Ulrich Gärtner

Harbour seal (Phoca vitulina) PMN and monocytes release extracellular traps to capture the apicomplexan parasite Toxoplasma gondii.

Extracellular traps (ETs) are composed of nuclear DNA as backbone adorned with histones, cytoplasmic antimicrobial peptides/proteins which are released from a range of vertebrate and invertebrate host immune cells in response to several invading pathogens. Until now this ancient novel innate defence mechanism has not been demonstrated in any marine mammal. Interactions of harbour seal (Phoca vitulina)-PMN and -monocytes with viable tachyzoites of Toxoplasma gondii were investigated in this respect in vitro. For the demonstration and quantification of harbour seal PMN- and monocyte-derived ETs, extracellular DNA was stained with Sytox Orange. Fluorescence assays as well as scanning electron microscopy (SEM) analyses demonstrated PMN- and monocyte-promoted ET formation rapidly being induced upon contact with T. gondii-tachyzoites. The co-localisation of extracellular DNA decorated with histones (H3), neutrophil elastase (NE) and myeloperoxidase (MPO) in parasite entrapping structures confirmed the classical characteristics of PMN- and monocyte-promoted ETs. Exposure of harbour seal PMN and monocytes to viable tachyzoites resulted in a significant induction of ETs when compared to negative controls. Harbour seal-ETs were efficiently abolished by DNase I treatment and were reduced after PMN and monocytes pre-incubation with the NADPH oxidase inhibitor diphenilane iodondium. Tachyzoites of T. gondii were firmly entrapped and immobilised within harbour seal-ET structures. To our best knowledge, we here report for the first time on T. gondii-induced ET formation in harbour seal-PMN and -monocytes. Our results strongly indicate that PMN- and monocyte-triggered ETs represent a relevant and ancient conserved effector mechanism of the pinniped innate immune system as reaction against the pathogenic protozoon T. gondii and probably against other foreign pathogens occurring in the ocean environment.

Gastropod-derived haemocyte extracellular traps entrap metastrongyloid larval stages of Angiostrongylus vasorum , Aelurostrongylus abstrusus and Troglostrongylus brevior

BackgroundPhagocyte-derived extracellular traps (ETs) were recently demonstrated mainly in vertebrate hosts as an important effector mechanism against invading parasites. In the present study we aimed to characterize gastropod-derived invertebrate extracellular phagocyte trap (InEPT) formation in response to larval stages of important canine and feline metastrongyloid lungworms. Gastropod haemocytes were isolated from the slug species Arion lusitanicus and Limax maximus, and the snail Achatina fulica, and exposed to larval stages of Angiostrongylus vasorum, Aelurostrongylus abstrusus and Troglostrongylus brevior and investigated for gastropod-derived InEPT formation.ResultsPhase contrast as well as scanning electron microscopy (SEM) analyses of lungworm larvae-exposed haemocytes revealed ET-like structures to be extruded by haemocytes thereby contacting and ensnaring the parasites. Co-localization studies of haemocyte-derived extracellular DNA with histones and myeloperoxidase in larvae-entrapping structures confirmed classical characteristics of ETs. In vivo exposure of slugs to A. vasorum larvae resulted in InEPTs being extruded from haemocytes in the slug mucous extrapallial space emphasizing the pivotal role of this effector mechanism against invasive larvae. Functional larval entrapment assays demonstrated that almost half of the haemocyte-exposed larvae were contacted or even immobilized by released InEPTs. Overall, as reported for mammalian-derived ETs, different types of InEPTs were here observed, i.e. aggregated, spread and diffused InEPTs.ConclusionsTo our knowledge, this study represents the first report on metastrongyloid lungworm-triggered ETosis in gastropods thereby providing evidence of early mollusc host innate immune reactions against invading larvae. These findings will contribute to the better understanding on complex parasite-intermediate host interactions since different gastropod species bear different transmitting capacities for metastrongyloid infections.

Bovine Polymorphonuclear Neutrophils Cast Neutrophil Extracellular Traps against the Abortive Parasite Neospora caninum

Neospora caninum represents a relevant apicomplexan parasite causing severe reproductive disorders in cattle worldwide. Neutrophil extracellular trap (NET) generation was recently described as an efficient defense mechanism of polymorphonuclear neutrophils (PMN) acting against different parasites. In vitro interactions of bovine PMN with N. caninum were analyzed at different ratios and time spans. Extracellular DNA staining was used to illustrate the typical molecules of NETs [i.e., histones (H3), neutrophil elastase (NE), myeloperoxidase (MPO), pentraxin] via antibody-based immunofluorescence analyses. Functional inhibitor treatments were applied to reveal the role of several enzymes [NADPH oxidase (NOX), NE, MPO, PAD4], ATP-dependent P2Y2 receptor, store-operated Ca++entry (SOCE), CD11b receptor, ERK1/2- and p38 MAPK-mediated signaling pathway in tachyzoite-triggered NETosis. N. caninum tachyzoites triggered NETosis in a time- and dose-dependent manner. Scanning electron microscopy analyses revealed NET structures being released by bovine PMN and entrapping tachyzoites. N. caninum-induced NET formation was found not to be NOX-, NE-, MPO-, PAD4-, ERK1/2-, and p38 MAP kinase-dependent process since inhibition of these enzymes led to a slight decrease of NET formation. CD11b was also identified as a neutrophil receptor being involved in NETosis. Furthermore, N. caninum-triggered NETosis depends on Ca++ influx as well as neutrophil metabolism since both the inhibition of SOCE and of P2Y2-mediated ATP uptake diminished NET formation. Host cell invasion assays indicated that PMN-derived NETosis hampered tachyzoites from active host cell invasion, thereby inhibiting further intracellular replication. NET formation represents an early and effective mechanism of response of the innate immune system, which might reduce initial infection rates during the acute phase of cattle neosporosis.

Müller glial cells of the primate foveola: An electron microscopical study

Abstract Previous studies on the ultrastructure of the primate foveola suggested the presence of an inverted cone‐like structure which is formed by 25–35 specialized Müller cells overlying the area of high photoreceptor density. We investigated the ultrastructure of the Müller cells in the foveola of a human and macaque retina. Sections through the posterior poles of an eye of a 40 years‐old human donor and an eye of an adult cynomolgus monkey (Macaca fascicularis) were investigated with transmission electron microscopy. The foveola consisted of an inner layer (thickness, 5.5–12 &mgr;m) which mainly contained somata (including nuclei) and inner processes of Müller cells; this layer overlaid the central Henle fibers and outer nuclear layer. The inner layer contained numerous watery cysts and thin lamelliform and tubular Müller cell processes which spread along the inner limiting membrane (ILM). The cytoplasm of the outer Müller cell processes became increasingly dispersed and electron‐lucent in the course towards the outer limiting membrane. The ILM of the foveola was formed by a very thin basal lamina (thickness, <40 nm) while the basal lamina of the parafovea was thick (0.9–1 &mgr;m). The data show that there are various conspicuous features of foveolar Müller cells. The numerous thin Müller cell processes below the ILM may smooth the inner surface of the foveola (to minimize image distortion resulting from varying light refraction angles at an uneven retinal surface), create additional barriers to the vitreous cavity (compensating the thinness of the ILM), and provide mechanical stability to the tissue. The decreasing density of the outer process cytoplasm may support the optical function of the foveola. HighlightsThe human and macaque foveola contains specialized Müller cells.The basal lamina of the ILM is very thin in the foveola.The inner foveolar layer contains somata and thin processes of the Müller cells.The outer Müller cell processes are increasingly electron‐lucent near the OLM.Foveolar Müller cells may have optical, barrier, and structural functions.

Novel approach to study gastropod-mediated innate immune reactions against metastrongyloid parasites

The anthropozoonotic metastrongyloid nematodes Angiostrongylus cantonensis and Angiostrongylus costaricensis, as well as Angiostrongylus vasorum, Crenosoma vulpis, Aelurostrongylus abstrusus and Troglostrongylus brevior are currently considered as emerging gastropod-borne parasites and have gained growing scientific attention in the last years. However, the knowledge on invertebrate immune responses and on how metastrongyloid larvae are attacked by gastropod immune cells is still limited. This work aims to describe an in vitro system to investigate haemocyte-derived innate immune responses of terrestrial gastropods induced by vital axenic metastrongyloid larvae. We also provide protocols on slug/snail management and breeding under standardized climate conditions (circadian cycle, temperature and humidity) for the generation of parasite-free F0 stages which are essential for immune-related investigations. Adult slug species (Arion lusitanicus, Limax maximus) and giant snails (Achatina fulica) were maintained in fully automated climate chambers until mating and production of fertilized eggs. Newly hatched F0 juvenile specimens were kept under parasite-free conditions before experimental use. An improved protocol for gastropod haemolymph collection and haemocyte isolation was established. Giemsa-stained haemolymph preparations showed adequate haemocyte isolation in all three gastropod species. Additionally, a protocol for the production of axenic first and third stage larvae (L1, L3) was established. Haemocyte functionality was tested in haemocyte-nematode-co-cultures. Scanning electron microscopy (SEM) and light microscopy analyses revealed that gastropod-derived haemocytes formed clusters as well as DNA-rich extracellular aggregates catching larvae and decreasing their motility. These data confirm the usefulness of the presented methods to study haemocyte-mediated gastropod immune responses to better understand the complex biology of gastropod-borne diseases.

Metabolic Maturation during Muscle Stem Cell Differentiation Is Achieved by miR-1/133a-Mediated Inhibition of the Dlk1-Dio3 Mega Gene Cluster

Muscle stem cells undergo a dramatic metabolic switch to oxidative phosphorylation during differentiation, which is achieved by massively increased mitochondrial activity. Since expression of the muscle-specific miR-1/133a gene cluster correlates with increased mitochondrial activity during muscle stem cell (MuSC) differentiation, we examined the potential role of miR-1/133a in metabolic maturation of skeletal muscles in mice. We found that miR-1/133a downregulate Mef2A in differentiated myocytes, thereby suppressing the Dlk1-Dio3 gene cluster, which encodes multiple microRNAs inhibiting expression of mitochondrial genes. Loss of miR-1/133a in skeletal muscles or increased Mef2A expression causes continuous high-level expression of the Dlk1-Dio3 gene cluster, compromising mitochondrial function. Failure to terminate the stem cell-like metabolic program characterized by high-level Dlk1-Dio3 gene cluster expression initiates profound changes in muscle physiology, essentially abrogating endurance running. Our results suggest a major role of miR-1/133a in metabolic maturation of skeletal muscles but exclude major functions in muscle development and MuSC maintenance.

Molecular analyses on Neospora caninum-triggered NETosis in the caprine system

Abstract Neospora caninum is an obligate intracellular protozoan parasite causing serious reproductive disorders in large and small ruminants worldwide. Polymorphonuclear neutrophils (PMN) react against multiple invading pathogens through different mechanisms including the release of neutrophil extracellular traps (NETs). Here, in vitro interactions of caprine PMN and N. caninum tachyzoites were studied. Scanning electron microscopic‐ and immunofluorescence‐analyses demonstrated that caprine PMN undergo NETosis upon contact with tachyzoites of N. caninum, extruding filaments that entrap parasites. Detailed co‐localization studies of N. caninum tachyzoite‐induced NETs revealed the presence of PMN‐derived DNA being decorated with histones (H1, H2A/H2B, H3,H4) and neutrophil elastase (NE) corroborating the molecular characteristics of classical mammalian NETs. As a new result for parasite‐induced NETosis, we identified pentraxin and cathepsin B in N. caninum‐triggered NETs. Nonetheless, functional inhibition assays revealed that during caprine NET formation triggered by N. caninum different molecular signaling pathways are induced, when compared to other apicomplexan parasites or host species. As such, N. caninum‐induced NETosis appears to be influenced by MPO but independent of NADPH oxidase, SOCE, ERK1/2 and p38 MAPK activities. Furthermore, the inhibition of PMN autophagy via blockage of the PI3K‐mediated signaling pathway failed to influence tachyzoite‐induced NETosis. Since N. caninum‐tachyzoites induced caprine NETosis, this effector mechanism should be considered as an early host immune response during acute caprine neosporosis. HighlightsCaprine PMN‐stimulated NETs were rapidly generated upon contact with N. caninum.Histones, neutrophil elastase, myeloperoxidase, pentraxin and cathepsin B were involved in N. caninum‐triggered NETs.N. caninum‐induced NETosis in goats is a NOX‐, SOCE‐, ERK1/2‐ and p38 MAPK –independent process.Suppression of PMN autophagy via PI3K‐blockage failed to influence NETosis.

Inhomogeneous Distribution of Alzheimer Pathology Along the Isocortical Relief – Are Cortical Convolutions an Achilles Heel of Evolution?

Alzheimers disease (AD) is neuropathologically characterized by neuritic plaques and neurofibrillary tangles. Progression of both plaques and tangles throughout the brain follows a hierarchical distribution which is defined by intrinsic cytoarchitectonic features and extrinsic connectivity patterns. What has less well been studied is how cortical convolutions influence the distribution of AD pathology. Here, the distribution of both plaques and tangles within subsulcal gyral components (fundi) to components forming their top regions at the subarachnoidal brain surface (crowns) by stereological methods in seven different cortical areas was systematically compared. Further, principle differences in cytoarchitectonic organization of cortical crowns and fundi that might provide the background for regionally selective vulnerability were attempted to identify. It was shown that both plaques and tangles were more prominent in sulcal fundi than gyri crowns. The differential distribution of pathology along convolutions corresponds to subgyral differences in the vascular network, GFAP‐positive astrocytes and intracortical and subcortical connectivity. While the precise mechanisms accounting for these differences remain open, the presence of systematic inhomogeneities in the distribution of AD pathology along cortical convolutions indicates that the phylogenetic shaping of the cortex is associated with features that render the human brain vulnerable to AD pathology.

Bordetella pseudohinzii targets cilia and impairs tracheal cilia-driven transport in naturally acquired infection in mice

Several species of the Gram-negative genus Bordetella are the cause of respiratory infections in mammals and birds, including whooping cough (pertussis) in humans. Very recently, a novel atypical species, Bordetella pseudohinzii, was isolated from laboratory mice. These mice presented no obvious clinical symptoms but elevated numbers of neutrophils in bronchoalveolar lavage fluid and inflammatory signs in histopathology. We noted that this species can occur at high prevalence in a mouse facility despite regular pathogen testing according to the FELASA-recommendations. Affected C57BL/6 J mice had, in addition to the reported pulmonary alterations, tracheal inflammation with reduced numbers of ciliated cells, slower ciliary beat frequency, and largely (>50%) compromised cilia-driven particle transport speed on the mucosal surface, a primary innate defence mechanism. In an in vitro-model, Bordetella pseudohinzii attached to respiratory kinocilia, impaired ciliary function within 4 h and caused epithelial damage within 24 h. Regular testing for this ciliotropic Bordetella species and excluding it from colonies that provide mice for lung research shall be recommended. On the other hand, controlled colonization and infection with Bordetella pseudohinzii may serve as an experimental model to investigate mechanisms of mucociliary clearance and microbial strategies to escape from this primary innate defence response.

Dirofilaria immitis Microfilariae and Third-Stage Larvae Induce Canine NETosis Resulting in Different Types of Neutrophil Extracellular Traps

Heartworm disease is a zoonotic vector-borne disease caused by Dirofilaria immitis mainly affecting canids. Infectious third-stage larvae (L3) are transmitted to the definitive hosts via culicid mosquitoes; adult nematodes reside in the pulmonary arteries and in the right heart releasing unsheathed first-stage larvae (microfilariae) into the bloodstream leading to chronic and sometimes fatal disease. So far, early innate immune reactions triggered by these different D. immitis stages in the canine host have scarcely been investigated. Therefore, D. immitis microfilariae and L3 were analyzed for their capacity to induce neutrophil extracellular traps (NETs) in canine polymorphonuclear neutrophils (PMN). Overall, scanning electron microscopy analysis revealed both larval stages as strong inducers of canine NETosis. Co-localization of PMN-derived extracellular DNA with granulocytic histones, neutrophil elastase, or myeloperoxidase in parasite-entrapping structures confirmed the classical characteristics of NETosis. Quantitative analyses showed that both larval stages triggered canine NETs in a time-dependent but dose-independent manner. Moreover, parasite-induced NET formation was not influenced by the parasites viability since heat-inactivated microfilariae and L3 also induced NETs. In addition, parasite/PMN confrontation promoted significant entrapment but not killing of microfilariae and L3. Both, NETosis and larval entrapment was significantly reversed via DNase I treatments while treatments with the NADPH oxidase inhibitor diphenyleneiodonium failed to significantly influence these reactions. Interestingly, different types of NETs were induced by microfilariae and L3 since microfilarial stages merely induced spread and diffuse NETs while the larger L3 additionally triggered aggregated NET formation.