Intestinal bacterial colonization induces mutualistic regulatory T cell responses

Mammals harbor a dense commensal microbiota in the colon. Regulatory T (Treg) cells are known to limit microbe-triggered intestinal inflammation and the CD4+ T cell compartment is shaped by the presence of particular microbes or bacterial compounds. It is, however, difficult to distinguish whether these effects reflect true mutualistic immune adaptation to intestinal colonization or rather idiosyncratic immune responses. To investigate truly mutualistic CD4+ T cell adaptation, we used the altered Schaedler flora (ASF). Intestinal colonization resulted in activation and de novo generation of colonic Treg cells. Failure to activate Treg cells resulted in the induction of T helper 17 (Th17) and Th1 cell responses, which was reversed by wild-type Treg cells. Efficient Treg cell induction was also required to maintain intestinal homeostasis upon dextran sulfate sodium-mediated damage in the colon. Thus, microbiota colonization-induced Treg cell responses are a fundamental intrinsic mechanism to induce and maintain host-intestinal microbial T cell mutualism.

Bacterial meningitis impairs hippocampal neurogenesis

Bacterial meningitis causes persisting neurofunctional sequelae. Theoccurrence of apoptotic cell death in the hippocampal subgranular zone of the dentate gyrus characterizes the disease in patients and relates to deficits in learning and memory in corresponding experimental models. Here, we investigated why neurogenesis fails to regenerate the damage in the hippocampus associated with the persistence of neurofunctional deficits. In an infant rat model of bacterial meningitis, the capacity of hippocampal-derived cells to multiply and form neurospheres was significantly impaired comparedto that in uninfected littermates. In an in vitro model of differentiating hippocampal cells, challenges characteristic of bacterial meningitis (i.e. bacterial components, tumor necrosis factor [20 ng/mL], or growth factor deprivation) caused significantly more apoptosis in stem/progenitor cells and immature neurons than in mature neurons. These results demonstrate that bacterial meningitis injures hippocampal stem and progenitor cells, a finding that may explain the persistence of neurofunctional deficits after bacterial meningitis.

Mitochondrial preprotein translocase of trypanosomatids has a bacterial origin

Mitochondria are found in all eukaryotic cells and derive from a bacterial endosymbiont [1, 2]. The evolution of a protein import system was a prerequisite for the conversion of the endosymbiont into a true organelle. Tom40, the essential component of the protein translocase of the outer membrane, is conserved in mitochondria of almost all eukaryotes but lacks bacterial orthologs [3-6]. It serves as the gateway through which all mitochondrial proteins are imported. The parasitic protozoa Trypanosoma brucei and its relatives do not have a Tom40-like protein, which raises the question of how proteins are imported by their mitochondria [7, 8]. Using a combination of bioinformatics and in vivo and in vitro studies, we have discovered that T. brucei likely employs a different import channel, termed ATOM (archaic translocase of the outer mitochondria! membrane). ATOM mediates the import of nuclear-encoded proteins into mitochondria and is essential for viability of trypanosomes. It is not related to Tom40 but is instead an ortholog of a subgroup of the 0mp85 protein superfamily that is involved in membrane translocation and insertion of bacterial outer membrane proteins [9]. This suggests that the protein import channel in trypanosomes is a relic of an archaic protein transport system that was operational in the ancestor of all eukaryotes.

Susceptibility of primary human endothelial cells to C. perfringens beta-toxin suggesting similar pathogenesis in human and porcine necrotizing enteritis

Clostridium perfringens type C causes fatal necrotizing enteritis in different mammalian hosts, most commonly in newborn piglets. Human cases are rare, but the disease, also called pigbel, was endemic in the Highlands of Papua New Guinea. Lesions in piglets and humans are very similar and characterized by segmental necro-hemorrhagic enteritis in acute cases and fibrino-necrotizing enteritis in subacute cases. Histologically, deep mucosal necrosis accompanied by vascular thrombosis and necrosis was consistently reported in naturally affected pigs and humans. This suggests common pathogenetic mechanisms. Previous in vitro studies using primary porcine aortic endothelial cells suggested that beta-toxin (CPB) induced endothelial damage contributes to the pathogenesis of C. perfringens type C enteritis in pigs. In the present study we investigated toxic effects of CPB on cultured primary human macro- and microvascular endothelial cells. In vitro, these cells were highly sensitive to CPB and reacted with similar cytopathic and cytotoxic effects as porcine endothelial cells. Our results indicate that porcine and human cell culture based in vitro models represent valuable tools to investigate the pathogenesis of this bacterial disease in animals and humans.

Grafted neuronal precursor cells differentiate and integrate in injured hippocampus in experimental pneumococcal meningitis

Bacterial meningitis (BM) frequently causes persisting neurofunctional sequelae. Autopsy studies in patients dying from BM show characteristic apoptotic brain injury to the stem cell niche in the subgranular zone of the hippocampal dentate gyrus (DG), and this form of brain damage is associated with learning and memory deficits in experimental BM. With an eye to potential regenerative therapies, the survival, migration, and differentiation of neuronal precursor cells (NPCs) were evaluated after engraftment into the injured hippocampus in vitro and in vivo in an infant rat model of pneumococcal meningitis. Green fluorescent protein (GFP)-expressing NPCs were grafted into the DG of organotypic hippocampal slice cultures injured by challenge with live Streptococcus pneumoniae. Seven days after engraftment, NPCs had migrated from the site of injection into the injured granular layer of the DG and electro-functionally integrated into the hippocampal network. In vivo, GFP-expressing NPCs migrated within 1 week from the injection site in the hilus region to the injured granular layer of the hippocampal DG and showed neuronal differentiation at 2 and 4 weeks after transplantation. Hippocampal injury induced by BM guides grafted NPCs to the area of brain damage and provides a microenvironment for neuronal differentiation and functional integration.

Gelatinase B [matrix metalloproteinase (MMP)-9] and collagenases (MMP-8/-13) are upregulated in cerebrospinal fluid during aseptic and bacterial meningitis in children

We investigated the protein expression of gelatinases [matrix metalloproteinase (MMP)-2 and -9] and collagenases (MMP-8 and -13) in cerebrospinal fluid (CSF) from patients with bacterial (BM, n = 17) and aseptic (AM, n = 14) meningitis. In both, MMP-8 and -9 were increased in 100% of patients, whereas MMP-13 was detectable in 53% and 82% respectively. Three patients with clinical signs of meningitis, without CSF pleocytosis, scored positive for all three MMPs. MMP-8 appeared in two isoforms, granulocyte-type [polymorphonuclear cell (PMN)] and fibroblast/macrophage (F/M) MMP-8. Analysis of kinetic changes from serial lumbar punctures showed that these MMPs are independently regulated, and correlate only partly with CSF cytosis or levels of the endogenous inhibitor, tissue inhibitor of matrix metalloproteinase-1. In vitro, T cells, peripheral blood mononuclear cells (PBMCs) and granulocytes (PMN) release MMP-8 and -9, whereas MMP-13 could be found only in the former two cell types. Using models of exogenous (n-formyl-Met-Leu-Phe, T cell receptor cross-linking) and host-derived stimuli (interleukin-2), the kinetics and the release of the MMP-8, -9 and -13 showed strong variation between these immune cells and suggest release from preformed stocks. In addition, MMP-9 is also synthesized de novo in PBMCs and T cells. In conclusion, invading immune cells contribute only partially to MMPs in CSF during meningitis, and parenchymal cells are an equally relevant source. In this context, in patients with clinical signs of meningitis, but without CSF pleocytosis, MMPs seem to be a highly sensitive marker for intrathecal inflammation. The present data support the concept that broad-spectrum enzyme inhibition targeting gelatinases and collagenases is a potential strategy for adjunctive therapy in infectious meningitis.

Beta-D-glucoside utilization by Mycoplasma mycoides subsp. mycoides SC: possible involvement in the control of cytotoxicity towards bovine lung cells

BACKGROUND: Contagious bovine pleuropneumonia (CBPP) caused by Mycoplasma mycoides subsp. mycoides small-colony type (SC) is among the most serious threats for livestock producers in Africa. Glycerol metabolism-associated H2O2 production seems to play a crucial role in virulence of this mycoplasma. A wide number of attenuated strains of M. mycoides subsp. mycoides SC are currently used in Africa as live vaccines. Glycerol metabolism is not affected in these vaccine strains and therefore it does not seem to be the determinant of their attenuation. A non-synonymous single nucleotide polymorphism (SNP) in the bgl gene coding for the 6-phospho-beta-glucosidase (Bgl) has been described recently. The SNP differentiates virulent African strains isolated from outbreaks with severe CBPP, which express the Bgl isoform Val204, from strains to be considered less virulent isolated from CBPP outbreaks with low mortality and vaccine strains, which express the Bgl isoform Ala204. RESULTS: Strains of M. mycoides subsp. mycoides SC considered virulent and possessing the Bgl isoform Val204, but not strains with the Bgl isoform Ala204, do trigger elevated levels of damage to embryonic bovine lung (EBL) cells upon incubation with the disaccharides (i.e., beta-D-glucosides) sucrose and lactose. However, strains expressing the Bgl isoform Val204 show a lower hydrolysing activity on the chromogenic substrate p-nitrophenyl-beta-D-glucopyranoside (pNPbG) when compared to strains that possess the Bgl isoform Ala204. Defective activity of Bgl in M. mycoides subsp. mycoides SC does not lead to H2O2 production. Rather, the viability during addition of beta-D-glucosides in medium-free buffers is higher for strains harbouring the Bgl isoform Val204 than for those with the isoform Ala204. CONCLUSION: Our results indicate that the studied SNP in the bgl gene is one possible cause of the difference in bacterial virulence among strains of M. mycoides subsp. mycoides SC. Bgl does not act as a direct virulence factor, but strains possessing the Bgl isoform Val204 with low hydrolysing activity are more prone to survive in environments that contain high levels of beta-D-glucosides, thus contributing in some extent to mycoplasmaemia.

Cleavage of CXCR1 on neutrophils disables bacterial killing in cystic fibrosis lung disease

Interleukin-8 (IL-8) activates neutrophils via the chemokine receptors CXCR1 and CXCR2. However, the airways of individuals with cystic fibrosis are frequently colonized by bacterial pathogens, despite the presence of large numbers of neutrophils and IL-8. Here we show that IL-8 promotes bacterial killing by neutrophils through CXCR1 but not CXCR2. Unopposed proteolytic activity in the airways of individuals with cystic fibrosis cleaved CXCR1 on neutrophils and disabled their bacterial-killing capacity. These effects were protease concentration-dependent and also occurred to a lesser extent in individuals with chronic obstructive pulmonary disease. Receptor cleavage induced the release of glycosylated CXCR1 fragments that were capable of stimulating IL-8 production in bronchial epithelial cells via Toll-like receptor 2. In vivo inhibition of proteases by inhalation of alpha1-antitrypsin restored CXCR1 expression and improved bacterial killing in individuals with cystic fibrosis. The cleavage of CXCR1, the functional consequences of its cleavage, and the identification of soluble CXCR1 fragments that behave as bioactive components represent a new pathophysiologic mechanism in cystic fibrosis and other chronic lung diseases.

Lipopolysaccharides from atherosclerosis-associated bacteria antagonize TLR4, induce formation of TLR2/1/CD36 complexes in lipid rafts and trigger TLR2-induced inflammatory responses in human vascular endothelial cells

Infection with bacteria such as Chlamydia pneumonia, Helicobacter pylori or Porphyromonas gingivalis may be triggering the secretion of inflammatory cytokines that leads to atherogenesis. The mechanisms by which the innate immune recognition of these pathogens could lead to atherosclerosis remain unclear. In this study, using human vascular endothelial cells or HEK-293 cells engineered to express pattern-recognition receptors (PRRs), we set out to determine Toll-like receptors (TLRs) and functionally associated PRRs involved in the innate recognition of and response to lipopolysaccharide (LPS) from H. pylori or P. gingivalis. Using siRNA interference or recombinant expression of cooperating PRRs, we show that H. pylori and P. gingivalis LPS-induced cell activation is mediated through TLR2. Human vascular endothelial cell activation was found to be lipid raft-dependent and to require the formation of heterotypic receptor complexes comprising of TLR2, TLR1, CD36 and CD11b/CD18. In addition, we report that LPS from these bacterial strains are able to antagonize TLR4. This antagonistic activity of H. pylori or P. gingivalis LPS, as well as their TLR2 activation capability may be associated with their ability to contribute to atherosclerosis.

A global view of Staphylococcus aureus whole genome expression upon internalization in human epithelial cells

BACKGROUND: Staphylococcus aureus, a leading cause of chronic or acute infections, is traditionally considered an extracellular pathogen despite repeated reports of S. aureus internalization by a variety of non-myeloid cells in vitro. This property potentially contributes to bacterial persistence, protection from antibiotics and evasion of immune defenses. Mechanisms contributing to internalization have been partly elucidated, but bacterial processes triggered intracellularly are largely unknown. RESULTS: We have developed an in vitro model using human lung epithelial cells that shows intracellular bacterial persistence for up to 2 weeks. Using an original approach we successfully collected and amplified low amounts of bacterial RNA recovered from infected eukaryotic cells. Transcriptomic analysis using an oligoarray covering the whole S. aureus genome was performed at two post-internalization times and compared to gene expression of non-internalized bacteria. No signs of cellular death were observed after prolonged internalization of Staphylococcus aureus 6850 in epithelial cells. Following internalization, extensive alterations of bacterial gene expression were observed. Whereas major metabolic pathways including cell division, nutrient transport and regulatory processes were drastically down-regulated, numerous genes involved in iron scavenging and virulence were up-regulated. This initial adaptation was followed by a transcriptional increase in several metabolic functions. However, expression of several toxin genes known to affect host cell integrity appeared strictly limited. CONCLUSION: These molecular insights correlated with phenotypic observations and demonstrated that S. aureus modulates gene expression at early times post infection to promote survival. Staphylococcus aureus appears adapted to intracellular survival in non-phagocytic cells.

Blockade of NMDA receptor subtype NR2B prevents seizures but not apoptosis of dentate gyrus neurons in bacterial meningitis in infant rats

BACKGROUND: Excitotoxic neuronal injury by action of the glutamate receptors of the N-methyl-d-aspartate (NMDA) subtype have been implicated in the pathogenesis of brain damage as a consequence of bacterial meningitis. The most potent and selective blocker of NMDA receptors containing the NR2B subunit is (R,S)-alpha-(4-hydroxyphenyl)-beta-methyl-4-(phenylmethyl)-1-piperid inepropanol (RO 25-6981). Here we evaluated the effect of RO 25-6981 on hippocampal neuronal apoptosis in an infant rat model of meningitis due to Streptococcus pneumoniae. Animals were randomized for treatment with RO 25-6981 at a dosage of either 0.375 mg (15 mg/kg; n = 28) or 3.75 mg (150 mg/kg; n = 15) every 3 h or an equal volume of sterile saline (250 microl; n = 40) starting at 12 h after infection. Eighteen hours after infection, animals were assessed clinically and seizures were observed for a period of 2 h. At 24 h after infection animals were sacrificed and brains were examined for apoptotic injury to the dentate granule cell layer of the hippocampus. RESULTS: Treatment with RO 25-6981 had no effect on clinical scores, but the incidence of seizures was reduced (P < 0.05 for all RO 25-6981 treated animals combined). The extent of apoptosis was not affected by low or high doses of RO 25-6981. Number of apoptotic cells (median [range]) was 12.76 [3.16-25.3] in animals treated with low dose RO 25-6981 (control animals 13.8 [2.60-31.8]; (P = NS) and 9.8 [1.7-27.3] (controls: 10.5 [2.4-21.75]) in animals treated with high dose RO 25-6981 (P = NS). CONCLUSIONS: Treatment with a highly selective blocker of NMDA receptors containing the NR2B subunit failed to protect hippocampal neurons from injury in this model of pneumococcal meningitis, while it had some beneficial effect on the incidence of seizures.

Neurotoxicity of glia activated by gram-positive bacterial products depends on nitric oxide production

The present study examined the mechanism by which bacterial cell walls from two gram-positive meningeal pathogens, Streptococcus pneumoniae and the group B streptococcus, induced neuronal injury in primary cultures of rat brain cells. Cell walls from both organisms produced cellular injury to similar degrees in pure astrocyte cultures but not in pure neuronal cultures. Cell walls also induced nitric oxide production in cultures of astrocytes or microglia. When neurons were cultured together with astrocytes or microglia, the cell walls of both organisms became toxic to neurons. L-NAME, a nitric oxide synthase inhibitor, protected neurons from cell wall-induced toxicity in mixed cultures with glia, as did dexamethasone. In contrast, an excitatory amino acid antagonist (MK801) had no effect. Low concentrations of cell walls from either gram-positive pathogen added together with the excitatory amino acid glutamate resulted in synergistic neurotoxicity that was inhibited by L-NAME. The induction of nitric oxide production and neurotoxicity by cell walls was independent of the presence of serum, whereas endotoxin exhibited these effects only in the presence of serum. We conclude that gram-positive cell walls can cause toxicity in neurons by inducing the production of nitric oxide in astrocytes and microglia.

Neuroprotective effect of excitatory amino acid antagonist kynurenic acid in experimental bacterial meningitis

Sustained high-level exposure to glutamate, an excitatory amino acid neurotransmitter, leads to neuronal death. Kynurenic acid attenuates the toxic effects of glutamate by inhibition of neuronal excitatory amino acid receptors, including the N-methyl-D-aspartate subtype. To evaluate the role of glutamate in causing neuronal injury in a rat model of meningitis due to group B streptococci, animals were treated with kynurenic acid (300 mg/kg subcutaneously once daily) or saline beginning at the time of infection. Histopathologic examination after 24-72 h showed two distinct forms of neuronal injury, areas of neuronal necrosis in the cortex and injury of dentate granule cells in the hippocampus. Animals treated with kynurenic acid showed significantly less neuronal injury (P < .03) in the cortex and the hippocampus than did untreated controls. These results suggest an important contribution of glutamate to neurotoxicity in this animal model of neonatal meningitis.

Toxicity in neuronal cells caused by cerebrospinal fluid from pneumococcal and gram-negative meningitis

To identify neurotoxic factors in meningitis, a neuronal cell line (HN33.1) was exposed to cerebrospinal fluid (CSF) obtained from rabbits with pneumococcal meningitis or Escherichia coli meningitis or 2 h and 6 h after meningitis was induced by proinflammatory bacterial products (pneumococcal cell walls, endotoxin). CSF from all types of meningitis induced similar degrees of cytotoxicity. When a soluble tumor necrosis factor (TNF) receptor that completely blocked TNF-mediated toxicity at 10(-7) M was used, all toxicity in meningitis caused by E. coli, endotoxin, or pneumococcal cell wall administration (2 h afterwards) was mediated by TNF. In contrast, CSF from animals with meningitis caused by live pneumococci or pneumococcal cell wall injection (6 h afterwards) retained cytotoxicity in the presence of the TNF receptor. Thus, in established pneumococcal meningitis, but not in the other forms of meningitis, TNF is not the only component toxic in this neuronal cell line.

Good manufacturing practice-compliant validation and preparation of BM cells for the therapy of acute myocardial infarction

BACKGROUND: Intracoronary application of BM-derived cells for the treatment of acute myocardial infarction (AMI) is currently being studied intensively. Simultaneously, strict legal requirements surround the production of cells for clinical studies. Thus good manufacturing practice (GMP)-compliant collection and preparation of BM for patients with AMI was established by the Cytonet group. METHODS: As well as fulfillment of standard GMP requirements, including a manufacturing license, validation of the preparation process and the final product was performed. Whole blood (n=6) and BM (n=3) validation samples were processed under GMP conditions by gelafundin or hydroxyethylstarch sedimentation in order to reduce erythrocytes/platelets and volume and to achieve specifications defined in advance. Special attention was paid to the free potassium (<6 mmol/L), some rheologically relevant cellular characteristics (hematocrit <0.45, platelets <450 x 10(6)/mL) and the sterility of the final product. RESULTS: The data were reviewed and GMP compliance was confirmed by the German authorities (Paul-Ehrlich Institute). Forty-five BM cell preparations for clinical use were carried out following the validated methodology and standards. Additionally three selections of CD34+ BM cells for infusion were performed. All specification limits were met. Discussion In conclusion, preparation of BM cells for intracoronary application is feasible under GMP conditions. As the results of sterility testing may not be available at the time of intracoronary application, the highest possible standards to avoid bacterial and other contaminations have to be applied. The increased expense of the GMP-compliant process can be justified by higher safety for patients and better control of the final product.