Neutrophil extracellular traps downregulate lipopolysaccharide-induced activation of monocyte-derived dendritic cells.

Polymorphonuclear neutrophils (PMN) play a central role in inflammation and participate in its control, notably by modulating dendritic cell (DC) functions via soluble mediators or cell-cell contacts. Neutrophil extracellular traps (NETs) released by PMN could play a role in this context. To evaluate NET effects on DC maturation, we developed a model based on monocyte-derived DC (moDC) and calibrated NETs isolated from fresh human PMN. We found that isolated NETs alone had no discernable effect on moDC. In contrast, they downregulated LPS-induced moDC maturation, as shown by decreased surface expression of HLA-DR, CD80, CD83, and CD86, and by downregulated cytokine production (TNF-α, IL-6, IL-12, IL-23), with no increase in the expression of tolerogenic DC genes. Moreover, the presence of NETs during moDC maturation diminished the capacity of these moDC to induce T lymphocyte proliferation in both autologous and allogeneic conditions, and modulated CD4(+) T lymphocyte polarization by promoting the production of Th2 cytokines (IL-5 and IL-13) and reducing that of Th1 and Th17 cytokines (IFN-γ and IL-17). Interestingly, the expression and activities of the lymphoid chemokine receptors CCR7 and CXCR4 on moDC were not altered when moDC matured in the presence of NETs. Together, these findings reveal a new role for NETs in adaptive immune responses, modulating some moDC functions and thereby participating in the control of inflammation.

Afa/Dr diffusely adhering Escherichia coli strain C1845 induces neutrophil extracellular traps that kill bacteria and damage human enterocyte-like cells.

We recently documented the neutrophil response to enterovirulent diffusely adherent Escherichia coli expressing Afa/Dr fimbriae (Afa/Dr DAEC), using the human myeloid cell line PLB-985 differentiated into fully mature neutrophils. Upon activation, particularly during infections, neutrophils release neutrophil extracellular traps (NETs), composed of a nuclear DNA backbone associated with antimicrobial peptides, histones, and proteases, which entrap and kill pathogens. Here, using fluorescence microscopy and field emission scanning electron microscopy, we observed NET production by PLB-985 cells infected with the Afa/Dr wild-type (WT) E. coli strain C1845. We found that these NETs were able to capture, immobilize, and kill WT C1845 bacteria. We also developed a coculture model of human enterocyte-like Caco-2/TC7 cells and PLB-985 cells previously treated with WT C1845 and found, for the first time, that the F-actin cytoskeleton of enterocyte-like cells is damaged in the presence of bacterium-induced NETs and that this deleterious effect is prevented by inhibition of protease release. These findings provide new insights into the neutrophil response to bacterial infection via the production of bactericidal NETs and suggest that NETs may damage the intestinal epithelium, particularly in situations such as inflammatory bowel diseases.

Afa/Dr-expressing, diffusely adhering Escherichia coli strain C1845 triggers F1845 fimbria-dependent phosphatidylserine externalization on neutrophil-like differentiated PLB-985 cells through an apoptosis-independent mechanism.

The enterovirulent Escherichia coli strains potentially involved in inflammatory bowel diseases include diffusely adherent strains expressing Afa/Dr fimbriae (Afa/Dr DAEC). We have previously observed type 1 pilus-mediated interleukin-8 (IL-8) hyperproduction in infected neutrophils. As pathogen induction of host cell death programs and clearance of apoptotic infected cells are crucial for innate immune system homeostasis and host integrity, we examined modulation of neutrophil cell death by Afa/Dr DAEC. Using the human PLB-985 cell line differentiated into fully mature neutrophils, we found that the wild-type enterovirulent E. coli strain C1845 and the recombinant strain DH5alpha/pF1845 (expressing the fimbrial adhesin F1845) similarly induced time-dependent phosphatidylserine (PS) externalization, suggesting a major specific role of this virulence factor. Using small interfering RNA (siRNA) decay-accelerating factor (DAF)-transfected PLB-985 cells, we then showed that this PS externalization was triggered in part by glycosylphosphatidylinositol (GPI)-anchored DAF receptor engagement (leading to tyrosine kinase and protein kinase C activation) and that it required cytoskeleton and lipid raft architectural integrity. PS externalization under these conditions was not dependent on caspases, mitochondria, lysosomes, or reactive oxygen or nitrogen species. F1845-mediated PS externalization was sufficient to enable macrophage engulfment of infected differentiated PLB-985 cells. These findings provide new insights into the neutrophil response to Afa/Dr DAEC infection and highlight a new role for F1845 fimbriae. Interestingly, although apoptosis pathways were not engaged, C1845-infected PLB-985 cells displayed enhanced removal by macrophages, a process that may participate in the resolution of Afa/Dr DAEC infection and related inflammation.

Escherichia coli type 1 pili trigger late IL-8 production by neutrophil-like differentiated PLB-985 cells through a Src family kinase- and MAPK-dependent mechanism.

The innate immune response to enteropathogenic bacteria includes chemokine-induced polymorphonuclear neutrophil (PMN) migration across mucosal epithelia leading to bacterial clearance and resolution of infection. Among these bacteria, diffusely adherent Escherichia coli expressing Afa/Dr fimbriae (Afa/Dr DAEC), causing childhood diarrhea, can promote IL-8-dependent PMN transmigration across cultured intestinal epithelial cell monolayers via MAPK pathway activation. However, interactions between PMN and Afa/Dr DAEC are poorly documented and constitute the aim of the present study. Using the human PLB-985 cell line differentiated into fully mature PMN, we described the coordinated response to various E. coli. The rapid and strong release of reactive oxygen species and preformed intragranular mediators (myeloperoxidase and IL-8) is followed by a later TNF-alpha, IL-1beta, and IL-8 synthesis. The use of wild-type (IH11128, C1845, LF82), control (AAEC185), and recombinant (AAEC185 bearing Dr or F1845 fimbriae, AdLF82, or type 1 pili) bacterial strains allowed us to demonstrate that late IL-8 hyperproduction is triggered by type 1 pili but not by Dr or F1845 fimbriae; MAPKs (p38, ERK, Src) and NF-kappaB activations are implicated in this response. Thus, in the course of Afa/Dr DAEC intestinal infection, epithelium- and neutrophil-derived IL-8 could, at least in part, control the flow of neutrophils through the lamina propria. Afa/Dr DAEC-induced IL-8 hyperproduction by PMN might thus be important for inducing and perpetuating local inflammation, and this self-amplifying loop might play a role in the pathogenesis of inflammatory bowel diseases such as Crohn's disease.

Molecular analysis of the digestive microbiota in a gnotobiotic mouse model during antibiotic treatment: Influence of Saccharomyces boulardii.

The probiotic Saccharomyces boulardii is a non-pathogenic yeast that has been proven efficient in the prevention of antimicrobial-associated diarrhea and of Clostridium difficile associated colitis. We evaluated the influence of the administration of S. boulardii on the composition of the fecal microbiota in a human microbiota-associated mouse model. This evaluation was run before, during and after a 7-day oral treatment with amoxicillin clavulanic acid. Predominant groups of bacteria were quantified with fluorescence in situ hybridization combined with flow cytometry using group-specific 16S rRNA targeted oligonucleotide probes designed for the Eubacteria, Bacteroides-Porphyromonas-Prevotella, Clostridium coccoides-Eubacterium rectale, Faecalibacterium prausnitzii, Clostridium histolyticum, Lactobacillus-Enterococcus and Enterobacteriaceae groups and Bifidobacterium species. S. boulardii did not quantitatively alter the total anaerobic microbiota nor the dominant bacterial groups. During the antibiotic treatment in the two groups of mice receiving the yeast or not, the level of Enterobacteriaceae and Bacteroides groups increased when the C. coccoides-E. rectale group decreased dramatically. After the antibiotic treatment was discontinued, the return to the initial level was reached more rapidly in the S. boulardii-treated mice than in the control mice (p<0.05) for the C. coccoides-E. rectale and Bacteroides-Porphyromonas-Prevotella groups. This quicker recovery of normal intestinal microbiota equilibrium after antibiotic therapy could be a mechanism for S. boulardii preventive effect on antibiotic-associated diarrhea in humans.

An improved strategy to recover large fragments of functional human neutrophil extracellular traps.

Netosis is a recently described neutrophil function that leads to the release of neutrophil extracellular traps (NETs) in response to various stimuli. NETs are filaments of decondensed chromatin associated with granular proteins. In addition to their role against microorganisms, NETs have been implicated in autoimmunity, thrombosis, and tissue injury. Access to a standardized source of isolated NETs is needed to better analyze the roles of NETs. The aim of this study was to develop a procedure yielding soluble, well-characterized NET preparations from fresh human neutrophils. The calcium ionophore A23187 was chosen to induce netosis, and the restriction enzyme AluI was used to prepare large NET fragments. DNA and proteins were detected by electrophoresis and specific labeling. Some NET proteins [histone 3, lactoferrin (LF)] were quantified by western blotting, and double-stranded DNA (dsDNA) was quantified by immunofluorescence. Co-existence of dsDNA and neutrophil proteins confirmed the quality of the NET preparations. Their biological activity was checked by measuring elastase (ELA) activity and bacterial killing against various strains. Interindividual differences in histone 3, LF, ELA, and dsDNA relative contents were observed in isolated NETs. However, the reproducibility of NET preparation and characterization was validated, suggesting that this interindividual variability was rather related to donor variation than to technical bias. This standardized protocol is suitable for producing, isolating, and quantifying functional NETs that could serve as a tool for studying NET effects on immune cells and tissues.

Glycodendrimeric phenylporphyrins as new candidates for retinoblastoma PDT: blood carriers and photodynamic activity in cells.

Photodynamic therapy (PDT) has recently been proposed as a possible indication in the conservative treatment of hereditary retinoblastoma. In order to create photosensitizers with enhanced targeting ability toward retinoblastoma cells, meso-tetraphenylporphyrins bearing one glycodendrimeric moiety have been synthesized. The binding properties to plasma proteins and photodynamic activity of two monodendrimeric porphyrins bearing three mannose units via monoethylene glycol (1) or diethylene glycol (2) linkers have been compared to that of the non-dendrimeric tri-substituted derivative [TPP(p-Deg-O-α-ManOH)(3)]. The dendrimeric structure was found to highly increase the binding affinity to plasma proteins and to modify to some extent plasma distribution. HDL and to a lesser extent LDL have been shown to be the main carriers of dendrimeric and non-dendrimeric compounds. The phototoxicity observed for the two glycodendrimers (1) and (2) (LD(50)=0.5 µM) in Y79 cells is of the same order of magnitude that for TPP(p-Deg-O-α-ManOH)(3) (LD(50)=0.7 µM), with a similar cellular uptake level for (1) and a lower for (2). A serum content increase from 2% to 20% (v/v) in the incubation medium was found to inhibit both cellular uptake and photoactivity of dendrimeric derivatives, whereas those of TPP(p-Deg-O-α-ManOH)(3) remained little affected. Specificities of glycodendrimeric porphyrins, combining a lower cellular uptake together with a higher affinity toward plasma proteins, make these derivatives possible candidates for a vascular targeting PDT.

Rotavirus induces apoptosis in fully differentiated human intestinal Caco-2 cells.

Rotaviruses, which are the main cause of viral gastroenteritis in young children, induce structural and functional damages in infected mature enterocytes of the small intestine. To investigate a relationship between rotavirus infection and cell death by apoptosis, we used the human intestinal Caco-2 cell line. We demonstrated by several methods including TUNEL and ELISA detection of cytoplasmic histone-associated DNA fragments that the infection of fully differentiated Caco-2 cells by the RRV rotavirus strain induces apoptosis. Rotavirus infection leads to the loss of mitochondrial membrane potential and the release of cytochrome C from mitochondria. We showed that rotavirus-induced apoptosis was dependent of the multiplicity of infection and increased with time from 4 h to 24 h of infection. Flow cytometric analysis showed that DNA fragmentation occurs in productively infected cells, suggesting that rotavirus induces apoptosis by a direct mechanism. We also demonstrated that non-replicative RRV particles are not sufficient to induce apoptosis and viral gene expression seems required. Intracellular calcium plays a role in RRV-induced apoptosis because treatment with an intracellular calcium ion chelator (BAPTA-AM) partially inhibited apoptosis.