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Furthermore, we also observed functional inhibition of uninfected bystander cells. This inhibition was mediated, in part, by a heat-stable bacterial component. Together, these data show that type A F. This suggests that immune dysregulation by F. Francisella tularensis is a gram-negative, facultative intracellular bacterium and the causative agent of tularemia. Although the bacteria was identified nearly years ago, the nature of its interaction with host cells and tissues has remained largely undefined until recent times.
The unfortunate realization of the potential of F. Much of our recent understanding of tularemia pathogenesis has come from manipulation of the mouse model of Francisella infections.
While these murine studies have yielded many important advances in our understanding of tularemia pathogenesis, very little is understood about how Francisella , especially the most virulent type A strains such as Schu S4, interacts with human cells.
Dendritic cells DCs serve as a central cell type in the immune system, bridging the innate and adaptive immune response to effectively eradicate invading pathogens. Thus, it is unsurprising that many microbes have developed mechanisms by which to modulate these cells to evade host immune responses 4 , For example, in the mouse model, both attenuated and virulent strains of F. However, only virulent F.
It is not known if virulent F. A few recently published studies have focused on the attenuated F. However, both of these subspecies are attenuated in humans.
Due to this attenuation, it is unsurprising that following infection with these strains, human DCs and monocytes are activated to produce proinflammatory cytokines and upregulate receptors associated with antigen presentation and that DCs can easily control bacterial replication when exposed to the appropriate stimulus following infection 22 , 26 , 27 , However, this does not necessarily reflect how human cells, in particular DCs, might behave when infected with a more formidable and virulent strain of F.
Here, we provide data supporting our hypothesis that, unlike more attenuated Francisella species and despite exponential replication, type A F. As observed in the murine model, this suppression of cytokine secretion mediated by F. Furthermore, we made the surprising observation that F. However, our data suggest that Schu S4 LPS present in tissue culture medium was not the primary mediator of the suppression observed in human DCs.
Rather, an undefined, moderately heat-stable molecule shed or secreted by Schu S4 significantly inhibited human DC responsiveness to secondary stimuli. Furthermore, our evidence suggests that modulation of DCs by Schu S4 components in the extracellular space is different from that observed among infected cells. Together, these results correlate with our in vivo observation that Schu S4 potently suppresses cells critical for initiation of innate immunity.
Data presented herein also represent an important step forward in understanding how virulent F. Titers were determined in frozen stocks by enumerating viable bacteria from serial dilutions plated on MMH agar as previously described 9 , Anti-LPS Ft and isotype antibodies were labeled using an AlexaFluor monoclonal labeling kit Invitrogen according to manufacturer's instructions. Cultures were collected and filter sterilized through a 0.
Ultrapure E. Human myeloid DCs were differentiated from peripheral blood monocytes that had been subjected to apheresis as previously described 6 , Briefly, monocytes that had been subjected to apheresis were enriched using Ficoll-Paque GE Healthcare. On day 2 of culture, 0. All cells were used on day 4 of culture. Human blood cells were collected from anonymous volunteers under a protocol reviewed and approved by the NIH Institutional Biosafety Committee.
DCs were removed from their original cultures and pelleted. The resulting DC-conditioned medium was reserved for replating of cells. Cells treated with medium alone served as negative controls. This concentration has been previously shown to be the MIC for control of F.
In these experiments, immediately prior to the addition of TLR agonist, an aliquot of the medium was collected, serially diluted, and plated onto MMH agar to enumerate extracellular bacteria.
Cells were then washed extensively and lysed following incubation in water for 5 min. The last wash from each sample was plated onto MMH agar to confirm depletion of residual extracellular bacteria. There were no bacteria detected in these samples in each experiment tested. DCs were incubated overnight, followed by addition of ultrapure LPS as described above.
DCs were then stained for intracellular cytokines as described below. Experimental and control groups were all performed in triplicate. The standard error of the mean SEM and statistical significance between treatment groups were determined by analysis of variance, followed by Tukey-Kramer's comparison of means.
Samples were then placed on a Dynal MPC magnet Invitrogen to immobilize complexed beads, and the resulting supernatant was collected. Blots were washed and incubated with horseradish peroxidase HRP -conjugated anti-mouse IgG to detect primary antibody bound to the membrane. Plates were washed with PBS Plates were incubated for 1 h at room temperature and washed as above. Plates were washed, incubated with HRP-conjugated donkey anti-mouse antibody Jackson ImmunoResearch , and washed again.
GFP-Schu S4 and intracellular cytokines were detected by flow cytometry as previously described After incubation, cells were washed once and resuspended in Flow Cytometry Staining buffer fluorescence-activated cell sorter [FACS] buffer; eBioscience. Cells were incubated with cytokine or isotype control antibodies for 20 min at room temperature. Cells were washed with PBS and stained for cleaved caspase-3 using rabbit anti-cleaved caspase-3 antibody Cell Signal Technology and then AlexaFluor anti-rabbit IgG, following the manufacturer's instructions exactly.
Samples were visualized using a Carl Zeiss LSM confocal scanning laser microscope for quantitative analysis and image acquisition. As a facultative intracellular pathogen, F. We along with others have shown that more attenuated LVS replicates exponentially in both mouse and human DCs 5 , 8. However, the efficiency of Schu S4 replication in human myeloid DCs has not been described. Since infection and intracellular replication are likely to be an integral element of Schu S4 pathogenesis, we first determined if human DCs were permissive to uptake and growth of Schu S4.
Due to the low numbers of bacteria recovered from the earliest time point, it was unlikely that all DCs in the cultures were infected. GFP-Schu S4 detected by flow cytometry was primarily intracellular since the addition of trypan blue prior to analysis did not significantly change the percentage of infected cells data not shown.
Schu S4 replicates in human DCs. Intracellular replication was monitored over time. A Schu S4 replicated logarithmically in human DCs throughout the h course of infection. Error bars represent SEM. Data are representative of four experiments of similar design.
One DC function is antigen presentation to T cells. One evasion strategy employed by successful pathogens is to interrupt this phenotypic maturation. However, this increase was significantly less than that observed with cells stimulated with E.
This increase in receptor expression reflects changes in the entire cell population uninfected and infected cells. Schu S4 elicits moderate increased expression of DC activation markers. DCs from Schu S4-infected cultures had moderate, but significant, increased expression of each marker analyzed compared to uninfected controls.
Data are representative of four experiments of similar designs. In addition to phenotypic maturation, another primary function of DCs is secretion of cytokines to aid in alerting or regulating the immune response to invading pathogens.
DCs recognize microbial products via an array of pathogen recognition receptors and secrete either pro- or anti-inflammatory cytokines following ligation of these receptors. Thus, we next determined if human DCs could respond to infection with Schu S4 via secretion of various cytokines previously shown to be crucial in developing effective immune responses against F. As expected, DCs responded to E.
Schu S4 does not elicit production of proinflammatory cytokines from DCs. Uninfected DCs treated with E. Uninfected untreated DCs served as negative controls. Given the striking lack of cytokine secretion in Schu S4-infected DCs, we next determined if Schu S4 was simply evading detection by human DCs or if it actively suppressed the ability of human DCs to respond to pathogen stimuli.
At various time points after infection, we stimulated DCs with ultrapure E. A similar pattern of poor responsiveness was observed when infected cultures were exposed to zymosan and Pam 3 CSK 4 , suggesting that the inhibition among infected DCs was not limited to responsiveness to E. Schu S4 inhibits DC responsiveness to E. As indicated, 48 h after infection cells were treated with E. Uninfected DCs served as negative controls.
Percentages in each quadrant represent the percentage of total gated forward scatter and side scatter DCs in that quadrant. To determine if live Schu S4 cells were required for the observed inhibition of responsiveness to E. This suggested that either metabolically active Schu S4, a molecule sensitive to paraformaldehyde degradation, or a molecule removed from bacterial preparations following washing of Schu S4 after fixation was responsible for suppression of DCs.
Bacteria were then washed three times to remove paraformaldehyde prior to use.
Practice for Perfection
Play along with guitar, ukulele, or piano with interactive chords and diagrams. Includes transpose, capo hints. Ludu Kaplanski Duration: — Size: Mb. Kaplanski, E. Lynch, K. Ye, E.