Martin Krueger

Origin, fate and dynamics of macrophages at central nervous system interfaces

Perivascular, subdural meningeal and choroid plexus macrophages are non-parenchymal macrophages that mediate immune responses at brain boundaries. Although the origin of parenchymal microglia has recently been elucidated, much less is known about the precursors, the underlying transcriptional program and the dynamics of the other macrophages in the central nervous system (CNS). It was assumed that they have a high turnover from blood-borne monocytes. However, using parabiosis and fate-mapping approaches in mice, we found that CNS macrophages arose from hematopoietic precursors during embryonic development and established stable populations, with the notable exception of choroid plexus macrophages, which had dual origins and a shorter life span. The generation of CNS macrophages relied on the transcription factor PU.1, whereas the MYB, BATF3 and NR4A1 transcription factors were not required.

Blood-Brain Barrier Breakdown after Embolic Stroke in Rats Occurs without Ultrastructural Evidence for Disrupting Tight Junctions

The term blood-brain barrier (BBB) relates to the ability of cerebral vessels to hold back hydrophilic and large molecules from entering the brain, thereby crucially contributing to brain homeostasis. In fact, experimental opening of endothelial tight junctions causes a breakdown of the BBB evidenced as for instance by albumin leakage. This and similar observations led to the conclusion that BBB breakdown is predominantly mediated by damage to tight junction complexes, but evidentiary ultrastructural data are rare. Since functional deficits of the BBB contribute to an increased risk of hemorrhagic transformation and brain edema after stroke, which both critically impact on the clinical outcome, we studied the mechanism of BBB breakdown using an embolic model of focal cerebral ischemia in Wistar rats to closely mimic the essential human pathophysiology. Ischemia-induced BBB breakdown was detected using intravenous injection of FITC-albumin and tight junctions in areas of FITC-albumin extravasation were subsequently studied using fluorescence and electron microscopy. Against our expectation, 25 hours after ischemia induction the morphology of tight junction complexes (identified ultrastructurally and using antibodies against the transcellular proteins occludin and claudin-5) appeared to be regularly maintained in regions where FITC-albumin massively leaked into the neuropil. Furthermore, occludin signals along pan-laminin-labeled vessels in the affected hemisphere equaled the non-affected contralateral side (ratio: 0.966 vs. 0.963; P = 0.500). Additional ultrastructural analyses at 5 and 25 h after ischemia induction clearly indicated FITC-albumin extravasation around vessels with intact tight junctions, while the endothelium exhibited enhanced transendothelial vesicle trafficking and signs of degeneration. Thus, BBB breakdown and leakage of FITC-albumin cannot be correlated with staining patterns for common tight junction proteins alone. Understanding the mechanisms causing functional endothelial alterations and endothelial damage is likely to provide novel protective targets in stroke.

Structure of a methyl-coenzyme M reductase from Black Sea mats that oxidize methane anaerobically.

The anaerobic oxidation of methane (AOM) with sulphate, an area currently generating great interest in microbiology, is accomplished by consortia of methanotrophic archaea (ANME) and sulphate-reducing bacteria. The enzyme activating methane in methanotrophic archaea has tentatively been identified as a homologue of methyl-coenzyme M reductase (MCR) that catalyses the methane-forming step in methanogenic archaea. Here we report an X-ray structure of the 280 kDa heterohexameric ANME-1 MCR complex. It was crystallized uniquely from a protein ensemble purified from consortia of microorganisms collected with a submersible from a Black Sea mat catalysing AOM with sulphate. Crystals grown from the heterogeneous sample diffract to 2.1 Å resolution and consist of a single ANME-1 MCR population, demonstrating the strong selective power of crystallization. The structure revealed ANME-1 MCR in complex with coenzyme M and coenzyme B, indicating the same substrates for MCR from methanotrophic and methanogenic archaea. Differences between the highly similar structures of ANME-1 MCR and methanogenic MCR include a F430 modification, a cysteine-rich patch and an altered post-translational amino acid modification pattern, which may tune the enzymes for their functions in different biological contexts.

Effector T-cell trafficking between the leptomeninges and the cerebrospinal fluid

In multiple sclerosis, brain-reactive T cells invade the central nervous system (CNS) and induce a self-destructive inflammatory process. T-cell infiltrates are not only found within the parenchyma and the meninges, but also in the cerebrospinal fluid (CSF) that bathes the entire CNS tissue. How the T cells reach the CSF, their functionality, and whether they traffic between the CSF and other CNS compartments remains hypothetical. Here we show that effector T cells enter the CSF from the leptomeninges during Lewis rat experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis. While moving through the three-dimensional leptomeningeal network of collagen fibres in a random Brownian walk, T cells were flushed from the surface by the flow of the CSF. The detached cells displayed significantly lower activation levels compared to T cells from the leptomeninges and CNS parenchyma. However, they did not represent a specialized non-pathogenic cellular sub-fraction, as their gene expression profile strongly resembled that of tissue-derived T cells and they fully retained their encephalitogenic potential. T-cell detachment from the leptomeninges was counteracted by integrins VLA-4 and LFA-1 binding to their respective ligands produced by resident macrophages. Chemokine signalling via CCR5/CXCR3 and antigenic stimulation of T cells in contact with the leptomeningeal macrophages enforced their adhesiveness. T cells floating in the CSF were able to reattach to the leptomeninges through steps reminiscent of vascular adhesion in CNS blood vessels, and invade the parenchyma. The molecular/cellular conditions for T-cell reattachment were the same as the requirements for detachment from the leptomeningeal milieu. Our data indicate that the leptomeninges represent a checkpoint at which activated T cells are licensed to enter the CNS parenchyma and non-activated T cells are preferentially released into the CSF, from where they can reach areas of antigen availability and tissue damage.

Blood–brain barrier breakdown involves four distinct stages of vascular damage in various models of experimental focal cerebral ischemia

Ischemic stroke not only impairs neuronal function but also affects the cerebral vasculature as indicated by loss of blood—brain barrier (BBB) integrity. Therefore, therapeutical recanalization includes an enhanced risk for hemorrhagic transformation and bleeding, traditionally attributed to a ‘reperfusion injury’. To investigate the mechanisms underlying ischemia-/reperfusion-related BBB opening, we applied multiple immunofluorescence labeling and electron microscopy in a rat model of thromboembolic stroke as well as mouse models of permanent and transient focal cerebral ischemia. In these models, areas exhibiting BBB breakdown were identified by extravasation of intravenously administered fluorescein isothiocyanate (FITC)-albumin. After 24 hours, expression of markers for tight and adherens junctions in areas of FITC-albumin leakage consistently remained unaltered in the applied models. However, lectin staining with isolectin B4 indicated structural alterations in the endothelium, which were confirmed by electron microscopy. While ultrastructural alterations in endothelial cells did not differ between the applied models including the reperfusion scenario, we regularly identified vascular alterations, which we propose to reflect four distinct stages of BBB breakdown with ultimate loss of endothelial cells. Therefore, our data strongly suggest that ischemia-related BBB failure is predominantly caused by endothelial degeneration. Thus, protecting endothelial cells may represent a promising therapeutical approach in addition to the established recanalizing strategies.

A novel microglial subset plays a key role in myelinogenesis in developing brain

Microglia are resident macrophages of the central nervous system that contribute to homeostasis and neuroinflammation. Although known to play an important role in brain development, their exact function has not been fully described. Here, we show that in contrast to healthy adult and inflammation‐activated cells, neonatal microglia show a unique myelinogenic and neurogenic phenotype. A CD11c+ microglial subset that predominates in primary myelinating areas of the developing brain expresses genes for neuronal and glial survival, migration, and differentiation. These cells are the major source of insulin‐like growth factor 1, and its selective depletion from CD11c+ microglia leads to impairment of primary myelination. CD11c‐targeted toxin regimens induced a selective transcriptional response in neonates, distinct from adult microglia. CD11c+ microglia are also found in clusters of repopulating microglia after experimental ablation and in neuroinflammation in adult mice, but despite some similarities, they do not recapitulate neonatal microglial characteristics. We therefore identify a unique phenotype of neonatal microglia that deliver signals necessary for myelination and neurogenesis.

The blood-brain barrier.

The concept of a blood-brain barrier (BBB) dates back to experiments performed by Paul Ehrlich. Using “intravital tracers” which change their color depending on their oxidative state, he intended to estimate the oxygen consumption of the bodily organs. An important prerequisite of this approach, however, would have been an equal distribution of these tracers at the beginning of the experiment, but this was not what he found: Hydrophilic dyes uniformly did not reach the parenchyma, which led his student, the Berlin physician Lewandowski to claim that the capillary wall provides a barrier for certain molecules in the brain, but it was not before the golden era of electron microscopy that Reese and Karnovsky detected what they called “morphological barriers” of the BBB. In this article, we provide an overview of what maintains barrier function for blood-molecules, clarify that a BBB for solutes is neither mechanistically equal to a barrier for immune cells nor in regard to the sites of entry (capillaries versus post-capillary venules), formulate areas of lack of knowledge and consequently, raise open questions to be addressed in the future.

CD11c-positive cells from brain, spleen, lung, and liver exhibit site-specific immune phenotypes and plastically adapt to new environments.

The brains immune privilege has been also attributed to the lack of dendritic cells (DC) within its parenchyma and the adjacent meninges, an assumption, which implies maintenance of antigens rather than their presentation in lymphoid organs. Using mice transcribing the green fluorescent protein under the promoter of the DC marker CD11c (itgax), we identified a juxtavascular population of cells expressing this DC marker and demonstrated their origin from bone marrow and local microglia. We now phenotypically compared this population with CD11c/CD45 double‐positive cells from lung, liver, and spleen in healthy mice using seven‐color flow cytometry. We identified unique, site‐specific expression patterns of F4/80, CD80, CD86, CX3CR1, CCR2, FLT3, CD103, and MHC‐II. Furthermore, we observed the two known CD45‐positive populations (CD45high and CD45int) in the brain, whereas liver, lung, and spleen exhibited a homogeneous CD45high population. CD11c‐positive microglia lacked MHC‐II expression and CD45high/CD11c‐positive cells from the brain have a lower percentage of MHC‐II‐positive cells. To test whether phenotypical differences are fixed by origin or specifically develop due to environmental factors, we transplanted brain and spleen mononuclear cells on organotypic slice cultures from brain (OHSC) and spleen (OSSC). We demonstrate that adaption and ramification of MHC‐II‐positive splenocytes is paralleled by down‐regulation of MHC‐II, whereas brain‐derived mononuclear cells neither ramified nor up‐regulated MHC‐II in OSSCs. Thus, brain‐derived mononuclear cells maintain their MHC‐II‐negative phenotype within the environment of an immune organ. Intraparenchymal CD11c‐positive cells share immunophenotypical characteristics of DCs from other organs but remain unique for their low MHC‐II expression. GLIA 2015;63:611–625

Stroke-induced blood–brain barrier breakdown along the vascular tree – No preferential affection of arteries in different animal models and in humans:

Stroke-induced blood–brain barrier breakdown promotes complications like cerebral edema and hemorrhagic transformation, especially in association with therapeutical recanalization of occluded vessels. As arteries, capillaries and veins display distinct functional and morphological characteristics, we here investigated patterns of blood–brain barrier breakdown for each segment of the vascular tree in rodent models of embolic, permanent, and transient middle cerebral artery occlusion, added by analyses of human stroke tissue. Twenty-four hours after ischemia induction, loss of blood–brain barrier function towards FITC-albumin was equally observed for arteries, capillaries, and veins in rodent brains. Noteworthy, veins showed highest ratios of leaky vessels, whereas capillaries exhibited the most and arteries the least widespread perivascular tracer extravasation. In contrast, human autoptic stroke tissue exhibited pronounced extravasations of albumin around arteries and veins, while the pericapillary immunoreactivity appeared only faint. Although electron microscopy revealed comparable alterations of the arterial and capillary endothelium throughout the applied animal models, structural loss of arterial smooth muscle cells was only observed in the translationally relevant model of embolic middle cerebral artery occlusion. In light of the so far available concepts of stroke treatment, the consideration of a differential vascular pathophysiology along the cerebral vasculature is likely to allow development of novel effective treatment strategies.

Fatty Acid Oxidation Compensates for Lipopolysaccharide-Induced Warburg Effect in Glucose-Deprived Monocytes

Monocytes enter sites of microbial or sterile inflammation as the first line of defense of the immune system and initiate pro-inflammatory effector mechanisms. We show that activation with bacterial lipopolysaccharide (LPS) induces them to undergo a metabolic shift toward aerobic glycolysis, similar to the Warburg effect observed in cancer cells. At sites of inflammation, however, glucose concentrations are often drastically decreased, which prompted us to study monocyte function under conditions of glucose deprivation and abrogated Warburg effect. Experiments using the Seahorse Extracellular Flux Analyzer revealed that limited glucose supply shifts monocyte metabolism toward oxidative phosphorylation, fueled largely by fatty acid oxidation at the expense of lipid droplets. While this metabolic state appears to provide sufficient energy to sustain functional properties like cytokine secretion, migration, and phagocytosis, it cannot prevent a rise in the AMP/ATP ratio and a decreased respiratory burst. The molecular trigger mediating the metabolic shift and the functional consequences is activation of AMP-activated protein kinase (AMPK). Taken together, our results indicate that monocytes are sufficiently metabolically flexible to perform pro-inflammatory functions at sites of inflammation despite glucose deprivation and inhibition of the LPS-induced Warburg effect. AMPK seems to play a pivotal role in orchestrating these processes during glucose deprivation in monocytes.