Neutrophil extracellular traps in CSF and serum of dogs with steroid-responsive meningitis-arteritis.

In steroid-responsive meningitis-arteritis (SRMA), inflammatory dysregulation is driven by neutrophilic granulocytes resulting in purulent leptomeningitis. Neutrophils can generate neutrophil extracellular traps (NET). Uncontrolled NET-formation or impaired NET-clearance evidently cause tissue and organ damage resulting in immune-mediated diseases. The aim of the study was to verify that NET-formation is detectable in ex vivo samples of acute diseased dogs with SRMA by visualizing and measuring NET-markers in serum and cerebrospinal fluid (CSF) samples. CSF-samples of dogs with acute SRMA (n = 5) and in remission (n = 4) were examined using immunofluorescence (IF)-staining of DNA-histone-1-complexes, myeloperoxidase and citrullinated Histone H3 (H3Cit). Immunogold-labeling of H3Cit and neutrophil elastase followed by transmission electron microscopy (TEM) were used to determine ultrastructural NET-formation in the CSF of one exemplary dog. H3Cit-levels and DNase-activity were measured in CSF and serum samples using an H3Cit-ELISA and a DNase-activity-assay, respectively in patients with the following diseases: acute SRMA (n = 34), SRMA in remission (n = 4), bacterial encephalitis (n = 3), meningioma with neutrophilic inflammation (n = 4), healthy dogs (n = 6). NET-formation was detectable with IF-staining in n = 3/5 CSF samples of dogs with acute SRMA but were not detectable during remission. Vesicular NET-formation was detectable in one exemplary dog using TEM. DNase-activity was significantly reduced in dogs suffering from acute SRMA compared to healthy control group (p < 0.0001). There were no statistical differences of H3Cit levels in CSF or serum samples of acute diseased dogs compared to dogs under treatment, dogs suffering from meningioma or bacterial encephalitis or the healthy control group. Our findings demonstrate that NET-formation and insufficient NET-clearance possibly drive the immunologic dysregulation and complement the pathogenesis of SRMA. The detection of NETs in SRMA offers many possibilities to explore the aetiopathogenetic influence of this defence mechanism of the innate immune system in infectious and non-infectious canine neuropathies.

d-Alanylation of Lipoteichoic Acids in Streptococcus suis Reduces Association With Leukocytes in Porcine Blood.

Streptococcus suis (S. suis) is a common swine pathogen but also poses a threat to human health in causing meningitis and severe cases of streptococcal toxic shock-like syndrome (STSLS). Therefore, it is crucial to understand how S. suis interacts with the host immune system during bacteremia. As S. suis has the ability to introduce d-alanine into its lipoteichoic acids (LTAs), we investigated the working hypothesis that cell wall modification by LTA d-alanylation influences the interaction of S. suis with porcine blood immune cells. We created an isogenic mutant of S. suis strain 10 by in-frame deletion of the d-alanine d-alanyl carrier ligase (DltA). d-alanylation of LTAs was associated with reduced phagocytosis of S. suis by porcine granulocytes, reduced deposition of complement factor C3 on the bacterial surface, increased hydrophobicity of streptococci, and increased resistance to cationic antimicrobial peptides (CAMPs). At the same time, survival of S. suis was not significantly increased by LTA d-alanylation in whole blood of conventional piglets with specific IgG. However, we found a distinct cytokine pattern as IL-1ß but not tumor necrosis factor (TNF)-α levels were significantly reduced in blood infected with the ΔdltA mutant. In contrast to TNF-α, activation and secretion of IL-1ß are inflammasome-dependent, suggesting a possible influence of LTA d-alanylation on inflammasome regulation. Especially in the absence of specific antibodies, the association of S. suis with porcine monocytes was reduced by d-alanylation of its LTAs. This dltA-dependent phenotype was also observed with a non-encapsulated dltA double mutant indicating that it is independent of capsular polysaccharides. High antibody levels caused high levels of S. suis-monocyte-association followed by inflammatory cell death and strong production of both IL-1ß and TNF-α, while the influence of LTA d-alanylation of the streptococci became less visible. In summary, the results of this study expand previous findings on d-alanylation of LTAs in S. suis and suggest that this pathogen specifically modulates association with blood leukocytes through this modification of its surface.

LPS Primes Brain Responsiveness to High Mobility Group Box-1 Protein.

High mobility group box (HMGB)1 action contributes to late phases of sepsis, but the effects of increased endogenous plasma HMGB1 levels on brain cells during inflammation are unclear. Here, we aimed to further investigate the role of HMGB1 in the brain during septic-like lipopolysaccharide-induced inflammation in rats (LPS, 10 mg/kg, i.p.). HMGB-1 mRNA expression and release were measured in the periphery/brain by RT-PCR, immunohistochemistry and ELISA. In vitro experiments with disulfide-HMGB1 in primary neuro-glial cell cultures of the area postrema (AP), a circumventricular organ with a leaky blood-brain barrier and direct access to circulating mediators like HMGB1 and LPS, were performed to determine the direct influence of HMGB1 on this pivotal brain structure for immune-to-brain communication. Indeed, HMGB1 plasma levels stayed elevated after LPS injection. Immunohistochemistry of brains and AP cultures confirmed LPS-stimulated cytoplasmatic translocation of HMGB1 indicative of local HMGB1 release. Moreover, disulfide-HMGB1 stimulation induced nuclear factor (NF)-κB activation and a significant release of interleukin-6, but not tumor necrosis factor α, into AP culture supernatants. However, only a few AP cells directly responded to HMGB1 with increased intracellular calcium concentration. Interestingly, priming with LPS induced a seven-fold higher percentage of responsive cells to HMGB1. We conclude that, as a humoral and local mediator, HMGB1 enhances brain inflammatory responses, after LPS priming, linked to sustained sepsis symptoms.

Analysis of Porcine Pro- and Anti-Inflammatory Cytokine Induction by S. suis In Vivo and In Vitro.

Weaning piglets are susceptible to the invasive Streptococcus (S.) suis infection, which can result in septicemia. The aim of this study was to investigate the cytokine profile induced upon S. suis infection of blood, to determine the cellular sources of those cytokines, and to study the potential effects of the induced cytokines on bacterial killing. We measured TNF-α, IL-6, IFN-γ, IL-17A and IL-10 after an experimental intravenous infection with S. suis serotype 2 in vivo, and analyzed whole blood, peripheral blood mononuclear cells (PBMC) and separated leukocytes to identify the cytokine-producing cell type(s). In addition, we used a reconstituted whole blood assay to investigate the effect of TNF-α on bacterial killing in the presence of different S. suis-specific IgG levels. An increase in IL-6 and IL-10, but not in IFN-γ or IL-17A, was observed in two of three piglets with pronounced bacteremia 16 to 20 h after infection, but not in piglets with controlled bacteremia. Our results confirmed previous findings that S. suis induces TNF-α and IL-6 and could demonstrate that TNF-α is produced by monocytes in vitro. We further found that IL-10 induction resulted in reduced secretion of TNF-α and IL-6. Rapid induction of TNF-α was, however, not crucial for in vitro bacterial killing, not even in the absence of specific IgG.

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.

IgM cleavage by Streptococcus suis reduces IgM bound to the bacterial surface and is a novel complement evasion mechanism.

Streptococcus suis (S. suis) causes meningitis, arthritis and endocarditis in piglets. The aim of this study was to characterize the IgM degrading enzyme of S. suis (IdeSsuis) and to investigate the role of IgM cleavage in evasion of the classical complement pathway and pathogenesis. Targeted mutagenesis of a cysteine in the putative active center of IdeSsuis abrogated IgM cleavage completely. In contrast to wt rIdeSsuis, point mutated rIdeSsuis_C195S did not reduce complement-mediated hemolysis indicating that complement inhibition by rIdeSsuis depends on the IgM proteolytic activity. A S. suis mutant expressing IdeSsuis_C195S did not reduce IgM labeling, whereas the wt and complemented mutant showed less IgM F(ab')2 and IgM Fc antigen on the surface. IgM cleavage increased survival of S. suis in porcine blood ex vivo and mediated complement evasion as demonstrated by blood survival and C3 deposition assays including the comparative addition of rIdeSsuis and rIdeSsuis_C195S. However, experimental infection of piglets disclosed no significant differences in virulence between S. suis wt and isogenic mutants without IgM cleavage activity. This work revealed for the first time in vivo labeling of S. suis with IgM in the cerebrospinal fluid of piglets with meningitis. In conclusion, this study classifies IdeSsuis as a cysteine protease and emphasizes the role of IgM cleavage for bacterial survival in porcine blood and complement evasion though IgM cleavage is not crucial for the pathogenesis of serotype 2 meningitis.