mDCs migration in highly confined microenvironments. Using microfluidic systems, we observed that during mDCs chemotaxis in 3D collagen gels under defined CCL21 gradients, MyoII activity was required to sustain their fast speed but not to orientate them toward the chemokine. Indeed, despite the fact that mDCs speed declined, these cells still migrated through the 3D gels, indicating that this molecular motor has a discrete function during their motility in this irregular microenvironment. Consistently, using microchannels of different sizes, we found that MyoII activity was essential to maintain fast cell speed specifically under strong confinement. Analysis of cell motility through micrometric holes further demonstrated that cell contractility facilitated mDCs passage only over very small gaps. Altogether, this work highlights that high contractility acts as an adaptation mechanism exhibited by mDCs to optimize their motility in restricted landscapes. Hence, MyoII activity ultimately facilitates their navigation in highly confined areas of structurally irregular tissues, contributing to the fine-tuning of their homing to LNs to initiate adaptive immune responses

Genetic identification of unique immunological responses in mice infected with virulent and attenuated Francisella tularensis

Francisella tularensis is a category A select agent based on its infectivity and virulence but disease mechanisms in infection remain poorly understood. Murine pulmonary models of infection were therefore employed to assess and compare dissemination and pathology and to elucidate the host immune response to infection with the highly virulent Type A F. tularensis strain Schu4 versus the less virulent Type B live vaccine strain (LVS). We found that dissemination and pathology in the spleen was significantly greater in mice infected with F. tularensis Schu4 compared to mice infected with F. tularensis LVS. Using gene expression rofiling to compare the response to infection with the two F. tularensis strains, we found that there were significant differences in the expression of genes involved in the apoptosis pathway, antigen processing and presentation pathways, and inflammatory response pathways in mice infected with Schu4 when compared to LVS. These transcriptional differences coincided with marked differences in dissemination and severity of organ lesions in mice infected with the Schu4 and LVS strains. Therefore, these findings indicate that altered apoptosis, antigen presentation and production of inflammatory mediators explain the differences in pathogenicity of F. tularensis Schu4 and LVS

Cdc42 controls the dilation of the exocytotic fusion pore by regulating membrane tension.

Membrane fusion underlies multiple processes, including exocytosis of hormones and neurotransmitters. Membrane fusion starts with the formation of a narrow fusion pore. Radial expansion of this pore completes the process and allows fast release of secretory compounds, but this step remains poorly understood. Here we show that inhibiting the expression of the small GTPase Cdc42 or preventing its activation with a dominant negative Cdc42 construct in human neuroendocrine cells impaired the release process by compromising fusion pore enlargement. Consequently the mode of vesicle exocytosis was shifted from full-collapse fusion to kiss-and-run. Remarkably, Cdc42-knockdown cells showed reduced membrane tension, and the artificial increase of membrane tension restored fusion pore enlargement. Moreover, inhibiting the motor protein myosin II by blebbistatin decreased membrane tension, as well as fusion pore dilation. We conclude that membrane tension is the driving force for fusion pore dilation and that Cdc42 is a key regulator of this force.journal articleresearch support, non-u.s. gov't2014 Oct 152014 08 20importe

Text-derived concept profiles support assessment of DNA microarray data for acute myeloid leukemia and for androgen receptor stimulation

BACKGROUND: High-throughput experiments, such as with DNA microarrays, typically result in hundreds of genes potentially relevant to the process under study, rendering the interpretation of these experiments problematic. Here, we propose and evaluate an approach to find functional associations between large numbers of genes and other biomedical concepts from free-text literature. For each gene, a profile of related concepts is constructed that summarizes the context in which the gene is mentioned in literature. We assign a weight to each concept in the profile based on a likelihood ratio measure. Gene concept profiles can then be clustered to find related genes and other concepts. RESULTS: The experimental validation was done in two steps. We first applied our method on a controlled test set. After this proved to be successful the datasets from two DNA microarray experiments were analyzed in the same way and the results were evaluated by domain experts. The first dataset was a gene-expression profile that characterizes the cancer cells of a group of acute myeloid leukemia patients. For this group of patients the biological background of the cancer cells is largely unknown. Using our methodology we found an association of these cells to monocytes, which agreed with other experimental evidence. The second data set consisted of differentially expressed genes following androgen receptor stimulation in a prostate cancer cell line. Based on the analysis we put forward a hypothesis about the biological processes induced in these studied cells: secretory lysosomes are involved in the production of prostatic fluid and their development and/or secretion are androgen-regulated processes. CONCLUSION: Our method can be used to analyze DNA microarray datasets based on information explicitly and implicitly available in the literature. We provide a publicly available tool, dubbed Anni, for this purpose

Patterns, receptors and signals:regulation of phagosome maturation

Recognition of microbial pathogens and dead cells and their phagocytic uptake by specialized immune cells are essential to maintain host homeostasis. Phagosomes undergo fusion and fission events with endosomal and lysosomal compartments, a process called 'phagosome maturation', which leads to the degradation of the phagosomal content. However, many phagocytic cells also act as antigen-presenting cells and must balance degradation and peptide preservation. Emerging evidence indicates that receptor engagement by phagosomal cargo, as well as inflammatory mediators and cellular activation affect many aspects of phagosome maturation. Unsurprisingly, pathogens have developed strategies to hijack this machinery, thereby interfering with host immunity. Here, we highlight progress in this field, summarize findings on the impact of immune signals, and discuss consequences for pathogen elimination