A New Methodology for the Oxygen Measurement in Lung Tissue of an Aged Ferret Model Proves Hypoxia During COVID-19.

Oxygen as a key element has a high impact on cellular processes. Infection with a pathogen such as SARS-CoV-2 and following inflammation may lead to hypoxic conditions in tissue that impact cellular responses. To develop optimized translational in vitro models for a better understanding of physiologic and pathophysiologic oxygen conditions, it is a prerequisite to determine oxygen levels generated in vivo. Our study objective was the establishment of an invasive method for oxygen measurements using a luminescence-based microsensor to determine the dissolved oxygen in the lung tissue of ferrets as animal models for SARS-CoV-2 research. In analogy to humans, aged ferrets are more likely to show clinical signs after SARS-CoV-2 infection compared to young animals. To investigate oxygen levels during a respiratory viral infection, we intratracheally infected nine aged (3-year-old) ferrets with SARS-CoV-2. The aged SARS-CoV-2 infected ferrets showed mild to moderate clinical signs associated with prolonged viral RNA shedding until 14 days post infection (dpi). SARS-CoV-2 infected ferrets showed histopathologic lung lesion scores that significantly negatively correlated with oxygen levels in lung tissue. At 4 dpi, oxygen levels in lung tissue were significantly lower (mean %O2 of 3.89 ≙ ≈ 27.78 mmHg) compared to the negative control group (mean %O2 of 8.65 ≙ ≈ 61.4 mmHg). In summary, we succeeded in determining the pathophysiologic oxygen conditions in the lung tissue of aged SARS-CoV-2-infected ferrets. This article is open access and distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives License 4.0 (http://creativecommons.org/licenses/by-nc-nd/4.0/). .

Assessment of associations between neutrophil extracellular trap biomarkers in blood and thrombi in acute ischemic stroke patients.

Inflammation including immunothrombosis by neutrophil extracellular traps (NETs) has important implications in acute ischemic stroke and can affect reperfusion status, susceptibility to stroke associated infections (SAI) as well as functional clinical outcome. NETs were shown to be prevalent in stroke thrombi and NET associated markers were found in stroke patients' blood. However, little is known whether blood derived NET markers reflect the amount of NETs in thrombi. Conclusions from blood derived markers to thrombus composition might open avenues for novel strategies in diagnostic and therapeutic approaches. We prospectively recruited 166 patients with acute ischemic stroke undergoing mechanical thrombectomy between March 2018 and May 2021. Available thrombi (n = 106) were stained for NET markers DNA-histone-1 complexes and myeloperoxidase (MPO). Cell free DNA (cfDNA), deoxyribonuclease (DNase) activity, MPO-histone complexes and a cytokine-panel were measured before thrombectomy and after seven days. Clinical data, including stroke etiology, reperfusion status, SAI and functional outcome after rehabilitation, were collected of all patients. NET markers were present in all thrombi. At onset the median concentration of cfDNA in blood was 0.19 µg/ml increasing to 0.30 µg/ml at 7 days. Median DNase activity at onset was 4.33 pmol/min/ml increasing to 4.96 pmol/min/ml at 7 days. Within thrombi DNA-histone-1 complexes and MPO correlated with each other (ρ = 0.792; p < 0.001). Moreover, our study provides evidence for an association between the amount of NETs and endogenous DNase activity in blood with amounts of NETs in cerebral thrombi. However, these associations need to be confirmed in larger cohorts, to investigate the potential clinical implications for individualized therapeutic and diagnostic approaches in acute ischemic stroke.

Phenotypical changes of satellite glial cells in a murine model of GM1 -gangliosidosis.

Satellite glial cells (SGCs) of dorsal root ganglia (DRG) react in response to various injuries in the nervous system. This study investigates reactive changes within SGCs in a murine model for GM1 -gangliosidosis (GM1 ). DRG of homozygous ß-galactosidase-knockout mice and homozygous C57BL/6 wild-type mice were investigated performing immunostaining on formalin-fixed, paraffin-embedded tissue. A marked upregulation of glial fibrillary acidic protein (GFAP), the progenitor marker nestin and Ki67 within SGCs of diseased mice, starting after 4 months at the earliest GFAP, along with intracytoplasmic accumulation of ganglioside within neurons and deterioration of clinical signs was identified. Interestingly, nestin-positive SGCs were detected after 8 months only. No changes regarding inwardly rectifying potassium channel 4.1, 2, 3-cyclic nucleotide 3-phosphodiesterase, Sox2, doublecortin, periaxin and caspase3 were observed in SGCs. Iba1 was only detected in close vicinity of SGCs indicating infiltrating or tissue-resident macrophages. These results indicate that SGCs of DRG show phenotypical changes during the course of GM1 , characterized by GFAP upregulation, proliferation and expression of a neural progenitor marker at a late time point. This points towards an important role of SGCs during neurodegenerative disorders and supports that SGCs represent a multipotent glial precursor cell line with high plasticity and functionality.

Endogenous Deoxyribonuclease Activity and Cell-Free Deoxyribonucleic Acid in Acute Ischemic Stroke: A Cohort Study.

BACKGROUND: Cell-free DNA (cfDNA) and endogenous deoxyribonuclease activity are opposing mediators and might influence the inflammatory response following acute ischemic stroke. In this cohort study, we investigated the relation between these markers, circulating inflammatory mediators and clinical course including occurrence of stroke-associated infections (SAI) in patients with acute stroke. METHODS: Ninety-two patients with stroke due to large vessel occlusion undergoing mechanical thrombectomy were prospectively recruited at Hannover Medical School from March 2018 to August 2019. Deoxyribonuclease activity, cfDNA, damage-associated molecular patterns, and circulating cytokines were measured in venous blood collected immediately before mechanical thrombectomy and 7 days later. Reperfusion status was categorized (sufficient/insufficient). Clinical outcome was evaluated using the modified Rankin Scale after 90 days, where a score of 3 to 6 was considered unfavorable. To validate findings regarding SAI, another stroke cohort (n=92) was considered with blood taken within 24 hours after stroke onset. RESULTS: Patients with unfavorable clinical outcome had higher cfDNA concentrations. After adjustment for confounders (Essen Stroke Risk Score, National Institutes of Health Stroke Scale, and sex), 7-day cfDNA was independently associated with clinical outcome and especially mortality (adjusted odds ratio: 3.485 [95% CI, 1.001-12.134] and adjusted odds ratio: 9.585 [95% CI, 2.006-45.790]). No association was found between reperfusion status and cfDNA or deoxyribonuclease activity. While cfDNA concentrations correlated positively, deoxyribonuclease activity inversely correlated with distinct biomarkers. Baseline deoxyribonuclease activity was lower in patients who developed SAI compared with patients without SAI. This association was confirmed after adjustment for confounding factors (adjusted odds ratio: 0.447 [95% CI, 0.237-0.844]). In cohort 2, differences of deoxyribonuclease activity between patients with and without SAI tended to be higher with higher stroke severity. CONCLUSIONS: The interplay of endogenous deoxyribonuclease activity and cfDNA in acute stroke entails interesting novel diagnostic and potential therapeutic approaches. We confirm an independent association of cfDNA with a detrimental clinical course after stroke due to large vessel occlusion. This study provides first evidence for lower endogenous deoxyribonuclease activity as risk factor for SAI after severe stroke.

Ferrets are valuable models for SARS-CoV-2 research.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), resulted in an ongoing pandemic with millions of deaths worldwide. Infection of humans can be asymptomatic or result in fever, fatigue, dry cough, dyspnea, and acute respiratory distress syndrome with multiorgan failure in severe cases. The pathogenesis of COVID-19 is not fully understood, and various models employing different species are currently applied. Ferrets can be infected with SARS-CoV-2 and efficiently transmit the virus to contact animals. In contrast to hamsters, ferrets usually show mild disease and viral replication restricted to the upper airways. Most reports have used the intranasal inoculation route, while the intratracheal infection model is not well characterized. Herein, we present clinical, virological, and pathological data from young ferrets intratracheally inoculated with SARS-CoV-2. Infected animals showed no significant clinical signs, and had transient infection with peak viral RNA loads at 4 days postinfection, mild to moderate rhinitis, and pulmonary endothelialitis/vasculitis. Viral antigen was exclusively found in the respiratory epithelium of the nasal cavity, indicating a particular tropism for cells in this location. Viral antigen was associated with epithelial damage and influx of inflammatory cells, including activated neutrophils releasing neutrophil extracellular traps. Scanning electron microscopy of the nasal respiratory mucosa revealed loss of cilia, shedding, and rupture of epithelial cells. The currently established ferret SARS-CoV-2 infection models are comparatively discussed with SARS-CoV-2 pathogenesis in mink, and the advantages and disadvantages of both species as research models for zoonotic betacoronaviruses are highlighted.

New Insights into Neutrophil Extracellular Trap (NETs) Formation from Porcine Neutrophils in Response to Bacterial Infections.

Actinobacillus pleuropneumoniae (A.pp, Gram negative) and Streptococcus (S.) suis (Gram positive) can cause severe diseases in pigs. During infection, neutrophils infiltrate to counteract these pathogens with phagocytosis and/or neutrophil extracellular traps (NETs). NETs consist of a DNA-backbone spiked with antimicrobial components. The NET formation mechanisms in porcine neutrophils as a response to both of the pathogens are not entirely clear. The aim of this study was to investigate whether A.pp (serotype 2, C3656/0271/11) and S. suis (serotype 2, strain 10) induce NETs by NADPH oxidase- or CD18-dependent mechanisms and to characterize phenotypes of NETs in porcine neutrophils. Therefore, we investigated NET induction in porcine neutrophils in the presence and absence of NET inhibitors and quantified NETs after 3 h. Furthermore, NETosis and phagocytosis were investigated by transmission electron microscopy after 30 min to characterize different phenotypes. A.pp and S. suis induce NETs that are mainly ROS-dependent. A.pp induces NETs that are partially CD18-dependent. Thirty minutes after infection, both of the pathogens induced a vesicular NET formation with only slight differences. Interestingly, some neutrophils showed only NET-marker positive phagolysosomes, but no NET-marker positive vesicles. Other neutrophils showed vesicular NETs and only NET-marker negative phagolysosomes. In conclusion, both of the pathogens induce ROS-dependent NETs. Vesicular NETosis and phagocytosis occur in parallel in porcine neutrophils in response to S. suis serotype 2 and A.pp serotype 2.

Phenotypical peculiarities and species-specific differences of canine and murine satellite glial cells of spinal ganglia.

Satellite glial cells (SGCs) are located in the spinal ganglia (SG) of the peripheral nervous system and tightly envelop each neuron. They preserve tissue homeostasis, protect neurons and react in response to injury. This study comparatively characterizes the phenotype of murine (mSGCs) and canine SGCs (cSGCs). Immunohistochemistry and immunofluorescence as well as 2D and 3D imaging techniques were performed to describe a SGC-specific marker panel, identify potential functional subsets and other phenotypical, species-specific peculiarities. Glutamine synthetase (GS) and the potassium channel Kir 4.1 are SGC-specific markers in murine and canine SG. Furthermore, a subset of mSGCs showed CD45 immunoreactivity and the majority of mSGCs were immunopositive for neural/glial antigen 2 (NG2), indicating an immune and a progenitor cell character. The majority of cSGCs were immunopositive for glial fibrillary acidic protein (GFAP), 2',3'-cyclic-nucleotide 3'-phosphodiesterase (CNPase) and Sox2. Therefore, cSGCs resemble central nervous system glial cells and progenitor cells. SGCs lacked expression of macrophage markers CD107b, Iba1 and CD204. Double labelling with GS/Kir 4.1 highlights the unique anatomy of SGC-neuron units and emphasizes the indispensability of further staining and imaging techniques for closer insights into the specific distribution of markers and potential colocalizations.

In vivo oxygen measurement in cerebrospinal fluid of pigs to determine physiologic and pathophysiologic oxygen values during CNS infections.

During infection and inflammation, a reduced oxygen level clearly affects cellular functions. Oxygen levels during CNS infections are unknown. Here we established and evaluated an in vivo measurement system to characterize the oxygen level in parallel with bacterial numbers (CFU/mL), the cell number and pH level inside the CSF of healthy compared to Streptococcus suis-infected pigs. The animals were anesthetized over a seven-hour period with isoflurane in air/oxygen at physiologic arterial partial pressure of oxygen. Oxygen levels in CSF of anesthetized pigs were compared to euthanized pigs. The detected partial pressure of oxygen in the CSF remained constant in a range of 47-63 mmHg, independent of the infection status (bacterial or cell number). In contrast, the pH value showed a slight drop during infection, which correlated with cell and bacterial number in CSF. We present physiologic oxygen and pH values in CSF during the onset of bacterial meningitis.

Ischaemic postconditioning reduces apoptosis in experimental jejunal ischaemia in horses.

BACKGROUND: Ischaemic postconditioning (IPoC) refers to brief periods of reocclusion of blood supply following an ischaemic event. This has been shown to ameliorate ischaemia reperfusion injury in different tissues, and it may represent a feasible therapeutic strategy for ischaemia reperfusion injury following strangulating small intestinal lesions in horses. The objective of this study was to assess the degree cell death, inflammation, oxidative stress, and heat shock response in an equine experimental jejunal ischaemia model with and without IPoC. METHODS: In this randomized, controlled, experimental in vivo study, 14 horses were evenly assigned to a control group and a group subjected to IPoC. Under general anaesthesia, segmental ischaemia with arterial and venous occlusion was induced in 1.5 m jejunum. Following ischaemia, the mesenteric vessels were repeatedly re-occluded in group IPoC only. Full thickness intestinal samples and blood samples were taken at the end of the pre-ischaemia period, after ischaemia, and after 120 min of reperfusion. Immunohistochemical staining or enzymatic assays were performed to determine the selected variables. RESULTS: The mucosal cleaved-caspase-3 and TUNEL cell counts were significantly increased after reperfusion in the control group only. The cleaved-caspase-3 cell count was significantly lower in group IPoC after reperfusion compared to the control group. After reperfusion, the tissue myeloperoxidase activity and the calprotectin positive cell counts in the mucosa were increased in both groups, and only group IPoC showed a significant increase in the serosa. Tissue malondialdehyde and superoxide dismutase as well as blood lactate levels showed significant progression during ischaemia or reperfusion. The nuclear immunoreactivity of Heat shock protein-70 increased significantly during reperfusion. None of these variables differed between the groups. The neuronal cell counts in the myenteric plexus ganglia were not affected by the ischaemia model. CONCLUSIONS: A reduced apoptotic cell count was found in the group subjected to IPoC. None of the other tested variables were significantly affected by IPoC. Therefore, the clinical relevance and possible protective mechanism of IPoC in equine intestinal ischaemia remains unclear. Further research on the mechanism of action and its effect in clinical cases of strangulating colic is needed.

Methylprednisolone Induces Extracellular Trap Formation and Enhances Bactericidal Effect of Canine Neutrophils.

Methylprednisolone is a glucocorticoid and can negatively influence immune defense mechanisms. During bacterial infections in the dog, neutrophils infiltrate infected tissue and mediate antimicrobial effects with different mechanisms such as phagocytosis and neutrophil extracellular trap (NET) formation. Here, we investigated the influence of methylprednisolone on canine NET formation and neutrophil killing efficiency of Gram positive and Gram negative bacteria. Therefore, canine blood derived neutrophils were treated with different concentrations of methylprednisolone over time. The survival factor of Staphylococcus pseudintermedius, Streptococcus canis or Escherichia coli was determined in presence of stimulated neutrophils. Additionally, free DNA and nucleosomes as NET marker were analyzed in supernatants and neutrophils were assessed for NET formation by immunofluorescence microscopy. Methylprednisolone concentrations of 62.5 and 625 µg/mL enhanced the neutrophil killing of Gram positive bacteria, whereas no significant influence was detected for the Gram negative Escherichia coli. Interestingly, higher amounts of free DNA were detected under methylprednisolone stimulation in a concentration dependency and in the presence of Streptococcus canis and Escherichia coli. The nucleosome release by neutrophils is induced by bacterial infection and differs depending on the concentration of methylprednisolone. Furthermore, immunofluorescence microscopy analysis identified methylprednisolone at a concentration of 62.5 µg/mL as a NET inducer. In summary, methylprednisolone enhances NET-formation and time-dependent and concentration-dependent the bactericidal effect of canine neutrophils on Gram positive bacteria.

Mesenchymal to epithelial transition driven by canine distemper virus infection of canine histiocytic sarcoma cells contributes to a reduced cell motility in vitro.

Sarcomas especially of histiocytic origin often possess a poor prognosis and response to conventional therapies. Interestingly, tumours undergoing mesenchymal to epithelial transition (MET) are often associated with a favourable clinical outcome. This process is characterized by an increased expression of epithelial markers leading to a decreased invasion and metastatic rate. Based on the failure of conventional therapies, viral oncolysis might represent a promising alternative with canine distemper virus (CDV) as a possible candidate. This study hypothesizes that a CDV infection of canine histiocytic sarcoma cells (DH82 cells) triggers the MET process leading to a decreased cellular motility. Immunofluorescence and immunoblotting were used to investigate the expression of epithelial and mesenchymal markers followed by scratch assay and an invasion assay as functional confirmation. Furthermore, microarray data were analysed for genes associated with the MET process, invasion and angiogenesis. CDV-infected cells exhibited an increased expression of epithelial markers such as E-cadherin and cytokeratin 8 compared to controls, indicating a MET process. This was accompanied by a reduced cell motility and invasiveness. Summarized, these results suggest that CDV infection of DH82 cells triggers the MET process by an increased expression of epithelial markers resulting in a decreased cell motility in vitro.

Survival of Streptococcus suis in Porcine Blood Is Limited by the Antibody- and Complement-Dependent Oxidative Burst Response of Granulocytes.

Bacteremia is a hallmark of invasive Streptococcus suis infections of pigs, often leading to septicemia, meningitis, or arthritis. An important defense mechanism of neutrophils is the generation of reactive oxygen species (ROS). In this study, we report high levels of ROS production by blood granulocytes after intravenous infection of a pig with high levels of S. suis-specific antibodies and comparatively low levels of bacteremia. This prompted us to investigate the working hypothesis that the immunoglobulin-mediated oxidative burst contributes to the killing of S. suis in porcine blood. Several S. suis strains representing serotypes 2, 7, and 9 proved to be highly susceptible to the oxidative burst intermediate hydrogen peroxide, already at concentrations of 0.001%. The induction of ROS in granulocytes in ex vivo-infected reconstituted blood showed an association with pathogen-specific antibody levels. Importantly, inhibition of ROS production by the NADPH oxidase inhibitor apocynin led to significantly increased bacterial survival in the presence of high specific antibody levels. The oxidative burst rate of granulocytes partially depended on complement activation, as shown by specific inhibition. Furthermore, treatment of IgG-depleted serum with a specific IgM protease or heat to inactivate complement resulted in >3-fold decreased oxidative burst activity and increased bacterial survival in reconstituted porcine blood in accordance with an IgM-complement-oxidative burst axis. In conclusion, this study highlights an important control mechanism of S. suis bacteremia in the natural host: the induction of ROS in blood granulocytes via specific immunoglobulins such as IgM.

Locality, time and heterozygosity affect chytrid infection in yellow-bellied toads.

The chytrid fungus Batrachochytrium dendrobatidis (Bd) infects numerous amphibian species worldwide and is suggested to drive population declines and extinction events. We report a study of Bd infection at the northernmost distribution of the European yellow-bellied toad Bombina variegata. A total of 577 individuals from ponds in 16 study sites were sampled for DNA and Bd throughout the breeding season. Microsatellite genotyping revealed 3 genetic clusters for the host B. variegata with an overall low genetic diversity. One of the clusters displayed a low microsatellite heterozygosity, a high inbreeding coefficient as well as high Bd infection prevalence and intensities. Multi-model estimates identified site, time of sampling, and heterozygosity to be important predictors of an individual's Bd infection status, and identified a strong effect of site on individual Bd infection intensity. The study site effects are suggestive of localized infection peaks, and the increase of individual Bd infection probabilities towards the end of the sampling period suggests cumulative infection during the breeding season. This study highlights the need for regular monitoring of Bd infection variables at multiple localities and times to gain insights into Bd dynamics. Due to the detected relationship between individual Bd infection status and heterozygosity, conservation measures should focus on the maintenance of high genetic diversity and connectivity within and among amphibian populations.

What Is the Evolutionary Fingerprint in Neutrophil Granulocytes?

Over the years of evolution, thousands of different animal species have evolved. All these species require an immune system to defend themselves against invading pathogens. Nevertheless, the immune systems of different species are obviously counteracting against the same pathogen with different efficiency. Therefore, the question arises if the process that was leading to the clades of vertebrates in the animal kingdom-namely mammals, birds, amphibians, reptiles, and fish-was also leading to different functions of immune cells. One cell type of the innate immune system that is transmigrating as first line of defense in infected tissue and counteracts against pathogens is the neutrophil granulocyte. During the host-pathogen interaction they can undergo phagocytosis, apoptosis, degranulation, and form neutrophil extracellular traps (NETs). In this review, we summarize a wide spectrum of information about neutrophils in humans and animals, with a focus on vertebrates. Special attention is kept on the development, morphology, composition, and functions of these cells, but also on dysfunctions and options for cell culture or storage.

Role of Bacterial and Host DNases on Host-Pathogen Interaction during Streptococcus suis Meningitis.

Streptococcus suis is a zoonotic agent causing meningitis in pigs and humans. Neutrophils, as the first line of defense against S. suis infections, release neutrophil extracellular traps (NETs) to entrap pathogens. In this study, we investigated the role of the secreted nuclease A of S. suis (SsnA) as a NET-evasion factor in vivo and in vitro. Piglets were intranasally infected with S. suis strain 10 or an isogenic ssnA mutant. DNase and NET-formation were analyzed in cerebrospinal fluid (CSF) and brain tissue. Animals infected with S. suis strain 10 or S. suis 10ΔssnA showed the presence of NETs in CSF and developed similar clinical signs. Therefore, SsnA does not seem to be a crucial virulence factor that contributes to the development of meningitis in pigs. Importantly, DNase activity was detectable in the CSF of both infection groups, indicating that host nucleases, in contrast to bacterial nuclease SsnA, may play a major role during the onset of meningitis. The effect of DNase 1 on neutrophil functions was further analyzed in a 3D-cell culture model of the porcine blood-CSF barrier. We found that DNase 1 partially contributes to enhanced killing of S. suis by neutrophils, especially when plasma is present. In summary, host nucleases may partially contribute to efficient innate immune response in the CSF.

Optimized cultivation of porcine choroid plexus epithelial cells, a blood-cerebrospinal fluid barrier model, for studying granulocyte transmigration.

The blood-cerebrospinal fluid barrier (BCSFB) plays important roles during the transport of substances into the brain, the pathogenesis of central nervous system (CNS) diseases, and neuro-immunological processes. Along these lines, transmigration of granulocytes across the blood-cerebrospinal fluid (CSF) barrier (BCSFB) is a hallmark of inflammatory events in the CNS. Choroid plexus (CP) epithelial cells are an important tool to generate in vitro models of the BCSFB. A porcine CP epithelial cell line (PCP-R) has been shown to present properties of the BCSFB, including a strong barrier function, when cultivated on cell culture filter inserts containing a membrane with 0.4 µm pore size. For optimal analysis of pathogen and host immune cell interactions with the basolateral side of the CP epithelium, which presents the physiologically relevant "blood side", the CP epithelial cells need to be grown on the lower face of the filter in an inverted cell culture insert model, with the supporting membrane possessing a pore size of at least 3.0 µm. Here, we demonstrate that PCP-R cells cultivated in the inverted model on filter support membranes with a pore size of 3.0 µm following a "conventional" protocol grow through the pores and cross the membrane, forming a second layer on the upper face. Therefore, we developed a cell cultivation protocol, which strongly reduces crossing of the membrane by the cells. Under these conditions, PCP-R cells retain important properties of a BCSFB model, as was observed by the formation of continuous tight junctions and a strong barrier function demonstrated by a high transepithelial electrical resistance and a low permeability for macromolecules. Importantly, compared with the conventional cultivation conditions, our optimized model allows improved investigations of porcine granulocyte transmigration across the PCP-R cell layer.

Neutrophil extracellular trap formation in the Streptococcus suis-infected cerebrospinal fluid compartment.

Streptococcus suis is an important meningitis-causing pathogen in pigs and humans. Neutrophil extracellular traps (NETs) have been identified as host defense mechanism against different pathogens. Here, NETs were detected in the cerebrospinal fluid (CSF) of S. suis-infected piglets despite the presence of active nucleases. To study NET-formation and NET-degradation after transmigration of S. suis and neutrophils through the choroid plexus epithelial cell barrier, a previously described model of the human blood-CSF barrier was used. NETs and respective entrapment of streptococci were recorded in the "CSF compartment" despite the presence of active nucleases. Comparative analysis of S. suis wildtype and different S. suis nuclease mutants did not reveal significant differences in NET-formation or bacterial survival. Interestingly, transcript expression of the human cathelicidin LL-37, a NET-stabilizing factor, increased after transmigration of neutrophils through the choroid plexus epithelial cell barrier. In good accordance, the porcine cathelicidin PR-39 was significantly increased in CSF of piglets with meningitis. Furthermore, we confirmed that PR-39 is associated with NETs in infected CSF and inhibits neutrophil DNA degradation by bacterial nucleases. In conclusion, neutrophils form NETs after breaching the infected choroid plexus epithelium, and those NETs may be protected by antimicrobial peptides against bacterial nucleases.

The inflammatory response and neuronal injury in Streptococcus suis meningitis.

BACKGROUND: Many of the currently used models of bacterial meningitis have limitations due to direct inoculation of pathogens into the cerebrospinal fluid or brain and a relatively insensitive assessment of long-term sequelae. The present study evaluates the utility of a Streptococcus (S.) suis intranasal infection model for the investigation of experimental therapies in meningitis. METHODS: We examined the brains of 10 piglets with S. suis meningitis as well as 14 control piglets by histology, immunohistochemistry and in-situ tailing for morphological alterations in the hippocampal dentate gyrus and microglial activation in the neocortex. RESULTS: In piglets with meningitis, the density of apoptotic neurons was significantly higher than in control piglets. Moreover, scoring of microglial morphology revealed a significant activation of these cells during meningitis. The slight increase in the density of dividing cells, young neurons and microglia observed in piglets suffering from meningitis was not statistically significant, probably because of the short time frame between onset of clinical signs and organ sampling. CONCLUSIONS: The morphological changes found during S. suis meningitis are in accordance with abnormalities in other animal models and human autopsy cases. Therefore, the pig should be considered as a model for evaluating effects of experimental therapeutic approaches on neurological function in bacterial meningitis.

Identification of a novel DNase of Streptococcus suis (EndAsuis) important for neutrophil extracellular trap degradation during exponential growth.

The porcine and human pathogen Streptococcus suis induces and degrades neutrophil extracellular traps (NETs) in vitro. In this study, we investigated the working hypothesis that NET degradation is mediated not only by the known secreted S. suis nuclease A (SsnA) but also by a so-far undescribed putative endonuclease A of S. suis (designated EndAsuis) homologous to the pneumococcal endonuclease A (EndA). Comparative analysis was conducted to identify differences in localization, expression and function of EndAsuis and SsnA. In contrast to ssnA, endAsuis RNA expression was not substantially different during exponential and stationary growth. Modelling of the 3D structure confirmed a putative DRGH-motif-containing ßßα-metal finger catalytic core in EndAsuis. Accordingly, nuclease activity of recombinant EndAsuis with a point-mutated H165 was rescued through imidazol treatment. In accordance with a putative membrane anchor, nuclease activity caused by endAsuis was not detectable in the supernatant. Importantly, endAsuis determined nuclease activity of S. suis prominently during exponential growth. This activity depended on the presence of Mg(2+) but, in contrast to SsnA activity, not on Ca(2+). A pH of 5.4 did not inhibit endAsuis-encoded nuclease activity during exponential growth. NET degradation of S. suis harvested during exponential growth was significantly attenuated in the endAsuis mutant. In contrast to SsnA, mutagenesis of endAsuis did not result in a significantly higher susceptibility against the antimicrobial effect mediated by NETs. As degradation of bacterial DNA caused by S. suis depended on ssnA and endAsuis, further functions of both factors in the host-pathogen interaction might be envisioned.

Streptococcus suis DNase SsnA contributes to degradation of neutrophil extracellular traps (NETs) and evasion of NET-mediated antimicrobial activity.

Streptococcus suis is an important cause of different pathologies in pigs and humans, most importantly fibrinosuppurative meningitis. Tissue infected with this pathogen is substantially infiltrated with neutrophils, but the function of neutrophil extracellular traps (NETs) - a more recently discovered antimicrobial strategy of neutrophils - in host defence against Strep. suis has not been investigated. The objective of this work was to investigate the interaction of Strep. suis with NETs in vitro. Strep. suis induced NET formation in porcine neutrophils and was entrapped but not killed by those NETs. As the amount of NETs decreased over time, we hypothesized that a known extracellular DNase of Strep. suis degrades NETs. Though this nuclease was originally designated Strep. suis-secreted nuclease A (SsnA), this work demonstrated surface association in accordance with an LPXTG cell wall anchor motif and partial release into the supernatant. Confirming our hypothesis, an isogenic ssnA mutant was significantly attenuated in NET degradation and in protection against the antimicrobial activity of NETs as determined in assays with phorbol myristate acetate (PMA)-stimulated human neutrophils. Though assays with PMA-stimulated porcine neutrophils suggested that SsnA also degrades porcine NETs, phenotypic differences between wt and the isogenic ssnA mutant were less distinct. As SsnA expression was crucial for neither growth in vitro nor for survival in porcine or human blood, the results indicated that SsnA is the first specific NET evasion factor to be identified in Strep. suis.