Microglia cells protect neurons by direct engulfment of invading neutrophil granulocytes: a new mechanism of CNS immune privilege

Microglial cells maintain the immunological integrity of the healthy brain and can exert protection from traumatic injury. During ischemic tissue damage such as stroke, peripheral immune cells acutely infiltrate the brain and may exacerbate neurodegeneration. Whether and how microglia can protect from this insult is unknown. Polymorphonuclear neutrophils (PMNs) are a prominent immunologic infiltrate of ischemic lesions in vivo. Here, we show in organotypic brain slices that externally applied invading PMNs massively enhance ischemic neurotoxicity. This, however, is counteracted by additional application of microglia. Time-lapse imaging shows that microglia exert protection by rapid engulfment of apoptotic, but, strikingly, also viable, motile PMNs in cell culture and within brain slices. PMN engulfment is mediated by integrin- and lectin-based recognition. Interference with this process using RGDS peptides and N-acetyl-glucosamine blocks engulfment of PMNs and completely abrogates the neuroprotective function of microglia. Thus, engulfment of invading PMNs by microglia may represent an entirely new mechanism of CNS immune privilege

Bruton's tyrosine kinase inhibition attenuates disease progression by reducing renal immune cell invasion in mice with hemolytic-uremic syndrome

Hemolytic-uremic syndrome (HUS) can occur as a complication of an infection with Shiga-toxin (Stx)-producing Escherichia coli . Patients typically present with acute kidney injury, microangiopathic hemolytic anemia and thrombocytopenia. There is evidence that Stx-induced renal damage propagates a pro-inflammatory response. To date, therapy is limited to organ-supportive strategies. Bruton’s tyrosine kinase (BTK) plays a pivotal role in recruitment and function of immune cells and its inhibition was recently shown to improve renal function in experimental sepsis and lupus nephritis. We hypothesized that attenuating the evoked immune response by BTK-inhibitors (BTKi) ameliorates outcome in HUS. We investigated the effect of daily oral administration of the BTKi ibrutinib (30 mg/kg) and acalabrutinib (3 mg/kg) in mice with Stx-induced HUS at day 7. After BTKi administration, we observed attenuated disease progression in mice with HUS. These findings were associated with less BTK and downstream phospholipase-C-gamma-2 activation in the spleen and, subsequently, a reduced renal invasion of BTK-positive cells including neutrophils. Only ibrutinib treatment diminished renal invasion of macrophages, improved acute kidney injury and dysfunction (plasma levels of NGAL and urea) and reduced hemolysis (plasma levels of bilirubin and LDH activity). In conclusion, we report here for the first time that BTK inhibition attenuates the course of disease in murine HUS. We suggest that the observed reduction of renal immune cell invasion contributes – at least in part – to this effect. Further translational studies are needed to evaluate BTK as a potential target for HUS therapy to overcome currently limited treatment options

The infectious propagules of Aspergillus fumigatus are coated with antimicrobial peptides

Fungal spores are unique cells that mediate dispersal and survival in the environment. For pathogenic fungi encountering a susceptible host, these specialised structures may serve as infectious particles. The main causative agent of the opportunistic disease aspergillosis, Aspergillus fumigatus, produces asexual spores, the conidia, that become dissipated by air flows or water currents but also serve as propagules to infect a susceptible host. We demonstrate that the defX gene of this mould encodes putative antimicrobial peptides resembling cysteine‐stabilised (CS)$_{αβ}$ defensins that are expressed in a specific spatial and temporal manner in the course of asexual spore formation. Localisation studies on strains expressing a fluorescent proxy or tagged defX alleles expose that these antimicrobial peptides are secreted to coat the conidial surface. Deletion mutants reveal that the spore‐associated defX gene products delay the growth of Gram‐positive Staphylococcus aureus and demonstrate that the defX gene and presumably its encoded spore‐associated defensins confer a growth advantage to the fungal opponent over bacterial competitors. These findings have implications with respect to the ecological niche of A. fumigatus that serves as a ‘virulence school’ for this human pathogenic mould; further relevance is given for the infectious process resulting in aspergillosis, considering competition with the host microbiome or co‐infecting microorganisms to break colonisation resistance at host surfaces

Grazing protozoa and the evolution of the Escherichia coli O157:H7 Shiga toxin-encoding prophage

Humans play little role in the epidemiology of Escherichia coli O157:H7, a commensal bacterium of cattle. Why then does E. coli O157:H7 code for virulence determinants, like the Shiga toxins (Stxs), responsible for the morbidity and mortality of colonized humans? One possibility is that the virulence of these bacteria to humans is coincidental and these virulence factors evolved for and are maintained for other roles they play in the ecology of these bacteria. Here, we test the hypothesis that the carriage of the Stx-encoding prophage of E. coli O157:H7 increases the rate of survival of E. coli in the presence of grazing protozoa, Tetrahymena pyriformis. In the presence but not the absence of Tetrahymena, the carriage of the Stx-encoding prophage considerably augments the fitness of E. coli K-12 as well as clinical isolates of E. coli O157 by increasing the rate of survival of the bacteria in the food vacuoles of these ciliates. Grazing protozoa in the environment or natural host are likely to play a significant role in the ecology and maintenance of the Stx-encoding prophage of E. coli O157:H7 and may well contribute to the evolution of the virulence of these bacteria to colonize humans

Rapid Immunomagnetic Negative Enrichment of Neutrophil Granulocytes from Murine Bone Marrow for Functional Studies In Vitro and In Vivo

Polymorphonuclear neutrophils (PMN) mediate early immunity to infection but can also cause host damage if their effector functions are not controlled. Their lack or dysfunction is associated with severe health problems and thus the analysis of PMN physiology is a central issue. One prerequisite for PMN analysis is the availability of purified cells from primary organs. While human PMN are easily isolated from peripheral blood, this approach is less suitable for mice due to limited availability of blood. Instead, bone marrow (BM) is an easily available reservoir of murine PMN, but methods to obtain pure cells from BM are limited. We have developed a novel protocol allowing the isolation of highly pure untouched PMN from murine BM by negative immunomagnetic isolation using a complex antibody cocktail. The protocol is simple and fast (∼1 h), has a high yield (5–10*106 PMN per animal) and provides a purity of cells equivalent to positive selection (>80%). Most importantly, cells obtained by this method are non-activated and remain fully functional in vitro or after adoptive transfer into recipient animals. This method should thus greatly facilitate the study of primary murine PMN in vitro and in vivo

Towards Translational ImmunoPET/MR Imaging of Invasive Pulmonary Aspergillosis: The Humanised Monoclonal Antibody JF5 Detects Aspergillus Lung Infections In Vivo

This is the final published versionAvailable from Ivyspring International Publisher via the DOI in this recordInvasive pulmonary aspergillosis (IPA) is a life-threatening lung disease of hematological malignancy and bone marrow transplant patients caused by the ubiquitous environmental fungus Aspergillus fumigatus. Current diagnostic tests for the disease lack sensitivity as well as specificity, and culture of the fungus from invasive lung biopsy, considered the gold standard for IPA detection, is slow and often not possible in critically ill patients. In a previous study, we reported the development of a novel non-invasive procedure for IPA diagnosis based on antibody-guided positron emission tomography and magnetic resonance imaging (immunoPET/MRI) using a [64Cu]DOTA-labeled mouse monoclonal antibody (mAb), mJF5, specific to Aspergillus. To enable translation of the tracer to the clinical setting, we report here the development of a humanised version of the antibody (hJF5), and pre-clinical imaging of lung infection using a [64Cu]NODAGA-hJF5 tracer. The humanised antibody tracer shows a significant increase in in vivo biodistribution in A. fumigatus infected lungs compared to its radiolabeled murine counterpart [64Cu]NODAGA-mJF5. Using reverse genetics of the pathogen, we show that the antibody binds to the antigenic determinant 1,5-galactofuranose (Galf) present in a diagnostic mannoprotein antigen released by the pathogen during invasive growth in the lung. The absence of the epitope Galf in mammalian carbohydrates, coupled with the enhanced imaging capabilities of the hJF5 antibody, means that the [64Cu]NODAGA-hJF5 tracer developed here represents an ideal candidate for the diagnosis of IPA and translation to the clinical setting.This work was supported by the European Union Seventh Framework Programme FP7/2007-2013 under Grant 602820, the Deutsche Forschungsgemeinschaft (Grant WI3777/1-2 to SW), and the Werner Siemens Foundation. We thank Sven Krappman for use of the A. fumigatustdTomato strain, and acknowledge the Imaging Centre Essen (IMCES) for assistance with optical imaging of lungs

Antibody-guided in vivo imaging of Aspergillus fumigatus lung infections during anti-fungal azole treatment

This is the final version. Available on open access from Nature Research via the DOI in this recordData availability: Due to their large size, the raw imaging data that support the findings of this study are directly available from the corresponding authors upon reasonable request. Derived data have been compiled in the Source Data file provided with this paper. Any remaining data supporting the findings from this study are available from the corresponding author upon reasonable request.Invasive pulmonary aspergillosis (IPA) is a life-threatening lung disease of immunocompromised humans, caused by the opportunistic fungal pathogen Aspergillus fumigatus. Inadequacies in current diagnostic procedures mean that early diagnosis of the disease, critical to patient survival, remains a major clinical challenge, and is leading to the empiric use of antifungal drugs and emergence of azole resistance. A non-invasive procedure that allows both unambiguous detection of IPA, and its response to azole treatment, is therefore needed. Here, we show that a humanised Aspergillus-specific monoclonal antibody, dual labelled with a radionuclide and fluorophore, can be used in immunoPET/MRI in vivo and 3D light sheet fluorescence microscopy ex vivo to quantify early A. fumigatus lung infections and to monitor the efficacy of azole therapy. Our antibody-guided approach reveals that early drug intervention is critical to prevent complete invasion of the lungs by the fungus, and demonstrates the power of molecular imaging as a non-invasive procedure for tracking IPA in vivo.Ministry of Culture and Science of North Rhine-WestphaliaGoverning Mayor of Berlin including Science and ResearchFederal Ministry of Education and ResearchEuropean Union FP7Werner Siemens Foundatio

Identification and Characterization of Microcin S, a New Antibacterial Peptide Produced by Probiotic Escherichia coli G3/10

Escherichia coli G3/10 is a component of the probiotic drug Symbioflor 2. In an in vitro assay with human intestinal epithelial cells, E. coli G3/10 is capable of suppressing adherence of enteropathogenic E. coli E2348/69. In this study, we demonstrate that a completely novel class II microcin, produced by probiotic E. coli G3/10, is responsible for this behavior. We named this antibacterial peptide microcin S (MccS). Microcin S is coded on a 50.6 kb megaplasmid of E. coli G3/10, which we have completely sequenced and annotated. The microcin S operon is about 4.7 kb in size and is comprised of four genes. Subcloning of the genes and gene fragments followed by gene expression experiments enabled us to functionally characterize all members of this operon, and to clearly identify the nucleotide sequences encoding the microcin itself (mcsS), its transport apparatus and the gene mcsI conferring self immunity against microcin S. Overexpression of cloned mcsI antagonizes MccS activity, thus protecting indicator strain E. coli E2348/69 in the in vitro adherence assay. Moreover, growth of E. coli transformed with a plasmid containing mcsS under control of an araC PBAD activator-promoter is inhibited upon mcsS induction. Our data provide further mechanistic insight into the probiotic behavior of E. coli G3/10

Antigen-Engaged B Cells Undergo Chemotaxis toward the T Zone and Form Motile Conjugates with Helper T Cells

Interactions between B and T cells are essential for most antibody responses, but the dynamics of these interactions are poorly understood. By two-photon microscopy of intact lymph nodes, we show that upon exposure to antigen, B cells migrate with directional preference toward the B-zone–T-zone boundary in a CCR7-dependent manner, through a region that exhibits a CCR7-ligand gradient. Initially the B cells show reduced motility, but after 1 d, motility is increased to approximately 9 μm/min. Antigen-engaged B cells pair with antigen-specific helper T cells for 10 to more than 60 min, whereas non-antigen-specific interactions last less than 10 min. B cell–T cell conjugates are highly dynamic and migrate extensively, being led by B cells. B cells occasionally contact more than one T cell, whereas T cells are strictly monogamous in their interactions. These findings provide evidence of lymphocyte chemotaxis in vivo, and they begin to define the spatiotemporal cellular dynamics associated with T cell–dependent antibody responses