Differential and Temporal Immunomodulation of alpha4 Integrins on CD4+ Memory Cells by Bordetella pertussis and Bordetella parapertussis

Pertussis, caused by Bordetella pertussis (B. pertussis), is reemerging worldwide due to vaccine inefficacy. The hallmarks of infection are extreme lymphocytosis and delayed recovery, which are partially associated with pertussis toxin. Lymphocytes migrate to infected tissues using trafficking receptors. Specific combinations of these lymphocyte trafficking receptors are identified for skin and gut but are not well established for lung. This study focused on the effect of pertussis toxin on lung-associated trafficking receptors and tested the hypothesis that pertussis toxin alters dendritic cell imprinting of lung trafficking receptors on T cells, thus delaying resolution of the infection. B. pertussis-infected mice were compared with pertussis toxin-deficient strains. Imprinting of trafficking receptors on allogeneic T cells by dendritic cells derived from Bordetella-infected mice was analyzed by flow cytometry. Mice infected with Bordetella strains showed an increase in mature dendritic cells on day 5 post-infection. Despite their mature phenotype, dendritic cells from B. pertussis infection, were compromised in their ability to imprint lung trafficking receptors on allogenic T cells. These results indicated a pertussis toxin-dependent defect in dendritic cell imprinting of lung trafficking receptors on T cells. In conclusion, this study provides important data for future vaccine development against respiratory pathogens

Differential Expression of Alpha 4 Integrins on Effector Memory T Helper Cells during Bordetella Infections. Delayed Responses in Bordetella pertussis

Bordetella pertussis (B. pertussis) is the causative agent of whooping cough, a respiratory disease that is reemerging worldwide. Mechanisms of selective lymphocyte trafficking to the airways are likely to be critical in the immune response to this pathogen. We compared murine infection by B. pertussis, B. parapertussis, and a pertussis toxin-deleted B. pertussis mutant (BpΔPTX) to test the hypothesis that effector memory T-helper cells (emTh) display an altered pattern of trafficking receptor expression in B. pertussis infection due to a defect in imprinting. Increased cell recruitment to the lungs at 5 days post infection (p.i.) with B. parapertussis, and to a lesser extent with BpΔPTX, coincided with an increased frequency of circulating emTh cells expressing the mucosal-associated trafficking receptors α4β7 and α4β1 while a reduced population of these cells was observed in B. pertussis infection. These cells were highly evident in the blood and lungs in B. pertussis infection only at 25 days p.i. when B. parapertussis and BpΔPTX infections were resolved. Although at 5 days p.i., an equally high percentage of lung dendritic cells (DCs) from all infections expressed maturation markers, this expression persisted only in B. pertussis infection at 25 days p.i. Furthermore, at 5 days p.i with B. pertussis, lung DCs migration to draining lymph nodes may be compromised as evidenced by decreased frequency of CCR7+ DCs, inhibited CCR7-mediated in vitro migration, and fewer DCs in lung draining lymph nodes. Lastly, a reduced frequency of allogeneic CD4+ cells expressing α4β1 was detected following co-culture with lung DCs from B. pertussis-infected mice, suggesting a defect in DC imprinting in comparison to the other infection groups. The findings in this study suggest that B. pertussis may interfere with imprinting of lung-associated trafficking receptors on T lymphocytes leading to extended survival in the host and a prolonged course of disease

Mathematical programming and game theory

This book discusses recent developments in mathematical programming and game theory, and the application of several mathematical models to problems in finance, games, economics and graph theory. All contributing authors are eminent researchers in their respective fields, from across the world. This book contains a collection of selected papers presented at the 2017 Symposium on Mathematical Programming and Game Theory at New Delhi during 9–11 January 2017. Researchers, professionals and graduate students will find the book an essential resource for current work in mathematical programming, game theory and their applications in finance, economics and graph theory. The symposium provides a forum for new developments and applications of mathematical programming and game theory as well as an excellent opportunity to disseminate the latest major achievements and to explore new directions and perspectives

Characterization of lung CD11c⁺ cells during infection with <i>B. pertussis</i> and <i>B. parapertussis</i>.

<p>(<b>A</b>) Enumeration of lung CD11c⁺ cells during the early phase of infection with <i>Bordetella sp</i>. Mononuclear cells were isolated from lungs and enriched for DCs (CD11c⁺) as detailed. After column enrichment, the total number of CD11c⁺ cells per mouse was estimated using a hemocytometer. (<b>B</b>) CD11c⁺ enriched lung mononuclear cells were gated on singlets and then on CD11c⁺ cells (DCs). The frequency of CD11c⁺ cells expressing maturation markers was assessed at 5 days p.i. (<b>C</b>) and at 25 days p.i. (<b>D</b>), and normalized to the respective cells in uninfected control mice (1.0 by definition). In addition, the frequency of lung CD11c⁺ cells expressing CCR7 and CXCR4 at 5 days p.i. was measured (<b>E</b>). The bar charts (<b>C, D, E</b>) represent compiled data with error bars showing the SEM of four independent experiments. In each experiment, six mice were used per treatment group. *: p<0.05, and no asterisk indicates p<0.05 as analyzed by a two-tailed student's <i>t</i>-test.</p

<i>B. pertussis</i> induces a longer course of infection than <i>B. parapertussis</i>.

<p>BALB/c mice were infected intranasally with 5×10⁶ CFU of <i>Bordetella</i> strains in 20 µl of PBS, or mock infected (as detailed in the Methods section). Bacterial load in the lungs was assessed at the time points shown above. 3 mice were used at each time point. This graph represents data from two independent experiments.</p

Antibodies used to stain dendritic cells.

<p>Antibodies used to stain dendritic cells.</p

α4β7 and α4β1 imprinting on allogeneic Thy1.1⁺/CD4⁺/CD38⁺⁺ cells.

<p>Lung DCs from 5 days p.i. with one of four infection types—<i>B. pertussis</i>, <i>B. parapertussis</i>, <i>BpΔPTX</i>, or uninfected controls—were co-cultured for 4 days with naïve allogeneic Thy1.1⁺/CD4⁺ splenocytes at a 1∶5 ratio. (<b>A</b>) Lung DCs from <i>Bordetella</i> infections were used to stimulate purified naïve Thy1.1⁺/CD4⁺ splenocytes labeled with CFSE. Histograms depict CFSE dilution profiles. Approximately four cell cycles of division are observed in each system as estimated by FlowJo proliferation analysis. Low mRMS values obtained in every allogeneic system confirmed the accuracy of the analysis. (<b>B</b>) Co-cultured cells were gated on lymphocytes, followed by gating on singlets, and then on Thy1.1⁺/CD4⁺/CD38⁺⁺ cells. (<b>C</b>) Proportion of Thy1.1⁺/CD4⁺/CD38⁺⁺ cells expressing α4β7 or α4β1 following co-culture with lung DCs derived from <i>Bordetella</i> infections was measured and normalized to the respective cells from the uninfected control co-culture (1.0 by definition). The bar charts represent compiled data with error bars showing the SEM of three independent experiments. In each experiment, five mice were used per treatment group. *: p≤0.05, no asterisk: p<0.05.</p

Functional analyses of lung DCs derived from <i>Bordetella</i> infections.

<p>(<b>A</b>) <b>Transwell CCR7 dependent migration of lung DCs</b>. 5×10⁵ isolated lung DCs from <i>Bordetella</i> infections were placed in the transwell upper chamber and allowed to migrate through a 5 µm polycarbonate membrane in the presence or absence of chemokines. Migrated cells were harvested, stained for CD11c & MHC-II, and enumerated by flow cytometry. Chemotaxis index data are calculated as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052903#s2" target="_blank">Materials and Methods</a>. Depicted are combined data of three experiments. In each experiment, five mice were used per treatment group. *: p<0.05 as analyzed by a two-tailed student's <i>t</i>-test. (<b>B</b>) <b>Enumeration of CD11c⁺ cells in the mediastinal lymph nodes.</b> The mediastinal lymph nodes were harvested, stained with CD11c and CD45, and enumerated with Trubeads by flow cytometry. The absolute number of CD11c⁺ cells from each treatment was normalized to that from <i>B. pertussis</i> (1.0 by definition) as detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052903#s2" target="_blank">Materials and Methods</a>. The bar chart represents compiled data with error bars showing the SEM of three independent experiments. In each experiment, three mice were used per treatment group. *: p<0.05, and no asterisk indicates p<0.05 as analyzed by the one sample two-tailed student's <i>t</i>-test.</p

Leukocyte recruitment to the lungs of mice infected with <i>Bordetella</i> strains.

<p>(<b>A</b>) Frozen sections of lungs from BALB/c mice infected with <i>B. pertussis</i>, <i>B. parapertussis</i>, <i>BpΔPTX</i>, or uninfected controls at 5 and 25 days p.i. were H&E stained, and ten independent fields per infection were photographed under 400× magnification. Three independent experiments were performed. (<b>B</b>) Frozen lung sections from mice infected with <i>B. pertussis</i>, <i>B. parapertussis</i>, <i>BpΔPTX</i>, or uninfected controls at 5 and 25 days p.i. were stained sequentially with F4/80 (green) for macrophages, followed by CD45R/B220 (red) for B cells. Alternatively, the slides were sequentially incubated with CD3 (green) for T cells, followed by Gr-1 (red) for neutrophils. Slides were counterstained with DAPI for cell nuclei. Staining with isotype control mAbs revealed no false positive cells (not depicted). Ten separate fields were photographed from each treatment at 100× magnification on a confocal microscope.</p