Role of Nucleotide-Binding Oligomerization Domain-Containing (NOD) 2 in Host Defense during Pneumococcal Pneumonia

Streptococcus (S.) pneumoniae is the most common causative pathogen in community-acquired pneumonia. Nucleotide-binding oligomerization domain-containing (NOD) 2 is a pattern recognition receptor located in the cytosol of myeloid cells that is able to detect peptidoglycan fragments of S. pneumoniae. We here aimed to investigate the role of NOD2 in the host response during pneumococcal pneumonia. Phagocytosis of S. pneumoniae was studied in NOD2 deficient (Nod2 ^-/-) and wild-type (Wt) alveolar macrophages and neutrophils in vitro. In subsequent in vivo experiments Nod2 ^-/- and Wt mice were inoculated with serotype 2 S. pneumoniae (D39), an isogenic capsule locus deletion mutant (D39Δcps) or serotype 3 S. pneumoniae (6303) via the airways, and bacterial growth and dissemination and the lung inflammatory response were evaluated. Nod2 ^-/- alveolar macrophages and blood neutrophils displayed a reduced capacity to internalize pneumococci in vitro. During pneumonia caused by S. pneumoniae D39 Nod2 ^-/- mice were indistinguishable from Wt mice with regard to bacterial loads in lungs and distant organs, lung pathology and neutrophil recruitment. While Nod2 ^-/- and Wt mice also had similar bacterial loads after infection with the more virulent S. pneumoniae 6303 strain, Nod2 ^-/- mice displayed a reduced bacterial clearance of the normally avirulent unencapsulated D39Δcps strain. These results suggest that NOD2 does not contribute to host defense during pneumococcal pneumonia and that the pneumococcal capsule impairs recognition of S. pneumoniae by NOD2.     

Impact of Anti-Retroviral Treatment and Cotrimoxazole Prophylaxis on Helminth Infections in HIV-Infected Patients in Lambaréné, Gabon

Background

Foci of the HIV epidemic and helminthic infections largely overlap geographically. Treatment options for helminth infections are limited, and there is a paucity of drug-development research in this area. Limited evidence suggests that antiretroviral therapy (ART) reduces prevalence of helminth infections in HIV-infected individuals. We investigated whether ART exposure and cotrimoxazole preventive therapy (CTX-P) is associated with a reduced prevalence of helminth infections.

Methodology and Principal Findings

This cross-sectional study was conducted at a primary HIV-clinic in Lambaréné, Gabon. HIV-infected adults who were ART-naïve or exposed to ART for at least 3 months submitted one blood sample and stool and urine samples on 3 consecutive days. Outcome was helminth infection with intestinal helminths, Schistosoma haematobium, Loa loa or Mansonella perstans. Multivariable logistic regression was used to assess associations between ART or CTX-P and helminth infection. In total, 408 patients were enrolled. Helminth infection was common (77/252 [30.5%]). Filarial infections were most prevalent (55/310 [17.7%]), followed by infection with intestinal helminths (35/296 [11.8%]) and S. haematobium (19/323 [5.9%]). Patients on CTX-P had a reduced risk of Loa loa microfilaremia (adjusted odds ratio (aOR) 0.47, 95% CI 0.23-0.97, P = 0.04), also in the subgroup of patients on ART (aOR 0.36, 95% CI 0.13-0.96, P = 0.04). There was no effect of ART exposure on helminth infection prevalence.

Conclusions/Significance

CTX-P use was associated with a decreased risk of Loa loa infection, suggesting an anthelminthic effect of antifolate drugs. No relation between ART use and helminth infections was established.     

The Pneumococcal Serine-Rich Repeat Protein Is an Intra-Species Bacterial Adhesin That Promotes Bacterial Aggregation In Vivo and in Biofilms

The Pneumococcal serine-rich repeat protein (PsrP) is a pathogenicity island encoded adhesin that has been positively correlated with the ability of Streptococcus pneumoniae to cause invasive disease. Previous studies have shown that PsrP mediates bacterial attachment to Keratin 10 (K10) on the surface of lung cells through amino acids 273–341 located in the Basic Region (BR) domain. In this study we determined that the BR domain of PsrP also mediates an intra-species interaction that promotes the formation of large bacterial aggregates in the nasopharynx and lungs of infected mice as well as in continuous flow-through models of mature biofilms. Using numerous methods, including complementation of mutants with BR domain deficient constructs, fluorescent microscopy with Cy3-labeled recombinant (r)BR, Far Western blotting of bacterial lysates, co-immunoprecipitation with rBR, and growth of biofilms in the presence of antibodies and competitive peptides, we determined that the BR domain, in particular amino acids 122–166 of PsrP, promoted bacterial aggregation and that antibodies against the BR domain were neutralizing. Using similar methodologies, we also determined that SraP and GspB, the Serine-rich repeat proteins (SRRPs) of Staphylococcus aureus and Streptococcus gordonii, respectively, also promoted bacterial aggregation and that their Non-repeat domains bound to their respective SRRPs. This is the first report to show the presence of biofilm-like structures in the lungs of animals infected with S. pneumoniae and show that SRRPs have dual roles as host and bacterial adhesins. These studies suggest that recombinant Non-repeat domains of SRRPs (i.e. BR for S. pneumoniae) may be useful as vaccine antigens to protect against Gram-positive bacteria that cause infection.     

Dendritic cell function can be modulated through cooperative actions of TLR ligands and invariant NKT cells

The quality of signals received by dendritic cells (DC) in response to pathogens influences the nature of the adaptive response. We show that pathogen-derived signals to DC mediated via TLRs can be modulated by activated invariant NKT (iNKT) cells. DC maturation induced in vivo with any one of a variety of TLR ligands was greatly improved through simultaneous administration of the iNKT cell ligand alpha-galactosylceramide. DC isolated from animals treated simultaneously with TLR and iNKT cell ligands were potent stimulators of naive T cells in vitro compared with DC from animals treated with the ligands individually. Injection of protein Ags with both stimuli resulted in significantly improved T cell and Ab responses to coadministered protein Ags over TLR stimulation alone. Ag-specific CD8(+) T cell responses induced in the presence of the TLR4 ligand monophosphoryl lipid A and alpha-galactosylceramide showed faster proliferation kinetics, and increased effector function, than those induced with either ligand alone. Human DC exposed to TLR ligands and activated iNKT cells in vitro had enhanced expression of maturation markers, suggesting that a cooperative action of TLR ligands and iNKT cells on DC function is a generalizable phenomenon across species. These studies highlight the potential for manipulating the interactions between TLR ligands and iNKT cell activation in the design of effective vaccine adjuvants.     

Crystal structure of human insulin‐regulated aminopeptidase with specificity for cyclic peptides

Insulin‐regulated aminopeptidase (IRAP or oxytocinase) is a membrane‐bound zinc‐metallopeptidase that cleaves neuroactive peptides in the brain and produces memory enhancing effects when inhibited. We have determined the crystal structure of human IRAP revealing a closed, four domain arrangement with a large, mostly buried cavity abutting the active site. The structure reveals that the GAMEN exopeptidase loop adopts a very different conformation from other aminopeptidases, thus explaining IRAP's unique specificity for cyclic peptides such as oxytocin and vasopressin. Computational docking of a series of IRAP‐specific cognitive enhancers into the crystal structure provides a molecular basis for their structure–activity relationships and demonstrates that the structure will be a powerful tool in the development of new classes of cognitive enhancers for treating a variety of memory disorders such as Alzheimer's disease.     

Fracture prevention in COPD patients; a clinical 5-step approach

Although osteoporosis and its related fractures are common in patients with COPD, patients at high risk of fracture are poorly identified, and consequently, undertreated. Since there are no fracture prevention guidelines available that focus on COPD patients, we developed a clinical approach to improve the identification and treatment of COPD patients at high risk of fracture. We organised a round-table discussion with 8 clinical experts in the field of COPD and fracture prevention in the Netherlands in December 2013. The clinical experts presented a review of the literature on COPD, osteoporosis and fracture prevention. Based on the Dutch fracture prevention guideline, they developed a 5-step clinical approach for fracture prevention in COPD. Thereby, they took into account both classical risk factors for fracture (low body mass index, older age, personal and family history of fracture, immobility, smoking, alcohol intake, use of glucocorticoids and increased fall risk) and COPD-specific risk factors for fracture (severe airflow obstruction, pulmonary exacerbations and oxygen therapy). Severe COPD (defined as postbronchodilator FEV₁ < 50% predicted) was added as COPD-specific risk factor to the list of classical risk factors for fracture. The 5-step clinical approach starts with case finding using clinical risk factors, followed by risk evaluation (dual energy X-ray absorptiometry and imaging of the spine), differential diagnosis, treatment and follow-up. This systematic clinical approach, which is evidence-based and easy-to-use in daily practice by pulmonologists, should contribute to optimise fracture prevention in COPD patients at high risk of fracture.     

Inhibition of the β-carbonic anhydrase from Streptococcus pneumoniae by inorganic anions and small molecules: Toward innovative drug design of antiinfectives?

The Gram-positive bacterium Streptococcus pneumoniae is a human respiratory tract pathogen that contributes significantly to global mortality and morbidity. It was recently shown that this bacterial pathogen depends on a conserved ??-carbonic anhydrase (CA, EC 4.2.1.1) for in vitro growth in environmental ambient air and during intracellular survival in host cells. Hence, it is to be expected that this pneumococcal carbonic anhydrase (PCA) contributes to transmission and pathogenesis of the bacterium, making it a potential therapeutic target. In this study, purified recombinant PCA has been further characterized kinetically and for inhibition with a series of inorganic anions and small molecules useful as leads. PCA has appreciable activity as catalyst for the hydration of CO₂ to bicarbonate, with a k_cat of 7.4 × 10⁵ s⁻¹ and k_cat/K_m of 6.5 × 10⁷ M⁻¹ s⁻¹ at an optimum pH of 8.4. Inorganic anions such as chloride, bromide, iodide, cyanate, selenocyanate, trithiocarbonate, and cyanide were effective inhibitors of PCA (K_Is of 21-98 ??M). Sulfamide, sulfamic acid, phenylboronic, phenylarsonic acid, and diethyldithiocarbamate showed inhibition constants in the low micromolar/submicromolar range (K_Is of 0.61-6.68 ??M), whereas that of the sulfonamide acetazolamide was in the nanomolar range (K_Is 89 nM). In conclusion, our results show that PCA can effectively be inhibited by a range of molecules that could be interesting leads for obtaining more potent PCA inhibitors. PCA might be a novel target for designing antimicrobial drugs with a new mechanism of action.     

Extremely low frequency electromagnetic field exposure does not modulate toll-like receptor signaling in human peripheral blood mononuclear cells

The effects of extremely low frequency electromagnetic fields (ELF-EMF) on human health remain unclear. It has been reported that ELF-EMF may modulate the innate immune response to microorganisms in animal models and mammalian cell-lines. With the recently gained insight in innate immune signaling and the discovery of pattern recognition, we aim to study whether ELF-EMF modulates innate inflammatory signaling pathways. We used human peripheral blood mononuclear cells (PBMCs), isolated from blood from healthy volunteers, which we stimulated with specific TLR2 and TLR4 ligands, and with several microorganisms. The cells were subsequently exposed in B(dc)=3 μT to a highly controlled and standardized ELF-EMF signal (20-5000Hz, B(ac)=5 μT, 30 min) and cytokine production was measured at different time points after stimulation. No significant difference in immune response, as reflected by IL-1β, IL-6, TNFα, IL-8 and IL-10 production, could be detected after stimulation with LPS (TLR4 ligand), Pam3Cys (TLR2 ligand) or a panel of heat killed microorganisms: Mycobacterium tuberculosis, Salmonella typhimurium, Candida albicans, Aspergillus fumigatus and Staphylococcus aureus (multiple TLR ligands). We therefore conclude that under our experimental conditions, ELF-EMF does not modulate the innate immune response of human primary cells after TLR stimulation in vitro.     

Exploiting the role of endogenous lymphoid-resident dendritic cells in the priming of NKT cells and CD8+ T cells to dendritic cell-based vaccines

Transfer of antigen between antigen-presenting cells (APCs) is potentially a physiologically relevant mechanism to spread antigen to cells with specialized stimulatory functions. Here we show that specific CD8+ T cell responses induced in response to intravenous administration of antigen-loaded bone marrow-derived dendritic cells (BM-DCs), were ablated in mice selectively depleted of endogenous lymphoid-resident langerin+ CD8α+ dendritic cells (DCs), suggesting that the antigen is transferred from the injected cells to resident APCs. In contrast, antigen-specific CD4+ T cells were primed predominantly by the injected BM-DCs, with only very weak contribution of resident APCs. Crucially, resident langerin+ CD8α+ DCs only contributed to the priming of CD8+ T cells in the presence of maturation stimuli such as intravenous injection of TLR ligands, or by loading the BM-DCs with the glycolipid α-galactosylceramide (α-GalCer) to recruit the adjuvant activity of activated invariant natural killer-like T (iNKT) cells. In fact, injection of α-GalCer-loaded CD1d-/- BM-DCs resulted in potent iNKT cell activation, suggesting that this glycolipid antigen can also be transferred to resident CD1d+ APCs. While iNKT cell activation per se was independent of langerin+ CD8α+ DCs, some iNKT cell-mediated activities were reduced, notably release of IL-12p70 and transactivation of NK cells. We conclude that both protein and glycolipid antigens can be exchanged between distinct DC species. These data suggest that the efficacy of DC-based vaccination strategies may be improved by the incorporation of a systemic maturation signal aimed to engage resident APCs in CD8+ T cell priming, and α-GalCer may be particularly well suited to this purpose.     

Molecular and functional characterisation of glutamate transporters in rat cortical astrocytes exposed to a defined combination of growth factors during in vitro differentiation

In vitro culture of astroglial progenitors can be obtained from early post-natal brain tissues and several methods have been reported for promoting their maturation into differentiated astrocytes. Hence, a combination of several nutriments/growth factors -- the G5 supplement (insulin, transferrin, selenite, biotin, hydrocortisone, fibroblast growth factor and epidermal growth factor) -- is widely used as a culture additive favouring the growth, differentiation and maturation of primary cultured astrocytes. Considering the key role played by glial cells in the clearance of glutamate in the synapses, cultured astrocytes are frequently used as a model for the study of glutamate transporters. Indeed, it has been shown that when tested separately, growth factors influence the expression and activity of the GLAST and GLT-1. The present study aimed at characterising the functional expression of these transporters during the time course of differentiation of cultured cortical astrocytes exposed to the supplement G5. After a few days, the vast majority of cells exposed to this supplement adopted a typical stellate morphology (fibrous or type II astrocytes) and showed intense expression of the glial fibrillary acidic protein. Both RT-PCR and immunoblotting studies revealed that the expression of both GLAST and GLT-1 rapidly increased in these cells. While this was correlated with a significant increase in specific uptake of radiolabelled aspartate, fluorescence monitoring of the Na+ influx associated with glutamate transporters activity revealed that the exposure to the G5 supplement considerably increased the percentage of cells participating in the uptake. Biochemical and pharmacological studies revealed that this activity did not involve GLT-1 but most likely reflected an increase in GLAST-mediated uptake. Together, these data indicate that the addition of this classical combination of growth factors and nutriments drives the rapid differentiation toward a homogenous culture of fibrous astrocytes expressing functional glutamate transporters.