Role of CFTR expressed by neutrophils in modulating acute lung inflammation and injury in mice

Objective and designCystic fibrosis transmembrane conductance regulator (CFTR) regulates infection and inflammation. In this study, we investigated whether a lack of functional CFTR in neutrophils would promote lipopolysaccharide (LPS)-induced lung inflammation and injury.Materials and methodsCFTR-inhibited or F508del-CFTR-mutated neutrophils were stimulated with LPS and cultured to evaluate production of cytokines and NF-κB activation. Wild-type mice were reconstituted with F508del neutrophils or bone marrow and then intratracheally challenged with LPS to observe lung inflammatory response.ResultsPharmacologic inhibition and genetic mutation of CFTR in neutrophils activated NF-κB and facilitated macrophage inflammatory protein-2 (MIP-2) and tumor necrosis factor-α (TNF-α) production. Wild-type mice reconstituted with F508del neutrophils and bone marrow had more severe lung inflammation and injury after LPS challenge compared to wild-type mice receiving wild-type neutrophils or bone marrow reconstitution.ConclusionsLack of functional CFTR in neutrophils can promote LPS-induced acute lung inflammation and injury

The invasome of Salmonella Dublin as revealed by whole genome sequencing

Background Salmonella enterica serovar Dublin is a zoonotic infection that can be transmitted from cattle to humans through consumption of contaminated milk and milk products. Outbreaks of human infections by S. Dublin have been reported in several countries including high-income countries. A high proportion of S. Dublin cases in humans are associated with invasive disease and systemic illness. The genetic basis of virulence in S. Dublin is not well characterized. Methods Whole genome sequencing was applied to a set of clinical invasive and non-invasive S. Dublin isolates from different countries in order to characterize the putative genetic determinants involved in the virulence and invasiveness of S. Dublin in humans. Results We identified several virulence factors that form the bacterial invasome and may contribute to increasing bacterial virulence and pathogenicity including mainly Gifsy-2 prophage, two different type 6 secretion systems (T6SSs) harbored by Salmonella pathogenicity islands; SPI-6 and SPI-19 respectively and virulence genes; ggt and PagN. Although Vi antigen and the virulence plasmid have been reported previously to contribute to the virulence of S. Dublin we did not detect them in all invasive isolates indicating that they are not the main virulence determinants in S. Dublin. Conclusion Several virulence factors within the genome of S. Dublin might contribute to the ability of S. Dublin to invade humans’ blood but there were no genomic markers that differentiate invasive from non-invasive isolates suggesting that host immune response play a crucial role in the clinical outcome of S. Dublin infection

Role of persistent CMV infection in configuring T cell immunity in the elderly

Ageing is associated with declines in many physiological parameters, including multiple immune system functions. The rate of acceleration of the frequency of death due to cardiovascular disease or cancer seems to increase with age from middle age up to around 80 years, plateauing thereafter. Mortality due to infectious disease, however, does not plateau, but continues to accelerate indefinitely. The elderly commonly possess oligoclonal expansions of T cells, especially of CD8 cells, which, surprisingly, are often associated with cytomegalovirus (CMV) seropositivity. This in turn is associated with many of the same phenotypic and functional alterations to T cell immunity that have been suggested as biomarkers of immune system aging. Thus, the manner in which CMV and the host immune system interact is critical in determining the "age" of specific immunity. We may therefore consider immunosenescence in some respects as an infectious state. This implies that interventions aimed at the pathogen may improve the organ system affected. Hence, CMV-directed anti-virals or vaccination may have beneficial effects on immunity in later life

Risk Factors for and Clinical Outcome of Congenital Cytomegalovirus Infection in a Peri-Urban West-African Birth Cohort

BACKGROUND: Congenital cytomegalovirus (CMV) infection is the most prevalent congenital infection worldwide. Epidemiology and clinical outcomes are known to vary with socio-economic background, but few data are available from developing countries, where the overall burden of infectious diseases is frequently high. METHODOLOGY/PRINCIPAL FINDINGS: As part of an ongoing birth cohort study in The Gambia among term infants, urine samples were collected at birth and tested by PCR for the presence of CMV DNA. Risk factors for transmission and clinical outcome were assessed, including placental malaria infection. Babies were followed up at home monthly for morbidity and anthropometry, and at one year of age a clinical evaluation was performed. The prevalence of congenital CMV infection was 5.4% (40/741). A higher prevalence of hepatomegaly was the only significant clinical difference at birth. Congenitally infected children were more often first born babies (adjusted odds ratio (OR) 5.3, 95% confidence interval (CI) 2.0-13.7), more frequently born in crowded compounds (adjusted OR 2.9, 95%CI 1.0-8.3) and active placental malaria was more prevalent (adjusted OR 2.9, 95%CI 1.0-8.4). These associations were corrected for maternal age, bed net use and season of birth. During the first year of follow up, mothers of congenitally infected children reported more health complaints for their child. CONCLUSIONS/SIGNIFICANCE: In this study, the prevalence of congenital CMV among healthy neonates was much higher than previously reported in industrialised countries, and was associated with active placental malaria infection. There were no obvious clinical implications during the first year of life. The effect of early life CMV on the developing infant in the Gambia could be mitigated by environmental factors, such as the high burden of other infections.Journal ArticleResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe

Differential effects of age, cytomegalovirus-seropositivity and end-stage renal disease (ESRD) on circulating T lymphocyte subsets

The age- and cytomegalovirus (CMV)-seropositivity-related changes in subsets and differentiation of circulating T cells were investigated in end-stage renal disease (ESRD) patients (n = 139) and age-matched healthy individuals. The results show that CMV-seropositivity is associated with expansion of both CD4+ and CD8+ memory T cells which is already observed in young healthy individuals. In addition, CMV-seropositive healthy individuals have a more differentiated memory T cell profile. Only CMV-seropositive healthy individuals showed an age-dependent decrease in CD4+ naïve T cells. The age-related decrease in the number of CD8+ naïve T cells was CMV-independent. In contrast, all ESRD patients showed a profound naïve T-cell lymphopenia at every decade. CMV-seropositivity aggravated the contraction of CD4+ naïve T cells and increased the number of differentiated CD4+ and CD8+ memory T cells. In conclusion, CMV-seropositivity markedly alters the homeostasis of circulating T cells in healthy individuals and aggravates the T cell dysregulation observed in ESRD patients

Intravenous Immunoglobulin Prevents Murine Antibody-Mediated Acute Lung Injury at the Level of Neutrophil Reactive Oxygen Species (ROS) Production

Transfusion-related acute lung injury (TRALI) is a leading cause of transfusion-associated mortality that can occur with any type of transfusion and is thought to be primarily due to donor antibodies activating pulmonary neutrophils in recipients. Recently, a large prospective case controlled clinical study of cardiac surgery patients demonstrated that despite implementation of male donors, a high incidence of TRALI still occurred and suggested a need for additional interventions in susceptible patient populations. To examine if intravenous immunoglobulin (IVIg) may be effective, a murine model of antibody-mediated acute lung injury that approximates human TRALI was examined. When BALB/c mice were injected with the anti-major histocompatibility complex class I antibody 34-1-2s, mild shock (reduced rectal temperature) and respiratory distress (dyspnea) were observed and pre-treatment of the mice with 2 g/kg IVIg completely prevented these symptoms. To determine IVIg's usefulness to affect severe lung damage, SCID mice, previously shown to be hypersensitive to 34-1-2s were used. SCID mice treated with 34-1-2s underwent severe shock, lung damage (increased wet/dry ratios) and 40% mortality within 2 hours. Treatment with 2 g/kg IVIg 18 hours before 34-1-2s administration completely protected the mice from all adverse events. Treatment with IVIg after symptoms began also reduced lung damage and mortality. While the prophylactic IVIg administration did not affect 34-1-2s-induced pulmonary neutrophil accumulation, bone marrow-derived neutrophils from the IVIg-treated mice displayed no spontaneous ROS production nor could they be stimulated in vitro with fMLP or 34-1-2s. These results suggest that IVIg prevents murine antibody-mediated acute lung injury at the level of neutrophil ROS production and thus, alleviating tissue damage

In Vivo CD8+ T-Cell Suppression of SIV Viremia Is Not Mediated by CTL Clearance of Productively Infected Cells

The CD8+ T-cell is a key mediator of antiviral immunity, potentially contributing to control of pathogenic lentiviral infection through both innate and adaptive mechanisms. We studied viral dynamics during antiretroviral treatment of simian immunodeficiency virus (SIV) infected rhesus macaques following CD8+ T-cell depletion to test the importance of adaptive cytotoxic effects in clearance of cells productively infected with SIV. As previously described, plasma viral load (VL) increased following CD8+ T-cell depletion and was proportional to the magnitude of CD8+ T-cell depletion in the GALT, confirming a direct relationship between CD8+ T-cell loss and viral replication. Surprisingly, first phase plasma virus decay following administration of antiretroviral drugs was not slower in CD8+ T-cell depleted animals compared with controls indicating that the short lifespan of the average productively infected cell is not a reflection of cytotoxic T-lymphocyte (CTL) killing. Our findings support a dominant role for non-cytotoxic effects of CD8+ T-cells on control of pathogenic lentiviral infection and suggest that cytotoxic effects, if present, are limited to early, pre-productive stages of the viral life cycle. These observations have important implications for future strategies to augment immune control of HIV

Primary Human mDC1, mDC2, and pDC Dendritic Cells Are Differentially Infected and Activated by Respiratory Syncytial Virus

Respiratory syncytial virus (RSV) causes recurrent infections throughout life. Vaccine development may depend upon understanding the molecular basis for induction of ineffective immunity. Because dendritic cells (DCs) are critically involved in early responses to infection, their interaction with RSV may determine the immunological outcome of RSV infection. Therefore, we investigated the ability of RSV to infect and activate primary mDCs and pDCs using recombinant RSV expressing green fluorescent protein (GFP). At a multiplicity of infection of 5, initial studies demonstrated ∼6.8% of mDC1 and ∼0.9% pDCs were infected. We extended these studies to include CD1c−CD141+ mDC2, finding mDC2 infected at similar frequencies as mDC1. Both infected and uninfected cells upregulated phenotypic markers of maturation. Divalent cations were required for infection and maturation, but maturation did not require viral replication. There is evidence that attachment and entry/replication processes exert distinct effects on DC activation. Cell-specific patterns of RSV-induced maturation and cytokine production were detected in mDC1, mDC2, and pDC. We also demonstrate for the first time that RSV induces significant TIMP-2 production in all DC subsets. Defining the influence of RSV on the function of selected DC subsets may improve the likelihood of achieving protective vaccine-induced immunity

The Binding of Triclosan to SmeT, the Repressor of the Multidrug Efflux Pump SmeDEF, Induces Antibiotic Resistance in Stenotrophomonas maltophilia

The wide utilization of biocides poses a concern on the impact of these compounds on natural bacterial populations. Furthermore, it has been demonstrated that biocides can select, at least in laboratory experiments, antibiotic resistant bacteria. This situation has raised concerns, not just on scientists and clinicians, but also on regulatory agencies, which are demanding studies on the impact that the utilization of biocides may have on the development on resistance and consequently on the treatment of infectious diseases and on human health. In the present article, we explored the possibility that the widely used biocide triclosan might induce antibiotic resistance using as a model the opportunistic pathogen Stenotrophomonas maltophilia. Biochemical, functional and structural studies were performed, focusing on SmeDEF, the most relevant antibiotic- and triclosan-removing multidrug efflux pump of S. maltophilia. Expression of smeDEF is regulated by the repressor SmeT. Triclosan released SmeT from its operator and induces the expression of smeDEF, thus reducing the susceptibility of S. maltophilia to antibiotics in the presence of the biocide. The structure of SmeT bound to triclosan is described. Two molecules of triclosan were found to bind to one subunit of the SmeT homodimer. The binding of the biocide stabilizes the N terminal domain of both subunits in a conformation unable to bind DNA. To our knowledge this is the first crystal structure obtained for a transcriptional regulator bound to triclosan. This work provides the molecular basis for understanding the mechanisms allowing the induction of phenotypic resistance to antibiotics by triclosan

Abundance of the Quorum-Sensing Factor Ax21 in Four Strains of Stenotrophomonas maltophilia Correlates with Mortality Rate in a New Zebrafish Model of Infection

Stenotrophomonas maltophilia is a Gram-negative pathogen with emerging nosocomial incidence. Little is known about its pathogenesis and the genomic diversity exhibited by clinical isolates complicates the study of pathogenicity and virulence factors. Here, we present a strategy to identify such factors in new clinical isolates of S. maltophilia, incorporating an adult-zebrafish model of S. maltophilia infection to evaluate relative virulence coupled to 2D difference gel electrophoresis to explore underlying differences in protein expression. In this study we report upon three recent clinical isolates and use the collection strain ATCC13637 as a reference. The adult-zebrafish model shows discrimination capacity, i.e. from very low to very high mortality rates, with clinical symptoms very similar to those observed in natural S. maltophilia infections in fish. Strain virulence correlates with resistance to human serum, in agreement with previous studies in mouse and rat and therefore supporting zebrafish as a replacement model. Despite its clinical origin, the collection strain ATCC13637 showed obvious signs of attenuation in zebrafish, with null mortality. Multilocus-sequence-typing analysis revealed that the most virulent strains, UV74 and M30, exhibit the strongest genetic similitude. Differential proteomic analysis led to the identification of 38 proteins with significantly different abundance in the three clinical strains relative to the reference strain. Orthologs of several of these proteins have been already reported to have a role in pathogenesis, virulence or resistance mechanisms thus supporting our strategy. Proof of concept is further provided by protein Ax21, whose abundance is shown here to be directly proportional to mortality in the zebrafish infection model. Indeed, recent studies have demonstrated that this protein is a quorum-sensing-related virulence factor