Susceptibility and Response of Human Blood Monocyte Subsets to Primary Dengue Virus Infection

Human blood monocytes play a central role in dengue infections and form the majority of virus infected cells in the blood. Human blood monocytes are heterogeneous and divided into CD16− and CD16+ subsets. Monocyte subsets play distinct roles during disease, but it is not currently known if monocyte subsets differentially contribute to dengue protection and pathogenesis. Here, we compared the susceptibility and response of the human CD16− and CD16+ blood monocyte subsets to primary dengue virus in vitro. We found that both monocyte subsets were equally susceptible to dengue virus (DENV2 NGC), and capable of supporting the initial production of new infective virus particles. Both monocyte subsets produced anti-viral factors, including IFN-α, CXCL10 and TRAIL. However, CD16+ monocytes were the major producers of inflammatory cytokines and chemokines in response to dengue virus, including IL-1β, TNF-α, IL-6, CCL2, 3 and 4. The susceptibility of both monocyte subsets to infection was increased after IL-4 treatment, but this increase was more profound for the CD16+ monocyte subset, particularly at early time points after virus exposure. These findings reveal the differential role that monocyte subsets might play during dengue disease

The CD14+/lowCD16+ monocyte subset is more susceptible to spontaneous and oxidant-induced apoptosis than the CD14+CD16− subset

Human monocytes can be classified into two subsets with distinctive characteristics. In this study, we report a difference in apoptotic potential between these two subsets with CD14+/lowCD16+ monocytes being more susceptible than CD14+CD16− monocytes to undergo spontaneous apoptosis and apoptosis induced by reactive oxygen species (ROS). By global transcriptomic and proteomic approaches, we observed that CD14+/lowCD16+ monocytes expressed higher levels of pro-apoptotic genes and proteins such as TNFα, caspase 3, Bax and cytochrome c and showed more caspases 3 and 7 activities. They also exhibited greater aerobic respiration resulting in a higher production of ROS from the mitochondria. CD14+CD16− monocytes, in contrast, showed higher expression of glutathione (GSH)-metabolizing genes such as GSH peroxidase and microsomal GSH S-transferase and were more resistant to oxidative stress than CD14+/lowCD16+ monocytes. The apoptosis of CD14+/lowCD16+ monocytes was ROS dependent as reducing ROS levels significantly reduced cell death. This is the first report of a differential apoptotic propensity of human monocyte subsets, and gaining a better understanding of this process may help to provide a better understanding of the roles of these subsets during homeostasis and under pathological conditions, particularly in situations in which high levels of oxidants are present

Exploring and challenging the network of angiogenesis

Angiogenesis is one of the hallmarks of cancer and, as such, one of the alternative general targets for anticancer therapy. Since angiogenesis is a complex process involving a high number of interconnected components, a network approach would be a convenient systemic way to analyse responses to directed drug attacks. Herein we show that, although the angiogenic network is easily broken by short combinations of directed attacks, it still remains essentially functional by keeping the global patterns and local efficiency essentially unaltered after these attacks. This is a clear sign of its high robustness and resilience and stresses the need of directed, combined attacks for an effective blockade of the process. The results of this theoretical study could be relevant for the design of new antiangiogenic therapies and the selection of their targets

Evolution and Dynamics of Regulatory Architectures Controlling Polymyxin B Resistance in Enteric Bacteria

Complex genetic networks consist of structural modules that determine the levels and timing of a cellular response. While the functional properties of the regulatory architectures that make up these modules have been extensively studied, the evolutionary history of regulatory architectures has remained largely unexplored. Here, we investigate the transition between direct and indirect regulatory pathways governing inducible resistance to the antibiotic polymyxin B in enteric bacteria. We identify a novel regulatory architecture—designated feedforward connector loop—that relies on a regulatory protein that connects signal transduction systems post-translationally, allowing one system to respond to a signal activating another system. The feedforward connector loop is characterized by rapid activation, slow deactivation, and elevated mRNA expression levels in comparison with the direct regulation circuit. Our results suggest that, both functionally and evolutionarily, the feedforward connector loop is the transitional stage between direct transcriptional control and indirect regulation

Schistosomes Induce Regulatory Features in Human and Mouse CD1dhi B Cells: Inhibition of Allergic Inflammation by IL-10 and Regulatory T Cells

Chronic helminth infections, such as schistosomes, are negatively associated with allergic disorders. Here, using B cell IL-10-deficient mice, Schistosoma mansoni-mediated protection against experimental ovalbumin-induced allergic airway inflammation (AAI) was shown to be specifically dependent on IL-10-producing B cells. To study the organs involved, we transferred B cells from lungs, mesenteric lymph nodes or spleen of OVA-infected mice to recipient OVA-sensitized mice, and showed that both lung and splenic B cells reduced AAI, but only splenic B cells in an IL-10-dependent manner. Although splenic B cell protection was accompanied by elevated levels of pulmonary FoxP3+ regulatory T cells, in vivo ablation of FoxP3+ T cells only moderately restored AAI, indicating an important role for the direct suppressory effect of regulatory B cells. Splenic marginal zone CD1d+ B cells proved to be the responsible splenic B cell subset as they produced high levels of IL-10 and induced FoxP3+ T cells in vitro. Indeed, transfer of CD1d+ MZ-depleted splenic B cells from infected mice restored AAI. Markedly, we found a similarly elevated population of CD1dhi B cells in peripheral blood of Schistosoma haematobium-infected Gabonese children compared to uninfected children and these cells produced elevated levels of IL-10. Importantly, the number of IL-10-producing CD1dhi B cells was reduced after anti-schistosome treatment. This study points out that in both mice and men schistosomes have the capacity to drive the development of IL-10-producing regulatory CD1dhi B cells and furthermore, these are instrumental in reducing experimental allergic inflammation in mice

High activity Rhenium-186 HEDP with autologous peripheral blood stem cell rescue: a phase I study in progressive hormone refractory prostate cancer metastatic to bone

We tested the feasibility and toxicity of high activities Rhenium-186 hydroxyethylidene diphosphonate, with peripheral blood stem cell rescue in patients with progressive hormone refractory prostate cancer metastatic to bone. Twenty-five patients received between 2500 and 5000 MBq of Rhenium-186 hydroxyethylidene diphosphonate followed 14 days later by the return of peripheral blood peripheral blood stem cells. Activity limiting toxicity was defined as grade III haematological toxicity, lasting at least 7 days, or grade IV haematological toxicity of any duration or any serious unexpected toxicity. Activity limiting toxicity occurred in two of six who received activities of 5000 MBq and maximum tolerated activity was defined at this activity level. Prostate specific antigen reductions of 50% or more lasting at least 4 weeks were seen in five of the 25 patients (20%) all of whom received more than 3500 MBq of Rhenium-186 hydroxyethylidene diphosphonate. The actuarial survival at 1 year is 54%. Administered activities of 5000 MBq of Rhenium-186 hydroxyethylidene diphosphonate are feasible using autologous peripheral blood peripheral blood stem cell rescue in patients with progressive hormone refractory prostate cancer metastatic to bone. The main toxicity is thrombocytopaenia, which is short lasting. A statistically significant activity/prostate specific antigen response was seen. We have now commenced a Phase II trial to further evaluate response rates

IL-22 Production Is Regulated by IL-23 During Listeria monocytogenes Infection but Is Not Required for Bacterial Clearance or Tissue Protection

Listeria monocytogenes (LM) is a gram-positive bacterium that is a common contaminant of processed meats and dairy products. In humans, ingestion of LM can result in intracellular infection of the spleen and liver, which can ultimately lead to septicemia, meningitis, and spontaneous abortion. Interleukin (IL)-23 is a cytokine that regulates innate and adaptive immune responses by inducing the production of IL-17A, IL-17F, and IL-22. We have recently demonstrated that the IL-23/IL-17 axis is required for optimal recruitment of neutrophils to the liver, but not the spleen, during LM infection. Furthermore, these cytokines are required for the clearance of LM during systemic infection. In other infectious models, IL-22 induces the secretion of anti-microbial peptides and protects tissues from damage by preventing apoptosis. However, the role of IL-22 has not been thoroughly investigated during LM infection. In the present study, we show that LM induces the production of IL-22 in vivo. Interestingly, IL-23 is required for the production of IL-22 during primary, but not secondary, LM infection. Our findings suggest that IL-22 is not required for clearance of LM during primary or secondary infection, using both systemic and mucosal models of infection. IL-22 is also not required for the protection of LM infected spleens and livers from organ damage. Collectively, these data indicate that IL-22 produced during LM infection must play a role other than clearance of LM or protection of tissues from pathogen- or immune-mediated damage

M-CSF Induces Monocyte Survival by Activating NF-κB p65 Phosphorylation at Ser276 via Protein Kinase C

Macrophage colony-stimulating factor (M-CSF) promotes mononuclear phagocyte survival and proliferation. The transcription factor Nuclear Factor-kappaB (NF-κB) is a key regulator of genes involved in M-CSF-induced mononuclear phagocyte survival and this study focused at identifying the mechanism of NF-κB transcriptional activation. Here, we demonstrate that M-CSF stimulated NF-κB transcriptional activity in human monocyte-derived macrophages (MDMs) and the murine macrophage cell line RAW 264.7. The general protein kinase C (PKC) inhibitor Ro-31-8220, the conventional PKCα/β inhibitor Gö-6976, overexpression of dominant negative PKCα constructs and PKCα siRNA reduced NF-κB activity in response to M-CSF. Interestingly, Ro-31-8220 reduced Ser276 phosphorylation of NF-κBp65 leading to decreased M-CSF-induced monocyte survival. In this report, we identify conventional PKCs, including PKCα as important upstream kinases for M-CSF-induced NF-κB transcriptional activation, NF-κB-regulated gene expression, NF-κB p65 Ser276 phosphorylation, and macrophage survival. Lastly, we find that NF-κB p65 Ser276 plays an important role in basal and M-CSF-stimulated NF-κB activation in human mononuclear phagocytes

Early Secreted Antigen ESAT-6 of Mycobacterium tuberculosis Promotes Protective T Helper 17 Cell Responses in a Toll-Like Receptor-2-dependent Manner

Despite its relatively poor efficacy, Bacillus Calmette-Guérin (BCG) has been used as a tuberculosis (TB) vaccine since its development in 1921. BCG induces robust T helper 1 (Th1) immune responses but, for many individuals, this is not sufficient for host resistance against Mycobacterium tuberculosis (M. tb) infection. Here we provide evidence that early secreted antigenic target protein 6 (ESAT-6), expressed by the virulent M. tb strain H37Rv but not by BCG, promotes vaccine-enhancing Th17 cell responses. These activities of ESAT-6 were dependent on TLR-2/MyD88 signalling and involved IL-6 and TGF-β production by dendritic cells. Thus, animals that were previously infected with H37Rv or recombinant BCG containing the RD1 region (BCG::RD1) exhibited improved protection upon re-challenge with virulent H37Rv compared with mice previously infected with BCG or RD1-deficient H37Rv (H37RvΔRD1). However, TLR-2 knockout (TLR-2-/-) animals neither showed Th17 responses nor exhibited improved protection in response to immunization with H37Rv. Furthermore, H37Rv and BCG::RD1 infection had little effect on the expression of the anti-inflammatory microRNA-146a (miR146a) in dendritic cells (DCs), whereas BCG and H37RvΔRD1 profoundly induced its expression in DCs. Consistent with these findings, ESAT-6 had no effect on miR146a expression in uninfected DCs, but dramatically inhibited its upregulation in BCG-infected or LPS-treated DCs. Collectively, our findings indicate that, in addition to Th1 immunity induced by BCG, RD1/ESAT-6-induced Th17 immune responses are essential for optimal vaccine efficacy