17β-Estradiol Enhances the Response of Plasmacytoid Dendritic Cell to CpG

Gender differences in immune capabilities suggest that sex hormones such as estrogens were involved in the regulation of the immunocompetence. Numerous studies also suggest that plasmacytoid dendritic cells (PDCs) play a pathogenic role in SLE. However, it is unclear whether estrogen can modulate the function of PDCs to influence the development of SLE. In the present study, PDCs from murine spleens were treated with 17β-estradiol (E2) and CpG respectively or both in vitro, then cell viability, costimulatory molecule expression, cytokine secretion of PDCs, as well as stimulatory capacity of PDCs to B cells were analyzed. Results showed that E2 and CpG increased the cell viability and costimulatory molecule expression on PDCs synergistically. Moreover, the intracellular and extracellular secretion of IFN-α was increased by E2 or E2 plus CpG. In addition, E2 and CpG also increased the stimulatory capacity of PDCs to B cells, and the viability of B cells was decreased after neutralizing IFN-α significantly. In the experiments in vivo, mice received daily s.c. injections of E2 and CpG respectively or both, then we found that the plasma concentration of IgM were elevated by E2 and CpG synergistically and the expression of IFN-α/β in spleens were noticeably increased by CpG plus E2 compared with the treatment of E2 or CpG only. This study indicates that E2 could exacerbate PDCs'' activation with CpG, which further activates B cells to upregulate susceptibility to autoantigens. IFN-α plays an important role in the stimulatory effect of PDCs on B cells. E2 stimulation of IFN-α production may result in female prevalence in autoimmune diseases such as SLE through activation of PDCs. This study provides novel evidence of relationship between estrogen and SLE and also sheds light on gender biases among SLE patients.     

The Response of Human Macrophages to β-Glucans Depends on the Inflammatory Milieu

Background

β-glucans are fungal cell wall components that bind to the C-type lectin-like receptor dectin-1. Polymorphisms of dectin-1 gene are associated with susceptibility to invasive fungal infection and medically refractory ulcerative colitis. The purpose of this study has been addressing the response of human macrophages to β-glucans under different conditions mimicking the composition of the inflammatory milieu in view of the wide plasticity and large range of phenotypical changes showed by these cells, and the relevant role of dectin-1 in several pathophysiological conditions.

Principal Findings

Serum-differentiated macrophages stimulated with β-glucans showed a low production of TNFα and IL-1β, a high production of IL-6 and IL-23, and a delayed induction of cyclooxygenase-2 and PGE₂ biosynthesis that resembled the responses elicited by crystals and those produced when phagosomal degradation of the phagocytic cargo increases ligand access to intracellular pattern recognition receptors. Priming with a low concentration of LPS produced a rapid induction of cyclooxygenase-2 and a synergistic release of PGE₂. When the differentiation of the macrophages was carried out in the presence of M-CSF, an increased expression of dectin-1 B isoform was observed. In addition, this treatment made the cells capable to release arachidonic acid in response to β-glucan.

Conclusions

These results indicate that the macrophage response to fungal β-glucans is strongly influenced by cytokines and microbial-derived factors that are usual components of the inflammatory milieu. These responses can be sorted into three main patterns i) an elementary response dependent on phagosomal processing of pathogen-associated molecular patterns and/or receptor-independent, direct membrane binding linked to the immunoreceptor tyrosine-based activation motif-bearing transmembrane adaptor DNAX-activating protein 12, ii) a response primed by TLR4-dependent signals, and iii) a response dependent on M-CSF and dectin-1 B isoform expression that mainly signals through the dectin-1 B/spleen tyrosine kinase/cytosolic phospholipase A₂ route.     

Susceptibility of Pseudomonas aeruginosa to catechol-substituted cephalosporin is unrelated to the pyochelin–Fe transporter FptA

Previously it has been postulated that the pyochelin–Fe outer membrane transporter, FptA, is involved in the uptake of catechol-substituted cephalosporins in Pseudomonas aeruginosa. Iron uptake and antibacterial activity studies on different mutants showed clearly that FptA is unable to bind and transport these antibiotics.     

Integrative Model of the Immune Response to a Pulmonary Macrophage Infection: What Determines the Infection Duration?

The immune mechanisms which determine the infection duration induced by pathogens targeting pulmonary macrophages are poorly known. To explore the impact of such pathogens, it is indispensable to integrate the various immune mechanisms and to take into account the variability in pathogen virulence and host susceptibility. In this context, mathematical models complement experimentation and are powerful tools to represent and explore the complex mechanisms involved in the infection and immune dynamics. We developed an original mathematical model in which we detailed the interactions between the macrophages and the pathogen, the orientation of the adaptive response and the cytokine regulations. We applied our model to the Porcine Respiratory and Reproductive Syndrome virus (PRRSv), a major concern for the swine industry. We extracted value ranges for the model parameters from modelling and experimental studies on respiratory pathogens. We identified the most influential parameters through a sensitivity analysis. We defined a parameter set, the reference scenario, resulting in a realistic and representative immune response to PRRSv infection. We then defined scenarios corresponding to graduated levels of strain virulence and host susceptibility around the reference scenario. We observed that high levels of antiviral cytokines and a dominant cellular response were associated with either short, the usual assumption, or long infection durations, depending on the immune mechanisms involved. To identify these mechanisms, we need to combine the levels of antiviral cytokines, including , and . The latter is a good indicator of the infected macrophage level, both combined provide the adaptive response orientation. Available PRRSv vaccines lack efficiency. By integrating the main interactions between the complex immune mechanisms, this modelling framework could be used to help designing more efficient vaccination strategies.     

Caspase-1 Cleavage of the TLR Adaptor TRIF Inhibits Autophagy and β-Interferon Production during Pseudomonas aeruginosa Infection

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Hyaluronan Synthesis Mediates the Fibrotic Response of Keratocytes to Transforming Growth Factor β

TGFβ induces fibrosis in healing corneal wounds, and in vitro corneal keratocytes up-regulate expression of several fibrosis-related genes in response to TGFβ. Hyaluronan (HA) accumulates in healing corneas, and HA synthesis is induced by TGFβ by up-regulation of HA synthase 2. This study tested the hypothesis that HA acts as an extracellular messenger, enhancing specific fibrotic responses of keratocytes to TGFβ. HA synthesis inhibitor 4-methylumbelliferone (4MU) blocked TGFβ induction of HA synthesis in a concentration-dependent manner. 4MU also inhibited TGFβ-induced up-regulation of α-smooth muscle actin, collagen type III, and extra domain A-fibronectin. Chemical analogs of 4MU also inhibited fibrogenic responses in proportion to their inhibition of HA synthesis. 4MU, however, showed no effect on TGFβ induction of luciferase by the 3TP-Lux reporter plasmid. Inhibition of HA using siRNA to HA synthase 2 reduced TGFβ up-regulation of smooth muscle actin, fibronectin, and cell division. Similarly, brief treatment of keratocytes with hyaluronidase reduced TGFβ responses. These results suggest that newly synthesized cell-associated HA acts as an extracellular enhancer of wound healing and fibrosis in keratocytes by augmenting a limited subset of the cellular responses to TGFβ.     

What It Takes to Be a Pseudomonas aeruginosa? The Core Genome of the Opportunistic Pathogen Updated

Pseudomonas aeruginosa is an opportunistic bacterial pathogen able to thrive in highly diverse ecological niches and to infect compromised patients. Its genome exhibits a mosaic structure composed of a core genome into which accessory genes are inserted en bloc at specific sites. The size and the content of the core genome are open for debate as their estimation depends on the set of genomes considered and the pipeline of gene detection and clustering. Here, we redefined the size and the content of the core genome of P. aeruginosa from fully re-analyzed genomes of 17 reference strains. After the optimization of gene detection and clustering parameters, the core genome was defined at 5,233 orthologs, which represented ~ 88% of the average genome. Extrapolation indicated that our panel was suitable to estimate the core genome that will remain constant even if new genomes are added. The core genome contained resistance determinants to the major antibiotic families as well as most metabolic, respiratory, and virulence genes. Although some virulence genes were accessory, they often related to conserved biological functions. Long-standing prophage elements were subjected to a genetic drift to eventually display a G+C content as higher as that of the core genome. This contrasts with the low G+C content of highly conserved ribosomal genes. The conservation of metabolic and respiratory genes could guarantee the ability of the species to thrive on a variety of carbon sources for energy in aerobiosis and anaerobiosis. Virtually all the strains, of environmental or clinical origin, have the complete toolkit to become resistant to the major antipseudomonal compounds and possess basic pathogenic mechanisms to infect humans. The knowledge of the genes shared by the majority of the P. aeruginosa isolates is a prerequisite for designing effective therapeutics to combat the wide variety of human infections.     

Site-specific integration of the phage ΦCTX genome into the Pseudomonas aeruginosa chromosome: characterization of the functional integrase gene located close to and upstream of attP

The site-specific integration of the phage ?CTX genome, which carries the gene for a pore-forming cytotoxin, into the Pseudomonas aeruginosa chromosome was analysed. The 1,167 by integrase gene, int, located immediately upstream of the attachment site, attP, was characterized using plasmid constructs, harbouring the integration functions, and serving as an integration probe in both P. aeruginosa and Escherichia coli. The attP plasmids p1000/p400 in the presence of the int plasmid pIBH and attP-int plasmids pINT/pINTS can be stably integrated into the P. aeruginosa chromosome. Successful recombination between the attP plasmid p1000 and the attB plasmid p5.1, in the presence of the int plasmid pIBH in E. coli HB101 showed that the int gene is active in trans in E. coli. The int gene product was detected as a 43 kDa protein in E. coli maxicells harbouring pINT. Proposed integration arm regions downstream of attP are not necessary for the integration process. pINT and phage ?CTX could be integrated together into P. aeruginosa chromosomal DNA, yielding double integrates.     

Induction of TGF-β1 Synthesis by Macrophages in Response to Apoptotic Cells Requires Activation of the Scavenger Receptor CD36

Background/Objective

Phosphatidylserine (PS) exposed on apoptotic cells has been shown to stimulate production of transforming growth factor-β (TGF-β) and promote anti-inflammatory responses. However, the PS receptor(s) responsible for this induction has not been clearly determined.

Methodology/Principal Findings

In the present study, using RAWTβRII cells in which a truncated dominant negative TGF-β receptor II was stably transfected in order to avoid auto-feedback induction of TGF-β, we show that TGF-β1 synthesis is initiated via activation of the scavenger receptor, CD36. The response requires exposure of PS on the apoptotic cell surface and was absent in macrophages lacking CD36. Direct activation of CD36 with an anti-CD36 antibody initiated TGF-β1 production, and signaling pathways involving both Lyn kinase and ERK1/2 were shown to participate in CD36-driven TGF-β1 expression.

Conclusion/Significance

Since CD36 has been previously implicated in activation of secreted latent TGF-β, the present study indicates its role in the multiple steps to generation of this important biological mediator.     

Differential Effects of β-catenin and NF-κB Interplay in the Regulation of Cell Proliferation,Inflammation and Tumorigenesis in Response to Bacterial Infection

Both β-catenin and NF-κB have been implicated in our laboratory as candidate factors in driving proliferation in an in vivo model of Citrobacter rodentium (CR)-induced colonic crypt hyper-proliferation and hyperplasia. Herein, we test the hypothesis that β-catenin and not necessarily NF-κB regulates colonic crypt hyperplasia or tumorigenesis in response to CR infection. When C57Bl/6 wild type (WT) mice were infected with CR, sequential increases in proliferation at days 9 and 12 plateaued off at day 19 and paralleled increases in NF-κB signaling. In Tlr4^−/− (KO) mice, a sequential but sustained proliferation which tapered off only marginally at day 19, was associated with TLR4-dependent and independent increases in NF-κB signaling. Similarly, increases in either activated or total β-catenin in the colonic crypts of WT mice as early as day 3 post-infection coincided with cyclinD1 and c-myc expression and associated crypt hyperplasia. In KO mice, a delayed kinetics associated predominantly with increases in non-phosphorylated (active) β-catenin coincided with increases in cyclinD1, c-myc and crypt hyperplasia. Interestingly, PKCζ-catalyzed Ser-9 phosphorylation and inactivation of GSK-3β and not loss of wild type APC protein accounted for β-catenin accumulation and nuclear translocation in either strain. In vitro studies with Wnt2b and Wnt5a further validated the interplay between the Wnt/β-catenin and NF-κB pathways, respectively. When WT or KO mice were treated with nanoparticle-encapsulated siRNA to β-catenin (si- β-Cat), almost complete loss of nuclear β-catenin coincided with concomitant decreases in CD44 and crypt hyperplasia without defects in NF-κB signaling. si-β-Cat treatment to Apc ^Min/+ mice attenuated CR-induced increases in β-catenin and CD44 that halted the growth of mutated crypts without affecting NF-κB signaling. The predominant β-catenin-induced crypt proliferation was further validated in a Castaneus strain (B6.CAST.11M) that exhibited significant crypt hyperplasia despite an attenuated NF-κB signaling. Thus, β-catenin and not necessarily NF-κB regulates crypt hyperplasia in response to bacterial infection.     

Tumor Necrosis Factor Receptor-associated Factor 6 (TRAF6) and TGFβ-activated Kinase 1 (TAK1) Play Essential Roles in the C-type Lectin Receptor Signaling in Response to Candida albicans Infection

Tumor necrosis factor receptor-associated factor 6 (TRAF6) and TGFβ-activated kinase 1 (TAK1) are considered as key intermediates in Toll-like receptor (TLR) signaling. However, the role of TRAF6 and TAK1 in C-type lectin receptors (CLRs) in response to fungal infection has not been studied. In this study, we have utilized macrophages derived from TRAF6 knock-out mice and myeloid-specific TAK1-deficient mice and determined the role of TRAF6 and TAK1 in CLR-induced signal transduction events. We demonstrate that TRAF6 and TAK1 are required for NF-κB and JNK activation, and expression of proinflammatory cytokines in response to Candida albicans infection. Our results highlight TRAF6 and TAK1 as key components in the signaling cascade downstream of C-type lectin receptors and as critical mediators of the anti-fungal immune response. Therefore, our studies provide a mechanistic understanding of the host immune response to C. albicans, which has a significant impact for the development of anti-fungal therapeutics and in understanding risk-factors and determining susceptibility to C. albicans infection.