Distribution of nerve fibers and nerve-immune cell association in mouse spleen revealed by immunofluorescent staining

The central nervous system regulates the immune system through the secretion of hormones from the pituitary gland and other endocrine organs, while the peripheral nervous system (PNS) communicates with the immune system through local nerve-immune cell interactions, including sympathetic/parasympathetic (efferent) and sensory (afferent) innervation to lymphoid tissue/organs. However, the precise mechanisms of this bi-directional crosstalk of the PNS and immune system remain mysterious. To study this kind of bi-directional crosstalk, we performed immunofluorescent staining of neurofilament and confocal microscopy to reveal the distribution of nerve fibers and nerve-immune cell associations inside mouse spleen. Our study demonstrates (i) extensive nerve fibers in all splenic compartments including the splenic nodules, periarteriolar lymphoid sheath, marginal zones, trabeculae, and red pulp; (ii) close associations of nerve fibers with blood vessels (including central arteries, marginal sinuses, penicillar arterioles, and splenic sinuses); (iii) close associations of nerve fibers with various subsets of dendritic cells, macrophages (Mac1+ and F4/80+), and lymphocytes (B cells, T helper cells, and cytotoxic T cells). Our data concerning the extensive splenic innervation and nerve-immune cell communication will enrich our knowledge of the mechanisms through which the PNS affects the cellular- and humoral-mediated immune responses in healthy and infectious/non-infectious states

Two-photon intravital imaging of lungs during anthrax infection reveals long-lasting macrophage-dendritic cell contacts.

International audience: Dynamics of the lung immune system at a microscopic level are largely unknown because of inefficient methods to rid chest motion during image acquisition. In this study, we developed an improved intravital method for two-photon lung imaging uniquely based on a posteriori parenchymal tissue motion correction. We took advantage of the alveolar collagen pattern given by second harmonic generation signal as a reference for frame registration. We describe here for the first time a detailed dynamic account of two major lung immune cell populations, alveolar macrophages and CD11b-positive dendritic cells, during homeostasis and infection by spores of Bacillus anthracis, the agent of anthrax. We show that after alveolar macrophages capture spores, CD11b-positive dendritic cells come in prolonged contact with infected macrophages. Dendritic cells are known to carry spores to the draining lymph nodes and elicit the immune response in pulmonary anthrax. The intimate and long-lasting contacts between these two lines of defense may therefore coordinate immune responses in the lung through an immunological synapse-like process

Functioning and Control of Phagocytosis

Phagocytosis is a very complex and versatile process that contributes to immunity through a series of events that is it’s sometimes referred to the Come and Eat me process. Due to the recognition ingestion and digestion then destruction. It’s also central to tissue homeostasis and remodeling by clearing dead cells. This ability of phagocytes to perform such diverse functions rests in large part on their vast repertoire of receptors. In this book chapter we looked at the processes used by phagocyte to perform there phagocytosis function. This is made possible by the binding of opsonins on the microbes like the C3b of the complement. This works as a chemo attractant to the phagocytes to come and initiate the process of eating. On recognition this microbe or dead cell interacts with the phagocyte with the help of a very big repertoire of receptors the microbe is engulfed with in the phagosome. As microbes interact with the phagocyte receptors a cascade of signaling events downstream that then activate phagocytosis. This membrane and cytoskeleton remodulation lead to the formation of pseudopods that cover the entire microbe forming a phagocytic cup which closes a few minutes to take up the microbe completely. The signal cascade is most known for the Fc receptor activities. Crosslinking of the Fc receptor on the surface of phagocyte activate phagocytosis and any other effector functions such as activation of the oxidative burst, degranulation, antibody dependent cell mediated cytotoxicity and activation of genes for cytokine/chemokine production that are beneficial in microbe destruction and initiation of inflammation. This starts once the interaction of phagocytes receptors and their ligands on the target microbes takes place appropriately. The phagocyte receptors will then aggregate to activate a series of pathways that regulate actin cytoskeleton which helps in the formation of a new vesicle which comes out of the membrane to enclose the microbe. In here a number of processes and stages take place all aimed at killing and denaturing the particle. They include early phagosome, intermediate phagosome, phagolysosome formation and the late phagosome all these participate in eliminating the phagocytized microbe. However with all the above phagocytic efficiency, some pathogens evade phagocytosis using different means and presence of certain capacities that facilitate evasion examples of organisms that evade phagocytosis include Mycobacterium tuberculosis, Listeria monocytogens Escherichia coli etc. all these use different means in evasion. Therefore the concept and science of Phagocytes used to be studied more to explore more pharmaceutical products based on the evasion mechanisms

Deciphering the functions of O-GlcNAc glycosylation in the brain: The role of site-specific quantitative O-GlcNAcomics

The dynamic posttranslational modification O-linked β-N-acetylglucosamine glycosylation (O-GlcNAcylation) is present on thousands of intracellular proteins in the brain. Like phosphorylation, O-GlcNAcylation is inducible and plays important functional roles in both physiology and disease. Recent advances in mass spectrometry (MS) and bioconjugation methods are now enabling the mapping of O-GlcNAcylation events to individual sites in proteins. However, our understanding of which glycosylation events are necessary for regulating protein function and controlling specific processes, phenotypes, or diseases remains in its infancy. Given the sheer number of O-GlcNAc sites, methods for identifying promising sites and prioritizing them for time- and resource-intensive functional studies are greatly needed. Revealing sites that are dynamically altered by different stimuli or disease states will likely go a long way in this regard. Here, we describe advanced methods for identifying O-GlcNAc sites on individual proteins and across the proteome and for determining their stoichiometry in vivo. We also highlight emerging technologies for quantitative, site-specific MS-based O-GlcNAc proteomics (O-GlcNAcomics), which allow proteome-wide tracking of O-GlcNAcylation dynamics at individual sites. These cutting-edge technologies are beginning to bridge the gap between the high-throughput cataloguing of O-GlcNAcylated proteins and the relatively low-throughput study of individual proteins. By uncovering the O-GlcNAcylation events that change in specific physiological and disease contexts, these new approaches are providing key insights into the regulatory functions of O-GlcNAc in the brain, including their roles in neuroprotection, neuronal signaling, learning and memory, and neurodegenerative diseases

Bioregulatory systems medicine: an innovative approach to integrating the science of molecular networks, inflammation, and systems biology with the patient\u27s autoregulatory capacity?

Bioregulatory systems medicine (BrSM) is a paradigm that aims to advance current medical practices. The basic scientific and clinical tenets of this approach embrace an interconnected picture of human health, supported largely by recent advances in systems biology and genomics, and focus on the implications of multi-scale interconnectivity for improving therapeutic approaches to disease. This article introduces the formal incorporation of these scientific and clinical elements into a cohesive theoretical model of the BrSM approach. The authors review this integrated body of knowledge and discuss how the emergent conceptual model offers the medical field a new avenue for extending the armamentarium of current treatment and healthcare, with the ultimate goal of improving population health

A role for the centrosome in regulating the rate of neuronal efferocytosis by microglia in vivo

During brain development, many newborn neurons undergo apoptosis and are engulfed by microglia, the tissue-resident phagocytes of the brain, in a process known as efferocytosis. A hallmark of microglia is their highly branched morphology characterized by the presence of numerous dynamic extensions that these cells use for scanning the brain parenchyma and engulfing unwanted material. The mechanisms driving branch formation and apoptotic cell engulfment in microglia are unclear. By taking a live-imaging approach in zebrafish, we show that while microglia generate multiple microtubule-based branches, they only successfully engulf one apoptotic neuron at a time. Further investigation into the mechanism underlying this sequential engulfment revealed that targeted migration of the centrosome into one branch is predictive of phagosome formation and polarized vesicular trafficking. Moreover, experimentally doubling centrosomal numbers in microglia increases the rate of engulfment and even allows microglia to remove two neurons simultaneously, providing direct supporting evidence for a model where centrosomal migration is a rate-limiting step in branch-mediated efferocytosis. Conversely, light-mediated depolymerization of microtubules causes microglia to lose their typical branched morphology and switch to an alternative mode of engulfment, characterized by directed migration towards target neurons, revealing unexpected plasticity in their phagocytic ability. Finally, building on work focusing on the establishment of the immunological synapse, we identified a conserved signalling pathway underlying centrosomal movement in engulfing microglia

A Drosophila-centric view of protein tyrosine phosphatases

AbstractMost of our knowledge on protein tyrosine phosphatases (PTPs) is derived from human pathologies and mouse knockout models. These models largely correlate well with human disease phenotypes, but can be ambiguous due to compensatory mechanisms introduced by paralogous genes. Here we present the analysis of the PTP complement of the fruit fly and the complementary view that PTP studies in Drosophila will accelerate our understanding of PTPs in physiological and pathological conditions. With only 44 PTP genes, Drosophila represents a streamlined version of the human complement. Our integrated analysis places the Drosophila PTPs into evolutionary and functional contexts, thereby providing a platform for the exploitation of the fly for PTP research and the transfer of knowledge onto other model systems

Deciphering the functions of O-GlcNAc glycosylation in the brain: The role of site-specific quantitative O-GlcNAcomics

The dynamic posttranslational modification O-linked β-N-acetylglucosamine glycosylation (O-GlcNAcylation) is present on thousands of intracellular proteins in the brain. Like phosphorylation, O-GlcNAcylation is inducible and plays important functional roles in both physiology and disease. Recent advances in mass spectrometry (MS) and bioconjugation methods are now enabling the mapping of O-GlcNAcylation events to individual sites in proteins. However, our understanding of which glycosylation events are necessary for regulating protein function and controlling specific processes, phenotypes, or diseases remains in its infancy. Given the sheer number of O-GlcNAc sites, methods for identifying promising sites and prioritizing them for time- and resource-intensive functional studies are greatly needed. Revealing sites that are dynamically altered by different stimuli or disease states will likely go a long way in this regard. Here, we describe advanced methods for identifying O-GlcNAc sites on individual proteins and across the proteome and for determining their stoichiometry in vivo. We also highlight emerging technologies for quantitative, site-specific MS-based O-GlcNAc proteomics (O-GlcNAcomics), which allow proteome-wide tracking of O-GlcNAcylation dynamics at individual sites. These cutting-edge technologies are beginning to bridge the gap between the high-throughput cataloguing of O-GlcNAcylated proteins and the relatively low-throughput study of individual proteins. By uncovering the O-GlcNAcylation events that change in specific physiological and disease contexts, these new approaches are providing key insights into the regulatory functions of O-GlcNAc in the brain, including their roles in neuroprotection, neuronal signaling, learning and memory, and neurodegenerative diseases

Dissection of virus-host cell interactions in the early response to infection

Stress granules (SG) are dynamic RNA/protein assemblies in the cytoplasm of the cell, formed under conditions of oxidative stress, heat shock or viral infections. These stress conditions trigger a sudden translational arrest, leading to a rapid switch of translation from housekeeping genes to stress-related factors. SGs fulfil multiple roles in the cell one of which is acting as triage centres for mRNA, where the mRNA is stored pending either degradation or reinitiation of translation. Many proteins are sequestered to SGs, among them signalling molecules, which make SGs signal centres to communicate a “state of emergency”. The importance of SGs is also underlined by the fact that they restrict viral propagation. The assembly of SGs is dependent on many RNA-binding proteins, one of which is G3BP (Ras-GAP SH3 domain binding protein). Semliki Forest virus (SFV) belongs to the alphaviruses, a large group of arthropod-borne animal viruses including several relevant human pathogens such as the re-emerging Chikungunya virus (CHIKV). Alphavirus infections lead to fever, rashes, arthralgia and can be lethal. Recent CHIKV outbreaks in the Caribbean area and the US, brings alphavirus research back on the agenda. Therefore there is a need to understand the molecular mechanisms how alphaviruses interact with their host. The aim of this thesis was to dissect virus-host cell interactions in the early response to alphavirus infection. Alphavirus infection leads to the formation of SGs at very early time points. Interestingly, they dissolve in the vicinity of viral replication complexes at later time points. In paper I, we showed that the non-structural protein nsP3 of SFV is responsible for sequestration of G3BP to replication complexes, by doing so, actively disassembling SGs and blocking their reformation. We mapped the binding site for G3BP to two C-terminal repeat domains of nsP3. A recombinant virus mutant lacking these repeats showed a longer and more persistent stress response and was attenuated in growth. In paper II, we extended this finding to the closely related CHIKV. Our results show that nsP3 of both SFV and CHIKV interact with G3BP via two C-terminal repeat domains and that the proline-rich region of nsP3 is dispensable for this interaction. In paper III we investigated the interaction between nsP3 and G3BP in molecular detail and determined that the residues FGDF in the C-terminal repeats of nsP3 are the G3BP binding motif. We further asked whether other proteins use the same mechanism as nsP3 to bind G3BP and whether this interaction inhibits the formation of SGs. We revealed that the phenylalanines and the glycine in the FGDF are essential for binding G3BP. We further demonstrated that the cellular ubiquitin-specific protease 10 (USP10) and the herpes simplex virus (HSV) protein ICP8 (infected cell protein 8) also bind G3BP via an FGDF motif. In addition we show that the FGDFmediated binding to G3BP leads to a negative regulation of G3BP’s SG-nucleating function. Lastly we present a 3D-model of G3BP bound to an FGDF-containing peptide, which we validated by site-directed mutagenesis. Our findings present a common FGDF motif to bind G3BP, which has a negative regulatory effect on the SG-nucleating function of G3BP. This molecular mechanism and the presented 3Dmodel demonstrate the therapeutic potential of targeting this interaction