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

Ischemic stroke and concomitant gastrointestinal complications- a fatal combination for patient recovery

Stroke is primarily a neurodegenerative disease but can also severely impact the functions of other vital organs and deteriorate disease outcomes. A malfunction of the gastrointestinal tract (GIT), commonly observed in stroke patients, is often characterized by severe bowel obstruction, intestinal microbiota changes and inflammation. Over-activated immune cells after stroke are the major contributors to endorse intestinal inflammation and may induce damage to single-layer epithelial cell barriers. The post-stroke leakage of intestinal barriers may allow the translocation and dissemination of resident microflora to systemic organs and cause sepsis. This overshooting systemic immune reaction fuels ongoing inflammation in the degenerating brain and slows recovery. Currently, the therapeutic options to treat these GIT-associated anomalies are very limited and further research is required to develop novel treatments. In this mini-review, we first discuss the current knowledge from clinical studies and experimental stroke models that provide strong evidence of the existence of post-stroke GIT complications. Then, we review the literature regarding novel therapeutic approaches that might help to maintain GIT homeostasis and improve neurological outcomes in stroke patients

Selective Detection of NADPH Oxidase in Polymorphonuclear Cells by Means of NAD(P)H-Based Fluorescence Lifetime Imaging

NADPH oxidase (NOX2) is a multisubunit membrane-bound enzyme complex that, upon assembly in activated cells, catalyses the reduction of free oxygen to its superoxide anion, which further leads to reactive oxygen species (ROS) that are toxic to invading pathogens, for example, the fungus Aspergillus fumigatus. Polymorphonuclear cells (PMNs) employ both nonoxidative and oxidative mechanisms to clear this fungus from the lung. The oxidative mechanisms mainly depend on the proper assembly and function of NOX2. We identified for the first time the NAD(P)H-dependent enzymes involved in such oxidative mechanisms by means of biexponential NAD(P)H-fluorescence lifetime imaging (FLIM). A specific fluorescence lifetime of 3670±140 picoseconds as compared to 1870 picoseconds for NAD(P)H bound to mitochondrial enzymes could be associated with NADPH bound to oxidative enzymes in activated PMNs. Due to its predominance in PMNs and due to the use of selective activators and inhibitors, we strongly believe that this specific lifetime mainly originates from NOX2. Our experiments also revealed the high site specificity of the NOX2 assembly and, thus, of the ROS production as well as the dynamic nature of these phenomena. On the example of NADPH oxidase, we demonstrate the potential of NAD(P)H-based FLIM in selectively investigating enzymes during their cellular function

Impact of the rpoS genotype for acid resistance patterns of pathogenic and probiotic Escherichia coli

BACKGROUND: Enterohemorrhagic E. coli (EHEC), a subgroup of Shiga toxin (Stx) producing E. coli (STEC), may cause severe enteritis and hemolytic uremic syndrome (HUS) and is transmitted orally via contaminated foods or from person to person. The infectious dose is known to be very low, which requires most of the bacteria to survive the gastric acid barrier. Acid resistance therefore is an important mechanism of EHEC virulence. It should also be a relevant characteristic of E. coli strains used for therapeutic purposes such as the probiotic E. coli Nissle 1917 (EcN). In E. coli and related enteric bacteria it has been extensively demonstrated, that the alternative sigma factor σ(S), encoded by the rpoS gene, acts as a master regulator mediating resistance to various environmental stress factors. METHODS: Using rpoS deletion mutants of a highly virulent EHEC O26:H11 patient isolate and the sequenced prototype EHEC EDL933 (ATCC 700927) of serotype O157:H7 we investigated the impact of a functional rpoS gene for orchestrating a satisfactory response to acid stress in these strains. We then functionally characterized rpoS of probiotic EcN and five rpoS genes selected from STEC isolates pre-investigated for acid resistance. RESULTS: First, we found out that ATCC isolate 700927 of EHEC EDL933 has a point mutation in rpoS, not present in the published sequence, leading to a premature stop codon. Moreover, to our surprise, one STEC strain as well as EcN was acid sensitive in our test environment, although their cloned rpoS genes could effectively complement acid sensitivity of an rpoS deletion mutant. CONCLUSION: The attenuation of sequenced EHEC EDL933 might be of importance for anyone planning to do either in vitro or in vivo studies with this prototype strain. Furthermore our data supports recently published observations, that individual E. coli isolates are able to significantly modulate their acid resistance phenotype independent of their rpoS genotype

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

Neutrophils self-limit swarming to contain bacterial growth in vivo

Neutrophils communicate with each other to form swarms in infected organs. Coordination of this population response is critical for the elimination of bacteria and fungi. Using transgenic mice, we found that neutrophils have evolved an intrinsic mechanism to self-limit swarming and avoid uncontrolled aggregation during inflammation. G protein–coupled receptor (GPCR) desensitization acts as a negative feedback control to stop migration of neutrophils when they sense high concentrations of self-secreted attractants that initially amplify swarming. Interference with this process allows neutrophils to scan larger tissue areas for microbes. Unexpectedly, this does not benefit bacterial clearance as containment of proliferating bacteria by neutrophil clusters becomes impeded. Our data reveal how autosignaling stops self-organized swarming behavior and how the finely tuned balance of neutrophil chemotaxis and arrest counteracts bacterial escape

Targeting the innate repair receptor axis via erythropoietin or pyroglutamate helix B surface peptide attenuates hemolytic-uremic syndrome in mice

Hemolytic-uremic syndrome (HUS) can occur as a systemic complication of infections with Shiga toxin (Stx)-producing Escherichia coli and is characterized by microangiopathic hemolytic anemia and acute kidney injury. Hitherto, therapy has been limited to organ-supportive strategies. Erythropoietin (EPO) stimulates erythropoiesis and is approved for the treatment of certain forms of anemia, but not for HUS-associated hemolytic anemia. EPO and its non-hematopoietic analog pyroglutamate helix B surface peptide (pHBSP) have been shown to mediate tissue protection via an innate repair receptor (IRR) that is pharmacologically distinct from the erythropoiesis-mediating receptor (EPO-R). Here, we investigated the changes in endogenous EPO levels in patients with HUS and in piglets and mice subjected to preclinical HUS models. We found that endogenous EPO was elevated in plasma of humans, piglets, and mice with HUS, regardless of species and degree of anemia, suggesting that EPO signaling plays a role in HUS pathology. Therefore, we aimed to examine the therapeutic potential of EPO and pHBSP in mice with Stx-induced HUS. Administration of EPO or pHBSP improved 7-day survival and attenuated renal oxidative stress but did not significantly reduce renal dysfunction and injury in the employed model. pHBSP, but not EPO, attenuated renal nitrosative stress and reduced tubular dedifferentiation. In conclusion, targeting the EPO-R/IRR axis reduced mortality and renal oxidative stress in murine HUS without occurrence of thromboembolic complications or other adverse side effects. We therefore suggest that repurposing EPO for the treatment of patients with hemolytic anemia in HUS should be systematically investigated in future clinical trials

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